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Materials and methods

Organic-lean, oligotrophic, and oxic sediments from the North Pond area in the 7 m.y. old western flank of the Mid-Atlantic Ridge at 23°N were sampled during the Expedition 336 site survey cruise with the German R/V Maria S. Merian in 2009 (MSM 11/1). Sediment cores (12 cm diameter) were successfully retrieved at six locations within the North Pond basin between 4040 and 4480 meters below sea level using gravity corers (Ziebis et al., 2012). These cores extended to 8 mbsf. Once recovered on deck, these cores were quickly cut into 1 m sections that were immediately carried to the cold storage room. Each 1 m section was subsequently split into half-core sections. From one half, samples for microbiological analysis were taken from the interior portion by excavation with a sterilized spoon, and the outer centimeter layer was left in the core liner to avoid contamination with seawater. In addition to the gravity cores, an additional 21 sediment samples from the shallow subsurface (0–15 cm depth) were taken with the remotely operated vehicle (ROV) Jason II (push cores) during another cruise of the Maria S. Merian in 2011 (MSM 20/5).

For total cell counting, 1 mL of each sediment sample was preserved in 9 mL of 0.2 µm sterile-filtered 2% formaldehyde in seawater. In the Federal Institute for Geosciences and Natural Resources (BGR) laboratory, 100 µL of fixed sediment was transferred in a 2 mL Eppendorf tube and suspended in 1 mL of 0.1 mM hydrochloric acid (after autoclaving sterile filtered) to dissolve carbonates. Samples were shaken for 5 min and centrifuged for 20 min at 16,000 g. Afterward, 1 mL of supernatant was carefully removed. The pellet was suspended in 1 mL tris-ethylenediaminetetraacetic acid (TE) buffer and centrifuged. This step was repeated. The pellet was suspended with 900 µL TE buffer followed by an ultrasonic treatment for 20 s (Weinbauer et al., 1998). A sample (100 µL) was stained on filters with SYBR Green as described elsewhere (Lunau et al., 2005). Cells were counted using fluorescence microscopes (Weinbauer et al., 1998).

For qPCR, samples were immediately frozen at –20°C after shipboard sampling. Samples were transported and stored frozen in the home laboratory. For DNA extraction from thawed samples, a published protocol for DNA extraction (Webster et al., 2003) with an additional preceding acid treatment step with iodic acid was applied to dissolve carbonates and to improve DNA extraction from cells. Kates et al. (1965) published a protocol to break ether bonds in cell walls of microorganisms using iodic acid. According to the described procedure, we applied iodic acid but in a lower concentration and a shorter incubation time (0.1 mol/L versus ~8 mol/L and 10 min versus 24 h). We assumed that the modified method dissolved the carbonates and/or partially disrupted the cell walls (in particular archaeal ether bonding) but did not lyse the cells. FAST-Prep tubes without matrix were filled with 0.5 g of sediment and centrifuged for 30 s at 14,000 g. Afterward, 1 mL of 0.1 µm filtered 0.1 M iodic acid was added and the pellet was suspended on a shaker for 10 min. The tubes were heated for 10 min at 80°C and centrifuged for 15 min at 16,000 g. A sample of 1 mL supernatant was removed, and 1 mL TE buffer was added to the pellet and vortexed. After centrifugation, this washing step was repeated. The previously removed matrix was added, and the further procedure followed the protocol of the manufacturer with addition of polyadenine (Webster et al., 2003). Empty tubes were used as a negative control. Extracted DNA was amplified in triplicate by qPCR using an ABI Prism 7000 (Applied Biosystems). Published assays for the quantification of the 16S rRNA gene copy numbers of Archaea (Takai and Horikoshi, 2000) and Bacteria (Nadkarni et al., 2002) were applied. 16S rRNA gene copy numbers were converted to cell numbers using conversion factors of 1.5 for Archaea and 4.1 for Bacteria, as previously done (Schippers et al., 2005).

The reliability of our new protocols was tested in spiking experiments in which known numbers of distinct organisms were added to a sediment sample and the recovery of cells was determined. Different numbers of cells of gram-negative (Escherichia coli), and gram-positive (Bacillus subtilis) Bacteria and Archaea (Methanohalobium evestigatum), were suspended in sediment samples, and the recovery of the cells was determined (Fig. F1). The new procedure considerably improved cell number recovery. In addition, the acid dissolution of carbonates was confirmed by scanning electron microscopy of samples before and after acid treatment (Fig. F2).