Y Ruffine

J.-P Carpentier

S Donval

V Ker

Riboulot

B Chazallon
email: bertrand.chazallon@univ-lille.fr

C T Rodriguez

L Ruffine

Y Carpentier

J.-P Donval

S Ker

V Riboulot

Characterizing the variability of natural gas hydrate composition

Keywords: Natural gas hydrates, Black Sea, methane, cage occupancy

de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction

Natural gas hydrates (NGH) are ice-like crystalline compounds in which hydrogenbonded water molecules form specific cages able to encase low molecular weight molecules.

They are usually stable at low temperature and high pressure. The water framework is called the "host" and the inserted molecule, the "guest". Previous studies have shown that gas hydrates (GH) are non-stoichiometric compounds in which not all the cages need to be filled with a gas molecule to stabilize the whole structure [START_REF] Sloan | Clathrate Hydrates of Natural Gases[END_REF]. Depending on the physico-chemical properties of the guest molecule, three main crystallographic structures can be found in nature [START_REF] Bourry | X-ray synchrotron diffraction study of natural gas hydrates from African margin[END_REF][START_REF] Bourry | Free gas and gas hydrates from the Sea of Marmara, Turkey Chemical and structural characterization[END_REF][START_REF] Chazallon | A comparative Raman spectroscopic study of natural gas hydrates collected at different geological sites[END_REF][START_REF] Khlystov | Gas hydrate of Lake Baikal: Discovery and varieties[END_REF][START_REF] Kida | Coexistence of structure I and II gas hydrates in Lake Baikal suggesting gas sources from microbial and thermogenic origin[END_REF][START_REF] Lu | Complex gas hydrate from the Cascadia margin[END_REF]: cubic structure I (sI) (lattice parameter a ~ 12 Å, cubic structure II (sII) (lattice parameter a ~ 17 Å) and hexagonal structure H (sH) (lattice parameter a ~12 Å and c ~10 Å). NGH are mainly composed of microbial methane, and thus sI is the most encountered structure in nature [START_REF] Lu | Complex gas hydrate from the Cascadia margin[END_REF][START_REF] Makogon | Natural gas-hydrates -A potential energy source for the 21st Century[END_REF], whereas structures sII [START_REF] Bourry | Free gas and gas hydrates from the Sea of Marmara, Turkey Chemical and structural characterization[END_REF][START_REF] Brooks | Thermogenic gas hydrates in the Gulf of Mexico[END_REF]Davidson et al., 1986a;[START_REF] Pape | Molecular and isotopic partitioning of lowmolecular-weight hydrocarbons during migration and gas hydrate precipitation in deposits of a high-flux seepage site[END_REF] and sH [START_REF] Lu | Complex gas hydrate from the Cascadia margin[END_REF][START_REF] Yousuf | Novel results on structural investigations of natural minerals of clathrate hydrates[END_REF] are less common, and require sources of thermogenic gases containing non-methane hydrocarbons to stabilize the structure.

NGH represent one of the largest methane reservoir on Earth with the total amount of carbon stored higher than all conventional hydrocarbon reserves [START_REF] Lu | A global survey of gas hydrate development and reserves: Specifically in the marine field[END_REF][START_REF] Ruppel | The interaction of climate change and methane hydrates[END_REF][START_REF] Wallmann | The Global Inventory of Methane Hydrate in Marine Sediments: A Theoretical Approach[END_REF]. Furthermore, it has been estimated that ~98-99 % of NGH are sequestered within marine sediments in the first tens to hundreds of meters of sediment on continental margins [START_REF] Boswell | Natural Gas Hydrates: Status of Potential as an Energy Resource[END_REF][START_REF] Kvenvolden | Methane hydrate -A major reservoir of carbon in the shallow geosphere?[END_REF], while other occurrences concern the permafrost region [START_REF] Paull | Natural Gas Hydrates: occurence, distribution and detection[END_REF]. The current substantial efforts in NGH research to better characterize these deposits [START_REF] Ruffine | Multiple gas reservoirs are responsible for the gas emissions along the Marmara fault network[END_REF][START_REF] Song | The status of natural gas hydrate research in China : A review[END_REF] is justified by the fact that marine methane hydrates are nowadays viewed as a doubleedged sword. On the one hand, a large proportion of the enclathrated methane might be produced in the future to meet the increasing world energy demand, and on the other hand, the temperature-sensitive hydrate deposits can be destabilized due to ocean warming. This would lead to the release of methane into the water column with potential input into the atmosphere as well as potential destabilization of the seafloor, hastening and perpetuating the cycle of global warming [START_REF] Kennett | Methane Hydrates in the Clathrate Gun Hypothesis[END_REF][START_REF] Maslin | Gas hydrates: past and future geohazard?[END_REF][START_REF] Ruppel | The interaction of climate change and methane hydrates[END_REF].

Accordingly, NGH may be an opportunity or a threat for modern societies [START_REF] Wang | Prevention of Potential Hazards Associated with Marine Gas Hydrate Exploitation: A Review[END_REF].

Elements of response for these two social issues may be obtained through the evaluation of the methane quantities stored in NGH deposits, and this requires in-depth microscale characterization of guest identification and distribution in hydrates. Up to now, most global estimates of gas trapped in NGH have often been calculated assuming (based on structure sI) a mean cage occupancy of 85%, i.e., a hydration number n of methane hydrate of ~6.75 which corresponds to a volumetric conversion factor of ~164 m 3 of methane per m 3 of hydrate [START_REF] Boswell | Current perspectives on gas hydrate resources[END_REF][START_REF] Kvenvolden | A primer on the geological occurrence of gas hydrate service Permission Subscribe[END_REF]. Actually, it turns out that both the cage occupancies ( LC and  SC being the large and small cage occupancies, respectively) and hydration numbers are key parameters to evaluate the volumetric conversion factor. These parameters ( LC ,  SC , n) can reliably be determined by spectroscopic technics (Raman or NMR-Nuclear Magnetic Resonance) [START_REF] Chazallon | A comparative Raman spectroscopic study of natural gas hydrates collected at different geological sites[END_REF][START_REF] Liu | Characterization of natural gas hydrate recovered from Pearl River Mouth basin in South China Sea[END_REF][START_REF] Lu | Occurrence and structural characterization of gas hydrates associated with a cold vent field, offshore Vancouver Island[END_REF][START_REF] Ripmeester | Structure and composition of hydrate in sediment recovered from Mallik 5L-38, Mackenzie Delta, NWT Canada: X-ray diffraction, Raman and Solid-state NMR spectroscopy[END_REF][START_REF] Uchida | Raman Spectroscopic Determination of Hydration Number of Methane Hydrates[END_REF]. However, it has been shown that methane cage occupancy can vary significantly in NGH with a ratio of  LC / SC ~ 0.86-1.51, depending on the area where they are collected. Even for NGH deposits for which methane is overwhelmingly present, a ratio of  LC / SC lower than 1 has already been observed (H. Lu et al., 2011b;[START_REF] Lu | Gas hydrate occurrences in the Qilian Mountain permafrost, Qinghai Province, China[END_REF][START_REF] Ripmeester | Structure and composition of hydrate in sediment recovered from Mallik 5L-38, Mackenzie Delta, NWT Canada: X-ray diffraction, Raman and Solid-state NMR spectroscopy[END_REF]. Similarly, a variability of the cage occupancy ratio ( LC / SC between ~2.38 and ~1.18) in NGH containing nearly pure methane was reported [START_REF] Yeon | Abnormal methane occupancy of natural gas hydrates in deep sea floor sediments[END_REF]. It was suggested that such abnormal methane occupancy (i.e., corresponding to a deviation from that usually found in synthetic pure CH 4 -hydrate) in NGH embedded in clay-rich deep-sea-sediments could be due to mobile ions (sodium cations) present in the clay interlayers [START_REF] Seo | Structure and Composition Analysis of Natural Gas Hydrates:13C NMR spectroscopic and gas uptake measurements of mixed gas hydrates[END_REF][START_REF] Yeon | Effect of Interlayer Ions on Methane Hydrate Formation in Clay Sediments[END_REF]. These latter could significantly affect the NGH stability, impact the CH 4 cage occupancy and the hydration number and thus the estimates of the quantity of gas trapped.

