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doi:10.2204/iodp.proc.336.102.2012

Microbiology

The primary microbiology objectives for Expedition 336 were to determine the microbial community composition and activity of the deep biosphere harbored in the buried basaltic oceanic crust at Sites 395A, U1382 (~50 m west of Hole 395A), and U1383 (prospectus Site NP-2) and near the sediment/​basement interface in Holes U1383D and U1383E (near the new deep Hole U1383C subseafloor borehole observatory [CORK]), U1382B (near Hole 395A), and U1384A (prospectus site NP-1) at the North Pond site on the western Mid-Atlantic Ridge flank. Strategies to reach these objectives included deployment of novel microbial colonization devices within CORK observatories (see Edwards et al., 2012; Orcutt et al., 2010, 2011), opportunistic sampling of biofilms on an old CORK recovered from Site 395, and collection of fresh crustal rocks by drilling in oceanic crust along the presumed flow path of formation fluids. These studies complement and expand upon similar work conducted on the eastern flank of the Juan de Fuca Ridge (Cowen et al., 2003; Engelen et al., 2008; Fisher et al., 2005, 2011; Nakagawa et al., 2006; Orcutt et al., 2010, 2011; Steinsbu et al., 2010), although the Juan de Fuca sites represent a reduced and warm hydrothermal setting as compared to the cool hydrothermal conditions within North Pond oceanic crust. These studies also build upon previous decades of work studying fluid flow in basement at North Pond and a recent site survey cruise with the R/V Merian (Ziebis et al., 2012).

A secondary microbiology objective of this expedition was to collect sediment from multiple sites around North Pond, including prospectus Sites NP-1 (Hole U1384A) and NP-2 (Holes U1382D and U1382E) and Site 395 (Hole U1382B), to examine the phylogenetic and functional connection between sediment microbial communities and those harbored within oceanic crust and the overlying seawater and to examine how these relationships may vary vertically (i.e., with distance from the sediment/​basement interface) and horizontally (i.e., along the presumed flow path of fluids within oceanic crust). Of interest was the evaluation of basement fluid flow on basal sediment biogeochemistry and microbial ecology, as has been evaluated elsewhere (Engelen et al., 2008; Lever et al., 2010).

This section focuses on the shipboard methods used for rock and sediment sample collection and handling for microbiological analyses; CORK-related microbiology experiments are described in Edwards et al., 2012. Briefly, samples of oceanic crust collected for microbiology were subsampled for environmental DNA and RNA extraction and analysis, cell counts, fluorescent in situ hybridization (FISH) studies, contamination tests, evaluation with the new deep ultraviolet (UV) fluorescence scanner for biofilm biomass (Bhartia et al., 2010), and several enrichment and culturing experiments. Sediment samples were also collected for DNA and RNA extraction and analysis, cell counts, FISH studies, contamination tests, lipid analysis, and several enrichment and culturing experiments to be conducted on shore.

Core handling and sampling

To examine potential contamination of hard rock and sediment core samples, slurries of yellow-green fluorescent microspheres (Fluoresbrite Carboxylate Microspheres; Polysciences, Inc., 15700) were sealed in plastic bags and placed inside the core catcher prior to deployment of the core barrel according to standard protocol (Smith et al., 2000). Microspheres were used in every RCB core during hard rock coring and also in every APC and XCB core during coring in sediments and across the sediment/​basement interface. Perfluorocarbon tracer contamination checks (Lever et al., 2006) were not conducted during this expedition.

