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doi:10.2204/iodp.proc.318.203.2015

Methodology

Shipboard

Amenable lipids were extracted using a method adapted from Bendle et al. (2007). Full details are given in the “Methods” chapter [Expedition 318 Scientists, 2011a; also see Figure F19 in that chapter]. In brief, sediments were freeze-dried and homogenized, and 2–20 g was hydrolyzed and extracted. The total lipid extract was separated into discrete chemical fractions using methods adapted from Bendle et al. (2007): aliphatic hydrocarbons/n-alkanes (N1), aromatic hydrocarbons (N2), aldehydes and ketones (N3), and alcohols (N4). Alcohols in fraction N4 were derivatized to trimethylsilyl ethers using bis(trimethylsilyl)trifluoroacetamide (BSTFA) reagent. Acidic components were extracted with methylene chloride from the remaining solution after acidifying (pH = 1) with HCl. The acidic fraction was concentrated and the carboxylic acids were derivatized to methyl esters with 14% BF₃/methanol (MeOH). The individual lipid fractions were analyzed, and structural identification was confirmed using an HP 5973 gas chromatography mass spectrometer (GC-MS) equipped with a 7683 autosampler and fused silica capillary column (DB-1 60 m × 0.317 mm; internal diameter × 1.50 µm) and mass selective detector (MSD). The GC oven temperature was programmed to ramp at 30°C/min from 50° to 120°C and then ramp at 5°C/min to 300°C, with a final hold time of 22 min.

Shore-based extraction and clean-up

Organic compounds were extracted from 100 powdered and freeze-dried sediment samples at the Royal Netherlands Institute for Sea Research (NIOZ; Utrecht University, Netherlands), and the University of Glasgow (Scotland, United Kingdom) using methods described by Pross et al. (2012). Lipids were extracted by dichloromethane (DCM)/MeOH (9:1, v/v) using the an accelerated solvent extractor (Dionex). The total lipid extract was separated into apolar, ketone, ethyl acetate (EtOAc), and polar fractions over an activated Al₂O₃ column using hexane: DCM (9:1, v/v), hexane:DCM (1:1, v/v), EtOAc:DCM (1:1 v/v), and DCM:MeOH (1:1, v/v), respectively.

Ketone fraction GC-MS analyses

Fifty ketone fraction samples (every other downcore sample from the 100 shore-based extractions) were analyzed using an Agilent GC 7890B coupled to a 5977A MSD GC-MS and equipped with a 7693 autosampler and fused silica capillary column (BP-1 60 m × 0.25 mm internal diameter × 0.25 µm) and mass selective detector at the Birmingham Molecular Climatology Laboratory. The GC oven temperature was programmed to hold for 1 min at 60°C, ramp at 30°C/min from 60° to 130°C and then ramp at 3°C/min to 345°C, with a final hold time of 15 min. The interface temperature was 340°C. The detector was operated in electron impact mode, scanning from 50 to 800 m/z.

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