In the present study, we combined two different techniques (gas chromatography and micro-Raman spectroscopic imaging) to characterize samples of natural hydrates collected during the Ghass cruise (2015) in the Romanian water sector of the Western Black Sea, which is known as the largest anoxic and methane-rich water mass on Earth. The Black sea contains several identified and inferred NGH deposits along its margin [START_REF] Colin | Irregular BSR: Evidence of an Ongoing Reequilibrium of a Gas Hydrate System[END_REF][START_REF] Haeckel | Gas H ydrate Occurrences in the Black Sea -New Observations from the German SUGAR Project[END_REF][START_REF] Heeschen | Quantifying in-situ gas hydrates at active seep sites in the eastern Black Sea using pressure coring technique[END_REF][START_REF] Minshull | Hydrate occurrence in Europe: A review of available evidence[END_REF][START_REF] Nasif | Seismic identification of gas hydrates: A case study from Sakarya Canyon, western Black Sea[END_REF][START_REF] Pape | High-intensity gas seepage causes rafting of shallow gas hydrates in the southeastern Black Sea[END_REF][START_REF] Popescu | Seismic expression of gas and gas hydrates across the western Black Sea[END_REF][START_REF] Zander | New insights into geology and geochemistry of the Kerch seep area in the Black Sea[END_REF]. Our molecular-scale approach allowed to investigate the variability in cage occupancy and the impact of the presence of minor guest components on the overall methane content.

Geological setting and sampling

The study area in the Black Sea is characterized by a large number of widespread gas seeps, [START_REF] Egorov | Methane seeps in the Black Sea: discovery , quantification and environmental assessment[END_REF][START_REF] Naudts | Geological and morphological setting of 2778 methane seeps in the Dnepr paleo-delta, northwestern Black Sea[END_REF][START_REF] Schmale | Methane emission from high-intensity marine gas seeps in the Black Sea into the atmosphere[END_REF][START_REF] Schmale | Response of the Black Sea methane budget to massive short-term submarine inputs of methane[END_REF] with a clear link between the pattern of seep distribution and the gas hydrate stability zone (GHSZ) [START_REF] Riboulot | Control of the geomorphology and gas hydrate extent on widespread gas emissions offshore Romania[END_REF]. Indeed, the gas flares detected in the water column from ship-borne multi-beam echo-sounding are primarily located on the shelf and outside the area delimited by GHSZ, suggesting that the hydrate deposits act as a barrier preventing intense methane release at locations where hydrates are stable (Fig. 1). [START_REF] Ker | Anomalously Deep BSR Related to a Transient State of the Gas Hydrate System in the Western Black Sea[END_REF]. The blue dots highlight the acoustic gas flares identified in the water column during the GHASS cruise [START_REF] Riboulot | Control of the geomorphology and gas hydrate extent on widespread gas emissions offshore Romania[END_REF]. They are mostly located above the GHSZ but can also be found at the landward termination of the GHSZ and within the GHSZ in the Crest line area. B. Close-up view of the study area with location of the Calypso core GAS-CS14 recovered at the summit of the crest line close to gas flares. This core, where the gas hydrates were sampled, is located at the limit of the shallow gas hydrate stability zone identified previously [START_REF] Riboulot | Freshwater lake to salt-water sea causing widespread hydrate dissociation in the Black Sea[END_REF]. This corresponds to the bounder area where gas hydrates are undergoing destabilization due to seawater infiltration within the sediment since the Black Sea reconnection with the Mediterranean Sea, leading to the increase of salinization of the pore water. [START_REF] Riboulot | Freshwater lake to salt-water sea causing widespread hydrate dissociation in the Black Sea[END_REF] and [START_REF] Ker | Anomalously Deep BSR Related to a Transient State of the Gas Hydrate System in the Western Black Sea[END_REF] calculated the boundary of the GHSZ between 660 and 720 m (Fig. 1), as proposed previously for seeps in the Dnepr paleo-delta area in the NW Black Sea [START_REF] Naudts | Geological and morphological setting of 2778 methane seeps in the Dnepr paleo-delta, northwestern Black Sea[END_REF]. A large number of seeps were detected around this water depth range, and [START_REF] Riboulot | Freshwater lake to salt-water sea causing widespread hydrate dissociation in the Black Sea[END_REF] suggest that the occurrence of these gas seeps is related to the dissociation of hydrates due to the ongoing salt intrusion from today's Black Sea marine bottom waters into the lacustrine sediment since the reconnection with the Mediterranean Sea about 9000 years ago [START_REF] Soulet | A revised calendar age for the last reconnection of the Black Sea to the global ocean[END_REF]. While seeps' density was much less pronounced inside the GHSZ, seeps were found only at specific locations where faults reach the seafloor along a crest line area [START_REF] Ker | Anomalously Deep BSR Related to a Transient State of the Gas Hydrate System in the Western Black Sea[END_REF]. The hydrates were collected from coring operations on this crest line (Fig. 1), where several persistent and focused gas flares were detected from the shipborne echo-sounder during the Ghass cruise. Thus, this site is classified as being a high-flux fluid system. The hydrate-bearing core, GAS-CS14 (N43°56.361'; E30°51.044'), was retrieved using a long Calypso piston corer at 738 m water depth. It was the unique core containing hydrates from a series of >20 cores, and it was mainly composed of Unit 3 lacustrine sediment [START_REF] Ross | Recent Sediments of Black Sea[END_REF]. The youngest sediments deposited during the Holocene, representing the lacustrine-marine transition (Unit II) and the marine stage (Unit I), were not clearly identifiable; possibly due to a loss of the uppermost part of the core as explained by [START_REF] Ruffine | Effects of postglacial seawater intrusion on sediment geochemical characteristics in the Romanian sector of the Black Sea[END_REF]. Thus, hydrates were located within the lacustrine sediments. This is in agreement with [START_REF] Pape | Molecular and isotopic partitioning of lowmolecular-weight hydrocarbons during migration and gas hydrate precipitation in deposits of a high-flux seepage site[END_REF] who found a higher concentration of hydrates in Unit III from cores collected off Georgia. The hydrate-bearing sediment samples were collected in the core GAS-CS14 at ~738 m water depth and ~9 °C, which corresponds to the Black Sea bottom water temperature (see Fig. 2 for further details on the description of the core and location of hydrate samples). Core GAS-CS14 has a length of 542 cm, and was subdivided in four lithofacies as described in Fig. 2. Its topmost part highlights 10 cm of light brownish clay with numerous small carbonate concretions. This carbonate-contained section is followed by a sedimentary sequence dominated by grey clay that turns to sediment dark and reworked, and such darkness and reworking become more pronounced with increasing depth. The sediment is highly unstructured and reworked within the hydrate-bearing interval as shown in the photo of Fig. 2. X-ray diffraction applied to the sediment in another study [START_REF] Ruffine | Effects of postglacial seawater intrusion on sediment geochemical characteristics in the Romanian sector of the Black Sea[END_REF] reveals that clays and mica represent between 53 and 67 wt.% of the matrix, with illite being by far the dominant species. The remaining minerals are mainly silicates (feldspar, plagioclase and quartz) and carbonates. A thin layer of few centimeters of light brownish clay with small carbonate concretions was identified at the uppermost part of the core followed by a light grey clay containing organic matter, whereas the hydrate-bearing zone consists of dark grey clay.

Evidence of core disturbances increases when moving towards the hydrate bearing-zone (Fig. 2). Sulfate concentration was < 1 mM within the first decimeter of sediment [START_REF] Ruffine | Effects of postglacial seawater intrusion on sediment geochemical characteristics in the Romanian sector of the Black Sea[END_REF].

Experimental methods

After recovery, the hydrate-bearing core were brought to the ship laboratory and split lengthwise. The core consists of centimeter scale hydrate nodules and lens. Small hydrate samples were immediately prepared for gas analysis, and the remaining hydrates stored into liquid nitrogen as quickly as possible, without determining their depth location. However, for the molecular and isotopic ( 13 C and D) composition analyses of the hydrate-bound gases, the samples were taken from the first hydrate-bearing core section identified visually, thus they were located within the 4-5 m depth interval. Hydrate nodule samples of more than 5 cm size were taken (Fig. 2) and cleaned of sediment, and placed in 10 mL glass vials which were then air vacuumed. The coexistence of free gas trapped during hydrate formation cannot firmly be excluded. However, such remaining pore gas will be a minor fraction of the gases collected from the hydrate-sample dissociation. Therefore, we believe that the collected samples are representative of the hydrate-bound gases. Two different hydrate samples were analyzed. Molecular composition analysis was performed at the Laboratoire des Cycles Géochimiques et ressources (LCG) of Ifremer using a gas chromatograph GC R3000 from SRA equipped with a Thermal Conductivity Detector and a 8m, 0.32 mm PoraPlot U capillary column to quantify both methane and carbon-dioxide. The concentrations of heavier hydrocarbons, from ethane to n-hexane, were measured on a gas chromatograph Agilent 7890A equipped with a 32 m, 0.32 mm x 30µm Porapak Q column. The configuration of the chromatograph is not suitable for hydrogen sulfide. The instrument was calibrated against standard gas mixtures, and both the relative accuracy and standard deviation were of ±2% for methane and carbon dioxide concentrations, and ±4% for the heavier hydrocarbons.

