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Sediment samples for microbiological studies were obtained primarily from Hole U1365C, using the advanced piston coring (APC) system. Because recovery in Core 329-U1365C-2H was low (0.43 m; possibly caused by blocking of the core liner by a manganese nodule), we also sampled two additional cores from Hole U1365D. To quantify contamination, PFT was injected into the drilling fluid during APC coring of Holes U1365B–U1365D. Hole U1365B was the source of samples for quantifying cell abundance; these samples were taken from the cut cores facing interstitial water whole-round samples on the Catwalk Deck. Microbiological whole-round cores were generally taken at a high depth resolution from the first core (i.e., Core 329-U1365C-1H). We also collected microbiological samples with high frequency from the two cores above the sediment/basalt interface (8H and 9H). Because the uppermost sediment (to ~0.50 mbsf) of Section 329-U1365C-1H-1 appeared to be disturbed during coring, no microbiological whole-round cores were obtained from this short interval.


Basalt samples were obtained by RCB coring system from Hole U1365E. Samples included altered and fresh basalt pieces, pieces with reddish oxidized surfaces, and pieces with mineral-filled fractures. Although contamination of RCB cores from seawater-based drilling fluids is unavoidable, its extent can be estimated. To estimate the contamination, we used fluorescent microspheres and PFT (see “Microbiology” in the “Methods” chapter [Expedition 329 Scientists, 2011]). For the first approach, a bag of fluorescent microspheres was placed in the core catcher of each core from Core 329-U1365E-2R onward. For the second approach, PFT was continuously injected into the drilling fluid. After core retrieval and sampling, all microbiology samples were checked for the presence of microspheres. Potential contaminant sources, such as surface seawater, bottom water, and the drilling fluid used for RCB coring were collected as reference samples for contamination monitoring. After each core recovery, all core sections in Hole U1365E were immediately transferred from the catwalk to the cold room (~7–10°C) on the Hold Deck. Prior to microbiological sampling, petrological characteristics were described. It took 8–18 h until subsequent microbiological sampling of the rock pieces in the cold room. A set of subsamples for fluorescent microspheres was prepared from exterior to interior parts of the massive basalt sample. To avoid cross-contamination, the core exterior was washed and then briefly flamed. We could not evaluate PFT concentrations because of the flame-induced volatilization. For cell enumeration, molecular analysis, and cultivation, the inner portion was powdered using a flame-sterilized percussion mortar and pestle. For biomineralogical analyses with stable isotope incubation, intact core pieces were stored for further processing.

In addition, small pieces of the altered uppermost basalt at Site U1365 were sampled from sediment/basalt interfaces in the core catcher at Holes U1365A– U1365C. During APC coring, no fluorescent microsphere beads were used because the fluorescent spectrum of beads may prevent shore-based image-based cell enumeration analysis and fluorescent in situ hybridization analysis. These core catcher samples were subsampled for cell counts, cultivation experiments, and mineralogical and molecular analyses.

Cell abundance

Cell abundance was determined by direct counting with an epifluorescence microscope. For shipboard analysis of sediment, subcore samples (2 cm3) were aseptically taken using tip-cut syringes from Hole U1365B and processed using the cell extraction method described in “Microbiology” in the “Methods” chapter (Expedition 329 Scientists, 2011). For shore-based analysis, 5 cm whole-round core samples were taken from Hole U1365C in the cold room and frozen at –80°C. Seventy-eight 2 cm3 syringe samples (Table T17) and nine whole-round core samples (Sections 329-U1365C-1H-2 through 1H-4, 3H-2, 5H-2, 8H-1, 8H-2 [10–20 and 140–150 cm], and 8H-3) were taken for cell enumeration.

