containe reported whole c
%) in th mixed l an 8-10 of clay quartz t organic 25%) do a signif (18%) a (Fig. IV therefor
comprises minated) de ed carbonat d for both u core.
-1: Litholo cated in TTR15; se to U3 indi grey shade lithostratigr he upper 36 layer clays
% quartz fr minerals (5 to phyllosili rich layer ( ominated ov ficant miner at 191 cmbs V-1B). The re shifts the
late Pleisto eposited und te-cemented units. No o
ogical, mine the south-w ee Akhmetz icate the typ ed area desi raphy is ref 6 cm (Unit (16-25%) a raction (cf. F 50-52%), wh icates is low (Fig. IV-1B ver kaolinit ralogical ch sf, while the e clay cont
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1) derived and a total c Fig. IV-1B) hile the qua west in Uni B). In Unit e and smect hange. The
e clay conte ent increase yllosilicate r
arly Holoce ne condition up to 1 cm ns of gas hy
nd geochem ack Sea (l al., 2007 an Sea lithostr sulfate-redu he solid pha
from cocco clay minera ). The sapro artz fraction it 2 with the 2, especiall tite (2-4, an e relative pr ent is lowes es in the lo ratio to lowe
ene homoge ns (Ross an thickness.
ydrates or fl
mical charac long. 41°45 d Table II-1 ratigraphic u uction zone.
ases. The h olith ooze ( al fraction o opel Unit 2 n was the sa e exception ly illite (9-3 nd 7-10%, re
roportion of st (24-28%) ower sectio er values (F
enous clay nd Degens 1 A hydroge luid flow w
cterization o 5.55, lat. 2 1 in chapter units (see te .
high calcite (not shown) of 40-44% a contained t ame as in U
of the upp 39%) and m espectively) f quartz clim ) in the upp
n, while qu ig. IV-1B).
(partly mm 1974). Unit
n sulfide sm were observe
of the study 28°55.50, B r II for deta ext for detai
content (19 ). High con are accompa the highest Unit 1. The per most par mixed layer c
). Unit 3 re mbs to a m er section o uartz decrea
m to cm-t 1 and 2 mell was ed in the
y site lo-BS340G, ails). U1 ils). The
9-30 wt.-ntents of anied by contents e ratio of rt of this clays (0-epresents maximum of Unit 3 ases and
The content of TOC gradually increases from the seafloor to the base of Unit 2 from 2.7 to 4.6%, while it swiftly dropped to ~0.5% and remained constant throughout Unit 3 (data not shown). The interstitial water volume occupied 88-80% of the sample volume in the upper 120 cm of the core, and accounted for 69-67% below 150 cmbsf (Fig. IV-1B).
The BET-minerals surface area showed constant values of ~13 m² g-1 in the upper 36 cmbsf, dropped to 6 m² g-1 within Unit 2, and then gradually increased to the maximum of 30 m² g-1 at the base of the Unit 3 (data not shown). The EGME-mineral surface area stayed rather constant within Unit 1 and 2 ranging from 253-306 m² g-1, with a slight minimum at 95 cmbsf which coincided with a drop in TOC (data not shown). The slight gradual increase in mineral surface area in Unit 3 ranged from 164 to 209 m² g-1.
Sulfate reduction was concentrated in the top 30 cm, where dissolved sulfate concentra-tions decreased from 9.4 to < 1 mM. Dissolved sulfate remained at t290 μM over the 3-m core (Fig. IV-1C). Below 30 cmbsf, dissolved methane concentrations increased rapidly to about 12 mM in the upper methanogenesis zone, dropped within Unit 2, showed a second maximum (10.7 mM) between 160 and 203 cmbsf, before declining to ~3 mM (Fig. IV-1C).
The two methane maxima occurred in zones with elevated quartz/phyllosilicate ratios, whe-reas minima were associated with lower ratios (Fig. IV-1B). The dissolved methane concen-trations exceeded ambient but not in situ solubility values (Duan et al., 1992). The mound-like structural anomaly of the study site motivated to test for the presence of gas hydrate.