Hard rock cores

Hard rock samples for microbiology originated from RCB coring in Holes U1382A and U1383C and from ACP and XCB coring in Holes U1383D, U1383E, U1382B, and U1384A. Priority was given to large (>10 cm in length) intact pieces or samples with interesting lithology. Nominally one sample was collected per section during RCB coring. Immediately following delivery of core on deck and cutting of the core liner into 1.5 m sections, rocks were exposed for subsampling in the core splitting room by shaking the recovered rocks into another split core liner (which, because of frequent splitting blade breakage, was much faster than trying to split the recovered core liner). Rocks for microbiological sampling were identified immediately, photographed in place, and then collected using combusted aluminum foil for transport to the microbiology laboratory. During ACP and XCB coring, some rocks were handled in the above manner, when appropriate; otherwise, rock and sediment matrix material was transferred via sterile spatula on the catwalk into a sterile Whirl-Pak bag for subsequent subsampling. All sample handlers wore gloves to reduce contamination. In the laboratory, whole-round rock pieces were transferred to sterile Whirl-Pak bags containing 10 mL of sterile filtered seawater for gentle rinsing and removal of any microspheres and other contaminates. The rinse was collected into a 15 mL conical vial and stored at 4°C until processing. The rinsing process was repeated three times. Next, the rock was transferred to a flame-sterilized rock processing box (Expedition 327 Scientists, 2011) and broken into smaller pieces using flame-sterilized chisels and forceps. Subsampling was done as rapidly as possible (5–15 min) to minimize oxygen exposure and cell degradation. Rock fragments were then split into aliquots for the following analyses, depending on available sample volume:

1. Fixed for shore-based FISH or Raman confocal microscopy assays:
1. Fixed in cold 3.7% [w/v] paraformaldehyde in 1× phosphate-buffered saline (PBS; 150 mM NaCl per liter of 10 mM sodium phosphate, pH 7.2) for 1–4 h, washed in 1× PBS, and then stored at –20°C in 1:1 1× PBS:ethanol;
2. Rinsed in 5% sodium hypochlorite and sterile seawater (to remove any exterior contaminants), fixed in 2% [v/v] formaldehyde (methanol free, Ultra Pure; Polysciences, Inc.) in 1× PBS, and then rinsed and stored as above; or
3. Rinsed in 5% sodium hypochlorite and sterile seawater and then fixed and stored in cold 50% [v/v] ethanol in double-deionized water.
2. Preserved for shore-based DNA and RNA analysis and archival either by immediately freezing at –80°C in sterile sample bags or by first rinsing the exterior in 2%–3% sodium hypochlorite and sterile seawater prior to freezing.
3. Transferred to baked aluminum foil (450°C to remove organics) and stored at 4°C for deep UV fluorescence scanning.
4. Transferred to baked aluminum foil (450°C) and frozen at –20°C for stable isotopic analysis of carbonate-¹³C and oxygen isotopes.
5. Rinsed in 5% sodium hypochlorite solution and sterile seawater.
6. Prepared for enrichment and culturing experiments (described below).

Leftover rock material was washed in deionized water, dried, and returned to shore-based laboratories for use as substrate in future colonization experiments.

Sediment cores

Sediments for microbiological analysis were collected from four holes as whole-round core or syringe samples. APC and XCB cores were cut on the catwalk using sterilized tools (autoclaved spatulas and bleach-cleaned end caps). Everyone assisting in the core cutting and sediment sampling wore gloves to minimize contamination. Each core was inspected before being cut to determine the integrity of the sediment and the potential for disturbance during drilling or recovery. A predetermined sampling plan was followed for each core and section to maximize sampling efficiency and accuracy. Samples were collected from almost every section recovered provided the section was of sufficient quality. A general sampling plan is described below; the full sampling plan is included as Figures F8, F9, F10, and F11.

