Molecular and Isotopic Composition of Volatiles in Gas Hydrates and in Sediment from the Joetsu Basin, Eastern Margin of the Japan Sea

Abstract

Hydrate-bearing sediment cores were retrieved from the Joetsu Basin (off Joetsu city, Niigata Prefecture) at the eastern margin of the Japan Sea during the MD179 gas hydrates cruise onboard R/V Marion Dufresne in June 2010. We measured molecular and stable isotope compositions of volatiles bound in the gas hydrates and headspace gases obtained from sediments to clarify how the minor components of hydrocarbons affects to gas hydrate crystals. The hydrate-bound hydrocarbons at Umitaka Spur (southwestern Joetsu Basin) primarily consisted of thermogenic methane, whereas those at Joetsu Knoll (northwestern Joetsu Basin, about 15 km from Umitaka Spur) contained both thermogenic methane and a mixture of thermogenic and microbial methane. The depth concentration profiles of methane, ethane, propane, CO₂, and H₂S in the sediments from the Joetsu Basin area showed shallow sulfate–methane interface (SMI) and high microbial methane production beneath the SMI depth. Relatively high concentrations of propane and neopentane (2,2-dimethylpropane) were detected in the headspace gases of the hydrate-bearing sediment cores obtained at Umitaka Spur and Joetsu Knoll. Propane and neopentane cannot be encaged in the structure I hydrate; therefore, they were probably excluded from the hydrate crystals during the structure I formation process and thus remained in the sediment and/or released from the small amounts of structure II hydrate that can host such large gas molecules. The lower concentrations of ethane and propane in the sediment, high δ¹³C of propane and isobutane, and below-detection normal butane and normal pentane at Umitaka Spur and Joetsu Knoll suggest biodegradation in the sediment layers.

1. Introduction

Gas hydrates are crystalline clathrate compounds consisting of water and gas molecules that form at low temperatures and high pressures [1]. Natural gas hydrates are found worldwide in continental margin sediments [2,3,4] and in near-surface sediments associated with active gas plume that vent from the seafloor [5,6,7]. Natural gas hydrates are considered potential energy resources and are large reservoirs of methane (C₁), and their dissociation may cause submarine geohazards and contribute to global warming [8,9,10].

Crystallographic structures of natural gas hydrate are usually either structure I (sI), which is composed of two 12-hedra and six 14-hedra with space group Pm3n, or structure II (sII), which is composed of sixteen 12-hedra and eight 16-hedra with space group Fd3m [1]. C₁ and ethane (C₂) are both known to form sI hydrates; however, certain compositions of C₁ and C₂ will form sII hydrates [11,12], whereas propane (C₃) and isobutane (i-C₄) is incorporated only in sII. The concentrations of C₂ and C₃ in hydrate-bound gas from the Gulf of Mexico represent 3%–5% and >15% of the total gas, respectively [13]. Gas hydrate from the Sea of Marmara contained high concentrations of C₃ (18.8%) and i-C₄ (9.5%) [14]. Mixed-gas (C₁ and C₂) hydrates in Lake Baikal belonged to the sII hydrate and contained 0.026%–0.064% of neopentane (neo-C₅, 2,2-dimethylpropane) [15,16], which can be encaged in the large cages of sII [17].

Shallow gas hydrates were recently found in the Joetsu Basin at the eastern margin of the Japan Sea, where gas venting was observed on echo-sounder images [18,19]. Gas hydrate was recovered from the sea floor [20], and δ¹³C of hydrate-bound C₁ was from −37.3‰ to −37.1‰ [20], suggesting thermogenic origin in the criteria of the Bernard plot [21]. However, C₂ and C₃ concentrations of hydrate-bound gas at Umitaka Spur and Joetsu Knoll (Figure 1) were very low; the molar ratio C₁/(C₂ + C₃) ranged from 1000 to 10,000 [22]. The Sado Nansei Oki drillings at Umitaka Spur conducted by the Ministry of Economy, Trade and Industry, Japan revealed C₁/(C₂ + C₃) values less than 100 at depths 1143−2016 m below the sea floor [20], indicating either that C₂ and C₃ concentrations are reduced in the ascending fluid during migration or that the addition of microbial C₁ is significant in higher strata. The depth profiles of sediment gases at Umitaka Spur and Joetsu Knoll were reported in our previous work [23]; however, molecular and isotopic compositions of hydrate-bound gas were not reported yet and the effect of low concentration of higher hydrocarbons on gas hydrates has not been discussed. In this study, we investigate the molecular and stable isotope compositions of hydrate-bound gas and gas in sediment cores (headspace gas) retrieved from Umitaka Spur and Joetsu Knoll and focus on C₂, C₃, and higher hydrocarbons, those may change crystallographic structure of the shallow gas hydrates in these areas.

Figure 1. Locations of the coring sites in the Joetsu Basin area, Japan Sea. The sites of hydrate-bearing cores are highlighted.

Figure 1. Locations of the coring sites in the Joetsu Basin area, Japan Sea. The sites of hydrate-bearing cores are highlighted.