Model calculations after Tishchenko et al. (2005), suggests that dissolved methane concentra-tions of ~88 mM are necessary oversaturate porewaters with methane in order to induce hy-drate formation at this site. No macroscopic and geochemical indications point towards me-thane hydrates, as concentration profiles of chloride (340-345 mM) and bromide (516-526 μM), and 18O of pore water at the bottom of the core (data not shown) were stable and un-suspicious. Besides methane, small amounts of C2 to C6 hydrocarbon gases were liberated from the pore water ranging from 7-27 μM in concentration (no profiles shown).
The profile of sorbed methane concentration correlates with the dissolved methane apart from the upper section of the core. The same two maxima and minima can be observed at 74 and 203 cmbsf (Fig. IV-1D). The sorbed methane profile does not mirror the dissolved me-thane in the upper 60 cm comprising the sulfate-reduction and upper methanogenesis zone (Fig. IV-1D). In sulfate-reducing sediments sorbed methane was significant (up to 37 μmol kg-1). In the upper methanogenesis zone (between 32-74 cmbsf), sorbed methane increased to
310 μmol kg-1, before declining to 26 μmol kg-1 in the lower sapropelic Unit 2, and rising again to its maximum of 1620 μmol kg-1 at 203 cmbsf (Fig. IV-1D). Hence, minima and maxima of sorbed methane concentrations are associated with low and high quartz/phyllosilicate ratios, respectively as did the dissolved methane concentration (Fig.
IV-1C). The base leaching time for most of the samples amounted to 180 days, whereas for the methane-rich interval at 203 cmbsf only 30 days were necessary to obtain 93% of the “to-tal” amount sorbed (data not shown). No higher hydrocarbon gases were liberated from the solid phase.
The carbon isotopic compositions of dissolved and sorbed methane are similar in trend and magnitude. values ranged from –51 to –69‰ for the dissolved phase, and from –57 to – 72‰ for the sorbed fraction, while both gas pools showed 13C-enrichement with sediment depth (Fig. IV-1E). In most of the core, sorbed methane is depleted in 13C relative to the dis-solved methane pool, with the strongest depletion in the upper zone of methanogenesis (60-74 cmbsf); there are, however, also horizons in the sulfate-reduction zone where it is enriched in
13C (Fig. IV-1E).
Functional gene diversity and abundance
The minimum copy number of functional genes increased significantly from the seafloor to the upper part of the methanogenesis zone at 30-33 cmbsf, remained stable to 74 cmbsf, and decreased below, with the sharpest decrease appearing in the sapropelic Unit 2 (Fig. IV-2A).
With the exception of the uppermost sample (0-3 cmbsf), the copy number of ANME-1-mcrA genes appeared to equal or exceed that of all other mcrA genes combined (Fig. IV-2A).
The diversity of the methanotrophic/methanogenic community increases from the sulfate-reduction zone to the methanogenesis zone. At the seafloor, genes of the reportedly methano-trophic ANME-1 and -3 clades (Orphan et al., 2001b); (Niemann and Amann, 2006), and the Unidentified Rice Field Soil mcrA (URFS) were detected (Table IV-1). At the base of the SMTZ at 30-33 cmbsf, genes of Methanogenium, URFS mcrA, Fen Cluster and the ANME-1, -2 and -3 clades were detected (Table IV-1). The highest diversity of mcrA genes occurred at 70-73 cmbsf which is the upper most part of Unit 2 (Table IV-1). Here, Methanogenium, the Rice Cluster I, Methanosaeta, and Methanosarcina phylotypes occurred along with the URFS mcrA, Fen Cluster and ANME-1 and -2 (Table IV-1). The diversity declined considerably within Unit 2, and was restricted to genes of ANME-1 and the genera Methanosaeta and Rice Cluster I (Table IV-1). In close spatial vicinity of the elevated methane contents (Fig. IV-1) was the biodiversity as low as in the superimposed horizon, but differed in the occurrence of
Methanoculleus that joined Methanosaeta and ANME-1 genes (Table IV-1). The same gene-ra were present in the lowermost section, with the exception that the same URFS phylotype from above was detected again (Table IV-1). The phylogenetic relationship of detected me-thanogenic/methanotrophic phylotypes are displayed in the appendix (Appendix III, Fig:
Fig. IV-2: Vertical distribution of universal and specific primer pairs and intact Archaeal membrane lipids at the study site (SW Black Sea: long. 41°45.55, lat. 28°55.50, BS340G, TTR15; see Akhmetzhanov, et al., 2007 and Table II-1 in chapter II for de-tails).