Isotopic analyses were performed on two hydrate samples at Isolab B.V. (Neerijnen, The Netherlands) for methane, ethane, propane and carbon dioxide using gas chromatographisotope ratio-mass spectrometers (GC-IR-MS). Labco® vials of 12 mL were filled with the hydrate-bound gases at ~0.2 MPa. Carbon ( 13 C) and hydrogen (D) stable isotope ratios were measured when appropriate. An Agilent 6890N gas chromatograph interfaced to a Finigan Delta S IRMS and a Finigan GC-C II interface (Thermo) was used for the analysis of the stable carbon isotopes of methane. The GC is equipped with a 12m, 0.32mm molsieve column (Agilent) and an injection valve. Stable carbon isotopes of heavier hydrocarbons and carbon dioxide were measured on an Agilent 7890A gas chromatograph interfaced with a MAT 253 IRMS using a GC-Isolink or a Finigan GC-C III interface. The GC is equipped with a 25m, 0.32mm Porabond-Q column (Agilent) and an injection valve. D measurement was carried out on an Agilent 7890A gas chromatograph equipped with a 25m, 0.32mm molsieve column (Agilent) and an injection valve, and interfaced with a MAT 253 IRMS and using a GC-Isolink interface from Thermo. For calibration purpose, the instruments were regularly checked with a calibration standard. Results are as part per thousand (‰) relative to the Viennia PeeDee Belemnite Standard (VPDB) and the Vienna Standard Mean Ocean Water (VSMOW) for  13 Cand D, respectively. The uncertainties in the  13 C and D measurements were given as ±0.1-0.3‰ and ±2‰, respectively.

NGH recovered from core GAS-CS14 were further analyzed by optical microscopy and Raman spectroscopy. Since the hydrates likely underwent partial decomposition during the recovery onboard and before storage in liquid nitrogen at 77 K, focus was made on central pieces from five hydrate subsamples (typically ~5 mm long) in a well-preserved state. For that, a small part of the NGH was placed in a mortar filled with liquid nitrogen and it was carefully crushed into smaller pieces. Each piece was placed in a cooling stage set at a working temperature of 128 K, in an atmosphere saturated with nitrogen gas. A more detailed description of our sample loading protocol and hydrate probing using Raman systems can be found in [START_REF] Bourry | Free gas and gas hydrates from the Sea of Marmara, Turkey Chemical and structural characterization[END_REF][START_REF] Charlou | Physical and chemical characterization of gas hydrates and associated methane plumes in the Congo-Angola Basin[END_REF][START_REF] Chazallon | A comparative Raman spectroscopic study of natural gas hydrates collected at different geological sites[END_REF][START_REF] Chazallon | Spectroscopy of Gas Hydrates: from fundamental aspects to Chemical Engineering[END_REF]. Note that we exclude any sample degradation during the long-term storage of NGH samples in liquid nitrogen because the NGH remains frozen. More specifically, nitrogen from the liquid does not interfere with the composition of the sample due to the higher volatility of N 2 in comparison with CH 4 , thus avoiding any exchange reaction with methane. Also, permeation processes of N 2 in the sample can be excluded as these would be too slow to occur within the time frame of this study [START_REF] Salamatin | Diffusion Model for Gas Replacement in an Isostructural CH4-CO2 Hydrate System[END_REF]. Raman measurements were performed at PhLAM laboratory (Lille, France) using an InVia Reflex Raman spectroscope (Renishaw).

The spectral resolution was set to ~ 4 cm 1 with a holographic grating of 1800 grooves/mm and a Peltier cooled front illuminated CCD detector. The excitation radiation (λ = 514.5 nm) was produced by a DPSS laser (Cobolt®). An Olympus free-space microscope was coupled to the spectrometer and Raman spectra were collected through a 50× objective (0.5 N.A.) that provides a circular beam spot smaller than ~1.5 µm diameter. The probed sample volume depends on several factors related to the NGH refraction index and the properties of the collection optics; it is estimated at several tens of µm 3 to 100 µm 3 for the most transparent part of the sample. The laser power focused onto the sample was typically adjusted to 3-4 mW, as measured by a Lasercheck power-meter (Coherent). High signal-to-noise-ratios (S/N) of individual spectra collected in this work were obtained with integration times of typically 60 s in one or two accumulations. A set of 48 Raman maps were collected in synchroscan mode (section 4.3) and representative information was extracted from two of them (see section 4.3 and supplementary material for further details). Errors in the measured integrated intensity are estimated from the lowest values found for the different components. It gives for CH 4 and H 2 O a maximal relative error of respectively ~2% and 0.2%, whereas for N 2 and H 2 S the maximal relative error is estimated at ~12%. The detection limit was estimated from the standard deviation ( b ) of the blank signal in a region free of Raman peaks. The minimal (S/N) of CH 4 and H 2 O were found at least two orders of magnitude higher than the adopted criterion of 3 b . For the components present at lower concentrations, the minimal cut-off of the contour lines of the Raman map of N 2 and H 2 S was set to 75 in order to adopt a high confidence level of detection for these species (see section 4.3). In addition, the errors on the guest concentrations derived from error propagation were found between 0.1 mol% (for the highest S/N ratio) and 1.7 mol% (for the lowest S/N ratio).

Results and discussion

Nature and source of the hydrate-forming light hydrocarbons

The analysis of the gases from the two decomposed hydrate samples indicates that they primarily contain methane (> 99.5 mol%), followed by nitrogen (> 0.29 mol%) and carbon dioxide (> 0.041 mol%). Their compositions are slightly different from one sample to another.

Ethane and propane are in trace amount (Table 1). Methane and ethane are highly depleted in 13 C, with values around -70 and -63 ‰, respectively, whereas the propane is more enriched in 11 13 C ( 13 C-C 3 H 8 around -27 ‰). [START_REF] Pape | Formation pathways of light hydrocarbons in deep sediments of the Danube deepsea fan, Western Black Sea[END_REF] analyzed void gases collected in sediments from deeper depths at the same study area, and they measured similar  13 C values for methane. They also found that ethane and propane were slightly enriched in 13 C with values ranging between -55.7 and -23.2 ‰ and -23.2 and -19.8 ‰, respectively. Both molecular and isotopic compositions of the light hydrocarbons were measured to construct the classical geochemical diagrams used for source determination (Fig. 3). [START_REF] Bernard | Light hydrocarbons in recent Texas continental shelf and slope sediments[END_REF] and [START_REF] Milkov | Organic Geochemistry Worldwide distribution and significance of secondary microbial methane formed during petroleum biodegradation in conventional reservoirs[END_REF]), b)  2 H-CH 4 vs  13 C-CH 4 space (modified after [START_REF] Bernard | Light hydrocarbons in recent Texas continental shelf and slope sediments[END_REF]), c)  13 C-CH 4 vs. CH 4 /(C 2 H 6 + C 3 H 8 ) space (modified after [START_REF] Bernard | Light hydrocarbons in recent Texas continental shelf and slope sediments[END_REF] and [START_REF] Milkov | Revised genetic diagrams for natural gases based on a global dataset of >20,000 samples[END_REF] This is in agreement with previous studies that have shown that methane in the area of the Danube deep-sea fan of the Black Sea and off Crimea is sourced from carbonate reduction [START_REF] Pape | Formation pathways of light hydrocarbons in deep sediments of the Danube deepsea fan, Western Black Sea[END_REF][START_REF] Römer | Geological control and magnitude of methane ebullition from a high-flux seep area in the Black Sea-the Kerch seep area[END_REF][START_REF] Zander | New insights into geology and geochemistry of the Kerch seep area in the Black Sea[END_REF]. Moreover, [START_REF] Pape | Formation pathways of light hydrocarbons in deep sediments of the Danube deepsea fan, Western Black Sea[END_REF] show that the relatively depleted D-CH 4 observed for methane is inherited from the formation water generated during relatively cold periods of the Black Sea history. The origin of propane is unclear, and in the absence of clear evidence of thermogenic gases, one may be tempted to think that it is also generated from microbial processes. However, thermogenic gases were found in both the Eastern and Western Black Sea [START_REF] Pape | Molecular and isotopic partitioning of lowmolecular-weight hydrocarbons during migration and gas hydrate precipitation in deposits of a high-flux seepage site[END_REF][START_REF] Reitz | Sources of fluids and gases expelled at cold seeps offshore Georgia, eastern Black Sea[END_REF][START_REF] Rusakov | Mantle origin of methane in the Black Sea[END_REF], and the trace of propane measured in the hydrate samples may also come from leaking thermogenic sources. Without information on the D-propane or the concentration measurement of hydrocarbons from butane to hexane, the origin of the propane remains elusive.

