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Methods and materials

Samples were taken during Expedition 320/321 from March to July 2009. Because of the rather soft nature of the sediment, it was possible to use hydraulic piston coring for most cores. This technique is known to produce the least contaminated cores because the actual coring does not involve the use of drilling fluids (Kallmeyer, 2011; Smith et al., 2000). Samples from the center of a freshly cut core section are rarely contaminated, so despite the fact that these samples were drilled without contamination control, the obtained cell count profiles should be reasonably unaffected by contamination through infiltrating drilling fluid.

A cut-off sterile 3 cm3 plastic syringe was inserted into a freshly cut core section alongside the methane safety sample (Pälike, Lyle, Nishi, Raffi, Gamage, Klaus, and the Expedition 320/321 Scientists, 2010) and exactly 2 cm3 of sediment was extruded into a vial filled with 8 mL of 2.5% NaCl solution with 2 vol% formalin as a fixative. The vial was quickly closed and thoroughly shaken to form a homogeneous suspension. The sediment slurries were stored at +4°C until analysis in Potsdam, Germany. Analysis was completed within about 7 months after the expedition. Experience has shown that formalin-fixed slurries maintain their original cell concentration for at least 1 y as long as they are not diluted and kept cold. From each slurry, between two and four replicate filters were prepared and counted.

With the exception of samples from the upper few meters, cell concentrations were too low to be detected by direct counting, in which a small aliquot of slurry is put on a filter without any further treatment, stained with a DNA-specific stain (usually Acridine Orange or SYBR Green I), and counted manually. The minimum detection limit of this “classical” counting technique, which has been used on almost all ODP and IODP expeditions so far, is around 105 cells/cm3 (Kallmeyer, 2011). For deeper samples with lower cell abundances, a cell extraction that separates the cells from the mineral matrix is necessary. This technique can be used to lower the detection limit to levels of ~103 cells/cm3. Although cell extraction does not recover 100% of the cells, values are usually within 1 standard deviation of the classical counting method (Kallmeyer et al., 2008, 2012a). Moreover, cell counts from extracts usually have a much smaller standard deviation than classical counts because of the higher number of cells counted.

All slurries were first checked for carbonate content by mixing a droplet of slurry with hydrochloric acid on a glass slide and looking for any mineral dissolution under a low-magnification microscope. Carbonates interfere with the cell extraction and drastically lower its efficiency. If carbonates were present, they were removed by dissolution through addition of a sodium acetate–acetic acid buffer containing 20 mL/L (0.43 M) glacial acetic acid and 35 g/L (0.43 M) sodium acetate. The carbonate dissolution mix has a high acidity but a moderate pH (4.6) in order to avoid any damage to the cells but to rapidly dissolve any carbonate minerals. Samples with high carbonate content were only treated with a carbonate dissolution step and filtered directly onto 0.22 µm pore size polycarbonate filters, stained with SYBR Green I, and counted under a fluorescence microscope (Leica DM2500; Leica EL 6000 light source; BP 480/40 excitation filter; 505 dichromatic mirror; 527/30 suppression filter, 100× objective). Through dissolution of the carbonate minerals, the volume of slurry to be used for a single analysis can be increased by more than 1 order of magnitude. Samples with a higher percentage of noncarbonate minerals, usually clays and biogenic silica, were treated according to the cell extraction protocol of Kallmeyer et al. (2008), by which the cells are first detached from the mineral particles and then separated by density centrifugation.

Depth spacing of the cell count samples is between 3 and 10 m. Although an exact stratigraphic correlation between the different holes is of utmost importance for high-resolution paleoceanographical work, the low resolution of the cell count data makes such efforts superfluous. Therefore, the uncorrected depth data were used.

Many of the cell counts were close to the minimum detection limit or even below. When dealing with such low values, a critical assessment of the background becomes an absolute necessity. Blank samples were processed alongside each sample processing run. On each sample and blank filter, 200 fields of view were counted. A sample was only considered a valid count if the total number of cells on 200 fields of view minus the average number of cells on the corresponding blank filters (i.e., the mean blank) was equal to or higher than three times the standard deviation of the mean blank (solid circles in Fig. F1). If the sample was lower than three times the standard deviation but still positive, the values were reported as below detection (open circles in Fig. F1). Some samples had negative cell counts after subtraction of the mean blank; those are reported as below detection (BD) in the tables or as open circles on the y-axes of Figure F1.