1. Whole-round samples were cut from the bottom of each section and then capped and stored according to submitted sample requests. A portion of the sediment was dedicated to pore water geochemistry (see “Inorganic geochemistry”). Samples were collected for the following microbiology procedures:
1. Culture independent analysis: whole-round samples for shore-based DNA and RNA were cut from each core. In upper and middle cores, three 10 cm whole-round cores were collected for both DNA and RNA analysis. In the deepest cores, a whole-round core sample was collected from each section for both DNA and RNA analysis. Additional samples were collected for polar lipid fatty acid and metagenome analysis at a frequency of one per core. All whole-round cores for culture independent analysis were stored at –80°C in a sterile sample bag.
2. Culturing: whole-round samples were collected for multiple ship- and shore-based culturing projects. Higher resolution sampling occurred in shallow and deep cores, with fewer samples collected in mid-depth cores. Enrichment cultures were established for nitrogen, iron, sulfur, and carbon-cycling functional groups. Additional culture assays will be constructed on shore to target heterotrophs, fermenters, phosphate cycling, and acetate turnover. Anaerobic and aerobic strategies were employed for both ship- and shore-based samples, with anaerobic samples being stored in silver Mylar bags under nitrogen headspace. Temperatures for incubations ranged from 8°–20°C, and whole rounds were stored at 9°C.
2. Multiple syringe samples were collected from the tops of each core section, focusing on the interior of the core. A syringe sampling diagram is included in Figure F12 and follows the above sediment sampling plan (Figs. F8, F9, F10, F11). One set of 10 mL syringe samples was collected for shore-based DNA characterizations of Bacteria and Archaea populations. In addition, 2 cm³ of sediment was collected via syringe and transferred to 20 mL glass serum vials with crimp caps for onboard methane concentration analysis. Sediment was also collected via toothpick from the edge and center of the top of the core section for microsphere contamination analysis. These samples were mixed with sterile filtered PBS solution and stored for shore-based analysis.

Storage and shipment conditions

All samples for shore-based DNA/RNA extraction and analysis were stored and shipped frozen (–80°C), whereas samples for shore-based FISH and enrichment studies were stored and shipped cold (–20°–4°C).

Analytical methods

Cell counts, FISH, and spectroscopy of hard rock materials

Sample fixation for FISH is described above. Washed samples will be used for shore-based FISH analyses, whereas unwashed samples will be used for cell counting with either SYBR Green I or acridine orange fluorescent dye using previously described methods (Morono et al., 2009). On the basis of results from DNA extraction and analysis, microbial groups of interest will be investigated using group-specific FISH primers according to published protocols (Biddle et al., 2006).

Dried samples for micro- and nano-imaging (with X-ray, electron, and UV-visible spectroscopies) were transferred for shore-based analysis with flame-sterilized pliers to sterile centrifuge tubes without any treatment. Manually polished sections (using gloves and absolute ethanol) or freshly broken fragments will be characterized for mineralogical and organic matter content using Fourier transform infrared spectroscopy and Raman microspectroscopy (Beyssac et al., 2003; van Zuilen et al., 2007; Marshall et al., 2010) and scanning electron microscopy with energy dispersive spectrometry and X-ray microscopy (Rommevaux-Jestin and Menéz, 2010; Ménez et al., 2007). Ultrathin sections will be prepared using a focused ion beam as described by Benzerara et al. (2005) for observations using scanning X-ray microscopy and transmission electron microscopy (Benzerara et al., 2006).

Nucleic acid extraction and analysis

DNA will be extracted in shore-based laboratories using a variety of methods, depending on sample type and analytical laboratory, to maximize cross-comparison of methods. Genes of interest, including the 16S rRNA gene as well as functional genes, will be amplified using multiple amplification strategies.

In one shore-based laboratory, DNA will be extracted from hard rock samples using the MO BIO DNA extraction kit for soil (MO BIO Laboratories, Inc.), following the manufacturer’s protocol with minor modification. An archaeal 16S rRNA gene amplicon library will be prepared, and the resulting library will be sequenced using the 454 GS FLX Titanium pyrosequencing platform (454 Life Sciences, Roche). The taxonomic affiliation of each read will be resolved as described elsewhere (Lanzén et al., 2011). In addition, archaeal and bacterial 16S rRNA genes will be quantified by quantitative polymerase chain reaction (qPCR) following a previously described protocol (Roalkvam et al., 2011). Specific archaeal groups such as Marine Benthic Group B and Marine Group I may be quantified by qPCR with group-specific primers, depending on results from the amplicon library.

