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typi-minerals out under (Widdel, ate tubes nd stored
under anoxic conditions for 24 hrs. 10 ml of a reduced, buffered artificial sea water medium as used for cultivation of sulfate-reducing bacteria (Widdel, 1992), containing 15 mM metha-nol as a substrate for methanogens was transferred into the mineral-containing incubation vials. Inoculation occurred from a 99% pure, macroscopic dense culture of Methanococ-coides burtonii by transferring 200 μl volume.
Parallel to the biotic incubations, the same number of abiotic experiments was prepared.
Instead of inoculation with viable biomass, a methane headspace (0.5 bar overpressure) was applied and handled in the same way as the biotic incubations.
High hydrostatic pressure (200 bar) was applied using a high-pressure incubation device (Fig. II-5). Slow increase of the surrounding water pressure in the steel cylinder by means of a hydrostatic pump forced medium from the syringe by means of the piston into the culture tube. The culture tube (total volume 20 ml) contains a methane headspace in the case of the abiotic incubation, and a CO2/N2 headspace in the case of the biotic incubation. The volume of the medium in the syringe is slightly larger (approximately 1.2-fold) than the initial gas volume in the tube. This set-up allows complete dissolution of the gas phase and avoids im-plosion of the tube because the inner pressure can equilibrate with any external hydrostatic pressure applied. The tube was in horizontal position during incubation.
The expected final concentration of dissolved methane resulting from the methylotrophic methanogenesis amounts to 20 mM, whereas not the full methane partial pressure is expected to occur due to growth time, and ~10% is expected to used up for biomass built up. The bio-tic incubation therefore lacks the full partial gas pressure from the beginning of the experi-ment, whereas the minerals react with the full partial gas pressure from the onset of the abiotic incubation experiments.
Results of the experiments are summarized in the appendix (Appendix II).
Fig. II-5: Device for biotic and abiotic incubation of pure minerals (Sed) in seawater medium at 12 °C (Nauhaus et al., 2002). Note: headspace was CO2/N2 for biotic incubations and methane for abiotic incubations. [W=water; SC=steel cylinder; HP=hydrostatic pressure; M=medium; S=syringe; P=piston; T=tube; pi=inner pressure; pe=external
Mineralogy
II.1.9. Mineral assemblage analysis
The X-ray diffraction data were collected on a Philips/Panalytical X’Pert Pro multipurpose diffractometer equipped with a Cu-tube ((K) 1.5418 , 45 kV, 40 mA), a fixed divergence slit of ¼°, a secondary monochromator and the X’Celerator detector system. The measure-ments have been performed as a continuous scan from 3–85° 2, with output intensities recal-culated for a step size of 0.016° 2 with a data collection of 100 seconds per step. Dried bulk samples of composites and chars have been ground to a fine powder (<20μm particle size) and prepared with the Philips/Panalytical backloading system. The full pattern quantification software QUAX was applied (c.f. Vogt et al., 2002). A thorough preparation commonly in-creases reproducibility of the results, however, the standard deviation given by (Moore, 1989) of ±5% can be considered as a general guideline for mineral groups with >20% clay fraction.
In addition the determination of well crystallized minerals like quartz, calcite or aragonite can be done with better standard deviations (Tucker, 1988; Vogt et al., 2002).
II.1.10.Mineral surface area
Mineral surface areas (MSA) of sediments were determined by (i) nitrogen adsorption ac-cording to Brunauer-Emmet-Teller (BET) and (ii) by applying ethylene glycerol mono ether (EGME). On a Quantachrome Quantasorb, specific grain surface area was determined by nitrogen adsorption in a flow-through cell. Changes in heat conductivity within the N2/He gas mixture (30% N2 4.6 quality, 70% He) were measured after nitrogen adsorption to dry sedi-ments (< 5 g) at liquid nitrogen temperature followed by desorption at room temperature.
Calibration was achieved by the simulation of desorption peaks via the injection of appropri-ate amounts of N2 into the N2/He gas stream. The detection limit was 0.2 m² absolute surface area.
Sediment splits were analyzed for EGME-MSA by the method of (Cihacek, 1975). After freeze drying, 0.5g sediment was soaked in 1 ml liquid EGME and evaporated in the presence of granular CaCl2 and liquid EGME in an evacuated chamber, allowed to come to equilibrium over 3 to 5 days, and assumed to record a monolayer coating of EGME (multiple weighings insured equilibrium was achieved). In order to avoid batch-to-batch variation in EGME par-tial pressure within the evacuated chamber (which might affect molecule orientation on the surface), samples were run using identical conditions in batches of 30 samples. Standards
were used to calibrate between batches. Up to 4 replicates of samples were analyzed and av-eraged for a final value. A factor of 3.2 was used to convert mg adsorbed EGME to m2 sur-face area (Kennedy et al., 2002).
II.1.11.Sediment porosity
Porosity was determined by weight difference, before and after freeze drying the wet sedi-ment sample and subsequently, transferred into a volume ratio (volume of porewater / volume of bulk sediment) assuming a dry sediment density of 2.5 g cm-3 and a water density of 1.0 g cm-3.