Generally, cell abundance was very low throughout the core. In the uppermost sample (0.4 mbsf), cell density was ~5 × 105 cells/cm3. Cell abundance decreased rapidly with increasing depth, and individual samples reached the minimum detection limit (MDL) for direct counts (103 cells/cm3) at ~15 mbsf. Between 15 mbsf and the chert layer at 44 mbsf (top of lithologic Unit II), cell abundances of some samples were above the MDL, whereas other samples were below the MDL. Below Unit II, all samples were below the MDL (Fig. F61; Table T17). In order to improve counting statistics, the number of microscopic fields of view per filter was increased from 200 (for previous ODP and IODP expeditions) to 300, which led to a reduction in the number of samples that could be processed on board. Of 78 syringe samples, only 37 samples were analyzed on the ship. Eight blanks were counted, resulting in a mean blank of 5 × 102 cells/cm3 with a standard deviation of 2.98 × 102 cells/cm3, resulting in the MDL (blank plus three times standard deviation) of 1.4 × 103 cells/cm3. As the blanks did not vary much between sites, they were pooled. At the end of the expedition, a single MDL for all sites (1.4 × 103 cells/cm3) was calculated based on the extended database.

Some samples exhibited such low counts that they were below the mean blank. Several samples from the upper part of the core were counted without cell extraction. All were below the MDL for nonextracted counts; all other nonseparated cell counts were below the mean blank as well, except for one replicate of the uppermost sample (329-U1365B-1H-1, 40–50 cm; 0.45 mbsf), which contained a cell abundance of 8 × 103 cells/cm3.

In order to estimate cell abundance in basalt samples, powdered samples were fixed with 4% paraformaldehyde in Tris-buffered saline (TBS; pH 7.4). Cell numbers were enumerated onboard using epifluorescence microscope. The detailed protocol of cell counting for basaltic samples is described in “Microbiology” in the “Methods” chapter (Expedition 329 Scientists, 2011). Results from cell counting of the basaltic basement rocks are shown in Table T18. No reliable cell numbers were obtained from basalt samples. Although a basalt sample from the sediment/basalt interface in Hole U1365A (Sample 329-U1365A-25H-CC) contained SYBR Green I–stainable cells at 1.0 × 104 cells/cm3 (Table T18), this count is not significant because the MDL for basalt samples was ~4.9 × 104 cells/cm3 (equivalent to 5 cells per 300 fields of view).

Virus abundance

After sample preparation and observation of virus-like particles (VLPs) using an epifluorescence microscope, the first two filtered samples (from the Hole U1365A mudline and Section 329-U1365C-3H-3) were found to have a high background by using SYBR Green I staining. Subsequently, staining of VLPs was done using SYBR Gold fluorescent dye (see “Microbiology” in the “Methods” chapter [Expedition 329 Scientists, 2011]). This change in the staining protocol led to a reduction in the number of samples used for estimating viral abundance. For sediment samples from Sections 329-U1365C-1H-1, 3H-2, 5H-2, 7H-3, and 329-U1365D-1H-6, the number of VLPs was counted according to the protocol described in “Microbiology” in the “Methods” chapter (Expedition 329 Scientists, 2011). The remaining samples, including basalt samples from Sections 329-U1365E-2R-1 and 3R-4, were preserved at –80°C for shore-based analysis.

Viral abundance in the uppermost sediment sample (0.45 mbsf) was estimated to be ~5 × 106 VLP/cm3 with a VLP/cell ratio of ~10 (Table T19). VLP abundance decreases rapidly within the uppermost 20 m of the sediment column. The lowest count of ~6 × 104 VLP/cm3 is in the deepest sample analyzed (~65 mbsf) (Fig. F61)


Multiple cultivations were initiated on board using a variety of media for heterotrophic (both aerobic and anaerobic) and autotrophic microorganisms. The core samples were subsampled aseptically with tip-cut syringes to make slurries for inoculation in liquid media or on solid media (Table T20). Additional samples were stored either in N2-flushed serum bottles or in syringes packed in sterile foil packs stored at 4°C for future cultivation experiments (referred to as SLURRY in Table T20). For future cultivation efforts, filtered bottom water was transferred to sterile 50 mL serum bottles, sparged with N2 for 5 min, and capped with rubber stoppers and aluminum crimp caps. The bottles were stored at 4°C for preparing liquid media on shore. For basalt samples, multiple cultivations were also undertaken on samples from Sections 329-U1365E-3R-4, 5R-4, 7R-1, 8R-4, and 12R-2 (Table T20).

