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Materials and methods

Subsampling of cores

Pieces of rock cores collected for microbiology were subsampled for several analyses (see the “Methods” chapter [Expedition 336 Scientists, 2012b]), and a portion was used for this study. Each piece of rock (10–20 cm3 per piece) was immediately transferred into the anaerobic chamber settled in the onboard cold room after the subsampling and then smashed using a tungsten carbide cylinder mortar. The smashed rock was put into a 100 mL glass bottle and sealed tightly with a butyl rubber cap in the anaerobic chamber. The bottles were kept cool before starting shore-based cultivations.

A 10 cm whole-round sediment sample was cut from a sediment core on the catwalk for this study (see the “Methods” chapter [Expedition 336 Scientists, 2012b]) and kept at 4°C before further processing. The following process was promptly conducted on the onboard laboratory bench to minimize laboratory contamination and exposure to oxygen. The surface of the whole-round sample was trimmed with a stainless steel spatula, and 10 cm3 of the sediment was taken from the center with a cut syringe. The syringe sample was put into a 100 mL glass bottle, and the headspace was purged with nitrogen gas. The bottle was sealed tightly with a butyl rubber cap. The bottles were kept at 4°C before starting shore-based cultivations. Another portion of the sediment sample was taken for DNA analysis; a syringe sample (7–10 cm3) was put in a plastic tube, and frozen at –20°C and kept frozen before the shore-based analysis.

Preparation of growth media

Cultivation media used in this study are summarized in Table T1. For hydrogenotrophic methanogens, 20 mL of marine medium salts in artificial seawater (MMJ) medium was prepared in a 70 mL glass vial as previously reported (Takai et al., 2002). For other microorganisms, respective cultivation media were prepared using the basic seawater medium. The basic seawater medium was prepared by adding 0.06 g KH2PO4, 0.17 g Na2HPO4, and 0.25 g NH4NO3 in 1 L of commercial artificial seawater MARINE ART SF-1 (Osaka Yakken), which did not contain any organic compound. A 3 mL portion of each growth medium was prepared in a 15 mL test tube with a butyl rubber cap. Headspace was replaced with each gas composition shown in Table T1.

Enrichment cultures

Slurry was prepared using each core sample in an onshore laboratory after the expedition. The smashed rock sample was suspended with 20–40 mL of the anaerobic artificial seawater, which was injected in a glass bottle by a syringe. The sediment sample was suspended with 20 mL of the anaerobic artificial seawater. The total 33 rock cores and 30 sediment cores were subjected to enrichments. A 0.3 mL portion of slurry was inoculated into each medium, and enrichments were performed for 4–8 months at temperatures shown in Tables T2, T3, and T4.

Bacterial and archaeal rRNA gene analyses

Some microbial cells in the enrichment cultures were distinguished from rock or sediment particles by microscopy; however, exact cell counting of the enrichment cultures was difficult because of deceptive cell-like particles in the cultures. Genomic DNA was then extracted from 1 mL of every culture using ISOIL for Beads Beating (NIPPON GENE) according to the manufacturer’s instructions. The volume of the final DNA extract was 15 µL. Bacterial and archaeal 16S rRNA genes were amplified from the extracted DNA by polymerase chain reaction (PCR) in each 15 µL volume of reaction mixture containing the following:

  • 1xGC buffer I (Takara Bio),
  • 0.33 mM each dNTP (deoxynucleotide),
  • 0.33 µM each primer,
  • 1.25 U (units) of LA Taq polymerase (TaKaRa Bio), and
  • 3.7 µL of genomic DNA extract.

Primers were Bac27F and Uni1492R for bacteria and Arc21F and Uni1492R for archaea (DeLong, 1992; Lane, 1991). The amplification was performed using Veriti Thermal Cycler (Applied Biosystems) with the following program:

  1. 96°C for 1 min,
  2. 37 cycles of 96°C for 25 s,
  3. 50°C (archaea) or 53°C (bacteria) for 45 s,
  4. 72°C for 90 s, and
  5. 72°C for 10 min.

In the PCR amplification, a negative control containing no DNA extract was always examined to exclude contamination. In addition, contamination in the DNA extraction kit was sometimes examined by doing the extraction procedure without a sample and the following PCR amplification.

Aliquots of 2 µL of PCR products were analyzed by agarose gel electrophoresis and ethidium bromide staining. The amplified 16S rRNA gene fragments shown as clearly visible bands were sequenced by direct sequencing or after cloned into vector pCR2.1 with TA Cloning Kit (Invitrogen). Partial 16S rRNA gene sequences were analyzed by 3130×l Genetic Analyzer (Applied Biosystems). The obtained sequences were compared against the DNA database using BLAST (

Analysis of a fungal isolate

A fungal isolate was routinely cultivated on an agar plate prepared with 2 g yeast extract, 2 g peptone, 2 g maltose, and 15 g agar per 1 L of artificial seawater MARINE ART SF-1. DNA was extracted and analyzed as described above for bacterial and archaeal DNA analyses. DNA fragments including 18S, 5.8S, and 28S rRNA coding regions and the internal transcribed spacer (ITS) regions were obtained by PCR. PCR amplification was first conducted using the following three sets of primers (

  • EU347F and EUK-B (Medlin et al., 1988; Puitika et al., 2007),
  • ITS1 and ITS4 (White et al., 1990), and
  • LT0R and LR5.

After sequencing the PCR products, blank regions were determined by primer walking to extend the sequences. The obtained sequence was compared against the DNA database using BLAST and analyzed to construct a phylogenetic tree as described previously (Hirayama et al., 2013).

Measurement of methane production

Methane production in rock core enrichment cultures targeting methanogens was examined by injecting 0.1 mL of headspace gas from each vial into a gas chromatograph GC-3200 (GL Science) equipped with a thermal conductivity detector and a SHINCARBON ST 50/80 column (Shinwa Chemical Industries). The detection limit was approximately 10 µM.

Real-time PCR of the methyl coenzyme-M reductase subunit A gene (mcrA)

Genomic DNA was extracted from 1 g of frozen sediment using ISOIL for Beads Beating. A 2 µL portion of the total 20 µL DNA extract was used for a real-time PCR experiment to examine the presence of methanogens in the sediment cores by amplification of the methyl coenzyme-M reductase subunit A gene mcrA, which is the key enzyme gene of methanogens. SYBR green dye-based PCR amplification and detection were performed using SYBR Premix Ex Taq (TaKaRa Bio) and a 7500 Real-Time PCR system (Applied Biosystems) as described previously (Nunoura et al., 2008).