2. Study Sites and Sediment Cores

Umitaka Spur and Joetsu Knoll are located in the Joetsu Basin off the city of Joetsu (Niigata Prefecture, Figure 1). High P-wave velocities at the mounds and pockmarks on the Umitaka Spur seafloor were reported, suggesting the existence of gas hydrate [24]. Echo sounding of the sea bottom at Umitaka Spur and Joetsu Knoll showed large-scale gas venting from the sea floor [18]. Depth profiles of sulfate in the pore water of the sediment cores indicated shallow (<5 m) sulfate–methane interface (SMI; where sulfate and methane are both consumed to depletion at the base of the sulfate reduction zone) in the Joetsu Basin [25], indicating high flux of hydrocarbons and other organic matter. High heat flow was observed in a restricted area on the sea floor around the gas seep sites at Umitaka Spur (maximum value of 1590 mW m⁻²) and Joetsu Knoll (maximum value of 519 mW m⁻²) compared to the average value obtained on a normal muddy seafloor in these areas (98 ± 13 mW m⁻²), indicating local fluid migration from the deep subsurface layer [26].

Previous study considered origin of hydrate-bound C₁ at Umitaka Spur and Joetsu Knoll as thermogenic one (C₁ δ¹³C ranged from −39.0 to −34.9‰ V-PDB) and mixed gas of microbial and thermogenic ones (C₁ δ¹³C ranged from −57.9 to −51.8‰ V-PDB), respectively [22]. Because the content of total organic carbon (TOC) ranged from 0.58% to 1.55% in the shallow sediment layer (0–9 m depth, [27]) and from 0.90% to 2.98% in the deep layer (1360–2088 m depth, mixed Type II/III kerogen, [20]), the potential of C₁ generation via microbial and thermogenic processes is high.

Sediment cores were retrieved from Umitaka Spur, Joetsu Knoll, Joetsu Basin and its peripheral area (Figure 1) during the MD179 cruise onboard R/V Marion Dufresne in June 2010. A Calypso giant piston corer enabled us to obtain sediment cores up to 40 m in length. A Calypso square box corer (CASQ, 25 cm × 25 cm × 12 m) was also used. Out of the 20 sediment cores obtained, four contained hydrates: MD179-3305G and MD179-3306 (located 300 m apart) from Umitaka Spur, and MD179-3317 and MD179-3318C (located 5 km apart) from Joetsu Knoll. Hydrates were virtually absent in the other sediment cores. MD179-3305G was retrieved by a gravity corer, and a few samples of the gas hydrates were taken for gas analysis. All the hydrate-bearing cores except MD179-3318C lost several meters of the top sediment layers because the lead weight section was disconnected from the core barrel due to a technical problem; the gas hydrates dissociated in the corer and the core top was displaced during the onboard recovery process. The top depths of the gas hydrates were estimated to be 4.5 mbsf (MD179-3306), 31.5 mbsf (MD179-3317), and 1.5 mbsf (MD179-3318C). The depth of gas hydrate in MD179-3305G was unknown.

3. Sampling Methods and Analysis

3.1. Onboard Gas Sampling

The gas sampling procedures for the hydrate-bound and headspace gases were the same as those used in our previous studies [28,29]. The hydrate-bound gases were collected using a 50-mL plastic syringe and stored in 5-mL vials sealed with butyl septum stoppers. Several samples were taken from each hydrate-bearing sediment core. First, we placed small pieces of hydrate sample into a 50-mL plastic syringe, pushed the cylinder to reduce the dead volume, and connected the syringe to a 5-mL vial with a needle. Another needle was connected to the vial to flush the air inside. Each vial was thus filled with hydrate-bound gas without sediment particles or water. Consequently, this method contributed to prevention of microbial activity during storage. The gas dissociated from hydrate flushed air in the system, and the concentration of air in the vial was less than 2%.

Headspace gas method was employed to know the depth profiles of each gas component in the sediment cores. 10 mL of sediment, 9.5 mL of saturated aqueous solution of NaCl, and 0.5 mL of preservative (10 wt % aqueous solution of benzalkonium chloride, [30]) were introduced into 25-mL vials to create a 5-mL headspace. The headspace was flushed with helium, the carrier gas used in the gas chromatography, to reduce air contamination. Although the flushing process may reduce gas concentrations from their native values, differences in concentration and stable isotopes (δ¹³C and δD) of C₁ between flushed and non-flushed samples were undetectable [31]. Hence, we can discuss the trend of their depth profiles using headspace gas data. The vials were shaken well and stored upside down until they were analyzed.

In some of the gas-rich cores (MD179-3296, 3299, 3301, 3304, 3308, 3313, and 3317, see Figure 1), voids formed in the plastic core liner during their retrieval from the core barrel. The gas in the voids was collected by modifying the method of [32]. The surface temperature of the plastic core liner was measured with an infrared thermal imaging camera to locate the voids. Small holes several meters apart were then drilled into the liner. Gas samples collected through these holes with a 50-mL plastic syringe were stored in evacuated 5-mL glass vials. Because the concentration of hydrocarbons in the void gas was considerably higher than that obtained by the headspace gas method, the void-gas method is suitable for stable carbon and hydrogen isotope analysis on non-C₁ hydrocarbons, which are typically present in low concentration. Isotopic difference between void and headspace gases was within 2‰ for C₁ δ¹³C and 3‰ for C₁ δD [23]. In this study, we obtained the stable carbon isotope signature of C₂ by the void-gas method.

3.2. Analytical Methods

The molecular composition of the gas samples was determined using a gas chromatograph (GC-14B, Shimadzu, Kyoto, Japan) equipped with a packed column (Shimadzu Sunpak-S; 2 m length, 3 mm ID), a thermal conductivity detector (TCD) for detecting CO₂, H₂S, and high concentrations (>0.1 mol% of the sample gas) of C₁, and a flame ionization detector (FID) for detecting low concentrations (<0.1 mol% of the sample gas) of hydrocarbons (C₁–C₅). The TCD and FID were connected in series. The detection limit was 0.00005 mol% (C₁–C₃) and 0.0005 mol% (C₄–C₅). The analytical error estimated by multiple injections of standard gases was less than 1.2% for each gas component.

