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doi:10.2204/iodp.proc.307.205.2009

Methods

Sampling

Sediment material from three sites on Challenger Mound (Holes U1317A and U1317D), downslope of Challenger Mound (Holes U1316B and U1316C), and upslope of Challenger Mound (Holes U1318A and U1318B) in the Belgica carbonate mound province was drilled and gathered taking strict precautions to avoid any microbial contamination. For details on contamination control, see the “Methods” chapter. For biomarker analysis, 12 cm whole-round cores (WRCs) were taken, capped, and packed into a gas-tight aluminium bag in a nitrogen atmosphere with an oxygen scrubber sachet (Merck “Aerocult A”). Approximately 5 cm WRCs were sampled from the mound site (U1317) for gas analysis. The sample material was removed from the liner and sealed in a tin after addition of 20 mL of a 1% sodium azide Milli-Q water solution to prevent subsequent microbial activity. All samples were stored at 4°C until finally sent to GFZ Potsdam in a cool box. Samples for biomarker analysis were subsampled using the inner coring technique (Kallmeyer et al., 2006).

Biomarker analysis

Intact phospholipids were analyzed using an extraction method modified after Bligh and Dyer (1954). Approximately 80–90 g of freeze-dried and ground sample aliquots were extracted using a flow blending system with a mixture of methanol/​dichloromethane/​ammonium acetate buffer (pH 7.6), 2:1:0.8 (v/v) for 5 min. For compound quantification an internal phospholipid standard (1-palmitoyl(D₃₁)-2-hydroxy-glycero-3-phosphocholine) was added prior to extraction. The solvent extract was separated from the residual sediment by centrifugation. After removal of the supernatant the sediment residue was reextracted ultrasonically twice using the same solvent mixture. The combined solvent extracts were then collected in a separation funnel and dichloromethane and water were added to achieve a ratio of 1:1:0.9 (v/v). After phase separation the organic phase was removed and the water phase was reextracted twice with 20 mL dichloromethane. The combined organic phases were evaporated to dryness For details of this method see Zink and Mangelsdorf (2004). The obtained sediment extract was separated into fractions of different polarity (low polar lipids, free fatty acids, glycolipids, and phospholipids) using a pure silica column and a Florisil column in sequence as well as solvents of different polarity (Zink and Mangelsdorf, 2004). Finally, the phospholipid fraction was measured using a liquid chromatography–mass spectrometry system (Shimadzu SCL-10a VP high performance liquid chromatography and a Finnigan MAT TSQ 7000 mass spectrometer). For details see Zink and Mangelsdorf (2004). Phospholipid concentrations were determined relative to the internal standard taking into account different response factors of the different phospholipid classes (Mangelsdorf et al., 2005; Zink et al., 2008).

For hopanoids and long-chain n-alkanes the low–polar lipid fraction was separated on a medium-pressure liquid chromatography (MPLC) system into fractions of different polarity (aliphatic, aromatic, and hetero component fractions). The aliphatic hydrocarbon fraction was analyzed for hopanoids and long-chain n-alkanes using a gas chromatographic–mass spectrometry system (Agilent 6890 Series GC and a Finnigan MAT 95 XL mass spectrometer). Compounds were quantified relative to an internal standard (5α-androstane) added prior to extraction. For details on the analytical method see Mangelsdorf et al. (2000).

Elemental parameters

Total organic carbon (TOC) was measured using an elemental analyzer (euro EA 3000, Hekatech). Bulk δ¹³C measurements of TOC were performed after dissolution of carbonates with 0.1 N HCl and subsequent drying of the samples overnight using an elemental analyzer coupled with an isotope ratio mass spectrometer (ThermoQuest delta plus XL).

Gas analysis

The gas-tight tins were sent to a laboratory in Norway (Applied Petroleum Technology AS) for headspace gas analysis to determine the gas compositions and carbon and hydrogen isotope data of the indigenous methane. The total tin volume was 470 cm³. Approximately 176 cm³ of wet sediment and ~215.5 cm³ Milli-Q water containing 20 mL of a 1% sodium azide solution were placed into the tins, resulting in a headspace volume of ~78.5 cm³. A volume of 176 cm³ of wet sediment corresponds on average to ~125 g dry sediment.

A septum was attached to each tin with a hose clip (metal band with a hole). The tins were shaken at a stirring board for 2 h. A headspace sample was taken with a syringe penetrating the tin through the septum. Aliquots of 0.1–1 mL were sampled with the syringe for gas chromatographic analysis on a Carlo Erba HRGC 5300 equipped with a Porabond Q column. The detection limit for the hydrocarbon gas components is 0.001 µL/mL using a flame ionization detector. The detection limit for CO₂ is 0.05 µL/mL using a thermal conductivity detector (TCD/HWD).

The carbon isotopic composition of the hydrocarbon gas components was determined by gas chromatography–isotope ratio–mass spectrometry (GC-C-IRMS) system. Aliquots were sampled with a syringe and analyzed on a Trace GC2000, equipped with a Poraplot Q column, connected to a Delta plus XP IRMS. The components were burnt to CO₂ and water in a 1000°C furnace over Cu/Ni/Pt. The water was removed by Nafion membrane separation. Repeated analyses of standards indicate that the reproducibility of δ¹³C values is better than 1‰ Peedee belemnite (PDB).

The hydrogen isotopic composition of methane was also determined by GC-C-IRMS. Aliquots were sampled with a syringe and analyzed on a Trace GC2000, equipped with a Poraplot Q column, connected to a Delta plus XP IRMS. The components were decomposed to H₂ and coke in a 1400°C furnace. The international standard National Geologic Society (NGS)-2 and an in-house standard (Std A) were used for testing accuracy and precision. The “true” value of NGS-2 is given as –172.5‰ (Vienna standard mean ocean water). Repeated analyses of standards indicate that the reproducibility of δD values is better than 10‰ PDB.

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