Properties of the hydrate samples from optical microscopy and single-point

Raman analysis

Fig. 4a shows a high-resolution image (min: 1600 x 1200 pixels) obtained with a 3Dmicroscope (Keyence) of a representative view of a selected small part of the NGH. The sample is maintained intact at liquid nitrogen temperature. It can be divided into three major zones where the GH was shown to be present in different relative amount (see next section):

1) a light-grey zone free of sediment, with white patches (A, Fig. 4, delineated by a white which is also a GH-rich zone where the brown color indicates the presence of iron-bearing minerals at an elevated concentration in the Black Sea [START_REF] Egger | Anaerobic oxidation of methane alters sediment records of sulfur, iron and phosphorus in the Black Sea[END_REF], as also shown in a complementary study [START_REF] Ruffine | Effects of postglacial seawater intrusion on sediment geochemical characteristics in the Romanian sector of the Black Sea[END_REF], 3) a dark-grey area with small white patches, which is a less rich GH-zone, at the bottom right-hand side (Fig. 4, C, delineated by a green dotted line) where solid hydrates (or ice) are intermixed with sediments.

4.2.a Raman signature of the sediment-rich zone

Single-point Raman spectra of typically four to five randomly-chosen locations at the surface of a freshly cut part of the NGH were collected over the range of 100 cm -1 to 3800 cm - known to exist widely in deep-seafloor natural products and have strong recognizable resonance Raman peaks [START_REF] Urmos | Characterization of some biogenic carbonates with Raman spectroscopy[END_REF][START_REF] Withnall | Raman spectra of carotenoids in natural products[END_REF][START_REF] Zhang | Development and deployment of a deep-sea Raman probe for measurement of pore water geochemistry[END_REF]. The position of the  1 band (at 1521 cm 1 ) depends on the effective conjugation length (number of double carbon-carbon lengths) and can be helpful to identify the corresponding conformer. The position of the  1 band is consistent with the presence of β-carotene in the NGH, and spectra collected at distinct positions indicate that it is distributed heterogeneously in the analyzed sample. This compound may result both from dissolved species and small solid particles of degraded shells or fine-grain organic material recovered from the seafloor.

Other peaks observed between 800 and 3100 cm 1 (Fig. 5, wave-numbers indicated in red) can be attributed to β-carotene [START_REF] Parker | Raman Spectrum of β-Carotene Using Laser Lines from Green (514.5 nm) to Near-Infrared (1064 nm): Implications for the Characterization of Conjugated Polyenes[END_REF]. The signal of CH 4 trapped in the NGH is detected concomitantly at ~2902 cm 1 and ~2913 cm 1 (see inset in Fig. 5). It is worth noting that the β-carotene signal may hinder minor contributions originating from SO 4 2 or CO 3 2 ions (respectively at 985 cm -1 and 1088 cm -1 ) of electrolytes that are often reported in NGHs [START_REF] Du | In situ Raman spectroscopy study of synthetic gas hydrate formed by cold seep flow in the South China Sea[END_REF][START_REF] Zhang | Development of a new deep-sea hybrid Raman insertion probe and its application to the geochemistry of hydrothermal vent and cold seep fluids[END_REF].

4.2.b Raman signature of the sediment-free zone

Two representative Raman spectra of the series (Fig. 6a trapped in both the large cage (LC) and the small cage (LC) of the NGH structure, respectively [START_REF] Chazallon | A comparative Raman spectroscopic study of natural gas hydrates collected at different geological sites[END_REF][START_REF] Sum | Measurement of Clathrate Hydrates via Raman Spectroscopy[END_REF] (Table 2). The discrimination between the large and the small cages for the nitrogen molecules is not achievable with the present spectral resolution [START_REF] Chazallon | Selectivity and CO2 capture efficiency in CO2-N2 clathrate hydrates investigated by in-situ Raman spectroscopy[END_REF]).

c Also reported in [START_REF] Chazallon | A comparative Raman spectroscopic study of natural gas hydrates collected at different geological sites[END_REF].

The peak frequencies (and for relative integrated intensity ratio, see below) indicate a structure sI which is in agreement with the compositional analysis of the hydrate-bound gases (see Table 1) with a strong methane dominance.

On some other parts of the investigated light-grey zone, single-point Raman spectra

show, in addition to methane peak, the band characteristic of nitrogen trapped in the NGH, which emerges at ~2323.5 cm 1 (Fig. 6a, inset N 2 ). This band is downshifted relative to that of free nitrogen gas occurring at ~ 2329 cm 1 [START_REF] Chazallon | Micro-Raman analysis of synthetic air clathrates[END_REF][START_REF] Chazallon | Selectivity and CO2 capture efficiency in CO2-N2 clathrate hydrates investigated by in-situ Raman spectroscopy[END_REF]) (Table 2). Nitrogen molecules are apparently heterogeneously distributed in the NGH as the signature is not systematically observed on the probed positions (Fig. 6a, inset N 2 upper spectrum with only free nitrogen gas).

Molecular H 2 S trapped in the NGH structure (Fig. 6b) can be identified at other distinct positions (see picture in Fig. 6b), with peaks observed at ~2593 cm 1 (LC) and 2604 cm 1 (SC) that appear simultaneously with the peaks of the trapped N 2 and CH 4 (Table 2). ). Nitrogen trapped in the NGH has a single peak at 2323 cm -1 (N 2 , green inset lower spectrum) and free N 2 gas peak (free N 2 gas from the background atmosphere) is at 2330 cm -1 (N 2 , green inset upper spectrum). Note also the occurrence of CH 4 band overtones (2 4 and 2 2 , [START_REF] Chazallon | A comparative Raman spectroscopic study of natural gas hydrates collected at different geological sites[END_REF]) (blue inset) and contribution of the water lattice observed in the range 3000-3800 cm 1 (O-H stretching, blue inset) and below 400 cm 1

(translational O---O vibration) (main spectrum). b) Single point Raman spectrum showing molecular H 2 S coincluded in the NGH (LC at 2593 cm -1 , SC at 2604 cm -1 ) with N 2 (2323 cm -1 ) (purple inset) and CH 4 (main spectrum) (see text for details and Table 2).

The interpretation of all the spectra suggests that the NGH has a heterogeneous

Spatial variability of the NGH's composition from 2D Raman images

4.3.a Spatial variability of the guest's distribution

2D-Raman mappings are applied on several parts of the sample surfaces to obtain a quantitative picture of the spatial variability of the NGH composition and guests' distribution.

This zone was selected independently from the position where the single point Raman spectra have been collected. First, a light-grey zone (represented typically in A Fig. 4) close to a sediment-rich zone (typically represented in C Fig. 4) with a relatively even surface is selected. Therefore, no specific cautions were taken to avoid the 'out of focus' zone during the mapping process of the laser beam, and the instrument was configured with a large depth of field (entrance slit of 65 µm). The zone is scanned in x and y directions with a step of 5 µm (20 s/spectra), using the objective x50. Depending on the size of the zone (typically of ~( 120x 85) µm²), maps of 400 to 700 complete spectra in synchro-scan mode are recorded. The color-coded images represent the values of a selected parameter (here, the integrated intensity) from a Raman band of each spectrum of the map. In Fig. 7, the Raman maps show the integrated intensity of the bands corresponding to CH 4 , H 2 S, N 2 , and H 2 O of the NGH. The signal of ethane and propane was too low to be detected with the present configuration of the instrument. Note that a baseline correction was applied automatically on each molecular spectral range of interest, and a post-processed verification was performed visually for each spectrum. Such maps highlight the spatial variability of the different components (see also supplementary material). As expected, methane and water are present over the whole investigated scanned area (Fig. 7a and7b). The single species map of CH 4 and H 2 O shows several maxima at certain points around (x:y, 605

to 620: -3410 to -3390), while blue tints at both the top and the bottom left of the CH 4 -map (Fig. 7a) are indicative of relatively lower CH 4 signal intensity. The N 2 signal is weaker but follows the same distribution as CH 4 , with less N 2 at the top and the bottom left of the map (blue color, Fig. 7c)).