Stable carbon and hydrogen isotopic ratios of the hydrocarbons and CO₂ were measured using a continuous-flow isotope-ratio mass spectrometer (CF-IRMS, DELTAplus XP, Thermo Fisher Scientific, Waltham, MA, USA) coupled with a gas chromatograph (Trace GC, Thermo Fisher Scientific) via a combustion/pyrolysis reactor (GC-C/TC III, Thermo Fisher Scientific). The gas chromatograph was equipped with a Carboxen-1006 PLOT capillary column (30 m length, 0.32 mm ID, 15 μm film thickness, Sigma-Aldrich, St. Louis, MO, USA). In the case of samples with low C₁ concentration, a Sigma-Aldrich Carboxen-1010 PLOT capillary column (30 m length, 0.32 mm ID, 15 μm film thickness) was also used to separate air components from hydrocarbons. A PoraPLOT Q capillary column (27.5 m length, 0.32 mm ID, 10 μm film thickness, Agilent Technologies, Santa Clara, CA, USA) was used for higher hydrocarbons (C₄–C₈). Stable isotope compositions are reported as δ values (‰):

$${\delta }_{sample}=\left(\frac{R_{sample}-R_{\text{standard}}}{R_{\text{standard}}}\right)\times 1000 (\text{‰})$$

(1)

where R denotes the ¹³C/¹²C or D/H ratio. δ¹³C and δD are given with reference to the V-PDB and V-SMOW standards, respectively, determined by using NIST RM8544 (NBS19) for δ¹³C and NIST RM8561 (NGS3) for δD. The analytical precision was 0.3‰ for δ¹³C of C₁–C₃, 1‰ for δ¹³C of C₄–C₈, and 2‰ for δD.

4. Results

Figure 2a–c show the depth profiles of the concentration (C₁, C₂, C₃, CO₂, and H₂S) of headspace gases, C₁/(C₂ + C₃) values calculated from the concentration of C₁, C₂, and C₃, and stable carbon and hydrogen isotopes of C₁, C₂, and CO₂. The concentration of C₁ drastically increased with depth toward the SMI, with the peak concentration appearing at around 5–8 mbsf and then decreasing slightly. The C₁ concentration profiles indicate that the SMI is around 0.5–5 mbsf in the Joetsu Basin area including Umitaka Spur and Joetsu Knoll. The trend of C₂ and C₃ concentration profiles was similar to that of C₁; however, their concentrations below the SMI differed between the sediment cores. C₁/(C₂ + C₃) values showed a peak near 5–8 m, which agrees with the peak of the C₁ concentrations. C₁/(C₂ + C₃) values below the SMI were less than 10,000 in the areas of Umitaka Spur (Figure 2a) and Joetsu Knoll (Figure 2b) except the cores MD179-3320G and 3324C, and more than 10,000 in the other cores of Joetsu Basin (Figure 2c). The CO₂ concentrations of the all cores simply increased with depth, and the H₂S concentrations showed peaks at their SMI depths.

The trend of C₁ δ¹³C profiles were similar to those of CO₂ δ¹³C, increasing with depth below the SMI. Negative C₁ δ¹³C and CO₂ δ¹³C peaks were observed around the SMI, which agreed with the high H₂S concentrations. High C₁ δ¹³C (more than –45‰ V-PDB) was observed in the hydrate-bearing cores MD179-3306 and 3318C. C₁ δ¹³C at the depth of 10 mbsf ranged from −66.7‰ to −57.8‰ at Umitaka Spur except the core MD-179-3306, from −74.5‰ to −58.3‰ at Joetsu Knoll, and from −83.5‰ to −77.4‰ at the other sites in Joetsu Basin. On the other hand, C₁ δD simply decreased with depth except the core MD179-3306. C₁ δD for MD179-3306 was distinctly large and ranged from −165.9‰ to −157.8‰, while those for other cores ranged from −207.4‰ to −175.8‰. C₁ δ¹³C, C₁ δD, and C₂ δ¹³C of hydrate-bound and headspace gases were almost the same with each other. On the contrary, distinct differences were found in CO₂δ¹³C; those of hydrate-bound gas was 10‰ lower (MD179-3306), 8‰ lower (MD179-3317), and 27‰ higher (MD179-3318C) than those of headspace gas around the same depth.

The concentration of C₂ in the hydrate-bearing cores (MD179-3306, 3317, and 3318C) below the SMI was nearly one order of magnitude higher than that in the nonhydrate-bearing cores. MD179-3297C, 3300C, 3304, and 3307C were also rich in C₂. The concentrations of C₃ in the three hydrate-bearing cores (MD179-3306, 3317, and 3318C) and in MD179-3304 were higher than in the nonhydrate-bearing cores. The C₂-rich cores (MD179-3304 and 3317) showed ¹³C enrichment in C₂, although C₃ δ¹³C is not available. C₂ δ¹³C of nonhydrate-bearing cores in Joetsu Basin was low, ranging from −62.8‰ to −52.6‰.

Figure 2. Depth profiles of C₁, C₂, C₃, CO₂, and H₂S concentrations, C₁/(C₂ + C₃) values, C₁ δ¹³C, C₁ δD, C₂ δ¹³C, and CO₂ δ¹³C in the headspace gas. (a) Umitaka Spur area; (b) Joetsu Knoll area; (c) Joetsu Basin and its peripheral area. These depth profiles of the headspace gases were already reported [23]. The C₁/(C₂ + C₃) values and stable carbon (C₁, C₂, and CO₂) and hydrogen (C₁) isotopic ratios in the hydrate-bound gas of the cores MD179-3306, 3317 and 3318C are plotted. The data of C₂ δ¹³C was obtained by the void-gas method and other data by the headspace gas method. The depth data of C₂ δ¹³C profile for MD179-3296 is unknown. The headspace gas data of MD179-3305G is missing due to sediment loss.

