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Materials and methods

Sediment cores from four sites (M0059, M0060, M0063, and M0065) collected during Integrated Ocean Drilling Program Expedition 347 were sampled for microbiology. To avoid contamination, samples for were only taken from the center of each sediment core using sterilized cut 5 mL syringes. Depth intervals for sampling were selected based on the quality of the cores with a higher depth resolution near the surface and a lower one at greater depth. The samples were put in anaerobic bags and sent to the Federal Institute for Geosciences and Natural Resources (BGR; Germany) at 2°–8°C and stored at 8°C until use for enrichments.

The media for enrichment of anaerobic, marine deep biosphere organisms were based on artificial seawater as described by Batzke et al. (2007). The salinity of the medium was adjusted by dilution with water to 1‰, 5‰, 10‰, 12‰, 20‰, 25‰, and 30‰ NaCl. For heterotrophic fermenting organisms, the media were amended with a polymer or monomer solution. The polymer solution contained chitin, cellulose, and peptone at 0.5 g/L each, whereas the monomer solution contained 36 different carbon sources like amino acids, fatty acids, organic acids, alcohols, and glucose (total final concentration of added organic carbon was 0.1 mM). HEPES buffer (4-[2-hydroxyetheyl]-1-piperazineethanesulfonic acid; 2.38 g/L) was added and the pH was adjusted to 7.2–7.4 by addition of NaOH before autoclaving. After autoclaving, the medium was cooled under N2 flow, and a solution of 10 vitamins (Balch et al., 1979) and sodium bicarbonate (0.2 g/L) was added from sterile stock solutions. For the enrichment of nitrate-, iron-, or manganese-reducing bacteria, KNO3, solid ferrihydrite, or manganese oxide, respectively, were added to reach final concentrations of 2 mM for nitrate and 10 mM for Mn and Fe. To obtain quantitative information for particular physiological groups, the most probable number (MPN) method with three parallel tubes for each 10-fold dilution was applied (Hungate, 1968). The enrichment cultures were incubated at 4°C for about 10 months. Growth of all cultures was checked by visual inspection (turbidity) and phase contrast microscopy. Tubes with ferrihydrite as the electron acceptor were counted positive when Fe(II) was higher than 5 mM, and tubes with manganese oxide as the electron acceptor were counted positive when decolorization of the black manganese mineral was clearly visible by eye. Tubes for testing nitrate-dependent Fe(II)-oxidation conditions were also set up and counted as positive when a clear precipitation of Fe(III) was visible.

To selectively enrich methanogens, autoclaved Hungate tubes were filled with 7 mL of sterile anoxic sulfate-free minimal medium (Widdel and Bak, 1992). Sediment slurries from each sample (1:1 mix of sediment and anaerobic medium) were anaerobically prepared, and 3 mL was added to the Hungate tubes. Then the tubes were purged with dinitrogen (100%) and sealed with sterile butyl rubber stoppers and screw caps. Anaerobic enrichments were set up under methanogenic conditions without the addition of electron acceptors in triplicate and incubated vertically in the dark at 8°C with or without additional carbon sources. As additional carbon sources, combinations of typical sedimentary substrates were added as monomer or polymer mix (Batzke et al., 2007), Spirulina extract (all: 50 µL of a 0.005% stock solution), or single compounds (betaine, palmitate, phytane, or pristane [25 µL of a 2% stock solution]). Substrate concentrations were kept low to resemble conditions in deep sediments. In addition to these more complex substrates, simple well-known substrates for methanogens were used (acetate [0.5 mM], methanol [0.1 mM], trimethylamine [TMA] [0.5 mM], and hydrogen [H2/CO2/N2 5%/5%/90%]). Furthermore, the potential for hydrocarbon degradation was studied in incubations with hexadecane (10 µL), ethylbenzene, dotriacontan (C32), or methylnaphthalene (20 µL of a 2% stock solution [v/v]), which were all added directly to the medium. The increase of methane in the headspace was measured monthly over a 12 month period as previously described (Krüger et al., 2002). Positive enrichment cultures had substantially higher methane concentrations in the headspace than not inoculated control tubes.