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Sediment samples for microbiological studies were obtained by APC, primarily from Holes U1367C and U1367D. Basalt core samples were collected from Holes U1367D and U1367F. During APC coring, sample contamination was monitored by PFT injection into the drilling fluid. Samples for sedimentary cell abundance were taken from the cut cores facing interstitial water whole-round cores. After core recovery on the catwalk, core sections were immediately transferred to the core refrigerator on the Hold Deck and subsampled. The temperature of the core refrigerator ranged from 7°–10°C. Microbiological whole-round cores were generally taken at a high depth resolution from the uppermost cores (329-U1367C-1H and 329-U1367D-1H), as well as the cores just above the sediment/basalt interface (Cores 329-U1367C-4H and 329-U1367D-4H). Because the lowermost cores from Holes U1367C and U1367E were less disturbed, oxygen measurements were taken from these samples prior to subsampling for microbiology (see “Biogeochemistry”). This required the core sections to be equilibrated to the temperature of the cold room prior to sampling.

Cell abundance

Microbial cells were enumerated by direct counting using epifluorescence microscopy (see “Microbiology” in the “Methods” chapter [Expedition 329 Scientists, 2011a]). Sediment subcores (2 cm3) were taken by tip-cut syringes from Holes U1367C and U1367D for shipboard analysis; for shore-based analysis 10 cm whole-round cores were taken from Hole U1367D and frozen at –80°C. Fifty-one 2 cm3 syringe samples (Table T14) and 8 whole-round cores (Sections 329-U1367D-1H-1, 1H-2, 1H-4, 2H-1, 2H-3, 2H-5, 3H-1, and 2H-4) were taken from Site U1367.

Four blanks were prepared and counted during processing of the samples from Site U1367, resulting in a mean blank of 4.1 × 102 cells/cm3 with a standard deviation of 2.3 × 102 cells/cm3. The minimum detection limit (MDL; blank plus three times standard deviation) was calculated to be 1.1× 103 cells/cm3. As the blanks did not vary much between sites, all data 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.

Cell abundance in the uppermost sample (329-U1367C-1H-1, 10–20 cm; 0.1 mbsf) was ~105 cells/cm3. Abundance decreased sharply to 103 cells/cm3 at 3.6 mbsf (Sample 1H-3, 60–70 cm). Below 3.6 mbsf, direct counts did not show any clear trend with depth and were mostly below the detection limit, with a few single replicates exhibiting values slightly more than 103 cells/cm3 (Fig. F39; Table T14).

Basalt samples were collected from the sediment/basalt interface (Section 329-U1367D-3H-CC) and the underlying basalt (Sections 329-U1367F-4R-1 and 6R-1). The whole-round core from Section 4R-1 was unaltered with no visible veins, whereas many veins associated with yellow, red, or green vein-filling material were in the whole-round core from Section 329-U1367D-6R-1 (for details, see “Lithostratigraphy”). The vein-bearing whole-round core from Section 6R-1 was manually fragmented across the vein during subsampling. A small basalt piece was collected from Section 329-U10367D-3H-CC and cleaned by washing and flaming before being crushed. Cell numbers for basaltic rock samples were directly counted without cell separation (see “Microbiology” in the “Methods” chapter [Expedition 329 Scientists, 2011a]). Table T15 contains cell count data for the basaltic samples. Although no SYBR-stainable cells were observed in the unaltered basement basalt (Section 329-U1367F-4R-1), the cell count of the vein-bearing basalt (Section 6R-1) is 3.9 × 104 cells/cm3. The cell number is higher (1.7 × 105 cells/cm3) in basalt pieces composed mainly of veins. However, particulate tracer (fluorescent microsphere) counts indicate that the vein was contaminated during coring or sample handling (see “Contamination assessment”). The interface basalt sample in Hole U1367D contains a relatively high number of cells, 1.1 × 105 cells/cm3, which is above our MDL (4.9 × 104 cells/cm3) for basalt. The SYBR-stainable cells in the basement and interface basalts will be carefully inspected by shore-based molecular biological and biomineralogical analyses in order to determine if they are distinguishable from contaminants.

Virus abundance

Unlike previous sites, most of the sediment column at Site U1367 contains abundant carbonate. Therefore, prior to extraction of virus-like particles (VLP), carbonate was dissolved to reduce background fluorescence during counting. Sodium acetate buffer (0.47 M, pH 4.7) was used instead of water during the extraction process. The carbonate was dissolved by gently shaking the buffered slurries for a few minutes. Further extraction of VLPs followed the protocol described in “Microbiology” in the “Methods” chapter (Expedition 329 Scientists, 2011a). Samples 329-U1367D-1H-1, 0–10 cm, and 120–130 cm, and Sections 2H-2, 3H-1, and 3H-5 (Table T16) were used for shipboard counting of VLPs. Additional samples were preserved at –80°C for shore-based analysis.

For the uppermost sample (329-U1367D-1H-1, 0–10 cm), 7.0 × 105 VLP/cm3 was identified. VLP abundance decreased rapidly within the upper meter of the sediment column (Fig. F40). Ship movement during transit prevented virus enumeration in samples from deeper horizons (Sections 2H-2, 3H-1, and 3H-5).


Sediment samples

Multiple cultivations were initiated onboard using a variety of media for heterotrophic (both aerobic and anaerobic) and autotrophic microorganisms. The whole-round cores were subsampled aseptically with tip-cut syringes to make slurries for inoculation in liquid or on solid media (Table T17). Additional samples were stored 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 T17). For future cultivation efforts, filtered bottom seawater was transferred to sterile 50 mL serum bottles, sparged for 5 min with N2, and capped with rubber stoppers and aluminum crimp caps. The bottles were stored at 4°C for preparing liquid media on shore.