The analysis of IPLs yielded one group of membrane lipids specific for Archaea. The iso-prenoidal glycerol dialkyl glycerol tetraethers (GDGTs) contained two glucose moieties as polar headgroup forming the 2-Gly-GDGTs. Their concentrations varied with sediment depth. The first occurrence at 60 cmbsf represents the maximum concentration of 14.6 μg kg
-1 dry sediment (Fig. IV-2B). A secondary maximum was observed in the upper methanoge-nesis zone at 72 cmbsf, before the concentrations declined and dropped to zero within the sapropelic Unit 2 (Fig. IV-2B). At 195 cmbsf, the intact tetraethers occurred again before their absence in the deepest parts of the core (Fig. IV-2B). The analysis of cleaved and trans-formed hydrocarbon chains upon HI treatment of the 2Gly-GDGTs did not yield enough ma-terial to obtain stable carbon isotopic values.
Table IV-1: Clone numbers of the methane-cycling microbial community with indicated me-tabolic methane formation pathways at the study site located in the south-western Black Sea (long. 41°45.55, lat. 28°55.50, BS340G, TTR15; see Akhmetzhanov, et al., 2007 and Table II-1 in chapter II for details).
Mineralogy and its impact on geochemistry and microbiota
The ratio of quartz to phyllosilicates indicates variations in mineral grain size, as quartz and phyllosilicates are commonly associated with different grain size fractions (Tucker, 1988;
Vogt, 1997). The observed changes correlate with the lithostratigraphy, and thus, with the Black Sea evolutionary history which is influenced by climatic variations affecting sedimenta-ry input due to eustatic sea-level changes, and changes in vegetation and glaciation of the hin-terlands (Ross and Degens 1974; Major, 2002; Bahr et al., 2005). However, the mineralogical changes do not correlate with surface area measurements via BET and EGME (cf. Fig. III-5).
Dissolved methane concentrations from methane-rich environments in great water depth have to be cautiously interpreted, as boundary conditions (temperature and pressure) changes severely during core retrieval. The sediment interstitial water dissolved gases will partition between dissolved and free (vapor) depending upon the new boundary conditions. The pre-sumably unreliable geochemical parameter of dissolved methane, however, becomes more plausible and valuable here, when compared to the changes of the quartz/phyllosilicate ratio and sorbed methane profile, as these mineralogical and geochemical parameters correlate (Fig. IV-1). Both methane pools indicate biogenic methane according to their carbon isotopic values (Fig. IV-1E). Hence, both pools and their quantitative variations seem to reflect changes in activity of the methanogenic microbial community which would consequently have been influenced by the inorganic particle composition/size or sediment permeability.
Both, increased permeability and grains increase in silty/sandy layers than in clay layers, and facilitates microbial activity (Krumholz et al., 1997; Rebata-Landa and Santamarina, 2006).
The distribution of sorbed methane in correlation with microbial activity limited by habitable pore space might point toward a similar pattern as observed for gas hydrate occurrences that
are strongly associated with permeable layers rather than fine grained sediment. A strong lithologic control of the gas hydrate occurrence with preferred gas hydrate formation in sand-rich sedimentary sections was observed in Cascadia Margin sediments (Riedel, 2006b).