In another shore-based laboratory, DNA will be extracted from hard rock samples using a “homemade” DNA extraction protocol utilizing phenol-chloroform extraction following published protocols (Lever et al., 2010; Orcutt et al., 2011). In addition, 16S rRNA genes will be amplified with published primer sets using Ion Torrent semiconductor sequencing (Rothburg et al., 2011) coupled with Sanger-style sequencing.

In another shore-based laboratory, DNA and RNA will be extracted from hard rock samples using the MO BIO PowerSoil DNA extraction kit (MO BIO Laboratories, Inc.) following the manufacturer’s protocol with minor modifications, as described in Gérard et al. (2009). Genes of interest, including the 16S rRNA gene and some functional genes, will be amplified using PCR with published protocols.

Total RNA will be extracted from sediment and crushed basalt in another shore-based laboratory using a method previously described in Mills et al. (2008) with modifications noted in Mills et al. (2012). Extracts will be treated with deoxyribonuclease (DNase) to remove residual DNA prior to reverse-transcription PCR. Initial amplifications will target the 16S rRNA gene transcripts using published primers. Amplicons will be sequenced using the 454 GS FLX Titanium pyrosequencing platform (454 Life Sciences, Roche). The metabolically active community structure will be determined by sequence annotation. Functional gene transcripts will be quantified on the basis of results of community structure analysis to determine community function.

To evaluate the preenrichment microbial community in sediments prior to cultivation, a preliminary analysis by PCR amplification will be conducted in a shore-based laboratory to examine the existence of methanogens, sulfate reducers, and methanotrophs. DNA will be extracted from each sediment sample using the PowerMax soil DNA isolation kit (MO BIO laboratories, Inc.) following the manufacturer’s protocol. The gene encoding methyl coenzyme M reductase (mcrA) of methanogens will be amplified using a PCR method, as described by Nunoura et al. (2008). The gene encoding dissimilatory sulfite reductase (dsrA) of sulfate reducers will be amplified using a PCR method, as described previously (Kondo et al., 2004). The gene encoding particulate methane monooxygenase (pmoA) will be amplified using a PCR method, as described previously by Tavormina et al. (2008).

Deep UV fluorescence scanning

To evaluate the presence of cells and organics on the surfaces of hard rock materials using deep UV fluorescence (Bhartia et al., 2008, 2010), rock fragments (1–2 cm³) were scanned with the new Deep Exploration Biosphere Investigative portable tool (DEBI-pt) similar to the Deep Exploration Biosphere Investigative tool (DEBI-t) downhole logging tool described in “Downhole logging.” The DEBI-pt combines a targeted ultraviolet chemical sensor (TUCS) (Photon Systems, Inc.) with an X-Y scanning stage. A 224.3 nm HeAg hollow-cathode laser induces fluorescence of organics and microbes. Detection uses six discrete, laser-gated PMT-based bands at 280, 300, 320, 340, 360, and 380 nm. The laser focuses on a 200 µm spot translated over the sample at a rate that satisfies Nyquist sampling. Using the motor encoders, the maps are displayed in millimeters and provide spatial coordinates from a registered set of points.

Samples for DEBI-pt analysis were carefully collected during rock subsampling to preserve the interior and exterior orientation of the material. Pieces were photo-documented before being wrapped in baked foil and stored at 4°C to enable detailed correlation of DEBI-pt scans with sample orientation. Samples collected from coring operations were placed on a stage below the TUCS. Care was taken to minimize exposure of the samples to potential contaminants. A charged-coupled device camera was used for targeting and focus. Once the sample was in focus, the x and y sample dimensions and the desired degree of overlap were input in the control software. When the scanning run was complete, the data were transferred to interpolation software that created a multidimensional array that was then viewed with a custom analysis package, which represented the data as a fluorescence map indicating the location and intensity of fluorescence for each of the bands. The scanned samples were then stored for shore-based thin sectioning for petrology to correlate fluorescence regions with the mineralogy of each sample.