Molecular analyses

Sediment samples

Whole-round core samples were taken throughout the entire sediment column and transferred to –80°C freezers for storage. These samples will be used to determine microbial community composition and the presence or absence of functional genes. Eight 10 cm whole-round core samples were taken as routine microbiology samples (RMS; curatorial code MBIO) and stored at –80°C at the core repositories for future biological sample requests.

Basalt samples

Powdered samples were homogenized by thorough mixing and distributed among investigators for molecular analyses. These samples were stored at –80°C.

Deep seawater control samples

Seawater samples from above the mudline in Holes U1365B–U1365D were pooled in a 20 L plastic bag and sterilized by filtration through a 0.22 µm pore sized polycarbonate filter to examine the microbial community in bottom water as a contamination control. The filter was stored at –80°C.

Fluorescence in situ hybridization analysis

Duplicate subcores or subsamples from sediment and basalt pieces were fixed as described in “Microbiology” in the “Methods” chapter (Expedition 329 Scientists, 2011) and stored at –20°C for shore-based fluorescence in situ hybridization analyses.

Radioactive and stable isotope tracer incubation experiments

Sediment samples

Whole-round core and/or syringe samples were taken for measuring potential rates of microbial carbon/nitrogen substrate incorporation and sulfate reduction. For sulfate reduction rate assays, triplicate subsamples (~2.5 mL) were collected directly from the whole-round core samples using a syringe-type Plexiglas plug and temporally stored in the core refrigerator on the Hold Deck (~7°–10°C) with both ends sealed. In the Isotope Isolation Van, 10 µCi (370 KBq in 5 µL) of 35S-sulfate were injected directly through a port into each respective syringe sample. The samples were placed in the incubator (4°C) for 50 days. Samples for blank experiments (i.e., incubation time “0”) were preserved in 10 mL zinc acetate (20%, v/v) immediately after radioisotope injection and kept frozen until distillation of sulfide in the shore-based laboratory.

Stable isotope (13C and 15N) experiments to measure carbon and nitrogen uptake activities were initiated on board in the Isotope Isolation Van. Sediment subcores (15 cm3) were taken from the inner part of 20 cm whole-round core samples, placed in a sterile glass vials, flushed with N2, sealed with a rubber stopper, and stored until processing in the core refrigerator on the Hold Deck (see “Microbiology” in the “Methods” chapter [Expedition 329 Scientists, 2011]). The six whole-round core samples processed for the stable isotope incubation experiments were from Sections 329-U1365C-1H-2, 3H-3, 4H-3, 5H-1, 8H-2, and 9H-3. We initiated eight parallel incubation experiments by injecting each of the following into a separate vial:

  • Six vials, each with a separate 13C-labeled carbon source (15 µM glucose, acetate, pyruvate, bicarbonate, and amino acids) and methane (1 atm headspace),

  • A negative control sample without isotope-labeled substrates, and

  • A sample with only 1.5 µM 15N-labeled ammonia.

Each vial with a labeled carbon source also included a labeled 15N-nitrogen source: 15 µM 15N-labeled amino acid for the amino acid experiment and 1.5 µM 15N-labeled ammonia for the other five experiments. The oxygen concentration in the headspace of each vial was set at 4% (v/v) (see “Microbiology” in the “Methods” chapter [Expedition 329 Scientists, 2011]). All stable isotope incubation experiments on subcore samples were carried out in the Isotope Isolation Van.

The following whole-round intervals from Site U1365 were used for slurry experiments on potential metabolic activities (i.e., autotrophic and heterotrophic assimilation and dissimilative respiration potentials) using radioisotopes and stable isotopes: Samples 329-U1365B-1H-2, 120–130 cm; 2H-4, 105–115 cm; 5H-4, 105–115 cm; and 9H-3, 120–130 (see “Microbiology” in the “Methods” chapter [Expedition 329 Scientists, 2011]). These incubation slurry samples from Sites U1365 were processed together with other samples from Sites U1366–U1368 in the Isotope Isolation Van. In addition, ~40 mL each of 1:5-diluted (v/v) slurry from three samples (329-U1365B-1H-2, 120–130 cm; 5H-4, 105–115 cm; and 9H-3, 120–130 cm) were used for cell viability studies with 14C-labeled compounds (ATP, leucine, and thymidine). 18O-labeled water (H218O; 2.5 mL) of was added to aliquots (5 mL) of the same slurries and incubated at 4°C.