Geochemistry Dissolved sulfate
Dissolved sulfate (SO4
2-) concentrations were determined on 2 ml sub-samples of filtered pore water by ion chromatography in the IFM-GEOMAR labs (Kiel, Germany). A Sykam-S was equipped with an anion exchange column (LCA A14). 7.5 mM Na2CO3-solution was used as an eluent at a flow rate of 1.75 ml min1. Samples were diluted by 1:54 with the elu-ent prior to injection.
Total organic carbon
Contents of total organic carbon (TOC) and total carbon (TC) were measured from dry and homogenized samples using a Leco CS 200 at the University Bremen, Germany. Prior to TOC analysis, the samples were treated with 12.5% HCl to remove inorganic carbon.
Intact membrane lipid analysis
II.1.12.Background to membrane lipids as biomarkers
Traces of past life are recorded in the rock record and in recent sedimentary material, either as morphological fossils and/or molecular fossils, biomarkers, that derived from biochemical precursors by reductive and oxidative processes (e.g. Brassell, 1992; Peters and Moldowan, 1993; Brocks and Summons, 2003). Biomarkers carry a wealth of information concerning the composition, ecology and diversity of past and recent microbial communities. Many biomark-ers encountered in sediments and oils have also been discovered in living organisms (land plants, algae, Bacteria, Archaea and heterotrophs), allowing recognition of their precursor lipids and the establishment of a biomarker connection (e.g. Peters and Moldowan, 1993;
Grice, 2001; Brocks and Summons, 2003). Lipids are the molecular components of cell mem-branes with examples including, sterols, hopanols, alcohols, phospholipids and ether-lipids.
Lipid biomarkers patterns can have taxonomic significance (Kaneda, 1991; Moore et al., 1994; Brocks and Pearson, 2005) and its isotopic analysis provides metabolic process infor-mation (e.g. Hayes et al., 1990; Brenna, 1997).
Phospholipid-based fatty acids (PLFAs) were validated as a measure for live bacterial biomass (White et al., 1979) because they are rapidly degraded after cell death. However, the taxonomic specificity is higher, when this biomarker concept is extended to the analysis of intact phospho- and glycolipids to also detect biomass from Archaea, which were discrimi-nated against by the PLFA approach (Sturt et al., 2004; Biddle et al., 2006; Lipp et al., 2008).
These intact polar lipids (IPLs) have a finite lifetime after cell death that is considered to be short on geological timescales (Harvey et al., 1986). IPLs provide a robust basis for estimat-ing biomass and broadly constrainestimat-ing the phylogenetic affiliation of the major contributors to the pool of biomass in natural ecosystems (Rütters et al., 2002; Zink et al., 2003; Sturt et al., 2004; Biddle et al., 2006, Lipp et al., 2008). Complementary isotopic analysis of microbial lipids sets constraints on the carbon metabolism of prokaryotes (e.g. Hinrichs et al., 1999;
Londry and Des Marais, 2003; Elvert et al., 2003; Zhang et al., 2003; Bühring et al., 2005;
Biddle et al., 2006).
Lipid-based biomarkers has been proven to be a powerful technique to indicate the process of AOM in recent and ancient marine sediments due to highly negative 13C values (Bian, 1994; Hinrichs et al., 1999; Elvert et al., 1999; Thiel et al., 1999; Elvert et al., 2000; Hinrichs et al., 2000a; Pancost et al., 2000; Hinrichs et al., 2003; Jiang et al., 2003; Birgel and Peck-mann, 2008). The biomarker approach can indicate the major prokaryotic groups involved in this process by multiple lipid signature analysis (Blumenberg et al., 2004; Rossel et al., 2008;
Niemann and Elvert, 2008), or in combination with molecular techniques (Hinrichs et al., 1999; Orphan et al., 2001a; Orphan et al., 2001b; Hinrichs et al., 2003). The genetic and me-tabolic similarity between methanogens and methanotrophs (Hallam et al., 2004) explains the observations that lipid biomarkers of both groups are almost identical. Lipid signatures indic-ative for particular ANME-Archaea include archaeol and sn2-hydroxyarchaeol, the C20 and C25, irregular tail-to-tail linked, isoprenoidal hydrocarbons 2,6,11,15-tetramethylhexadecane (crocetane) and PMI and their unsaturated homologues (crocetenes and pentamethylicosenes, PMID) as well as a variety of isoprenoidal glycerol dialkyl glycerol tetraethers (GDGTs;
Niemann and Elvert, 2008 and references therein). With the exception of crocetane, all of
these molecules were also found in methanogenic Archaea utilizing bicarbonate, acetate or methylated substrates (Niemann and Elvert, 2008 and references therein).
