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Sediment samples for microbiological studies were obtained by APC, primarily from Holes U1368C and U1368D. Basalt samples from the sediment/basalt interface were collected from Holes U1368B and U1368D using APC. The RCB system was used to obtain basalt samples from Hole U1368F. During APC coring, sample contamination was monitored by PFT injection into the drilling fluid. Samples for cell and virus-like particle (VLP) abundance were taken from the cut cores facing interstitial water whole-round samples. After core recovery on the catwalk, core sections were immediately transferred to the core refrigerator on the Hold Deck, where whole-round cores were collected for microbiological analyses. The temperature of the core refrigerator during subsampling was ~7°–10°C. Microbiological whole-round cores were generally taken at a high depth resolution from the first cores (1H), as well as from the core above the sediment/basalt interface (2H).

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 Hole U1368C for shipboard analysis. For shore-based analysis, 10 cm whole-round cores were taken from Hole U1368D and frozen at –80°C. Thirty-three 2 cm3 syringe samples (Table T14) and 7 whole-round cores (Sections 329-U1368D-1H-1, 1H-2, 1H-4, 2H-1, 2H-2, 2H-3 and 2H-4) were taken from Site U1368.

Four blanks were prepared and counted during processing of the samples from Site U1368, resulting in a mean blank of 6.7 × 102 cells/cm3 with a standard deviation of 4.8 × 102 cells/cm3. The minimum detection limit (MDL; blank plus three times standard deviation) was calculated to be 2.1 × 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.

As the sediment at Site U1368 contained calcium carbonate, a carbonate extraction step was employed prior to the actual cell extraction. Samples from Core 329-U1368C-1H were very stiff and did not suspend in the carbonate dissolution; therefore, an initial treatment with detergent mix (see “Microbiology” in “Methods” chapter [Expedition 329 Scientists, 2011a]) was used to disperse the particles before the carbonate dissolution. The order of sample processing therefore changed to

  1. Detergent mix/methanol,

  2. Carbonate dissolution, and

  3. Detergent mix/methanol.

Samples from Core 329-U1368C-2H downhole were treated in the conventional order. No obvious difference is seen in the data from either order of treatment.

Cell abundance in the uppermost sample (329-U1368C-1H-1, 10–15 cm) was ~6 × 104 cells/cm3. Numbers decreased sharply to ~103 cells/cm3 at 1.4 mbsf (Sample 1H-1, 135–140 cm) and remained near 103 cells/cm3 to the basalt with a few samples below MDL (Fig. F65).

A subset of the sediment samples was recounted by two additional shipboard microbiologists for cross-comparison. These counts were indistinguishable from the initial counts. Also, several samples from the upper part of the core were counted without cell extraction. In the uppermost sample (329-U1368C-1H-1, 10–15 cm), the nonextracted cell counts were almost one order of magnitude higher, suggesting that either the cell extraction did not retrieve all cells or the nonextracted cell numbers were overestimated because of mineral autofluorescence. Counting of nonextracted samples from deeper horizons exhibited cell abundances that were all below the detection limit with large standard deviations.

Virus abundance

As at Site U1367, most of the sediment samples from Site U1368 contain abundant carbonate. Because autofluorescence of carbonate minerals significantly hampers recognition of VLPs in sediment, we treated the samples with sodium acetate buffer (0.47 M; pH 4.7) instead of water during the extraction process and the carbonates were 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 from Hole U1368C were taken at a high depth resolution (Table T15). A subset of these samples was used for shipboard counting of VLPs. Additional samples were preserved at –80°C for shore-based analysis.

For the uppermost sample (329-U1368C-1H-1, 10–20 cm), 1.4 × 106 VLP/cm3 were observed. VLP abundance decreases rapidly within the upper meter of the sediment column (Fig. F65). VLP abundance in Sample 1H-2, 110–120 cm, is ~105 VLP/cm3, and VLP abundance decreases slightly with depth. Samples from deeper horizons (Sections 329-U136C-2H-1, 2H-3, and 2H-5) were not counted because the ship movement during transit prevented additional shipboard VLP abundance estimates from Site U1367.


Sediment samples

Multiple cultivations were initiated onboard using a variety of media for heterotrophic (both aerobic and anaerobic) and autotrophic microorganisms. The core samples were subsampled aseptically with sterilized tip-cut syringes to make slurries for inoculation in liquid or on solid media (Table T16). Additional samples were stored in N2-flushed serum bottles or in syringes packed in sterile foil packs and stored at 4°C (referred to as SLURRY in Table T16) for future shore-based cultivation experiments. For use in future cultivation experiments, the 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.

Surface seawater control sample

A surface seawater sample was collected from Site U1368 with a sterile 500 mL glass bottle immediately after the JOIDES Resolution arrived at the site. Cultivation of aerobic heterotrophic bacteria in the seawater sample was performed on marine agar and marine R2A plates and in liquid medium SPG-9 (see Table T8 in the “Methods” chapter [Expedition 329 Scientists, 2011a]) at 25°C. No visible colonies were observed on either the marine agar or R2A plates after 1 week of incubation, whereas very good growth was observed in the SPG-9 medium after 1 day of incubation. Highly motile, curved rods were observed under the phase-contrast microscope.