Furthermore, the H 2 S contribution is weaker than N 2 with maxima distributed heterogeneously on the map, whereas dark areas (black) imply the absence of the H 2 S signal (Fig. 7d). It should be noted that earlier work argues that the heterogeneous composition of NGH samples collected at the Cascadia margin was a result of a variation in the availability the hydrate-forming gas (CH 4 and H 2 S) which is closely related to hydrocarbon fluxes [START_REF] Schicks | Raman spectroscopic investigations on natural samples from the Integrated Ocean Drilling Program (IODP) Expedition 311: Indications for heterogeneous compositions in hydrate crystals[END_REF]. We also believe that such variability in the composition of the gases supplying the hydrate deposit may be responsible for the heterogeneity observed in our hydrate samples. The main processes that generate hydrogen sulfide in upper marine sediments, as either ionized species or free gas, are Anaerobic Oxidation of Methane (AOM)

and Sulfate Organo-clastic Reduction (OSR) [START_REF] Claypool | The origin and distribution of methane in marine sediments[END_REF][START_REF] Froelich | Early oxidation of organic matter in pelagic sediments of the eastern equatorial Atlantic: suboxic diagenesis[END_REF][START_REF] Reeburgh | Methane consumption in cariaco trench waters and sediments[END_REF] and they take place above the gas hydrate occurrence zone (GHOZ).

While H 2 S is involved in the precipitation of several iron-bearing minerals (especially pyrite, FeS 2 ) [START_REF] Canfield | The reactivity of sedimentary iron minerals toward sulfide[END_REF][START_REF] Jørgensen | Anaerobic methane oxidation and a deep H2S sink generate isotopically heavy sulfides in Black Sea sediments[END_REF][START_REF] Ruffine | Effects of postglacial seawater intrusion on sediment geochemical characteristics in the Romanian sector of the Black Sea[END_REF] show that H 2 S concentration of the pore water decreases near-zero values within the first meter of sediment and pyrite is present in very small amounts. Thus, H 2 S is largely consumed above the GHOZ and a small amount would remain to be enclathrated as hydrate, as the distance between AOM, OSR, and the GHOZ is short and less than 1 m. Besides, deeply sourced hydrogen sulfide can originate from thermogenic gas reservoirs [START_REF] Machel | Bacterial and thermochemical sulfate reduction in diagenetic settingsold and new insights[END_REF][START_REF] Machel | Products and distinguishing criteria of bacterial and thermochemical sulfate reduction[END_REF][START_REF] Ruffine | Multiple gas reservoirs are responsible for the gas emissions along the Marmara fault network[END_REF] or brines that are enriched in this compound [START_REF] Commeau | Chemistry and mineralogy of pyrite-enriched sediments at a passive margin sulfide brine seep: abyssal Gulf of Mexico[END_REF].

However, previous studies have shown that the limnic sediment is characterized by a lower salinity [START_REF] Ruffine | Effects of postglacial seawater intrusion on sediment geochemical characteristics in the Romanian sector of the Black Sea[END_REF][START_REF] Soulet | Glacial hydrologic conditions in the Black Sea reconstructed using geochemical pore water profiles[END_REF]. Therefore, the seepage of brine is unlikely in the study area. The contribution of thermogenic gas sources cannot be discarded since the origin of propane is unclear. Thus, the local heterogeneous distribution of H 2 S in the hydrates may result from changes in the migration process of this compound from its source to the hydrate occurrence zone.

4.3.b Spatial variability of the NGH's composition

From the intensity maps generated in Fig. 7, we derived quantitative information on the relative composition of the molecular guest components in the NGH as follows: (1)

𝑋 𝑖 =
where 𝑋 𝑖 is the molar fraction (mol%) of the guest in the NGH (with 𝑖 = 𝐶𝐻 4 , 𝑁 2 , 𝑜𝑟 𝐻 2 𝑆)

while the total cage occupancy of a certain species i derived from the Raman spectra is: This latter is assumed to be independent of the encaged guest species for the same pixel (spectrum) of the map and is therefore simplified in the calculation of the ratio of equation ( 1). Then, the relative F-factors (guest to water) 𝐹 𝑖 𝐹 𝑖 ℎ𝑜𝑠𝑡 ⁄ in (1) are taken from the tabulated values reported for N 2 (0.13), CH 4 (1.0685, average value) [START_REF] Qin | Calibration of Raman Quantification Factors of Guest Molecules in Gas Hydrates and Their Application to Gas Exchange Processes Involving N2[END_REF], and of H 2 S

𝜃 𝑖 = 𝐴 𝑖 ×46 8×𝐴
(1.372), (Yang et al., 2019). 𝐹 𝑖 (or 𝐹 𝑖 ℎ𝑜𝑠𝑡 ) represent the Raman quantification factor of the species i and gathers the information on the Raman cross section of i at a certain wavelength of the excitation laser source and on the instrumental efficiency of the optical and electronic response. Fig. 8 shows a color-coded map of the guest molar composition (mol%) of CH 4 and N 2 in the NGH using equation ( 1). Some parts of the maps clearly exhibit a relative depletion of the CH 4 molar fraction (< ~97 mol%) together with a relative enrichment with N 2 (> ~6 mol%). This can typically be seen in the yellow-green zone of Fig. 8a from (x:y, 520:-3360)

to (x:y, 630:-3360), or equivalently in the green-light blue zone of Fig. 8b. This indicates that while our NGH samples show an optically homogeneous appearance (typically a light-grey area as in Fig. 4 A), the relative guest composition shows a spatial variability (at least at the micron-scale).

To investigate how the presence of nitrogen may impact the stability of the NGH, the equilibrium line of a gas hydrate containing 6% N 2 and balanced CH 4 was simulated (Fig. 9, red line) using CSMGem [START_REF] Sloan | Clathrate Hydrates of Natural Gases[END_REF]). The desired nitrogen composition in the hydrate phase was obtained by processing the equilibrium points with different feed gas ratios N 2 :CH 4 . In the simulation, only pure water was used (no salinity). The result was compared with the equilibrium line of the pure CH 4 -hydrate (hydrate + vapor + liquid water equilibrium line (Fig. 9, blue triangles)). Also, the equilibrium line of the pure N 2 -hydrate was plotted (Fig. 9, black squares) for comparison.

Fig. 9. Simulated (CSMGem) equilibrium line of a gas hydrate containing 6% N 2 + 94% CH 4 (red line). Only pure water is considered for the simulation (no salinity). Comparison with the equilibrium line (hydrate + vapor + liquid water) of pure CH 4 -hydrate (dark-blue up-triangle), pure N 2 -hydrate (black squares): literature data from [START_REF] Adisasmito | Hydrates of carbon dioxide and methane mixtures[END_REF] and [START_REF] Van Cleef | Gas hydrates of nitrogen and oxygen[END_REF]. The green filled circle represents the conditions (282 K, 7.5 MPa) at the sea floor where the NGH was collected.

The presence of nitrogen shifts the dissociation line to higher pressures or lower temperatures (red area on the plot Fig. 9). In other words, the stability of an NGH containing 6% N 2 is lower than that of a gas hydrate containing 100% CH 4 . Note that the N 2 enrichment up to ~6 mol% is an upper concentration limit for N 2 (Fig. 8) at a certain location of the sample and the value of ~0.29 mol% N 2 reported above (section 4.1) constitutes an average concentration which will provide an equilibrium line (not calculated here) much closer to the CH 4 -hydrate equilibrium line and below the NGH point (green point in Fig. 9). Moreover, the formation of hydrates with (94% CH 4 + 6% N 2 ) requires a feed gas with an even higher nitrogen content (~20% N 2 + 80% CH 4 ) as the N 2 selectivity at the hydrate phase is assumed to be lower than that of CH 4 . It appears that the thermodynamic conditions of the NGH, which is expected to be that of the sea floor ~282 K and 7.38 MPa, falls outside the equilibrium line of a simulated mixed hydrate of composition (94 % CH 4 + 6% N 2 ). This suggests that the formation conditions of the NGH can be impacted by important but very local changes in temperature and pressure within the sediment, and/or in gas composition or even by the salt environment. Further work with synthetic binary (CH 4 +N 2 ) mixtures would be useful to elucidate how the guests are spatially distributed in the hydrate sample when starting from a finite amount of gas (water-saturated conditions) (see discussion in section 4.5).