Microbial biomass in the form of cell membrane lipids are of common interest for studying the microbial ecology as they are available for isotopic analysis upon solvent extraction of sediments allocating valuable information on element assimilation (e.g. Freeman et al., 1990;
Hayes et al., 1990; Brenna, 1997; Hayes, 2000; Boschker and Middelburg, 2002; Galimov, 2006).
II.1.13.Liquid chromatography-multiple mass spectrometry (LC-MSn)
Two quarters of the squeeze cake sediment material from the pore water separation were stored frozen (–20°C) and used for the analysis of intact polar lipids (IPLs) and functional genes. The freeze dried sediment was spiked with an internal standard (1-O-hexadecyl-2-acetyl-sn-glycero-3-phosphocholine; platelet activation factor, PAF) prior to the modified Bligh and Dyer extraction for microbial lipids including four steps (Sturt et al., 2004), fol-lowed by 10 min centrifugation at 800 × g. The combined supernatants were washed with water and evaporated to dryness. IPLs were analyzed at the University of Bremen, Germany, by high performance liquid chromatography/electrospray ionization-multiple stage-mass spec-trometry (HPLC/ESI-MSn). A LiChrospher® Diol column (125 mm × 2.1 mm, 5 μm; Alltech Associates Inc., Deerfield, IL, USA) was fitted with a 7.5 mm × 4 mm guard column of the same packing material and was used at 30 °C in a column oven using a ThermoFinnigan Sur-veyor HPLC system. The following linear gradient of eluants was used with a flow rate of 0.2 ml min-1: 100% A to 35% A: 65% B over 45 min, then back to 100% A for 1 hr to re-equilibrate the column for the next run (A= mixture of hexane / 2-propanol / formic acid / 14.8M NH3(aq) in the portions of 79:20:0.12:0.04 v/v; and B= 2-propanol / water / formic acid / 14.8M NH3(aq), 88:10:0.12:0.04 v/v). Multiple stage mass spectrometry experiments (MSn) were performed using a ThermoFinnigan LCQ Deca XP plus ion trap mass spectrome-ter (Thermo Finnigan, San Jose, CA, USA) with an electrospray ionization inspectrome-terface (ESI).
ESI settings derived from tuning with diester-C16-phosphatidylethanolamine have been de-scribed previously (Sturt et al., 2004). A typical mass range of 500-2000 m/z was scanned while fragmenting the base peaks up to MS3. Separate experiments for positive and negative ionization modes provided complementary structural information. Compound identification is based on characteristic molecular masses of ionized IPLs shown in the mass spectra, and product ions formed by loss of the neutrally charged headgroups, indicating ether or ester bonds between the hydrophilic headgroup and the core lipid with different numbers of carbon
atoms attached (Sturt et al., 2004; Biddle et al., 2006). Relative abundances and absolute concentrations of IPLs were obtained by integration of peak areas in mass chromatograms.
Therefore, individual molecular ions of each IPL class were extracted from the full scan chromatograms and compared with the peak area of the internal standard. A similar response factor for the internal standard was assumed as for IPLs.
Phylogenetic analysis
II.1.14.Background of molecular-genetic techniques
An extremely powerful technique used to identify microorganisms in the environment is the polymerase chain reaction (PCR) that is based on DNA or RNA extracts (e.g. Teske, 2008). Most of the current knowledge concerning deep subsurface biogeography has been obtained by PCR-based techniques (e.g. Inagaki et al., 2006). Unfortunately, a major limita-tion of PCR is that only phylotypes containing matching (or nearly matching) priming sites may be detected that causes a bias in the detection of environmentally relevant microorgan-isms (Teske, 2008). The detection of, e.g. subsurface archaeal lineages ultimately depends on the specificity of the archaeal PCR 16S rRNA primers that developments and improvements are crucial (Teske, 2008). Genomic markers for anaerobic microbial processes in marine se-diment such as sulfate reduction, methanogenesis, and anaerobic methane oxidation, reveal the structure of sulfate-reducing, methanogenic, and methane-oxidizing microbial communi-ties (including uncultured members); they allow inferences about the evolution of these an-cient microbial pathways; and they open genomic windows into extreme microbial habitats, such as deep subsurface sediments and hydrothermal vents, that are analogs for the early Earth and for extraterrestrial microbiota (Teske et al., 2003).
Recent metagenomic studies have identified a modified methyl-coenzyme M-reductase (MCR, “methanase”) which may catalyze the activation of methane under anoxic conditions (Hallam et al., 2003; Hallam et al., 2004; Krüger et al., 2003). It catalyzes the final step of methanogenesis in which the methyl group linked to coenzyme M is reduced with formation of methane (Ellermann, 1988; Deppenmeier, 2002). This enzyme is present in all known me-thanogens, and unlike many other enzymes in the methanogenic pathway, it is absent from non-methanogenic Archaea and Bacteria (e.g. Thauer, 1998; Bapteste, 2005). The functional alpha subunit of MCR (mcrA) is the key enzyme of methanogenesis and therefore a powerful tool to detect and differentiate methane-producing and consuming prokaryotes in
environmen-tal samples (Bokranz, 1988; Klein, 1988; Weil, 1989; Thauer, 1998; Lueders et al., 2001;
Krüger et al., 2003; Hallam et al., 2003).