The vertical profiles of dissolved and sorbed methane follow not only the abundance of in-organic components but also show a relationship with the geochemistry and methanogenic community. Sulfate-reducing bacteria outcompete methanogens for common substrates in the presence of significant sulfate concentrations (Kristjansson et al., 1982). Hence, significant methane production typically only occurs in sulfate-depleted sediment (Hoehler et al., 1994).
Nevertheless, significant amounts were leached from surface sediments that showed isotopic values affected by AOM. The increasing 13C-methane values with sediment depth (Fig.
IV-1E) might reflect a slight shift from predominantly hydrogenotrophic methanogenesis in the upper part of the methanogenesis zone to mostly acetrophic metabolism in the lower part, and would be consistent with the changing methanogenic community (Table IV-1) and estab-lished methane classification schemes (Whiticar, 1999). If this change is also a result of grain size, as observed in Cretaceous sand-shale formations or different permeable aquifers, in which a partitioning of fermentive and respiratory processes into fine and coarse layers, re-spectively, and subsequent diffusion of organic acids as substrate into coarse layers are pro-posed (Krumholz et al., 1997; McMahon and Chapelle, 1991), cannot be inferred.
In general, the biogenic sorbed methane content was quantitatively enriched compared to other oceanographic settings with very high concentration values at 203 cmbsf (Fig. IV-1D;
cf. chapter III). The specific surface areas of different mineral assemblages were not impor-tant for the uptake of methane, and particular sorbents were not apparent from the mineralogi-cal data set.
Interpretation of the vertical mcrA profile and lipid evidence
The highest mcrA copy numbers between 30 to 120 cmbsf suggest peak methanogenic and anaerobic methanotrophic activity in the upper methanogenesis zone (Fig. IV-2A). Intri-guingly, the putatively methanotrophic ANME-1 appear more abundant than all other metha-notrophic and methanogenic Archaea combined (Fig. IV-2A). At first glance, the absence of oligonucleotide degeneracies in the group-specific ANME-1-mcrI primer compared to the general mcrIRD primer pair may seem like a plausible explanation. However, amplification efficiency of the mcrIRD primer pair is only slightly lower that of non-degenerate group-specific primer pairs designed to amplify the individual target groups of the mcrIRD primer pair (Lever et al., in prep.). It therefore appears unlikely that the apparent dominance of the
ANME-1 clade was the result of higher PCR amplification rates with the ANME-1-mcrI pri-mer pair. More likely, ANME-1 Archaea are dominant members of the methane-cycling community at the site.
The mcrA abundance profile is consistent with the lipid biomarker pattern, although the synthesis of GDGT is not exclusive to methanogens or anaerobic methanotrophs. GDGTs are characteristic membrane lipids of cosmopolitan pelagic Crenarchaeota (Sinninghe Damste et al., 2002), such as marine group I Crenarchaea (Schouten et al., 2008). 2-Gly-GDGT lipids have been identified in a variety of environmentally relevant cultivated Cren- and Euryar-chaea such as Archaeoglobales, Methanobacteriales, Methanococcales and Thermoproteales Hinrichs et al., unpublished data), anaerobic methane-oxidizing ANME-1 mats (Thiel et al., 2007; Rossel et al., 2008), methanogen enrichments (Koga et al., 1993), and subseafloor se-diments (Sturt et al., 2004; Biddle et al., 2006; Lipp et al., 2008). The exact origin of 2-Gly-GDGTs could not be inferred, due to the lack of stable carbon isotopic values of ether-cleaved biphytanes that could have been used to discriminate between methanogenic and pelagic ori-gins. However, the similarity between the vertical distribution of GDGTs and mcrA genes suggests a microbial fingerprint of the active Archaeal population within the sediment col-umn, which could at least in part derive from actively methane-cycling populations.