Figure 2. Depth profiles of C₁, C₂, C₃, CO₂, and H₂S concentrations, C₁/(C₂ + C₃) values, C₁ δ¹³C, C₁ δD, C₂ δ¹³C, and CO₂ δ¹³C in the headspace gas. (a) Umitaka Spur area; (b) Joetsu Knoll area; (c) Joetsu Basin and its peripheral area. These depth profiles of the headspace gases were already reported [23]. The C₁/(C₂ + C₃) values and stable carbon (C₁, C₂, and CO₂) and hydrogen (C₁) isotopic ratios in the hydrate-bound gas of the cores MD179-3306, 3317 and 3318C are plotted. The data of C₂ δ¹³C was obtained by the void-gas method and other data by the headspace gas method. The depth data of C₂ δ¹³C profile for MD179-3296 is unknown. The headspace gas data of MD179-3305G is missing due to sediment loss.

Molecular compositions and stable isotope signatures of hydrate-bound gases at Umitaka Spur and Joetsu Knoll are summarized in Table 1. In the entire study sites C₁ was the main component of the hydrate-bound gas, comprising more than 98 mol% of the total volume; the concentrations of C₂, C₃, CO₂, and H₂S were 0.0148–0.0456 mol% (n = 17), 0.0001–0.0025 mol% (n = 17), 0.03–1.26 mol% (n = 16), and 0.03–0.77 mol% (n = 8), respectively. C₁ δ¹³C has a wide range (−57.1‰ to −43.9‰) at Joetsu Knoll (MD179-3317 and 3318C) and a narrow range (−37.3‰ to −34.6‰) at Umitaka Spur (MD179-3305G and 3306), whereas C₁/(C₂ + C₃) values concentrate from 2200 to 9700 at the both areas. C₂ δ¹³C ranged from −31.9‰ to −21.3‰ at Joetsu Knoll and from −18.4‰ to −17.9‰ at Umitaka Spur, respectively.

Table 1. Molecular compositions (mol% of the total components) and stable carbon (C_1–3 and CO₂) and hydrogen (C₁) isotopic ratios of hydrate-bound gases retrieved from Umitaka Spur (MD179-3305G and 3306) and Joetsu Knoll (MD179-3317 and 3318C), Japan Sea. Because H₂S is highly corrosive and reaction with sampling and analytical tools was apparently not prevented, a higher portion of H₂S is lost during core retrieval and gas analysis compared to hydrocarbons and CO₂.

Core No. Depth [mbsf]	Molecular composition						Isotopic composition
	C1 [mol%]	C2 [mol%]	C3 [mol%]	CO2 [mol%]	H2S [mol%]	C1/(C2+C3)	C1δ13C [‰V-PDB]	C2δ13C [‰V-PDB]	C3δ13C [‰V-PDB]	CO2δ13C [‰V-PDB]	C1δD [‰V-SMOW]
MD179-3305G unknown depth	98.1	0.0292	0.0019	1.10	0.77	3158	−36.6	−17.9	5.8	14.3	−167
	98.3	0.0270	0.0020	1.18	0.53	3389	−36.0	−18.2	5.7	18.0	−164
	98.2	0.0301	0.0017	1.26	0.53	3086	−36.0	−18.2	5.9	19.5	−164
	99.8	0.0331	0.0018	0.20	n.d.	2859	−34.6			17.3	−167
MD179-3306 4.5 [mbsf]	99.1	0.0292	0.0020	0.85	n.d.	3176	−37.3	−18.4	6.3	20.3	−164
	100.0	0.0221	0.0016			4213	−35.7	−18.1	4.8	23.8	−167
MD179-3317 31.5 [mbsf]	99.3	0.0171	0.0001	0.64	0.04	5783	−56.4	−31.7	−5.6	9.7	−194
	99.3	0.0153	0.0001	0.63	0.04	6446	−54.6	−31.8		11.6	−195
	99.4	0.0172	0.0001	0.57	n.d.	5758	−54.8	−31.9		8.5	−194
	99.9	0.0171	0.0001	0.11	n.d.	5810	−54.8				−194
	99.3	0.0205	0.0001	0.66	0.03	4819	−57.1	−31.4	−5.3	9.1	−194
	99.9	0.0211	0.0001	0.10	n.d.	4713	−55.4				−195
	100.0	0.0216	0.0001	0.03	n.d.	4603	−54.8				−194
MD179-3318C 1.5 [mbsf]	99.8	0.0148	0.0011	0.18	n.d.	6257	−44.0	−21.3	4.0		−189
	99.1	0.0421	0.0005	0.51	0.36	2328		−22.8	4.5	17.4	−188
	99.9	0.0456	0.0004	0.07	n.d.	2172	−44.3	−24.5			−190
	99.7	0.0078	0.0025	0.21	0.09	9682	−43.9	−21.4			n.d.

Notes: mbsf: meters below sea floor; blank: not measured; n.d.: not detected.