II.1.15.Protocol of DNA extraction and phylogenetic analysis
DNA was extracted following the protocol as outlined in (Biddle et al., 2008) except that the pH of the extraction buffer and phenol was raised to 8.0, the bead beating time reduced to 15 s, and the bead beating speed reduced to 4.0 (Qbiogene, Carlsbad, CA). Moreover, the DNAse incubation was omitted, and DNA purified with the PowerClean DNA Clean-Up Kit (MOBIO laboratories, Carlsbad, CA) rather than the RNeasy Mini Kit (Qiagen, Valencia, CA).
To target mcrA genes we used the previously designed general mcrIRD and the ANME-1-specific ANME-1-mcrI primer pairs (Lever et al., in prep.). The mcrIRD primer is a reduced-degeneracy version (Lever et al., in prep.) of the mcrI primer (Springer et al., 1995) that tar-gets mcrA genes of all known methanogens and anaerobic methanotrophs except ANME-1.
The mcrIRD primer had previously been found to have a lower detection limit and cover a wider phylogenetic breadth of mcrA genes than two previously published general mcrA pri-mer pairs, the mcrI (Springer et al., 1995), and ME1/ME2 (Hales et al., 1996) pripri-mer pairs.
Since neither the mcrIRD, nor the mcrI or ME1/ME2 primer pairs target ANME-1-mcrA genes with high sensitivity (Lever et al., in prep.), we used the ANME-1-specific ANME-mcrI primer pair in addition to the mcrIRD primer pair. PCR assays were performed using the Ta-kara SpeedSTAR HS DNA polymerase kit (TaKaRa Bio USA, Madison, WI) using the fol-lowing PCR protocol: (1) 1 u 2 min denaturation (98°C), (2) 40 u (a) 10 s denaturation (98°C), (b) 30 s annealing, (c) 1 min extension (72°C), and (3) 1u5 min extension (72°C).
To estimate minimum mcrA copy numbers, we performed PCR with dilutions of DNA ex-tracts (1:3, 1:10, 1:30, 1:100, etc.). The minimum mcrA copy number of a sample (per g se-diment) was then calculated from (1) the amount of sediment used in the extraction (in g), (2) the volume of the eluent in which extracted DNA was dissolved, (3) the highest dilution that rendered PCR detection, and (4) the volume of that dilution that was used in the PCR.
III. MA N U S C R I P T 1
The biogeochemical significance of gaseous hydrocarbons sorbed to marine sediments
Tobias F. Ertefai1,*, Verena B. Heuer1, Xavier Prieto-Mollar1, Christoph Vogt2, Sean Sylva3, Jeffrey Seewald3, Kai-Uwe Hinrichs1,4,*
1Organic Geochemistry Group, MARUM - Center for Marine Environmental Sciences and Department of Geosciences, University of Bremen, Leobener Str., D-28359 Bremen, Germa-ny
2Central Laboratory for Crystallography and Applied Material Sciences, Department of Geos-ciences, University of Bremen, Post Box 330440, D-28334 Bremen, Germany
3Woods Hole Oceanographic Institution, Department of Marine Chemistry and Geochemistry, Woods Hole, MA 02543, USA
4Woods Hole Oceanographic Institution, Department of Geology and Geophysics, Woods Hole, MA 02543, USA
*Corresponding authors: Tobias Ertefai, Tel.: (+49) 421-218-65709; email: to-bias.ertefai@uni-bremen.de; Kai-Uwe Hinrichs, Tel.: (+49) 218-65700; Fax: (+49) 421-218-65715; email: khinrichs@uni-bremen.de
(submission to Geochimica et Cosmochimica Acta)
___________________________________________________________________________
Abstract
Sorption of hydrocarbon gases (HCs) to marine sediments is a recognized phenomenon that has been investigated mostly in the context of petroleum exploration. However, little is known about the mechanisms of sorption and the importance of biologically produced HCs.
In this study, we sought to constrain quantities and sources of sorbed HCs, its major sorbents and sorption mechanisms and its potential importance for sedimentary biogeochemistry. We applied geochemical and mineralogical analysis to 431 sediment samples from different ocea-nographic settings and geochemical regimes, integrated efficiency tests of extraction protocols for sorbed HCs, and used high pressure equipment to experimentally infer sorption capacities of clay minerals. Significant amounts of biogenic methane were liberated from all samples,
regardless of the pore water geochemistry. Alkaline conditions mostly yielded more HCs than the established acidic extraction, whereas both methods indicated the importance of bio-logical methane production. Ethane to hexane were not restricted to cold seep settings or sul-fate-free sediments. C2+ HCs were selectively retained according to their carbon number, and therefore showed opposite abundance patterns compared to the dissolved gas phase. Metha-nogenic sediments with a high quartz/phyllosilicate ratio released particularly high amounts of sorbed methane (up to 2.1 mmol kg-1 dry sediment). Both, clay-rich or sulfate-reducing envi-ronments displayed strong partitioning of HCs in favor of the sorbed reservoir. The minera-logical data set did not show significant correlations with inorganic minerals or organic car-bon, whereas experimental sorption experiments pointed to the importance of hydrophobic siloxane patches of tetrahedral silicate sheets as sorbent sites. Microbial production and con-sumption of methane, even in sulfate-replete environments, affected the sorbed methane pool with resulting stable isotopic compositions ranging from –44 to –84‰ vs. VPDB (N=225) for carbon, and from –194 to –203‰ vs. SMOW (N=7) for hydrogen, respectively.