The concentration of H 2 S is too low to be estimated with sufficient accuracy in the present configuration. As mentioned above, the growth of the NGH likely took place in pore water with a fluctuating composition of the dissolved N 2 and H 2 S throughout the hydrate formation process. These two compounds are generated at variable rates and depth in the marine sediments. They can be more or less available to be enclathrated in the hydrates. Thus, the composition of the hydrates may change according to the fluctuation of pore water composition. Note finally that the estimated gas concentration from the single point Ramanspectra shown in Fig. 6b is 98.2 mol% CH 4 , 1.5 mol% N 2 , and 0.3 mol% H 2 S whereas, this reaches ~94.3 mol% CH 4 and ~5.7 mol% N 2 in the absence of H 2 S (Fig. 6a). This is in agreement with the concentration range covered by the map shown in Fig. 8 and collected on a distinct sample part.

Relative cage occupancies of the NGH

From the integrated intensity ratio of the C-H stretching bands of each spectrum collected in the Raman image, we derived a map of the spatial distribution of the relative cage occupancy of the CH 4 in the NGH (Fig. 10). Such results are valuable to future work aiming at improving the estimate of the amount of methane storage in the Western Black Sea hydrate deposit. Because the unit cell of sI has two small cages and six large cages with 46 water molecules, a relation between the Raman integrated intensity of the C-H bands (I LC and I SC )

and the cage occupancy ratio (𝜃 𝐿𝐶 𝜃 𝑆𝐶 ⁄ ) is obtained by the relation (2):

𝜃 𝐿𝐶 𝜃 𝑆𝐶 ⁄ = (𝐼 𝐿𝐶 3𝐼 𝑆𝐶 ⁄ ) × 𝐹 (2)
where F ~1.08 represents the Raman scattering cross-section ratio of methane trapped in the SC relative to the one of methane in the LC [START_REF] Qin | Quantitative Analysis of Gas Hydrates Using Raman Spectroscopy[END_REF].

It is noticeable to observe that N 2 -rich areas in Fig. 8b correspond to the low I LC /I SC ratio in Our reported values are the first micron-scale structural study of gas hydrate sample from the Black Sea, it is then interesting to compare the results with those obtained on other NGHs from the literature (Table S1 in supplementary material) or synthetic pure methane hydrates.

It has often been reported that in marine gas hydrates the LC is almost filled with cage occupancy higher than 90%, whereas the SC remains partially filled around 80% for most marine hydrates (see [START_REF] Liu | The Characteristics of Gas Hydrates Recovered from Shenhu Area in the South China Sea[END_REF] and reference therein). It is also found that the relative cage occupancy (𝜃 𝐿𝐶 𝜃 𝑆𝐶 ⁄ ) values vary strongly in NGH according to the geological area (Table S1) and the nature of the hydrocarbons co-encased in the structure. The analyses from offshore India, Ulleung Basin, Malik 5L-38, Mount Elbert Well, Gulf of Mexico, Lake Baikal indicate that the NGH consist of various guest molecules [START_REF] Bahk | Occurrence of near-seafloor gas hydrates and associated cold vents in the Ulleung Basin, East Sea[END_REF][START_REF] Kida | Natural gas hydrates with locally different cage occupancies and hydration numbers in Lake Baikal[END_REF][START_REF] Klapp | Mixed gas hydrate structures at the Chapopote Knoll, southern Gulf of Mexico[END_REF][START_REF] Kumar | Structural Characterization of Natural Gas Hydrates[END_REF]H. Lu et al., 2011aH. Lu et al., , 2011b;;[START_REF] Lu | Gas hydrate occurrences in the Qilian Mountain permafrost, Qinghai Province, China[END_REF][START_REF] Ripmeester | Structure and composition of hydrate in sediment recovered from Mallik 5L-38, Mackenzie Delta, NWT Canada: X-ray diffraction, Raman and Solid-state NMR spectroscopy[END_REF] In comparison, in artificial methane hydrates, it was found that 𝜃 𝐿𝐶 𝜃 𝑆𝐶 ⁄ decreases slightly (from ~1.15 to 1.10) as pressure increases from 3.5 to 15 MPa along isotherm 268 K, that is, when exploring the stability field of methane hydrates. This was attributed to a pressuredependent change of the relative size of the SCs and LCs [START_REF] Chazallon | In situ structural properties of N2-, O2-, and air-clathrates by neutron diffraction[END_REF][START_REF] Qin | Quantitative Analysis of Gas Hydrates Using Raman Spectroscopy[END_REF]. In contrast, the ratio 𝜃 𝐿𝐶 𝜃 𝑆𝐶 ⁄ was found to be independent of pressure along the three-phase equilibrium curve between 5 and 60 MPa, with I LC /I SC ~3.9 (i.e., 𝜃 𝐿𝐶 𝜃 𝑆𝐶 ⁄ ~ 1.28) [START_REF] Moryama | In Situ Raman Spectroscopic Studies on Small-Cage Occupancy of Methane in the Simple Methane and Methane + Deuterated Tetrahydrofuran Mixed Hydrates[END_REF]. In both cases, the SC occupancy was found to be lower (and constant (~80%) even below 20 MPa) than that of the LC with 100% occupancy. Since our NGH was collected in conditions (~282 K and ~7.5 MPa) close to the three-phase equilibrium curve of pure (sI structure) CH 4 -hydrate (~ 1.5 MPa above the equilibrium line), the expected I LC /I SC ratio should be close to that calculated by [START_REF] Moryama | In Situ Raman Spectroscopic Studies on Small-Cage Occupancy of Methane in the Simple Methane and Methane + Deuterated Tetrahydrofuran Mixed Hydrates[END_REF]. Any deviation from I LC /I SC ~3.9 is then attributed either to a variation in the relative amount of minor gas components mixed with methane (nitrogen in our case), or to a local change in thermodynamic conditions (i.e. exploring other deeper regions in the stability field, far from the three-phase equilibrium curve in the (p, T) space). seafloor. This is attributed to sodium ions that compete with methane to occupy the SC of structure sI. In contrast, the highest ratio measured here is I LC /I SC ~ 3.9 although clays (with illite > 90% w/w and kaolinite ~5% w/w) are the dominant minerals of the sediment at the area where the core was collected [START_REF] Ruffine | Effects of postglacial seawater intrusion on sediment geochemical characteristics in the Romanian sector of the Black Sea[END_REF], no evidence of montmorillonite was found. Therefore, an analogy with what has been observed with Ca-and Na-montmorillonite needs to be developed.

Furthermore, electrolyte solutions (3.5 wt% NaCl in a sediment-free solution) are known to have a negligible effect on the cage occupancy of CH 4 -hydrates (I LC /I SC ~3.8) [START_REF] Moryama | In Situ Raman Spectroscopic Studies on Small-Cage Occupancy of Methane in the Simple Methane and Methane + Deuterated Tetrahydrofuran Mixed Hydrates[END_REF]. Similarly to the case of hydrates formed from pure water [START_REF] Qin | Quantitative Analysis of Gas Hydrates Using Raman Spectroscopy[END_REF], the ratio slightly decreases (I LC /I SC ~3.2) at higher pressure (10 MPa and 274 K), i.e., deep in the stability field, in a salt environment (4 wt% NaCl) [START_REF] Shin | Temperature-and pressure-dependent structural transformation of methane hydrates in salt environments[END_REF]. Thus, sodium ions present in the original aqueous solution do not impact cage occupancy in artificial hydrates.