Table 1. Molecular compositions (mol% of the total components) and stable carbon (C_1–3 and CO₂) and hydrogen (C₁) isotopic ratios of hydrate-bound gases retrieved from Umitaka Spur (MD179-3305G and 3306) and Joetsu Knoll (MD179-3317 and 3318C), Japan Sea. Because H₂S is highly corrosive and reaction with sampling and analytical tools was apparently not prevented, a higher portion of H₂S is lost during core retrieval and gas analysis compared to hydrocarbons and CO₂.

Core No. Depth [mbsf]	Molecular composition						Isotopic composition
	C1 [mol%]	C2 [mol%]	C3 [mol%]	CO2 [mol%]	H2S [mol%]	C1/(C2+C3)	C1δ13C [‰V-PDB]	C2δ13C [‰V-PDB]	C3δ13C [‰V-PDB]	CO2δ13C [‰V-PDB]	C1δD [‰V-SMOW]
MD179-3305G unknown depth	98.1	0.0292	0.0019	1.10	0.77	3158	−36.6	−17.9	5.8	14.3	−167
	98.3	0.0270	0.0020	1.18	0.53	3389	−36.0	−18.2	5.7	18.0	−164
	98.2	0.0301	0.0017	1.26	0.53	3086	−36.0	−18.2	5.9	19.5	−164
	99.8	0.0331	0.0018	0.20	n.d.	2859	−34.6			17.3	−167
MD179-3306 4.5 [mbsf]	99.1	0.0292	0.0020	0.85	n.d.	3176	−37.3	−18.4	6.3	20.3	−164
	100.0	0.0221	0.0016			4213	−35.7	−18.1	4.8	23.8	−167
MD179-3317 31.5 [mbsf]	99.3	0.0171	0.0001	0.64	0.04	5783	−56.4	−31.7	−5.6	9.7	−194
	99.3	0.0153	0.0001	0.63	0.04	6446	−54.6	−31.8		11.6	−195
	99.4	0.0172	0.0001	0.57	n.d.	5758	−54.8	−31.9		8.5	−194
	99.9	0.0171	0.0001	0.11	n.d.	5810	−54.8				−194
	99.3	0.0205	0.0001	0.66	0.03	4819	−57.1	−31.4	−5.3	9.1	−194
	99.9	0.0211	0.0001	0.10	n.d.	4713	−55.4				−195
	100.0	0.0216	0.0001	0.03	n.d.	4603	−54.8				−194
MD179-3318C 1.5 [mbsf]	99.8	0.0148	0.0011	0.18	n.d.	6257	−44.0	−21.3	4.0		−189
	99.1	0.0421	0.0005	0.51	0.36	2328		−22.8	4.5	17.4	−188
	99.9	0.0456	0.0004	0.07	n.d.	2172	−44.3	−24.5			−190
	99.7	0.0078	0.0025	0.21	0.09	9682	−43.9	−21.4			n.d.

Notes: mbsf: meters below sea floor; blank: not measured; n.d.: not detected.

The molecular and stable carbon and hydrogen isotopic composition of volatiles in the headspace gas of MD179-3306 (Umitaka Spur) and 3318C (Joetsu Knoll) are summarized in Table 2. A high concentration of a compound (0.1–0.2 mol%) putatively assigned as neo-C₅ based on its relative retention time during gas chromatography was also detected. Although normal butane (n-C₄), i-C₄, isopentane (i-C₅), and normal pentane (n-C₅) were below the detection limit of the gas chromatograph, CF-IRMS (detection limit: 0.00006 mol%) detected only i-C₄ in the samples. δ¹³C of C₅–C₈ in MD179-3306 and 3318C was within −23‰ ± 5‰; however, C₃ δ¹³C and i-C₄ δ¹³C were exceptionally high (+1.1‰ to +8.8‰) compared with δ¹³C of the other hydrocarbons.

Table 2. Molecular and isotopic compositions of headspace gas of the hydrate-bearing cores (MD179-3306 and MD179-3318C).

Core	MD179-3306				MD179-3318C
Depth [mbsf]	4.50	5.00	5.95	7.00	1.50
	Molecular composition [mol%]
C1	40.7	42.3	41.4	28.4	86.9
C2	0.0211	0.0264	0.0255	0.0153	0.0289
C3	0.0037	0.0039	0.0037	0.0022	0.0063
neo-C5	0.1946	0.1298	0.1862	0.0830	0.0760
CO2	59.1	57.5	58.4	71.5	6.6
H2S	n.d.	n.d.	n.d.	n.d.	6.3
C1/(C2+C3)	1640	1393	1422	1624	2469
	Isotopic composition [δ13C ‰V-PDB]
C1	–33.9	–34.6	–34.4	–34.3	–45.3
C2	–47.3	–32.9	–21.5	–21.1	–24.8
C3	4.5	4.5	3.9	2.9	8.8
i-C4	2.6	4.4		2.3	1.1
neo-C5	–23.0	–23.3	–22.8	–23.2	–21.4
2,2DMB	–18.3	–19.0	–19.3	–18.6	–15.7
2,3DMB	–21.9	–20.5		–21.6
n-C6	–23.0	–22.6		–23.0
n-C7	–28.1	–26.6		–27.4
n-C8	–26.4
CO2	31.4	31.5	31.8	31.9	–10.2
	Isotopic composition [δD ‰V-SMOW]
C1	–162	–166	–158	–161	–183
neo-C5	–125	–124	–133	–125	–119

Notes: mbsf: meters below sea floor; blank: not measured; n.d.: not detected; DMB: dimethylbutane.

Table 2. Molecular and isotopic compositions of headspace gas of the hydrate-bearing cores (MD179-3306 and MD179-3318C).

