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doi:10.2204/iodp.proc.331.203.2016

Materials and methods

Overall, 120 sediment samples from 5 sites (C0013–C0017) to a maximum depth of 110 mbsf were taken onboard the D/V Chikyu for microbiological research at the Federal Institute for Geosciences and Natural Resources (BGR). The samples were taken from the centermost part of the sediment cores under aseptic conditions in an anaerobic box, put in anaerobic bags, and sent to BGR at 2°–8°C as previously described in the “Methods” chapter (Expedition 331 Scientists, 2011b; Shipboard Scientific Party, 2003) and stored at 8°C until use.

To determine microbial activity, the heat output in microwatts per gram for each sample from Sites C0014, C0015, and C0017 was measured in an isothermal microcalorimeter (Thermal Activity Monitor Thermostat type 2277; Thermometric, Sweden) within 1 month after the expedition. A measurement lasted for 2–4 h until a stable value was obtained. After measuring the total heat output (microbial and chemical) in a ~5 g sample at 25° and 90°C, the chemical heat output at 90°C was measured after chloroform treatment (addition of 0.5 mL chloroform and 24 h incubation and subsequent removal by vacuum evaporation) to kill the microorganisms, as described elsewhere (Schippers and Bosecker, 2005). Chemical heat output was only measured at 90°C. Measurements under aerobic conditions were made in air; for anaerobic conditions, glass ampoules containing samples were closed in a nitrogen atmosphere (anaerobic box). Microbial substrates were not added for measurements. Activity values were calculated per sample dry weight (determined as weight difference after drying). Microbial activity can be calculated as the difference between total and chemical heat output (Schippers et al., 1995; Schippers and Bosecker, 2005).

For enrichment of microorganisms, liquid culture media were inoculated in a flow hood with sediment (Shipboard Scientific Party, 2003) from Sites C0013, C0014, and C0017 within 2 months after Expedition 331. The media were based on artificial seawater as described by Batzke et al. (2007) and were amended either with a polymer or monomer solution. The polymer solution contained chitin, cellulose, and peptone at 0.5 per gram each, whereas the monomer solution contained 36 different carbon sources such as amino acids, fatty acids, organic acids, alcohols, and glucose (final concentration = 0.1 mM). Media for aerobic incubations were buffered with bicarbonate/CO₂. In media for anaerobic incubations, HEPES (2.38 per gram) was added, and pH was adjusted to 7.2–7.4 by addition of NaOH before autoclaving. After autoclaving, the media were cooled under N₂ flow, and a solution of 10 vitamins (Balch et al., 1979) and sodium bicarbonate (0.2 per gram) were added from sterile stock solutions (Batzke et al., 2007). For the enrichment of sulfide-, manganese-, or iron-oxidizing bacteria, 1 mL of a 100 mM sterilized filter and anoxic (0.2 µm) Na₂S, MnSO₄, or FeCl₂ solution was added in gradient tubes. The enrichment cultures were incubated at 12°, 60°, 70°, or 80°C.

Growth was continuously checked by visual inspection (turbidity) and/or phase contrast microscopy, and in case of growth, colonies were picked and transferred to fresh media. Several aerobic and anaerobic enrichments were obtained. To identify isolates, 16S rRNA gene sequencing and analysis was done. The amplified polymerase chain reaction products of bacterial gene fragments were purified and sequenced at Microsynth sequencing company, Switzerland. Primers GM3F (5′AGAGTTTGATCMTGGC3′) and GM4R (5′TACCTTGTTACGACTT3′) were used for sequencing. The sequences obtained were edited with Geneious 6 software and compared with the NCBI database through BLAST searches.

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