Introduction
Enormous amounts of gaseous hydrocarbons (HCs) can accumulate as dissolved, free and hydrate-bound gas in marine sediments due to the microbial degradation of organic matter (OM; e.g. Claypool and Kaplan, 1974; Claypool and Kvenvolden, 1983; Reeburgh, 2007).
Volatile HCs (C1 to C6) sorbed to the solid phase are not considered as quantitatively and bio-geochemically important. Sorbed HC gases have been analyzed for decades in surface geo-chemistry to infer type and maturity of OM of petroleum reservoirs under the premise that thermogenic gases migrate upwards and accumulate in surface sediments causing quantitative HC anomalies (e.g. Horvitz, 1972; Horvitz, 1985; Price, 1986; Abrams, 2005). Anomalies not macroscopically visible but detectable as micromolar concentrations, however, bear the risk of exploration failures due to secondary alteration processes that change the molecular and isotopic compositions of the reservoir gases extracted from surface sediments. The lack of understanding of migration processes, selective sorption and microbial decay of HC gases recurrently invoke studies on sorbed HCs (e.g. Prinzhofer and Pernaton, 1997; Zhang and Krooss, 2001; Cheng and Huang, 2004). Key issues to understand are molecular and isotopic fractionation due to natural processes and laboratory sample handling (Abrams, 2005). The analysis of sorbed HCs is controversially discussed and described as unconventional (e.g.
Price, 1986; Abrams, 2005). Only anomalies well above background HC concentrations safe-ly indicate microseepage of thermogenic HC reservoirs, whereas the background has to be
defined in each survey for every setting (Abrams, 2005). Despite extensive research efforts, quantities of sorbed HC gases in marine sediments, their sources in environments without petroleum reservoirs underneath, the major sorbents and sorption mechanisms, and the role of sorbed HCs in biogeochemical processes are still poorly constrained.
To liberate sorbed HCs, different techniques have been applied over the past 80 years, while all protocols have the uncertainty about the full recovery and originality of molecular and isotopic compositions in common (e.g. Horvitz, 1972; Pflaum, 1989; Abrams, 2005).
Horvitz (1972) was the first to establish a protocol for the extraction of sorbed HCs with hot phosphoric acid. This widely applied method is thought to quantitatively separate the easily escapable biogenic gas (e.g. dissolved and free methane) from the sorbed fraction in order to analyze the thermogenic gas composition (e.g. Horvitz, 1981; Faber and Stahl, 1983; Abrams, 1996a). Sorbed HCs are commonly obtained by acidification and heating under vacuum of the fine fraction (< 63 μm) of a sample obtained by wet sieving and prior removal of the free gas via pumping. Remarkably uniform results have been described ever since, mainly for geological settings associated with thermogenic reservoirs in the subsurface (e.g. Gulf of Mexico). Accordingly, sorbed HCs are thought to occur in low and narrow concentration ranges, with stable carbon isotopic compositions (13C) indicative of thermogenic gases (Hor-vitz, 1981; Faber and Stahl, 1983; Faber and Stahl, 1984; Whiticar and Faber, 1989; Whiticar and Suess, 1990a; Whiticar et al., 1995; Abrams, 1996a; Brekke et al., 1997; Knies et al., 2004). From these reports, sorbed methane concentrations typically range from 1 to 100 μmol kg-1 (16-1600 ppb by weight of wet sediment) accompanied by individual higher HCs below 6 μmol kg-1 (< 100 ppb) (e.g. Faber and Stahl, 1983; Whiticar and Faber, 1989; Whiticar and Suess, 1990a; Whiticar et al., 1995; Brekke et al., 1997). Maximum methane concentrations of 331 μmol per kg wet sediment were reported in two studies conducted in the northern North Sea and Arctic ocean (Brekke et al., 1997; Knies et al., 2004, respectively). In almost all instances, the sorbed methane pool was enriched in 13C with -values ranging from –45‰
to –22‰ (e.g. Whiticar and Suess, 1990a; Whiticar and Faber, 1989). Molecular ratios of C1
vs. C2+ gases were commonly below 100 (e.g. Faber and Stahl, 1984; Whiticar and Suess, 1990a). Negative carbon isotopic values indicative of biogenic methane were declared as artifacts and contaminations; biogenic methane was thought to be restricted to the early di-agenetic zones of methanogenesis right below sulfate-methane interfaces (SMIs; Whiticar and Suess, 1990b).