Core	MD179-3306				MD179-3318C
Depth [mbsf]	4.50	5.00	5.95	7.00	1.50
	Molecular composition [mol%]
C1	40.7	42.3	41.4	28.4	86.9
C2	0.0211	0.0264	0.0255	0.0153	0.0289
C3	0.0037	0.0039	0.0037	0.0022	0.0063
neo-C5	0.1946	0.1298	0.1862	0.0830	0.0760
CO2	59.1	57.5	58.4	71.5	6.6
H2S	n.d.	n.d.	n.d.	n.d.	6.3
C1/(C2+C3)	1640	1393	1422	1624	2469
	Isotopic composition [δ13C ‰V-PDB]
C1	–33.9	–34.6	–34.4	–34.3	–45.3
C2	–47.3	–32.9	–21.5	–21.1	–24.8
C3	4.5	4.5	3.9	2.9	8.8
i-C4	2.6	4.4		2.3	1.1
neo-C5	–23.0	–23.3	–22.8	–23.2	–21.4
2,2DMB	–18.3	–19.0	–19.3	–18.6	–15.7
2,3DMB	–21.9	–20.5		–21.6
n-C6	–23.0	–22.6		–23.0
n-C7	–28.1	–26.6		–27.4
n-C8	–26.4
CO2	31.4	31.5	31.8	31.9	–10.2
	Isotopic composition [δD ‰V-SMOW]
C1	–162	–166	–158	–161	–183
neo-C5	–125	–124	–133	–125	–119

Notes: mbsf: meters below sea floor; blank: not measured; n.d.: not detected; DMB: dimethylbutane.

5. Discussion

5.1. Depth Profiles of Headspace Gas

Shallow SMI was observed in the Joetsu Basin area including Umitaka Spur and Joetsu Knoll, suggesting that the hydrocarbon flux from great depth was primarily high. The peak of the C₁ concentrations and high C₁/(C₂ + C₃) values appeared around 5–8 mbsf, indicating high microbial C₁ production (methanogenesis) just beneath the SMI. Negative C₁ δ¹³C and CO₂ δ¹³C peaks and high H₂S concentrations were observed around the SMI (Figure 2a–c), which typically results from the anaerobic oxidation of methane (AOM). C₁ δ¹³C decreased around the SMI can be explained as a result of carbon recycling between the AOM and methanogenesis [33]. ANME-1 and ANME-2 groups were distinguished at Umitaka Spur and Joetsu Knoll [34] using the lipid biomarker signatures of the AOM communities [35]. In the same study area, the occurrence of AOM in the SMI zone is also supported by environmental DNA analysis [36]. Similar relations between the depth profiles of C₁ δ¹³C and CO₂ δ¹³C were also reported in the gassy sediment of Eckernförde Bay in the western Baltic Sea [37] and in the C₁-rich sediment near a gas chimney in northern Gulf of Mexico [38]. Although high C₁ δ¹³C was observed in the hydrate-bearing core MD179-3318C that indicated thermogenic origin according to empirical classifications [21,39], depleted C₁/(C₂+C₃), C₁ δ¹³C, and CO₂ δ¹³C around SMI were observed even in the MD179-3318C, suggesting that C₁ is basically thermogenic and the top layer around SMI depth is affected by microbial alternation.

The upward increase in the profiles of C₁ δ¹³C and CO₂ δ¹³C above the SMI is due to C₁ oxidation near the sea floor [39]. On the other hand, the increase in the C₁ δ¹³C below the SMI is explained by the concurrent increase in the CO₂ δ¹³C with depth, resulting from the reduction of CO₂ in the methanegenic zone and the Rayleigh process. Except for MD179-3306, the profiles of C₁ δD were almost identical and gradually decreased with depth. This trend agrees fairly well with previously published δD profiles of pore water (ambient H₂O) in the same cores [40]; this is because C₁ δD is primarily determined by δD of H₂O and H₂ in the ambient water in the case of microbial C₁ generation via CO₂ reduction [41]. In contrast, the profile of C₁ δD for MD179-3306 was distinctly larger than those of other cores, indicating that C₁ is of thermogenic origin.

The concentration of C₂ in the three hydrate-bearing cores below the SMI was nearly one order of magnitude higher than that in the nonhydrate-bearing cores (Figure 2a–c), and the other cores at Umitaka Spur were also rich in C₂. These C₂-rich cores showed ¹³C enrichment in C₂. The concentrations of C₃ in the hydrate-bearing cores and in MD179-3304 were also higher than in the nonhydrate-bearing cores. The results for C₂ and C₃ suggest two possibilities: (1) injections of thermogenic hydrocarbons from greater depth and (2) dissociation of tiny amounts of C₂- and C₃- rich sII hydrates during recovery process of sediment cores.

The concentration of C₃ in MD179-3317 was conspicuously higher (0.039–0.331 μM) than in the other cores, whereas the hydrate-bound C₃ in MD179-3317 was negligible (0.0001% of the hydrate-bound gas, Table 1). Because sI gas hydrates cannot encage C₃, it is reasonable to assume that C₃ was excluded from the hydrate formation. Similar molecular fractionations were described in previous studies [42,43,44].

5.2. Effect of Gas Characteristics on Hydrate Crystal

The relation between the molecular ratio of hydrate-bound hydrocarbons and the stable carbon and hydrogen isotopic ratio of individual hydrate-bound volatiles is shown in Figure 3. The Bernard plot (Figure 3a), which compares C₁ δ¹³C with C₁/(C₂ + C₃) [21], is useful for understanding the gas origin and its pathway. The data from stations MD179-3305G, 3306, and 3318C plot in the mixed gas field, whereas those from MD179-3317 plot near and within the field of microbial hydrocarbons (Figure 3a). These relation between C₁ δ¹³C and C₁/(C₂ + C₃) at Umitaka Spur and Joetsu Knoll agree well with those obtained in a previous study [22].

