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doi:10.2204/iodp.proc.314315316.122.2009

Microbiology

Core handling and sampling

Whole-round cores

Microorganisms in deep-sea sediments are expected to be sensitive to chemical and physical changes, particularly changes in oxygen, temperature, and pressure. Therefore, the core sections designated for microbiological sampling were transferred as quickly as possible from the rig floor to a refrigerator and were kept as whole sections until processed. However, because of the danger of explosion of core liners because of expanding sediment, some cores had to be kept on the rig floor for up to 3 h before X-ray CT scanning. Whole-round cores for microbiological analysis were cut based on X-ray CT scans. Our aim was to minimize potential contamination by selecting sections with minimal disturbance, such as voids or cracks. Three types of whole-round cores (10–20 cm) were taken for microbiological analysis. The first type was subsampled on board for cell counting, cultivation assays, and deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) isolation. The second type was taken for cultivation studies. These cores were brought quickly to an anaerobic glove box (Coy Laboratory Products, Inc.), placed into an anaerobic pouch (Mitsubishi Gas Chemical Co., Inc.) with AnaeroPack (Mitsubishi Gas Chemical Co., Inc.), sealed with a plastic clip, and stored at 4°C for shore-based studies. The third type was subsampled for cell counting and then immediately placed into a plastic bag and stored at –80°C for onshore DNA-, RNA-, and intact polar lipids–based molecular analyses.

Subsampling of soft and hard sediments

Subsampling of soft-sediment whole-round cores was conducted with autoclaved tip-cut 5 mL syringes after scraping off the sediment surface with an ethanol-wiped spatula. Subsampling was restricted to sediments at or near the center of the cores because core liners are not sterile and outer core surfaces are contaminated during drilling (Smith et al., 2000). For subsampling of semiconsolidated sediment, intact rock pieces were selected. The surface was washed with ethanol and/or scraped off with an ethanol-wiped spatula, and the rock was crushed with a hammer in an aluminum bag or ethanol-wiped aluminum folio.

Subsample treatment

Subsamples taken for DNA and RNA isolation were immediately frozen at –80°C. Subsamples taken for cell counting were fixed with 2% paraformaldehyde in phosphate-buffered saline (PBS; Invitrogen, pH adjusted to 7.6) overnight, washed two times with PBS, and finally stored in PBS:ethanol (1:1) at –20°C. Subsamples taken for cultivation assays were immediately placed into anaerobic serum bottles containing 35 g/L of Sigma sea salts (pH adjusted to 7.2) under counterflow of sterile-filtered (pore size = 0.22 µm) nitrogen. The slurries were homogenized by repeated shaking and vortexing and were stored at 4°C. Subsamples of the slurries were used for cultivating heterotrophic sulfate-reducing bacteria in a modified DSMZ medium 1040 (Table T8). Acetate, lactate, pyruvate, and ethanol (10 mM) were used as organic carbon and electron sources. Liquid cultures were incubated at different temperatures (9°, 37°, 50°, and 70°C) until the end of the expedition. Incubation will be continued on shore.

Drilling mud sampling

As it was not possible to do perfluorocarbon tracer contamination tests on board, preused drilling muds (seawater gel and kill mud) were used as control samples for microbiological analysis. Seawater gel (pH = 12.3) is a seawater-based bentonite mud used for drilling and coring throughout entire sections. It contains 0.5 m³ seawater, 0.5 m³ drill water, 60.0 kg bentonite, 2.0 kg caustic soda, and 2.0 kg lime. Kill mud is a freshwater-based, barite-weighted mud used to suspend or abandon the hole. It was not used before or during coring in this expedition. Kill mud (pH 11.3) contains 1 m³ drill water, 60.0 kg bentonite, 1.0 kg caustic soda, 2.0 kg XCD-polymer, and 40.0 kg barite. The density of both drilling muds is 1.05 g/cm³. Mud samples were taken into autoclaved glass bottles and subsampled for cell counting, culturing, and molecular analysis. Subsamples for cell counting were fixed as described above. Subsamples for culturing and molecular analysis were stored at 4°C and –80°C, respectively.

Cell detection by fluorescence microscopy

Selected samples of fixed cells were stained on board with double-stranded DNA-binding SYBR Green I stain for detecting cells (Lunau et al., 2005). Before staining, the fixed sediment slurry was vortexed and diluted in PBS:methanol (9:1). The mixture was sonicated at 50 W for 1 min with an ultrasonic homogenizer UH-50 (SMT Co., Ltd), and sediment particles were removed by centrifuging at 100 g for 2 min. An aliquot of the supernatant was mixed with 5 mL PBS and filtered through a black 0.2 µm pore-sized polycarbonate filter. The filter was washed twice with PBS and placed on an object glass. A coverslip was mounted on the filter with 8 µL of SYBR Green I staining solution. Cells were viewed with an epifluorescence microscope (ZEISS Axioplan 2 imaging microscope), and images were taken with a ZEISS AxioCam HRc camera and AxioVision AC software.

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