Zonation of the methane-cycling community
The analysis of mcrA genes demonstrates that methanogens and, at least in part, anaerobic methanotrophs occur from the seafloor through the sulfate-reduction zone into the methano-genesis zone. Methanotrophic ANME-1 occur throughout the sulfate-reduction zone and me-thanogenesis zone, suggesting that ANME-1 might be facultatively methanogenic. The dis-tribution of other methanotrophs, ANME-2 and -3, is also not restricted to sulfate-reducing sediment, but extends into the upper methanogenesis zone (30-33 and 70-73 cmbsf; Table IV-1). Phylogenetic and isotopic data have shown syntrophic associations of both, ANME-1 and ANME-2, with sulfate-reducing bacteria (Orphan et al., 2001b). The detection of known anaerobic methane oxidizers in the sulfate-depleted sediment is not new, however, both ANME-1 (Orcutt et al., 2005; Lever et al., in prep) and ANME-2 (Kendall et al., 2007) have previously been detected in horizons that were unambiguously located in methanogenesis zones. A plausible explanation is that both ANME-1 and ANME-2 are facultative methano-gens. The co-occurrence of ANME-1 and -3 with putatively methanogenic URFS in the sul-fate-reduction zone of this site is not a new observation (Lever et al., in prep), and may be evidence of a cryptic methane cycle where ANME-1 and -3 are anaerobically oxidizing
me-rence of URFS in the sulfate-reduction zone may come by surprise. Methanogens are be-lieved to be outcompeted by sulfate reducers for common substrates (H2, acetate) in the pres-ence of sulfate, due to higher energy yields of sulfate reduction (Kristjansson, 1982). None-theless, certain substrates not used by most sulfate reducers, e.g. methanol, trimethylamine, or dimethyl sulfide, are believed to allow certain, methylotrophic methanogens to coexist with sulfate reducers in the presence of elevated sulfate concentrations (Whitman et al., 2006). It would be consistent with this interpretation that URFS are (facultative) methylotrophs. Alter-natively, these methanogens may be able to gain energy by growing syntrophically with hete-rotrophic bacteria, and using H2 produced by syntrophic partners for hydrogenotrophic me-thanogenesis as proposed for sediments of Skan Bay, Alaska and other settings (Kendall et al., 2006).
The mcrA gene diversity is highest in the uppermost horizons of the methanogenesis zone that we examined (30-33 and 70-73 cmbsf). Given that (i) OM is likely to be more labile, (ii) methanogen substrate production rates are higher, and consequently (iii) substrate availability is less limiting here than in deeper horizons of the methanogenesis zone, we would expect competition for substrates to be the least intense and methanogen diversity highest here. Con-sistent with this interpretation, mcrA gene diversity is lower deeper in the methanogenesis zone. Relatives of known hydrogenotrophic and/or acetoclastic Methanoculleus, Methanoge-nium, Rice Cluster I, Methanosaeta and Methanosarcina (Table IV-1) (Whitman et al., 2006) are restricted to the methanogenesis zones, consistent with the well-documented competitive exclusion of methanogenesis by sulfate reduction for common substrates in the presence of high sulfate concentrations (Lovley and Goodwin, 1988).
A greater diversity of mcrA genes was detected than in cold deep sea environments (e.g.
Bidle et al., 1999; Marchesi et al., 2001; Reed et al., 2002; Newberry et al., 2004), or coastal sediments from Skan Bay, Alaska (Kendall et al., 2007). The only other site in marine sedi-ments with comparatively high diversity was located on the Juan de Fuca Ridge Flank (Lever et al., in prep.). In the latter and this study, the mcrIRD and ANME-1-mcrI primer pairs were used, in comparison to the other studies in which previously published mcrI (Springer et al., 1995) and ME1/ME2 (Hales et al., 1996) primer pairs were used. The latter PCR amplify at a lower rate (mcrI) and target a lower diversity (ME1/ME2) than the mcrIRD primer pair, and, like the mcrIRD primer pair, are biased against ANME-1 due to a large number of oligonuc-leotide mismatches.