For sorbed HCs and other HC reservoirs, distinct formation histories, and thus, sources are described. Past seepage and migration of thermogenic gases through faults are thought to
initially charge the sedimentary matrix with thermogenic HCs (e.g. Whiticar and Suess, 1990a; Knies et al., 2004). Biogenic HCs, especially methane which is produced in situ in the pore space of sediments during early diagenesis is described to overprint the signal of sorbed thermogenic HCs, as the latter gases are sorbed and therefore isolated and well protected from microbial alteration processes (e.g. Whiticar and Faber, 1989; Whiticar and Suess, 1990a;
Faber and Stahl, 1983; Abrams, 1996b). The biological formation of C2+ HC gases is known but described to account for minor fractions that are less relevant for sorption processes in surface geochemical surveys (e.g. Price, 1986; Abrams, 2005).
Sorption mechanisms and major sorbents for gaseous HCs are poorly constrained. The chemical properties of gaseous HCs restricts sorption mechanisms to physisorption and physi-cal entrapment that include van der Waals forces (London dispersion forces) and capillary condensation effects (e.g. Rouquerol, 1999). Frequently discussed sorbents are carbonate minerals, silicates/clays and OM. An occlusion of HCs by precipitating carbonate minerals, especially during the anaerobic oxidation of methane (AOM) has been discussed to cause a good preservation mechanism and lead to the acid extraction approach (Horvitz, 1972;
Thompson, 1974; Price, 1986; Pflaum, 1989). Specific carbonate minerals such as dolomites were suspected to merely trap HC gases (Thompson, 1974), whereas it was recently doubted that carbonates at all would account for recently described variable HC concentrations due to a lack of correlation with carbonate contents (Knies et al., 2004). It is doubtful that OM is a major sorbent for gaseous HCs. On the one hand, experimental work showed increasing iso-topic depletion of the heavier methane isotope (13C) during methane diffusion through shales with increasing TOC contents due to the different effective diffusion coefficients of 13C and
12C-methane (Zhang and Krooss, 2001). On the other hand, environmental samples did not show a correlation between the concentration of sorbed HC gases and OM in sediments off Peru (Whiticar and Suess, 1990b; Hinrichs et al., 2006). Clay minerals are considered impor-tant host phases of particulate and dissolved organic carbon (POC and DOC) (e.g. Ransom et al., 1997; Baldock and Skjemstad, 2000; Wattel-Koekkoek et al., 2001). Clays are common constituents of marine sediments and are classified according to the ratio of tetrahedral to oc-tahedral structural sheets. Smectite clays are composed of two tetrahedral and one ococ-tahedral layers and are termed 2:1 clays, whereas group of kaolinite is composed of only one of each layer type, 1:1 clay (e.g. Sposito et al., 1999). Particularly swellable clays such as smectites have an extensive accumulation potential for DOC (e.g. Kaiser and Guggenberger, 2000; Ka-wahigashi et al., 2006; Kennedy et al., 2002). In earlier studies, clay minerals were not thought to trap gaseous HCs efficiently (Horvitz, 1972; Stoessell, 1982; Pflaum, 1989;
Ab-rams, 1996a), but more recent findings support the idea of clay minerals as major sorbents for HC gases (Sposito et al., 1999; Sugimoto et al., 2003; Hinrichs et al., 2006). Interlayers of smectites are likely sites for the accumulation of HC molecules, and may even serve as nuc-leation sites for gas hydrate (Guggenheim and van Groos, 2003).
There is growing evidence that sorbed HC gases in marine environments are more diverse in their concentrations and isotopic compositions than known from petroleum-related surface geochemistry. Sizeable quantities of HCs may be bound to near-surface sediments (Fleischer, 2001; Knies et al., 2004). The occurrence of gaseous HCs in sulfate-rich deep subsurface sediments of the Peruvian Basin implies unusual metabolic pathways of microorganisms re-sulting in the biological formation of ethane and propane, and hence, a biogeochemical and geomicrobiological relevance (Hinrichs et al., 2006). The concept offered by Hinrichs et al.
(2006) might be valid for other settings and surface sediments, as higher HCs were found to be of biological origin in shelf sediments as well (Welhan et al., 1980; Oremland, 1981).
These indications of the importance of sorbed HCs motivated us to examine the pool of sorbed HC gases in the marine environment in order to (i) assess its quantity and distribution, sources and isotopic compositions in different geochemical regimes, (ii) clarify its role in bio-geochemical processes, and (iii) to reveal potential sorbents and sorption mechanisms. We tested experimentally whether clay minerals act as major sorbents. X-ray diffraction of the environmental samples was performed to investigate potential mineral controls on the amount of sorbed HCs extracted at alkaline conditions. This study is the first which systematically assesses the relationship of sorbed HC gases to other biogeochemical properties of the sedi-ment.