Figure 3. Relationship between molecular and isotopic composition of hydrate-bound hydrocarbons. (a) “Bernard plot” showing relationship between C₁ δ¹³C and C₁/(C₂ + C₃) values [21]. The data agree well with those obtained at the same area from 2005 to 2008 [22]. The headspace gas data obtained from deeper sediment layer of Umitaka Spur at depths 993−2016 m below the sea floor [20] are plotted; (b) Relationship between C₁ δ¹³C and C₂ δ¹³C, based on the classification of Milkov [4].

Figure 3. Relationship between molecular and isotopic composition of hydrate-bound hydrocarbons. (a) “Bernard plot” showing relationship between C₁ δ¹³C and C₁/(C₂ + C₃) values [21]. The data agree well with those obtained at the same area from 2005 to 2008 [22]. The headspace gas data obtained from deeper sediment layer of Umitaka Spur at depths 993−2016 m below the sea floor [20] are plotted; (b) Relationship between C₁ δ¹³C and C₂ δ¹³C, based on the classification of Milkov [4].

Figure 3b shows the relation between C₁ δ¹³C and C₂ δ¹³C. The boundaries between thermogenic and microbial origins are based on literature [4,45,46]. C₂ δ¹³C ranged from −31.9‰ to −17.9‰, plotted in the field of thermogenic C₂ [4,45,46]. Because microbial C₂ is generally a minor component of microbial gas (<0.1%, see Figure 3a) and is depleted in ¹³C [46], mixing of microbial and thermogenic hydrocarbons slightly decreases the C₂ δ¹³C and strongly decreases the C₁ δ¹³C.

The molecular ratios of hydrate-bound hydrocarbons and stable carbon isotope signatures of C₁ shown in Figure 3a indicate a mixed gas origin for the hydrate at Umitaka Spur, implying depletion of C₂ and C₃ from the thermogenic gas field in Figure 3a (C₁/(C₂ + C₃) < 100). C₁/(C₂ + C₃) values were less than 100 at depths 1143−2016 m below the sea floor at Umitaka Spur [20]. Although thermogenic gas might contain significant amounts of C₂ and C₃ (sometimes >10 mol%), as found in samples from the Gulf of Mexico [13,47], the northern Cascadia margin offshore Vancouver Island [48], and the Caspian Sea [6], the composition of C₂ and C₃ of hydrate-bound hydrocarbons at Umitaka Spur was less than 0.04% and 0.002%, respectively (Table 1). These results can be explained by molecular hydrocarbon fractionation during upward gas migration [49]. Several similar theories have been proposed, e.g., formation of C₂-rich gas hydrates in the deep layers and C₂ depletion in the residual migrating gas [22], and adsorption of C₂ and C₃ to the mineral matrix during migration [30]. Assuming steady-state upward migration of thermogenic hydrocarbons, we have to consider the consumption and/or degradation of C₂ and C₃ in the deeper sediment layers. Non-C₁ hydrocarbon degradation [43,50,51] likely selectively decreases C₂ and C₃.

Stable isotope signatures (C₁ δ¹³C, C₂ δD, and C₂ δ¹³C) of hydrate-bound and headspace gases were almost the same with each other (Figure 2a,b); however, distinct differences were found in CO₂ δ¹³C. Previous studies showed no or rather small differences in CO₂ δ¹³C between stable isotopes of hydrate-bound and headspace gases [42,52]. It remains unsettled and further studies are needed to find the reason why such large differences in CO₂ δ¹³C existed.

Although C₃ composition of hydrate-bound gas was very small (less than 0.0025 mol%, Table 1), we need to discuss the existence of C₃ in the hydrate-bound gas. C₁, C₂, CO₂, and H₂S can be encaged in the sI gas hydrate [1], but C₃ is exclusively found in the sII gas hydrate because of its large molecular size. It is possible that C₃ in these samples was a contaminant from the sediments attached to the hydrate during the preparation of the hydrate-bound gas. Based on the gas compositions, the gas hydrates investigated in this study were assumed to be mainly sI. This assumption is supported by the powder X-ray diffraction (PXRD) data for gas hydrates collected from Umitaka Spur and Joetsu Knoll in the past [53,54]. Nevertheless, we cannot exclude the potential coexistence of sI (C₁-rich) and sII (C₃ encaged in the large cages) gas hydrates, because PXRD cannot detect small amounts of sII. As encaged C₃ molecules in C₁-rich natural gas hydrate retrieved at the Mackenzie Delta (Onshore, Canada) were detected by Raman spectroscopy [55], heterogeneity of hydrocarbons in the different crystallographic structures is an attractive research target in the future from the viewpoint of crystal growth process of natural gas hydrates.

5.3. Neopentane and Non-Methane Hydrocarbons in the Headspace Gases

The presence of gem-dimethyl hydrocarbons was previously reported in the Deep Sea Drilling Project Holes 381 and 397 [56,57]. neo-C₅ is considered to form from the decomposition of gem-dimethylcycloalkanes derived from the terpenes of terrestrial organic matter [56]. Unusually high concentration of neo-C₅ in gases extracted from the Athabasca oil sands and proposed that neo-C₅ forms from the microbial degradation of bitumen [58]. Although the origin of neo-C₅ remains unknown, the potential involvement of microbes has been suggested [57]. Schaefer and Leythaeuser [59] explained that enrichment of neo-C₅ is caused by preferential diffusion due to the nearly spherical molecules and its diffusion coefficient, which is higher than that of less branched isomers.

