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Whole-round sampling procedure

WRC samples were taken at a frequency of ~1 sample per 4.5 m of core recovered. A total 150 cm3 of sediment or deposit was required for the subsequent microbiological investigation (see below), and a ~10 cm length of whole round was retrieved from a portion of the section showing minimal disruption by drilling after evaluation by X-ray CT scan imaging. All subsampling and processing were carried out within 60 min of core arriving on deck.

Sample preparation for microscopic observations

Approximately 1 cm3 of sediment or deposit sample was taken with a sterilized spatula. In a clean area of the laboratory, the samples were immediately fixed with 5 mL of 4% paraformaldehyde in a phosphate-buffered saline (PBS) buffer and stored at 4°C for 3 h. Fixed samples were preserved at –80°C. In addition to the chemically fixed samples, another 1 cm3 of subsample was collected by a sterilized spatula and directly frozen at –80°C.

Sample preparation for molecular analyses

Approximately 20 cm3 of sediment or deposit was collected with a sterilized spatula into Falcon 50 mL tubes for subsequent DNA/RNA analysis. These tubes were sealed in aluminum bags with oxygen absorbers and indicators in an anaerobic glove box and were stored at –80°C. Approximately 5 cm3 of sediment or deposit was subsampled by a sterilized spatula into a 15 mL Falcon tube for later RNA analysis. The tubes were frozen with liquid nitrogen and stored at –80°C. Approximately 50 cm3 of sediment or deposit was also collected into a Falcon 50 mL tube. The tubes were sealed in aluminum bags with oxygen absorbers and indicators in an anaerobic globe box and were then stored at –80°C.

Sample preparation for living microorganisms

Approximately 75 mL of sediment or deposit was subsampled by a sterilized spatula into a Schott 100 mL bottle and sealed with a black butyl rubber stopper in an anaerobic glove chamber. The gas phase of the bottle was exchanged with 100% nitrogen and stored at 4°C.

Contamination test

PFC (perfluoro-methylcyclohexane; MW 350.6) (Matrix Scientific, USA) was used as a chemical tracer for contamination assessment during coring operations. PFC tracer was added to the drilling mud tank every time a new batch of drilling mud fluid was prepared with surface seawater. After each new batch of drilling mud was prepared, a 50 cm3 sample was collected to determine the concentration of PFC so that the contamination of core samples by drilling fluid could be assessed. Quantification of PFC in the core samples and original drilling mud was carried out following standard IODP procedures (Smith et al., 2000) using an Agilent Technologies model 6890N GC with an electron capture detector. The GC was equipped with a HP-PLOT Al2O3 “M” deactivation column (30 m long; 0.53 mm inside diameter [ID]; 15 µm coating thickness). Helium and nitrogen were used as the carrier gas and make-up gas, respectively. The extent of contamination was estimated by comparison of values between the mud fluid in the tank and the subsampled sediment or deposits. Sediment or deposit showing PFC concentrations <0.1% of that in the mud tank fluid are used as standards for reliable samples free from external contamination (Takai, Mottl, Nielsen, and the Expedition 331 Scientists, 2011).