Seawater control sample

A surface seawater sample was collected from Site U1367 with a sterile 500 mL glass bottle immediately after the JOIDES Resolution arrived at the site. Aerobic heterotrophic bacteria were cultured on marine agar (MA) and marine R2A (MR2A) plates at 25°C for 1 week. The abundance of cultivable aerobic heterotrophic bacteria on MA and MR2A were ~30 and ~16.5 colony-forming units (cfu)/mL, respectively. Twenty-seven isolates were subcultured and stored at –80°C in 20% glycerol and will be identified by 16S rRNA gene sequencing on shore to serve as a contamination control in the future.

Molecular analyses

Sediment samples

Whole-round cores 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 cores were taken as routine microbiology samples (RMS; curatorial code MBIO) for storage at –80°C at the core repositories for future biological sample requests.

Deep seawater control sample

As a control sample for shore-based molecular analysis, 300 mL of bottom seawater was collected from Core 329-U1367D-1H in a sterilized plastic bag and immediately stored at 4°C in the Microbiology Laboratory. The bottom water sample was then filtered through 0.2 µm polycarbonate membrane filters under aseptic conditions and stored at –80°C for shore-based microbiological analyses.

Basalt samples

Samples of basement basalt (Sections 329-U1367F-4R-1 and 6R-1), as well as sediment, basalt, and alteration material from the sediment/basalt interface (Cores 329-U1367D-3H and 4H) were stored at –80°C. Before being frozen, each basalt piece was washed at least twice with 3% NaCl solution, flamed, and crushed into powder (see “Microbiology” in the “Methods” chapter [Expedition 329 Scientists, 2011a]). Some portions of these powdered samples were incubated at 4°C in RNAlater solution overnight and then frozen at –80°C for shore-based DNA and RNA analyses.

Fluorescence in situ hybridization analysis

Duplicate 10 cm3 subcores of sediment from Sections 329-U1367F-1H-1, 1H-2, 2H-2, 3H-1, and 3H-5 were fixed as described in “Microbiology” in the “Methods” chapter (Expedition 329 Scientists, 2011a) for shore-based fluorescence in situ hybridization analyses.

Radioactive and stable isotope tracer incubation experiments

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 cores, 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, 2011a]). From Site U1367, five whole-round cores (i.e., Sections 329-U1367D-1H-2, 2H-1, 2H-6, 3H-2, and 3H-6) were processed for incubation experiments, as described in “Microbiology” in the “Site U1365” chapter (Expedition 329 Scientists, 2011b).

The following whole-round intervals from Site U1367 were used for sediment slurry experiments on potential metabolic activities (i.e., assimilation and dissimilative respiration) using radio- and stable isotopes: Samples 329-U1367C-1H-2, 90–80 cm; 1H-5, 70–80 cm; and 3H-3, 80–90 cm (see “Microbiology” in the “Methods” chapter [Expedition 329 Scientists, 2011a]). These incubation slurry samples from Sites U1367 were processed together with other samples from Sites U1365, U1366, and U1368 in the Isotope Isolation Van.

Samples of basement basalt (Sections 329-U1367F-4R-1 and 6R-1) as well as sediment, basalt, and alteration material from the sediment/basalt interface (Cores 329-U1367D-3H and 4H) were stored at 4°C. Incubations of the sediment/basalt interface samples with 15N-labeled NO3 as a nitrogen source and 13C-labeled HCO3 or acetate as a carbon source were initiated onboard.

Contamination assessment

Perfluorocarbon tracer

We used perfluoromethylcyclohexane as PFT to monitor the level of drilling fluid contamination in sediment cores. PFT was continuously injected into drilling fluids during APC coring in Holes U1367C and U1367D. Subcore sediment samples (3 cm3) were taken from the catwalk and from whole-round cores in the cold room and stored in vials with 2 mL of water for postexpedition gas chromatography measurement (see “Microbiology” in the “Methods” chapter [Expedition 329 Scientists, 2011a]).

Particulate tracer

Fluorescent microspheres (0.5 µm in diameter) were used for contamination testing during basaltic basement coring (see “Microbiology” in the “Methods” chapter [Expedition 329 Scientists, 2011a]). This approach is not quantitative but does provide evidence for the occurrence of contamination, even in interior structures of basaltic samples (e.g., microfractures, veins). For detection of microspheres, small rock pieces and/or surface wash solutions were stored in 3% NaCl solution prior to microscopic examination.

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 of a 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 occurrence of microspheres using a fluorescence microscope.

Results from microscopic counting of microspheres in subsamples from each cleaning step are shown in Figure F40 and Table T15. The unit in the figure is either per cubic centimeter of rock for removed basalt pieces or per cubic centimeter post surface-wash solution. Two basalt sections (329-U1367F-4R-1 and 6R-1) were associated with high numbers of microspheres. Even after the cleaning steps, microspheres were detected in the interior of the vein-bearing core from Section 6R-1. However, no microspheres were detected in the interior of the unaltered core from Section 6R-1. The results from microscopic counting of microspheres in Hole U1365E are also shown for comparison. (Fig. F40) The basalt cores with hard vein material in Hole U1365E were not intruded with microspheres during drilling and laboratory cleaning. Therefore, it is likely that veins filled with soft materials are more susceptible to drilling contamination than those filled with hard materials.