Bottom seawater samples were collected from the uppermost cores (1H) in Holes U1368A–U1368D in a sterilized plastic bag and immediately stored at 4°C. Aerobic heterotrophic bacteria were cultured at 25°C for 3 days. The abundance of cultivable aerobic heterotrophic bacteria on marine agar and R2A plates were ~200 and ~150 colony-forming units/mL, respectively, which is in marked contrast to numbers obtained from surface seawater as described above.

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 several functional genes. Four 10 cm whole-round core samples were taken as routine microbiology samples (curatorial code MBIO) and stored at –80°C. These samples will be stored 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 the mudline cores (1H) of Holes U1368A–U1368D in a sterile plastic bag and immediately stored at 4°C in the Microbiology Laboratory. The bottom seawater 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-U1368F-2R-1, 2R-4, 4R-2, 5R-3, 7R-3, 9R-1, 13R-3, and 14R-1), as well as sediment, basalt, and alteration material from the sediment/basalt interface (Sections 329-U1368B-2H-1 and 3H-1 and 329-U1368D-2H-CC) were processed for microbiological and (bio-)mineralogical analyses. Before being frozen at –80°C, each basalt piece was washed at least twice with 3% NaCl solution, briefly 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-U1368F-1H-1, 1H-2, 1H-5, 2H-4, and 2H-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 U1368, four whole-round cores (Sections 329-U1368D-1H-2, 1H-5, 2H-4, and 2H-5) were processed for stable isotope tracer incubation experiments, as described in “Microbiology” in the “Site U1365” chapter (Expedition 329 Scientists, 2011b).

Slurries were prepared from the following whole-round intervals from Site U1368 for experiments investigating potential metabolic activities (i.e., assimilation and dissimilative respiration) using radio and stable isotopes: Samples 329-U1368C-1H-3, 90–100 cm; 2H-1, 80–90 cm; and 2H-5, 80–90 cm (see “Microbiology” in the “Methods” chapter [Expedition 329 Scientists, 2011a]). In addition, ~40 mL of 1/5 diluted (v/v) slurries from two samples (1H-3, 90–100 cm, and 2H-5, 80–90 cm) were used for cell viability studies with 14C-labeled compounds (ATP, leucine, and thymidine). Potential metabolic activity was also tested by adding 2.5 mL of 18O-labeled water (H218O) to aliquots (5 mL) of the same slurries and incubated at 4°C. These slurries from Site U1368 were processed together with other samples from Sites U1365, U1366, and U1367 in the Isotope Isolation Van.

For sulfate reduction rate measurements, four whole-round cores were collected from Hole U1368C (Samples 329-U1368C-1H-1, 85–90 cm; 1H-5, 85–90 cm; 2H-3, 85–90 cm; and 2H-5, 35–40 cm). Five to seven subsamples (~2.5 cm3) were collected directly from each whole-round core for shore-based distillation analysis of 35S-labeled reduced sulfur (see “Microbiology” in the “Methods” chapter [Expedition 329 Scientists, 2011a]).

Samples of basement basalt (Sections 329-U1368F-2R-1, 2R-4, 4R-2, 5R-3, 7R-3, 9R-1, 13R-3, and 14R-1) as well as sediment, basalt, and alteration material from the sediment/basalt interface (Sections 329-U1368B-2H-1 and 3H-1 and 329-U1368D-2H-CC) were stored at 4°C. Incubations of samples from the sediment/basalt interface 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 a PFT to monitor the level of drilling fluid contamination in sediment cores. PFT was constantly injected into drilling fluids during APC coring in Holes U1368C and U1368D. Sediment subcore samples (3 cm3) were taken on the catwalk and from whole-round cores in the Hold Deck refrigerator 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 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 provides evidence for the occurrence of contamination, even in interior structures of basaltic samples (e.g., microfractures and veins). For detection of microspheres, small rock pieces and/or surface wash solutions were stored in 3% NaCl solution for microscopic observation.

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 the wash solutions were pooled in a 50 mL centrifuge tube. After the washing step, the rock surface was briefly flamed. After flaming, the 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 using epifluorescence microscopy.

Microscopic counts of microspheres in subsamples at each cleaning step are shown in Figure F66 and Table T17. The unit in the figure is either per cubic centimeter of rock for crushed basalt pieces or per cubic centimeter of post–surface-wash solution. Eight basalt cores (Sections 329-U1368F-2R-1, 2R-4, 4R-2, 5R-3, 7R-3, 9R-1, 13R-3, and 14R-1) contained high abundances of microspheres, whereas no microspheres were detected from one untreated core (Section 5R-3) and two cores after washing and flaming (Sections 2R-1 and 2R-4). Even after the cleaning steps, microspheres were detected from the interior in the four lower cores (Sections 5R-3, 7R-3, 13R-3, and 14R-1). These results indicate that unlike the cores from Hole U1365E, many of the cores from Hole U1368F were susceptible to drilling fluid contamination.