DEBI-pt experiments were also conducted with smears of the microsphere solutions, pipe dopes, and drilling fluids used during coring to generate background spectra profiles for these materials that could be used for comparison with natural samples.

Culturing and enrichment

Several types of metabolic groups will be targeted by enrichment and cultivation using various techniques, as outlined below.

Target 1

• Metabolic group: hydrogen-utilizing microorganisms from basement (specifically from peridotite, gabbro, veins, and basalt glass), including hydrogenotrophic methanogens, anaerobic nitrate reducers, anaerobic sulfate reducers, and microaerobic and aerobic hydrogen oxidizers.
• Procedure: on board, rock fragments (20–30 cm³) were crushed inside a stainless cylinder mortar in the anaerobic chamber. The small rock fragments were transferred into a 100 mL glass bottle that was sealed tightly with a butyl rubber stopper and stored at 4°C until processing in the shore-based laboratory. The targeted microbial groups will be cultivated in a basic medium composition described elsewhere (Takai et al., 2003, 2008) and outlined in Table T4.
• For cultivation of methanogens, the basic medium will be supplemented with 0.2% (w/v) NaHCO₃, 0.03% (w/v) NH₄Cl, 0.5 mg/L of resazurin, 0.05% (w/v) sodium sulfide, 2% (w/v) metallic iron powder, and 1% (v/v) vitamin mix solution, and the pH will be adjusted to 6.5. The cultivation will be conducted in the test tube with the gas phase of H₂ (80%) + CO₂ (20%) (3 atm).
• For cultivation of other H₂-oxidizers, the basic medium will be supplemented with 0.1% (w/v) NaHCO₃, 0.025% (w/v) NH₄Cl, 0.025% (w/v) NaNO₃, and 0.1% (v/v) vitamin mix solution.
• For anaerobic H₂-oxidizers, 0.05% (w/v) NaNO₃ will also be added. The pH will be adjusted to ~7. The cultivation will be conducted in the test tubes with the gas phase of
1. H₂ (80%) + CO₂ (20%) (3 atm),
2. H₂ (79.5%) + CO₂ (20%) + O₂ (0.5%) (3 atm), and
3. H₂ (78%) + CO₂ (20%) + O₂ (2%) (3 atm). The incubation temperatures will be 4°–15°C.

Target 2

• Metabolic group: high pressure–adapted heterotrophs from Hole 395A CORK rust and thermistor cable.
• Procedure: rust samples (~100 µL) were mixed with 25 mL of Difco 2216 marine media or 1:10 diluted Difco 2216 marine media (for oligotrophic heterotrophs) in glass bottles. Samples were stored at 4°C until shore-based manipulation. Thermistor cable samples were stored at 4°C in sterile Whirl-Pak bags for shore-based manipulation. In the shore-based laboratory, high-pressure incubation will be used to enrich and select for high pressure–adapted microorganisms using pin-closure pressure vessels (Wang et al., 2009a). Isolated high pressure–adapted microorganisms will then be characterized and compared with other deep-sea high-pressure microorganisms isolated from different ocean provinces.