Basalt samples

For stable isotope incubation of basalt samples, small pieces (0.5 to 1.0 cm diameter) were stored at 4°C. Stable isotope incubation was initiated under microaerobic conditions (~4% O2 in headspace gas) in sterile bottom seawater amended with 100 µM 15N sodium nitrate and 100 µM 13C-labeled sodium acetate or 13C-labeled sodium. At given time points (~4 weeks, 6 months, and 2 y after starting incubation), vials will be opened and basalt pieces will be fixed with 4% paraformaldehyde in TBS solution or frozen for shore-based molecular and isotopic analyses using NanoSIMS.

Contamination assessment

Chemical tracer

PFT was used to monitor the level of drilling fluid contamination in sediment cores (see “Microbiology” in the “Methods” chapter [Expedition 329 Scientists, 2011]). PFT was continuously injected into drilling fluid during APC coring in Holes U1365C and U1365D. PFT tests were also conducted for basalt coring from Core 329-U1365E-3R. For the PFT measurement, subcores (3 cm3) of sediments were taken from whole-round cores in the cold room (see “Microbiology” in the “Methods” chapter [Expedition 329 Scientists, 2011]). A preliminary PFT quantification standard curve for the measurement using gas chromatography with an electrolytic conductivity detector was generated by a dilution series of PFT ranging from 10–13 to 10–9 using iso-octane as the solvent (Fig. F62). It was determined that the analysis was sensitive enough to detect the presence of ~1 µL seawater contamination; this is equivalent to ~1 microorganism. The first tests with standards containing PFT in dilution with iso-octane revealed that the PFT peak was at a retention time of 1.5 min.

For all sediment samples analyzed from Holes U1365C and U1365D with this new method, PFT peaks were comparable to the highest dilution of the standard curve (10–13). In other words, they were below the detection limit. However, PFT concentrations in the drilling fluid from Holes U1365C and U1365D were also always below the detection limit, suggesting that this new PFT extraction method using iso-octane and gas chromatography detection was not suitable for such onboard analysis.

PFT samples will be reanalyzed postcruise, using the approach described in “Microbiology” in the “Methods” chapter (Expedition 329 Scientists, 2011).

Particulate tracer

Fluorescent microspheres (0.5 µm diameter) were used for contamination testing during basement rock coring (see “Microbiology” in the “Methods” chapter [Expedition 329 Scientists, 2011]). This approach is not quantitative but provides evidence for the occurrence of contamination, even in interior structures of basaltic samples (e.g., microfracture and vein). Small rock pieces and/or post surface-wash solutions were stored in 3% NaCl solution for microscopic detection of microspheres.

Contamination was first examined on the untreated exterior by removing small pieces of rock using a flame-sterilized hammer and chisel. The rock surface was washed twice with 25 mL 3% NaCl solution in a sterile plastic bag. Small pieces of the washed exterior were removed using a flame-sterilized hammer and chisel, and wash solutions were pooled in a 50 mL centrifuge tube. After the washing step, the rock surface was flamed with a propane torch for a few seconds. The flamed rock was cracked open using a flame-sterilized hammer and chisel, and small pieces from the interior and exterior were separately inspected for the presence of microspheres.

Results from microscopic counting of microspheres in subsamples from each cleaning step are shown in Figure F63. The unit in the figure is either cubic centimeters of rock for removed basalt pieces or cubic centimeters of post surface-wash solution. It is clear from the figure that the uppermost basement basalt sections (329-U1365E-3R-4 and 5R-4) were associated with higher numbers of microspheres than the lowermost basalt sections (7R-2, 8R-3, and 12R-1). After the washing step with 3% NaCl solution, the number of microspheres decreased by one order of magnitude from the untreated exterior. Although flaming effects varied from one sample to another, the interiors contained no detectable microspheres. These results suggest that the interiors of cores are suitable for microbiological investigations.