Nevertheless, the Black Sea has a lower salinity level (~1 wt% measured in the pore water)

than the known mean value of sea water (~3.5 wt%) as a result of ongoing seawater infiltration within the limnic sediment of the Black Sea since the reconnection of this sea with the Atlantic-ocean. This progressive downward increase in pore-water salinity changes the chemical and mineral properties of sediment through ion-exchange and redox processes [START_REF] Ruffine | Effects of postglacial seawater intrusion on sediment geochemical characteristics in the Romanian sector of the Black Sea[END_REF]. These changes in sediment properties may also affect the mechanism of hydrate formation. Accordingly, a more detailed study of the impact of sediment property changes due to seawater infiltration on cage occupancy appears necessary in the Black Sea where salinity decreases downward from 20 to 2 psu at around 25 mbsf [START_REF] Riboulot | Freshwater lake to salt-water sea causing widespread hydrate dissociation in the Black Sea[END_REF][START_REF] Ruffine | Effects of postglacial seawater intrusion on sediment geochemical characteristics in the Romanian sector of the Black Sea[END_REF][START_REF] Soulet | Glacial hydrologic conditions in the Black Sea reconstructed using geochemical pore water profiles[END_REF]. This salinity value of 2 psu is lower than the 6 psu measured by [START_REF] Manheim | Interstitial Waters of Black Sea Sediments: New Data and Review[END_REF] within the first 8 m of sediment.

Hydration numbers and correlation with the estimated total amount of methane

From the results of the previous map measuring relative cage occupancy (Fig. 10), we derived the absolute values of cage occupancies using the van der Waals and Platteeuw theory (van der [START_REF] Van Der Waals | Clathrate solutions[END_REF]) assuming that the chemical potential difference between ice and the hypothesized empty clathrate lattice of sI is ∆𝜇 𝑤 °= 1297 𝐽/𝑚𝑜𝑙 (Davidson et al., 1986b) and the temperature is fixed at 282 K (the sea floor temperature). The map of Fig. 11a displays the spatial variation of the CH 4 occupancy in the SC that shows significant variation from ~75 % (dark color) to 100 % (red color) in the analyzed zone. It should be noted that the part of the map where the CH 4 -SC occupancy is between ~98-100 % (Fig. 11a) corresponds to an N 2 -rich area (Fig. 8b). In contrast the CH 4 -LC occupancy never reaches 100% (Fig. 11b) and the orange to brown-colored area with CH 4 -LC occupancy of ~93-95 % corresponds to the N 2 -depleted area (Fig. 8b). Moreover, the green-colored area (Fig. 11b) with CH 4 -LC occupancies of ~85 % corresponds to an N 2 -rich area (Fig. 8b). The comparison between the concentration map (Fig. 8) and the map of Fig. 11 suggests that the occurrence of N 2 guest molecules have an impact on the global methane cage occupancy with N 2 preferentially populating the LC of the structure I. This result is unexpected when considering the equilibrium cage occupancies obtained during the simulation by CSMGem (Fig. 9) of the equilibrium line trajectory with corresponds to equilibrium cage occupancies for CH 4 of  LC ~90% and  SC ~77%. The guest distribution in the NGH may compensate the less good stabilization due to the large cages being occupied by N 2 . Therefore, we suggest that the distinct guest distribution observed here may play a role in the stability of the N 2 -rich area of the NGH. It should be noted that the earlier work of [START_REF] Sum | Measurement of Clathrate Hydrates via Raman Spectroscopy[END_REF] does not show any variation in absolute methane cage occupancy. The authors started to form their synthetic mixed CH 4 -N 2 -hydrate samples from a CH 4 -N 2 feed gas composition as low as y CH4 ~ 58%.

This may be explained by the consumption of the milder hydrate former first (with a CH 4enriched hydrate), followed by the formation of spatially separated secondary pure hydrate crystals (N 2 -enriched hydrate). Further investigation of the influence of nitrogen on methane cage occupancy using synthetic hydrates would be helpful to elucidate the distribution of nitrogen in the cages of this mixed hydrate system. It should be noted that N 2 and CH 4 molecules possess similar sized properties with guest-to-cavity diameter ratios of ~0.8 (N 2 ) to 0.86 (CH 4 ) for the SC and ∼0.7 (N 2 ) to 0.74 (CH 4 ) for the LC of sI [START_REF] Sloan | Clathrate Hydrates of Natural Gases[END_REF].

According to the guest-to-cavity diameter rule (i.e., guest-to-cavity diameter ratio < 0.76 or > 1 does not allow cage stabilization), both molecules fit the SC better and are expected to occupy preferentially this cage type. This makes the prediction of the preferential partitioning within the cages relatively unsure using simple additive effects, specifically when a structure sI former (CH 4 ) is mixed with a sII former (N 2 ). The guest-host interactions likely change in the mixed system maintaining a preferential stable structure with adapted cage occupancy.

How this result will affect the global estimate of methane is a critical question. As mentioned previously, the global estimates of methane gas trapped in NGH are based on several parameters that are functions of geological conditions. One of these concerns the volumetric conversion factor that has often been calculated by assuming (based on structure sI) a mean cage occupancy of 85%, i.e. a hydration number of methane hydrates of ~6.75 (= 46/(0.85 x 8)), and therefore a value of ~164 m 3 of methane per m 3 of hydrate is being typically used [START_REF] Boswell | Current perspectives on gas hydrate resources[END_REF][START_REF] Kvenvolden | A primer on the geological occurrence of gas hydrate service Permission Subscribe[END_REF]. We derived an upper estimate of the volumetric conversion factor of our NGH which can contain up to ~6 mol% of N 2 according to the spectral map presented in Fig. 8. Given the CH 4 cage occupancy calculated in the map of Fig. 10 at the corresponding composition of 6% N 2 , one derives that 1 m 3 of such NGH will instead contain ~153 m 3 of CH 4 . For this calculation, we used the lattice constant of CH 4hydrate provided at 282 K by [START_REF] Kuhs | Ice perfection and onset of anomalous preservation of gas hydrates[END_REF] as a = 11.98 Å and a cage occupancy of 𝜃 𝐿𝐶 ~ 0.84, 𝜃 𝑆𝐶 ~ 0.99 (hydration number of ~6.55) (from Fig. 11). Note that both the lattice constant and the cage occupancies adopted in this calculation are critical parameters. A value of ~148 m 3 is obtained with the lattice constant a = 11.98 Å and the mean cage occupancy of 85% [START_REF] Boswell | Current perspectives on gas hydrate resources[END_REF]. To summarize, the spatial variation of the methane cage occupancy which is reminiscent of the presence of additional minor guest-gas components (mainly N 2 ) in the NGH may affect significantly the volumetric conversion factor and therefore the global estimate of methane in NGH.

Conclusion

NGH collected during the Ghass cruise (2015) in the Black Sea onboard the R/V

Pourquoi pas? have been analyzed using different complementary techniques: gas chromatography and micro-Raman spectroscopic imaging. The results showed that the hydrate-bound light hydrocarbons are of microbial origin, and generated from the carbondioxide reduction process. Methane is overwhelmingly present, followed by nitrogen (with some enrichment higher than 6 mol%). Carbon dioxide, as well as ethane and propane, are minor elements to traces. The Raman signal of a small amount of hydrogen sulfide (H 2 S) is also detected in the NGH, which supposes that it may be found locally in the surrounding water at a higher concentration level than ethane, propane, and carbon dioxide, as they were not identified in the hydrate phase. This important result indicates that hydrates are potential sinks for H 2 S generated from seawater-sulfate reduction. Single point Raman spectra allow the identification of distinct guest components in the NGH embedded in the sediment, while 2D-Raman images reveal the heterogeneous distribution of the guest concentration at the microscale, with methane being the main component. The spatial variability of the CH 4 cage occupancy is reported for the first time and can be correlated with the inclusion of nitrogen that is shown to preferentially occupy the LC of the structure I. Other components (carotenoids) often identified in pore water during field experiments [START_REF] Zhang | Development and deployment of a deep-sea Raman probe for measurement of pore water geochemistry[END_REF] are identified here in the well-preserved recovered samples. The small-scale heterogeneities of the sample composition, with addressed statistical issues, highlight the importance to conduct 2D-Raman images when characterizing natural gas hydrate samples. Furthermore, from the Raman map of the CH 4 cage occupancies in the NGH, it was derived that the occurrence of minor components, even at low concentrations, could impact the volumetric conversion factor of CH 4 gas. Typically, hydrates including N 2 at 6mol% have a lower volumetric conversion factor than that expected in a GH containing only methane. There is also a need to characterize the variation of the cage occupancy with depth within the sediment, and then compare the result with the zonation of geochemical reactions, which would help to decipher sources of minor guest molecules. Also, a systematic study on a larger number of NGH samples from the same area would be appropriate to identify the main factors that are responsible for the heterogeneity of cage occupancy and to provide meaningful insights into the hydrate formation mechanism. The overall results allow to define a statistical cage occupancy distribution, which, if coupled with seismic data, would allow us to refine the estimation of the amount of gas trapped in the hydrate deposit.