Target 3

• Metabolic group: aerobic and anaerobic CO₂, CH₄, acetate, NH₄, and NO₃-cycling microorganisms from hard rocks.
• Procedure: rock fragments were broken into smaller pieces using sterile Xpress hydraulic press devices. Smaller rock fragments (~50 cm³) were transferred to sterile 250 mL screw-cap Pyrex bottles with thick rubber stoppers and mixed with 50 mL of artificial seawater. A variety of aerobic and anaerobic enrichments were then created with mixtures of stable isotope–labeled substrates, with the labeled substrate used at 1–2 mM or 20% [v/v] headspace.
• Aerobic combinations included ¹³C-labeled sodium bicarbonate + ¹⁵N-labeled NH₄Cl, ¹³C-labeled sodium bicarbonate + ¹⁵N-labeled sodium nitrate, ¹³C-labeled methane + ¹⁵N-labeled ammonium chloride, and ¹³C-labeled sodium acetate + ¹⁵N-labeled ammonium chloride.
• Anaerobic combinations included ¹³C-labeled sodium bicarbonate + ¹⁵N-labeled NH₄Cl, ¹³C-labeled methane + ¹⁵N-labeled ammonium chloride, ¹³C-labeled methane + ¹⁵N-labeled sodium nitrate, and ¹³C-labeled sodium acetate + ¹⁵N-labeled ammonium chloride.
• After enrichment at ambient temperature in the dark for 6 weeks or more, the enrichments will be checked by microscopy to evaluate growth in the shore-based laboratory. Headspace gas in the enrichments will be analyzed by gas chromatography–mass spectrometry to evaluate the production or consumption of ¹³C-compounds. Enriched samples will be evaluated in the shore-based laboratory using taxonomic, metagenomic, or transcriptomic analyses of extracted DNA or RNA (Wang et al., 2009b; Xie et al., 2011). Based on the genetic results, oligonucleotide probes may be designed to target specific microbial groups using FISH (Pernthaler et al., 2002). Subsequently, FISH-targeted isotopically labeled cells may be evaluated by nanoscale secondary-ion mass spectrometry to link taxonomic groups with functional processes (Orphan et al., 2001). Further enrichment to isolation may be conducted, and single-cell capture with micromanipulator or flow cytometry followed by single-cell sequencing may also be performed (Zhang et al., 2006).

Target 4

• Metabolic group: aerobic autotrophic carbon fixers from basement rocks.
• Procedure: rock fragments were broken into smaller pieces using sterile Xpress hydraulic press devices. Smaller rock fragments (~5 cm³) were transferred to glass serum vials, covered with sterile filtered aerobic surface seawater amended with ¹³C-labeled sodium bicarbonate at a concentration of 1–2 mM, and sealed without headspace. Enrichments will be incubated for various time periods (up to 6 months) following methods described previously (Expedition 330 Scientists, 2012). At the end of each time period, samples will be collected for determining ¹³C incorporation into bulk organic matter. Samples exhibiting significant incorporation will subsequently be analyzed by molecular methods (density gradient centrifugation of extracted DNA) to identify active microbial populations.

Target 5

• Metabolic group(s): methanogens, sulfate reducers, sulfide oxidizers, and nitrate-reducing iron-oxidizing bacteria from basement.
• Procedure: rock fragments were broken into smaller pieces using a flame-sterilized metal rock crusher. Smaller rock fragments (~3 g) were transferred to 160 mL glass serum vials, covered with 60 mL of media, sealed with a butyl rubber stopper, and incubated in the dark at 10°C. Cultures were inoculated in the anaerobic chamber.
• Media for methanogenic enrichments consisted of 500 mL of autoclaved surface seawater (from Site 395) amended with 5 mL Wolfe’s mineral solution, 5 mL Wolfe’s vitamin solution, 10 mL methanol, and 5 mg resazurin.
• Media for sulfate reducers was the same, with the substitution of 1 g sodium acetate for the methanol. Media was boiled and then transferred to an anaerobic glove box to purge with O₂-free nitrogen gas.
• Media for sulfide oxidizers consisted of autoclaved seawater amended with potassium nitrate (0.5 g/L) and potassium phosphate (0.5 g/L), which was filter-sterilized and added to 160 mL serum vials containing a 20 mL sulfide-rich (1 mM) agar plug.
• Media for nitrate-reducing iron-oxidizing bacteria was similar to the base media for sulfide oxidizers (without the agar plug), with the addition of 0.5 g steel wool (as an iron source).