Although neo-C₅ can be encaged in the large cages of sII gas hydrates [17], only trace levels of neo-C₅ were detected in the samples of hydrate-bound gas, perhaps because of contamination with the sediment gas at their retrieval process. Similar to C₃, during the formation of sI gas hydrates, neo-C₅ is excluded and remains in sediment. However, negligible amounts of sII gas hydrates containing neo-C₅ could also be present, which dissociated during core retrieval. While C₃ and neo-C₅ in C₁-rich hydrocarbons decrease the equilibrium pressures and stabilize their hydrate phase, the dissociation behavior of the gas hydrate depends on the guest molecules. C₁ hydrate shows self-preservation phenomena [60] in the dissociation process; however, the hydrates with lower decomposition pressures (C₂ and C₃) do not show it [61]. Further investigations are needed to understand the behavior of the small amount of sII formers, C₃ and neo-C₅.

C₃ δ¹³C and i-C₄ δ¹³C were exceptionally high compared with δ¹³C of the other hydrocarbons as shown in Table 2. Possibly, ¹³C-depleted C₃ and i-C₄ were preferentially consumed as microbial substrates; hence, the residual C₃ and i-C₄ were enriched in ¹³C [30]. The concentration of n-C₄ was below the detection limit (0.00006 mol% of the original headspace sample) of the CF-IRMS; n-C₄ is easily transformed by hydrocarbon degraders, whereas C₂ and i-C₄ appear less affected or remain unaltered [42,50,51,62]. Therefore, it is reasonable to assume that n-C₄ was consumed primarily by biodegradation because it is more affected by biodegradation than i-C₄. δ¹³C of C₁–C₈ are plotted in Figure 4: the so-called “Natural gas plot” [63]. In the Umitaka Spur area, δ¹³C values of C₁–C₈ except C₃ and i-C₄ show a linear trend, indicating no microbial contribution in C₁. Moreover, the hydrate-bound C₁ comprised pure thermogenic gas.

Figure 4. δ¹³C of hydrocarbons plotted in the “Natural gas plot” adapted from [63], where n is the number of the carbon atoms of individual hydrocarbon molecules. δ¹³C of C₁, C₂, C₃, n-C₄, and n-C₅ were plotted in the original natural gas plot. However, we plotted the data of i-C₄ (instead of n-C₄), neo-C₅ (instead of n-C₅), and C_6–8 shown in Table 2. The concentrations of n-C₄, and n-C₅ were below the detection limit. Thick shaded lines show linear relation between C₂ and C₈ for MD179-3305G and 3306 (Umitaka Spur) and for MD179-3318C (Joetsu Knoll), except ¹³C-rich C₃ and i-C₄ due to potential biodegradation. C₂ δ¹³C of MD179-3306 has a large error bar because of wide distribution along with depth (Table 2).

Figure 4. δ¹³C of hydrocarbons plotted in the “Natural gas plot” adapted from [63], where n is the number of the carbon atoms of individual hydrocarbon molecules. δ¹³C of C₁, C₂, C₃, n-C₄, and n-C₅ were plotted in the original natural gas plot. However, we plotted the data of i-C₄ (instead of n-C₄), neo-C₅ (instead of n-C₅), and C_6–8 shown in Table 2. The concentrations of n-C₄, and n-C₅ were below the detection limit. Thick shaded lines show linear relation between C₂ and C₈ for MD179-3305G and 3306 (Umitaka Spur) and for MD179-3318C (Joetsu Knoll), except ¹³C-rich C₃ and i-C₄ due to potential biodegradation. C₂ δ¹³C of MD179-3306 has a large error bar because of wide distribution along with depth (Table 2).

In contrast, these gases mixed with microbial C₁ during migration to shallow sediment layers in the Joetsu Knoll area, as C₁ δ¹³C decreased from the anticipated thermogenic C₁ value for MD179-3318C.

6. Conclusions

Molecular and stable carbon and hydrogen isotopic compositions of hydrate-bound and pore-water gas were reported for samples retrieved from Umitaka Spur and Joetsu Knoll at the eastern margin of the Japan Sea. According to empirical classifications of gas data, the hydrate-bound gas from Umitaka Spur was of thermogenic origin and that from Joetsu Knoll partly contained microbial gas. C₁ concentration depth profiles showed shallow SMI in these areas, indicating high C₁ flux. The molecular composition of C₂ and C₃ for the headspace gas of hydrate-bound cores were around a thousandth smaller than those of hydrate-bound thermogenic gases across the world, and C₃ δ¹³C and i-C₄ δ¹³C were exceptionally high (+1.1‰ to +8.8‰) in the pore water, suggesting that biodegradation affects these hydrocarbons in hydrate-bearing sediment systems.

High concentration of neo-C₅ was detected in the pore water gases in MD179-3306 (Umitaka Spur) and 3318C (Joetsu Knoll), which suggests that neo-C₅ was excluded from the hydrate crystal during the formation process and thus remained in the pore water, because neo-C₅ cannot be encaged by sI gas hydrates. This is supported by the observation that the concentration of C₃ in the hydrate-bearing core was higher than that in the nonhydrate-bearing cores. However, small amounts of sII gas hydrate can possibly encage these heavier hydrocarbons. Because neo-C₅ forms from the microbial degradation of organic matter and is easy to diffuse in sediment layers, the accumulation of neo-C₅ in subsurface sediment can affect and change the crystallographic properties of gas hydrates in marine sediments.