Fig. 1 :

 1 Fig. 1: A Bathymetric map of the Romanian sector of the Black Sea showing the Danube canyon and the location of the study area on the continental slope inside the gas hydrate province. The upper limit of the predicted base of the Gas Hydrate Stability Zone (GHSZ) is indicated by the thick red line[START_REF] Ker | Anomalously Deep BSR Related to a Transient State of the Gas Hydrate System in the Western Black Sea[END_REF]. The

Fig. 2 :

 2 Fig. 2: Description of the hydrate-bearing core with localization of the hydrate zone within the sample.

Fig. 3 :

 3 Fig. 3: Source assessment of the light hydrocarbons of the hydrate-bound gases from geochemical diagrams in the a)  13 C-CH4 vs.  13 C-C 2 H 6 space (modified after[START_REF] Bernard | Light hydrocarbons in recent Texas continental shelf and slope sediments[END_REF] and[START_REF] Milkov | Organic Geochemistry Worldwide distribution and significance of secondary microbial methane formed during petroleum biodegradation in conventional reservoirs[END_REF]), b)  2 H-CH 4 vs

  dotted line) which corresponds to a GH-rich zone mixed with frozen water (ice phase) 2) a light-brown zone with dark patches (sediment) (B, Fig 4, delineated by an orange-dotted line)

Fig. 4 :

 4 Fig. 4: 3D microscopic image (objective x20) of the NGH collected in the Black Sea. The light-grey zone with white patches (A, delineated by a white-dotted line) indicates the presence of bulk ice and/or hydrates, a light brown zone (B, delineated by an orange-dotted line) with small dark patches (right corner of the orange-dotted line) consistent with the presence of iron-bearing minerals (see text for details), a dark-grey area at the bottom right-hand side with small white patches (C, delineated by a light-green dotted line) indicating solid hydrates (or ice) intermixed with sediments. The inset (top right) shows a macroscopic view of hydrates pieces extracted from the core.

Fig. 5 :

 5 Fig. 5: Optical image of a sediment-rich zone (typically represented in Fig 4, B, or C) and corresponding Raman spectrum in the 200-3600 cm 1 spectral region of the NGH sample. The point on the optical image corresponds to the position where the Raman spectrum (yellow inset) was collected. Wavenumbers indicated in red correspond to the contribution of β-carotene. The inset in the spectrum shows the characteristic peak of methane molecules trapped in the NGH.

  and 6b) collected at the surface of a light-grey zone (typically represented in Fig 4, A) show the well-known signature of methane with a band doublet at ~2903 cm 1 and 2914 cm 1 attributed to C-H stretch of CH 4

Fig. 6 :

 6 Fig. 6: Typical spectra collected from single-point Raman analysis of the NGH. The optical images are taken from a light-grey zone (sub-sample of the NGH's piece of Fig. 4 A) and the connected-lines show the positions at which the Raman spectra have been collected. The attribution of guests trapped in the large cage (LC) and small cage (SC) of the NGH structure I is shown in the insets (see text for details). All insets are baseline corrected: a) CH 4 (red inset) trapped into the NGH (LC peak at 2903 cm -1 and SC peak at 2914 cm -1 ). Nitrogen

Fig. 7 :

 7 Fig. 7: Intensity distribution (integrated intensity after baseline correction) images from a Raman map (120 x 85) µm² of a selected sub-sample of the NGH. Color scale on the right of each image represents the variation of the integrated intensity of selected Raman bands for (a) CH 4 (range: 2859-2950 cm 1 ), (b) H 2 O (2950-3700 cm 1 ), (c) N 2 (2321-2326 cm 1 ), (d) H 2 S (2590-2606 cm 1 ). Note that the value on the ranges (scale) are different despite the same color. See text for details.

  𝑖 ℎ𝑜𝑠𝑡 ×𝐹 𝑖 𝐹 𝑖 ℎ𝑜𝑠𝑡 ⁄ in structure I. 𝐴 𝑖 represents the integrated intensity of the Raman bands attributed to guest 𝑖 and 𝐴 𝑖 ℎ𝑜𝑠𝑡 the integrated intensity of the water contribution (O-H stretch).

Fig. 8 :

 8 Fig. 8: Spatial variation of the guest molar fraction (mol%) (see equation (1)) in the NGH from the Raman map (120 x 85) µm² of a selected sub-sample. The color scale on the right of each image represents the variation of the integrated intensity of the selected Raman bands for (a) CH 4 (range: 2859-2950 cm 1 ), (b) N 2 (2321-2326 cm 1 ). Note the yellow-green area in map (a) (from (x:y, 520:-3360) to (x:y, 630:-3360)) has its counterpart in map (b) zone (green-light blue).

Fig. 10 (

 10 Fig. 10 (dark-blue zone, Fig. 10). For example, I LC /I SC ~2.3 means that the cage occupancy ratio is 𝜃 𝐿𝐶 𝜃 𝑆𝐶 ⁄ ~ 0.82 and thus the LCs are less populated by CH 4 molecules than the SCs in structure sI. This is attributed to the fact that a certain percentage of the LC is occupied by N 2 molecules. When a very small amount (or no amount) of N 2 is detected, the I LC /I SC increases to ~3.5, i.e., a cage occupancy ratio of 𝜃 𝐿𝐶 𝜃 𝑆𝐶 ⁄ ~ 1.26.

Fig. 10 :

 10 Fig. 10: Spatial variability of the Raman integrated intensity ratio of the C-H bands attributed to methane in the LC and SC of the NGH containing methane and nitrogen as main gas components. See text for details.

Fig. 11 :

 11 Fig. 11: Spatial variation of cage occupancies in the NGH structure I derived from the Raman data and the van der Waals and Platteeuw model a) SC (small cage) occupancy, b) LC (large cage) occupancy. Note that the filling of the SC approaches 100% in most areas whereas the filling of the LC is around 85-95%. See text for details.

Table 1 :

 1 Molecular and isotopic compositions of the hydrate-bound gases from two different samples.

			N 2	CH 4	CO 2	C 2 H 6	C 3 H 8	CH 4 /
								(C 2 H 6 + C 3 H 8 )
	SAMPLE	mol.%	0,3796 99,5571	0,0560	0,0071	0,0002	13638
	1	 13 C (‰)		-69.3	-22.3	-63.6	-27.8
		D (‰)		-291		-250
	SAMPLE	mol.%	0,2933 99,6570	0,0416	0,0078	0,0003	12303
	2	 13 C (‰)		-69.4	-22.1	-63.6	-27.9
		D (‰)		-290		-250

Table 2 :

 2 Raman spectral positions of the molecules CH 4 , N 2 and H 2 S in the hydrate and the free gas phase

	Position of the Raman bands	CH 4	N 2	H 2 S
	(cm 1 )
	Hydrate	~2903 (Large cage) a	2323.5 b	2593 (Large cage) a, c
		~2914 (Small cage) a		2604 (Small cage) a, c
	Free gas	2916 c	2329 a	2611 c
	a Observed in the present study
	b

  . The lowest ratios reported are 𝜃 𝐿𝐶 𝜃 𝑆𝐶 ⁄ ~ 0.91 in Mount Elbert well, and Malik 5L-38 well Alaska (onshore) and ~1.09 in Pearl River Mouth basin (PRM basin, offshore south China).

. They show the predominance of methane gas trapped in the hydrate structure. In sedimentrich zones (typically represented in Fig.4by zone B or C), single-point Raman spectra show many peaks in the range of 800 cm 1 to 3100 cm 1 (Fig.5). Specifically, two main contributions occurred at ~1155 cm 1 and ~1521 cm 1 which are characteristic of the C-C stretching  3 and  1 of the main polyene chain of carotenoids, respectively. Carotenoids are

Acknowledgements

We thank the captain and his crew on-board the RV Pourquoi pas? for their technical support and advice. Thanks to Alison Chalm for proofreading the article. Financial support for the cruise was provided by Ifremer and the European project Midas (grant agreement 603418).

One author (C.T.R.) thanks Lille University and the Région Hauts-de-France for support through a Ph.D. grant n°17006757. This work is a contribution to the BLAME project sponsored by the French National Research Agency (ANR-18-CE01-0007). This study was undertaken in the frame of the French Research Consortium "GdR2026-Hydrates de gaz".