Results and discussion
Evaluation of alkaline extraction procedure
The extraction of sorbed HCs at alkaline conditions is a new method that was only recently introduced (Hinrichs et al., 2006). To test and evaluate this method, we compared its extrac-tion efficiency to the standard extracextrac-tion procedure which involves hot phosphoric acid solu-tion (Horvitz, 1981; Faber and Stahl, 1983). Autoclavasolu-tion of lake sediment to extract ga-seous HCs was previously reported (Sugimoto et al., 2003) and additionally compared to the alkaline approach in this study. We used a choice of samples from four marine provinces with at least 1800 m water depth that cover various methane contents, ranging from gas hy-drate-bearing sediments to the methane depleted sulfate-reducing zone of seep sediments (Table II-1).
Methane was the major compound of the extracted HC gas pool, independent of the ap-plied extraction conditions, but the amount of released methane differed substantially between the different protocols (Table III-1). Autoclavation always liberated significantly smaller amounts of methane than the cold NaOH- or hot H3PO4 protocol. This observation confirms the need for harsh chemical conditions to liberate sorbed HCs from solid matrices, as reported earlier (Horvitz, 1972; Pflaum, 1989). The alkaline solution released up to 2058 μmol of me-thane per kg of dry sediment (Table III-1). These concentrations of sorbed meme-thane exceed previously reported values substantially (cf. Faber and Stahl, 1983; Whiticar and Faber, 1989;
Whiticar and Suess, 1990a; Whiticar et al., 1995). The maximal methane yield of acid extrac-tion, 717 μmol kg-1 at the hydrate bearing IODP Site U1326 (Table III-1), was higher than any previous finding for sorbed methane extracted with phosphoric acid (Brekke et al., 1997;
Knies et al., 2004). In general, acidic conditions released less methane than alkaline condi-tions, but in a few cases, methane yields of the hot acid extraction were similar to those of the cold alkaline extraction or higher. Concentrations of higher HCs were generally below 5 μmol kg-1 with maxima ranging from 8 to 15 μmol kg-1; only one sample showed all alkanes from C1 to C6 (Table III-1). The alkaline slurry of the Dvurechenskii mud volcano (DMV) yielded exceptionally high amounts of n-hexane (214 μmol kg-1), an observation that remains unexplained so far.
Table III-1: Concentrations and carbon and hydrogen isotopic values of sorbed HC gases extracted from marine sediments at different conditions.
The release of gaseous HCs at alkaline conditions typically proceeded at a constant rate and was often complete after 30 days (Fig. III-1). Subsequent to the leaching that yielded 41 to 2058 μmol methane per kg dry sediment (cf. Table III-1), sediment slurries were subjected to hot phosphoric acid solution in order to test whether more HCs could be extracted. Me-thane of 0.07 to 4 μmol kg-1 dry sediment was detected that accounted for less than 0.2% of the alkaline yield (not shown). The reproducibility of both the alkaline and acid extraction was low and suggested a considerable variability in the extraction efficiency and/or strong sediment heterogeneities (Table III-1). Poor reproducibility and contradicting results when applying different protocols for the extraction of gaseous HCs have been described in pre-vious reports, and were commonly attributed to sediment heterogeneities and/or biases due to sediment handling, storage or extraction procedure (e.g. Brekke et al., 1997; Abrams, 2005).
Though the compared extraction protocols differed strongly with respect to methane yields, carbon and hydrogen isotopic compositions of the released methane were similar for all three methods and for repeated extractions. Differences were smaller than 4‰ for methane-carbon and 10‰ for methane-hydrogen. The only exception was the large difference of ~18‰ be-tween the acidic and alkaline protocol observed for methane-carbon of the Juan de Fuca Ridge sample material (Table III-1).
Fig. III-1: Kinetics of release of sorbed methane extracted at alkaline conditions from marine sediments (A) and clay minerals (B) after autoclave incubation with dissolved methane
in sorption experiments. Different release kinetics were observed within the group of smectite minerals, and in environmental samples containing high and low sorbed me-thane concentrations.
Experimental sorption of methane to minerals
In laboratory sorption experiments, we induced the uptake of dissolved methane by pure mineral phases in stainless steel containers (Fig. II-4), and subsequently extracted sorbed me-thane with the alkaline extraction protocol (Fig. II-2). Technical limitations (no stirring to facilitate solubilization of methane within pressure vessels) and the uncertainty of the equili-brium concentrations (concentrations of dissolved methane within the vessels did not reach fully the expected magnitude), required to relate maximal dissolved methane concentrations to sorbed concentrations. Hence, our data do not represent sorption capacities in the classical sense (e.g. Rouquerol, 1999), but indicate empirical distribution coefficients between the sorbed and dissolved gas phase. For example, the relationship of dissolved and sorbed me-thane obtained from Na-montmorillonite experiments, showed significant deviation from a type V isotherm indicating weak sorbate-sorbent interactions (not shown). Despite these limi-tations, our results provide useful information on the uptake and release of dissolved methane by clay minerals.