Similar to coastal marine subsurface sediments from the German tidal flat (Wilms et al., 2006) and the Juan de Fuca Ridge Flank (Lever et al., in prep.) the distribution of
methano-genic Archaea correlated with the sulfate profile; and like at the Juan de Fuca Ridge Flank, mcrA genes were not restricted to sulfate-free conditions (Table IV-1). Based on what is known about closest cultured relatives, the reduction of CO2 and fermentation of acetate are important methanogenic pathways in the methanogenesis zone of this Black Sea core, howev-er, an important role of methylotrophy cannot be inferred since we did not detect mcrA genes similar to those of cultivated methanogens.
Conclusions and implications
A profile of biogenic methane sorbed to a Black Sea sediment core exhibited distinct varia-tions across geochemical regimes, and correlated with the dissolved methane pool in quantity and carbon isotopic composition. The relations of the sorbed methane pool to mineralogical and microbial community changes were investigated in this study. Variations of dissolved and sorbed methane correlated with geochemical and mineralogical changes, and might indi-cate patches of enhanced methanogenic activity in sediment layers with larger grains. The importance of methanogenesis for methane sorption is indicated by the abundance and isotop-ic compositions of sorbed methane whisotop-ich reflects acetoclastisotop-ic and hydrogenotrophisotop-ic metha-nogenesis, and maybe methylotrophy in the sulfate-reduction zone.
The in situ methanogenic and methanotrophic community is vertically stratified; however, we observe no obvious link between community composition, mcrA gene abundance, or in-ferred pathway of methanogenesis, and methane concentrations or G13C-methane. The clone libraries revealed a mix of hydrogenotrophic and acetoclastic/methylotrophic Archaeal as-semblage in good accordance with the observed geochemical conditions and standard geo-chemical models. However, ANME-Archaea were not restricted to the sulfate-methane inter-face, but were also found in upper and deeper methanogenesis zones. ANME-1 specific genes dominate the methanogenic/methanotrophic signal in accordance with Archaeal intact membrane lipids and most likely reflect the active methane-cycling community.
From our results, a better understanding of dissolved and sorbed methane in relation to bio-logical, mineralogical and geochemical conditions was obtained. Large amounts of sorbed methane in quartz-rich inorganic matrices are in accordance with metabolic boundary condi-tions and occurrences of extensive methane volumes bound in hydrates of other settings.
Complex small scale interferences between mineralogical, geochemical and biochemical con-ditions rule methanogenic activity and subsequent patchy gas accumulation. We conclude that methane sorption benefits from enhanced methanogenic activity and/or facilitated distri-bution of its gaseous products in permeable layers which meliorates microbial life.
V. CO N C L U S I O N S A N D O U T L O O K
This PhD thesis provides insight into the biogeochemical role of sorbed gaseous HCs in marine sediments. It helps to understand the mechanisms which control the amount, source and molecular and isotopic composition of sorbed HCs, especially methane in surface and subsurface sediments. A further promising extraction protocol based on alkaline solution is presented which adds on various existing but controversial techniques.
Four major goals were addressed by this research effort:
x Determine the amount and vertical distribution of alkaline extractable sorbed HC gas-es in marine sediments from numerous oceanographic settings compared to the gas- estab-lished acid extraction.
x Determine the sources of sorbed HCs and relation to other marine gas reservoir by analyzing the molecular and isotopic compositions.
x Determine the major sorbents for gaseous HCs and involved sorption mechanisms.
x Determine the biological control on the amount and molecular and isotopic composi-tion of sorbed HCs.
The following key results are summarized:
1. Significant amounts of sorbed HC gases varying with sediment depth were success-fully extracted from marine sediments at alkaline conditions. In part, they were up to orders of magnitude higher compared to acidic extracted amounts. Biogenic and thermogenic HCs were extracted depending upon the geological setting.