Target 6

• Metabolic group(s): aerobic iron-oxidizing bacteria and anaerobic heterotrophs from Hole 395A CORK rust and thermistor cable.
• Procedure: six 150 mL serum bottles were filled with 60 mL autoclaved and filter-sterilized seawater. Each was stoppered, brought to a semirapid boil, and maintained at boiling for 5 min with a 22.5 gauge needle as a pressure outlet. After 5 min another needle was inserted, from which flowed nitrogen gas at 2–4 psi. The bottle was then flushed with nitrogen while boiling for 5 min. At this point, both needles were removed and the bottles were sealed and allowed to cool on the bench top. In an anaerobic chamber, a “tip of spatula” amount of material from the CORK remotely operated vehicle platform Area 9D and Packer 1 samples (see Tables T5, T7, both in the “Site 395” chapter [Expedition 336 Scientists, 2012b]) were added into two bottles each. Additionally, the ThermStr-5 sample, representing a mid-depth string sample, was shaken in a Whirl-Pak bag with 50 mL of autoclaved seawater. The bag was then vortexed for 90 s to shake loose any colonizing microbes on the external polyethylene rope. One milliliter of this water was used as inoculum in one bottle. After inoculation, 0.25 g of steel wool was added to all bottles, and 15% of headspace was replaced with pure O₂. One bottle was treated as a negative control.

Target 7

• Metabolic group(s): aerobic and anaerobic heterotrophic groups from sediment and basement.
• Procedure: basement material and sediment were used as inoculum for enrichment assays for heterotrophy. Prior to addition, basement samples were crushed inside a flame-sterilized stainless steel cylinder mortar to coarse grain size. A total of 2 g sediment or 2.5 g basement material was transferred in duplicate to 30 mL serum vials containing one of three autoclaved media types: 1× Difco 2216 marine media, 1/100× Difco 2216 marine media, or 1/100× Difco 2216 marine media with 0.5 g pyrite. An additional set of the pyrite-enriched media was made and autoclaved to provide a kill control to differentiate abiotic and biotic effects on the pyrite. A representative vial from each media type remained basalt free to provide a negative control. All vials were incubated in the dark at 4°C.

Target 8

• Metabolic group: methanogens, sulfate reducers, sulfur oxidizers, and methanotrophs in sediments.
• Procedure: two syringes of sediment were subsampled from each whole-round core (10 cm long). One syringe was used for cultivation and the other was used for DNA analysis as an advance inspection for cultivation. Before subsampling took place, the sectional surface was cut off. The syringe samples were taken from the central part of the core. A syringe sample (10 cm³) for cultivation was put into a 100 mL glass bottle and immediately flashed with nitrogen gas to remove oxygen. After the bottle was sealed tightly with a butyl rubber stopper, it was stored at 4°C until processing in the shore-based laboratory. The other syringe sample (5 cm³) for a DNA analysis was put into a 15 mL plastic tube and frozen at –80°C. Target microbial groups will be cultivated using the same methods as those used for the rock samples. The additional cultivation targets for sediment are aerobic methanotrophs, which will be cultivated as described elsewhere (Hirayama et al., 2007). The basic medium outlined in Table T4 will be used for methanotrophs, and the gas phase in the tubes will be prepared as CH₄ (45%) + N₂ (40%) + CO₂ (10%) + O₂ (5%) (2 atm). The incubation temperatures will be 4°–15°C.

Onshore sample requests

Sediment samples were collected for multiple onshore sample requests. Requests included both molecular and culture-based analysis. These requests were fulfilled when they complemented without overlapping the work being conducted by the onboard science party. These plans included nitrogen-based culturing, colony-forming units, lipid and fatty acid analysis, and phosphorus-cycling measurements. Sediment samples were collected approximately once per core for each of the four requests and preserved at either 4° or –80°C, as instructed (Figs. F8, F9, F10, F11). Additional samples were collected using a syringe from the top of selected sections (Fig. F12).

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