The yield of extracted methane subsequent to the abiotic autoclave incubation differed among the studied sorbents (Table III-2). Incubation time differed but did not show any cor-relation to sorbed methane concentrations suggesting that the uptake of methane was rapid during and/or after solubilization. The swelling clays saponite, Ca-bentonite and Na-montmorillonite and zeolite showed the highest concentrations of sorbed methane of ~2 mmol kg-1 dry sediment, followed by kaolinite, powdery CaCO3, illite and sand (Table III-2). This order of decreasing methane uptake/release is consistent with characteristic features of the studied sorbents. Swelling clays and zeolites are good sorbents because of their regular pore system within their crystal structures (e.g. Rouquerol, 1999). The very low grain size of the fine carbonate powder was most likely the reason for the enhanced uptake compared to illite or quartz minerals. The latter showed the lowest sorption affinity (Table III-2).
Table III-2: Experimentally derived distribution of dissolved and sorbed methane in the pres-ence of different inorganic minerals.
For comparison, three replicate Na-montmorillonite experiments directly subjected to alkaline solution without prior autoclave incubation showed methane concentrations of 103 ± 88 nmol kg-1 that equaled 0.1 to 0.5 ppmv in the headspace of the reaction containers (data not shown).
Similar to the environmental samples described above, alkaline leaching and subsequent release of methane from methane-charged clay phases proceeded at constant rates and was complete after 20 days for most clay experiments (Fig. III-1B). Duplicate Na-montmorillonite experiments, however, differed by up to 43 days (Fig. III-1B). The release rate of methane from pure clay minerals was similar to environmental samples (Fig. III-1A).
Environmental samples associated with high amounts of sorbed methane (in our data set >
300 μmol kg-1) exhibited the same rates as the clays, whereas samples within the same sedi-ment core of few meters length could differ in similar fashion as the clays (Fig. III-1). Mine-ralogical differences were too small to satisfactorily explain the strong quantitative variations of sorbed methane between sites and geochemical regimes.
Compared to previously published sorption capacities which were obtained from concen-tration changes in the headspace of pressure vessels (Cheng and Huang, 2004), but not from the extraction subsequent to the charging with aqueous hydrocarbons in autoclave containers, the presented average concentrations of sorbed methane extracted from 1:1 and 2:1 layered clays were a factor of 2 to 20 lower (Table III-2). The discrepancy likely represents the loose-ly bound gas fraction which easiloose-ly escapes upon the retrieval of material.
Experimental limitations and/or natural variability of sorption processes (or uncontrollable changes in experimental conditions) likely caused the low reproducibility of the sorption ca-pacities. Heterogeneous reactive surface areas are linked to different degrees of surface
sol-condensation of methane may have contributed to sorption of methane to saturated siloxane units of clay minerals. Alternatively, hydrophobic effects and capillary condensation might have been differently important in conjunction with hydrated surface areas. Our experiments indicate the potential of the siloxane sites, since the alkaline solution lead, first, to significant higher yields compared to other chemicals (formalin, HgCl2; data not shown), and, second, to the collapse of the smectite crystal lattices proved by the missing (001)-reflex of Na-montmorillonite during subsequent XRD-analysis.
Our experimental data show that clays are effective sorbents for gaseous HCs. This is in good agreement with theoretical observations (Sposito et al., 1999), other experimental work (Cheng and Huang, 2004), and sorbed HC distributions observed in clay-rich environments (Sugimoto et al., 2003; Hinrichs et al., 2006).
Sorbed methane in marine sediments
With the alkaline extraction of gaseous HCs from numerous environmental sediment sam-ples we intended to better quantify the natural abundance of sorbed HCs, to identify its rela-tion to sediment mineralogy and to evaluate its role in biogeochemical processes. Concentra-tions of sorbed methane ranged from 50 nmol to 2.1 mmol kg-1 dry sediment (N=431), with data significantly deviating from a normal distribution (positive skewness 1.95; p<0.005).
We observed (i) the presence of sorbed methane in every sample; (ii) quantitative differences between the studied settings, and (iii) changes in abundance that occurred vertically with se-diment depth.
In order to facilitate intra- and inter-site comparisons of sorbed methane pools, we related the concentration and carbon isotopic composition of sorbed methane to the dissolved me-thane pool (Fig. III-2). Our on-board analyses of dissolved meme-thane provide merely an esti-mate of the minimal in situ concentrations since depressurization during core recovery goes along with outgassing and loss of dissolved gases (Dickens et al., 1997; Paull and Ussler, 2001). However, depressurization does not alter the qualitative and isotopic composition of a gas mixture and is a common tool to determine the prevailing gas geochemistry at the site of interest (e.g. Claypool and Kaplan, 1974; Reeburgh, 1976; Alperin et al., 1988; Whiticar, 1999). We found a poor correlation between the concentrations of sorbed and dissolved me-thane (Fig. III-2A), while the stable carbon and hydrogen isotopic compositions of meme-thane were generally similar in both pools and only deviated in sulfate-reduction zones (Fig.
III-2B).