2. The comparison of different extraction protocols (acid, autoclave and base extrac-tion) revealed the requirement for chemicals to liberate sorbed HC gases. It is demonstrated that different techniques easily lead to different yields, molecular and isotopic compositions.
3. C2+ HC gases are selectively sorbed to marine sediments in the manner that mole-cules with increasing carbon numbers are stronger retained resulting in an opposite abundance pattern compared to the dissolved gas phase. No discrimination of C2+
HCs by alkaline extraction conditions was observed in both, sites with macrosee-page, and sites without known thermogenic gas sources.
4. Abiotic sorption experiments indicate the importance of clay minerals as sorbents for HCs, while in biotic incubations no metabolic stimulation of the methanogenic activity of Methanococcoides burtonii by clay minerals could be observed.
5. The mineralogical data set of environmental samples exhibits a lack of correlation between carbonate minerals and sorbed methane. Exceptionally high amounts of sorbed methane correlate with high quartz/phyllosilicate ratios.
6. There are indications that the quantity of sorbed methane records methanogenic ac-tivity. Patches of enhanced microbial activity of the methane-cycling community are indicated in accordance with dissolved and sorbed methane maxima that are as-sociated with beneficial sedimentological and lithological conditions.
7. Hydrogenotrophic and acetoclastic methanogenesis can be differentiated by carbon and hydrogen isotopic compositions of sorbed methane. No selective sorption of methane isotopologues can be inferred from the dataset.
8. Sorbed biogenic methane in sulfate-reducing sediments is a ubiquitous phenome-non, and suggests methylotrophic methanogenesis and/or biogeochemical processes beyond common thermodynamic models, and therefore indicates a lack of under-standing of biogeochemical processes in sulfate-replete sediments.
One of the main problems in analyzing sorbed HC gases is the fact that it is almost imposs-ible with any geochemical extraction procedure to ever know the ‘absolute’ amount or state of compounds extracted. Moreover, it has been demonstrated earlier that different techniques provide different gas quantities, compositions and compound specific isotope ratios (Abrams, 2005). To determine which method best represents the in situ gas composition and isotopic ratio requires additional study. One chance to completely extract gaseous HCs and to suffi-ciently discriminate between silicate and carbonate minerals as HC sorbents would be the application of hydrofluoric acid and subsequent phosphoric acid extraction. The strongly cor-rosive hydrofluoric acid reacts with silicate to gaseous silicon tetrafluoride (Spierings, 1993).
This harsh and dangerous chemical would therefore decompose silicate minerals in order to liberate sorbed HCs at faster rates than sodium hydroxide. To successfully decompose sili-cates and to trap released gas would require Teflon-made and sealed containers. The applica-tion of this chemical however, is not always straightforward, as decomposiapplica-tion of refractory material might additionally require high temperatures (Bernas, 1968).
There is evidence that microbial activity is enhanced in sedimentary matrices composed of silt/sand-sized grains, whereas metabolism appears limited in OM-rich and/or fine grained horizons (e.g. Krumholz et al., 1997; Rebata-Landa and Santamarina, 2006). In coarse-grained horizons, activity of methanogens can lead to extensive methane hydrate accumula-tions (Riedel et al., 2006b). Clay minerals are likely to act as sorbents or, at least, catalysts of microbial growth by providing cations as trace elements (e.g. Maqueda, 1998). The presented mineral data set indicates an association of high sorbed methane concentrations with interme-diate clay contents (cf. chapter III). Therefore, incubation experiments of methanogens might be conceivable, in which different clay contents are added to a sandy matrix and mixed with methanogens in anoxic growth media, in order to test the stimulation of habitable pore space with direct analysis of sorbed methane. The experiment could include porosity and permea-bility measurements by combining neutron and density logging tools or scanning electron microcopy to infer the quantitative orientation and micro-porosity of the sedimentary matrix (Tovey and Dadey, 2002). Via atomic force microscopy it may be possible to observe the interaction of gaseous HCs with the mineral edges of clays at an atomic level.
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