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Sediment samples for microbiological studies were obtained using the APC system, primarily from Hole U1366F. During APC coring, sample contamination was monitored by PFT injection into the drilling fluid. Samples for 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, where we subsampled microbiological whole-round cores. 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 core (329-U1366F-1H), as well as from the core just above the sediment/basalt interface (Core 4H). Because of disturbance in the lowermost cores in Holes U1366D and U1366E, oxygen measurements were taken from these cores prior to microbiological sampling, requiring the core sections to be equilibrated to the temperature of the Microbiology Laboratory cold room (8.5°C) prior to sampling.

Cell abundance

Microbial cells were enumerated using epifluorescence microscopy (see “Microbiology” in the “Methods” chapter [Expedition 329 Scientists, 2011a]). Sediment subcore samples (2 cm3) were taken from Hole U1366F cores using tip-cut syringes for shipboard analysis. For shore-based analysis, 10 cm whole-round cores were taken from Hole U1366F and frozen at –80°C. Seventy 2 cm3 syringe samples (Table T10) and nine whole-round cores (Sections 329-U1366F-1H-1, 1H-4, 2H-1, 2H-3, 2H-5, 3H-2, 3H-6, 4H-2, and 4H-4) were taken at Site U1366 for cell enumeration.

Four blanks were prepared and counted during processing of the samples from Site U1366, resulting in a mean blank of 6.6 × 102 cells/cm3 with a standard deviation of 2.4 × 102 cells/cm3. The minimum detection limit (MDL; blank plus three times standard deviation) was calculated to be 1.4 × 103 cells/cm3. As the blanks showed little variation between sites, they were pooled from all sites and the MDL for all sites calculated based on extended database at the end of the expedition. The overall MDL is 1.4 × 103 cells/cm3.

Cell counts did not show any clear trend with depth (Fig. F34). Between 3 and 25 mbsf, the data scatter from below the detection limit to maximum values close to 104 cells/cm3. In sediment deeper than 25 mbsf, all values are below the detection limit. From comparison with the cell count data from the site survey cruise (D’Hondt et al., 2009), the uppermost 40–50 cm of the sediment column might have been lost during drilling. Several samples from the upper part of the core were counted without cell extraction. All were below the mean blank for nonextracted counts (3.2 × 104 cells/cm3).

The sediment/basalt interface sample was collected in a core liner from Section 329-U1366F-4H-5, which included a large piece of basalt. Cell numbers in the basaltic rock sample were directly counted without cell separation. Before counting, the basalt sample was processed by washing and flaming and then ground into powder (see “Microbiology” in the “Methods” chapter [Expedition 329 Scientists, 2011a]). The cell count for the interface basalt in Hole U1366F is 2.3 × 104 cells/cm3. However, because our MDL is 4.9 × 104 cells/cm3 (equivalent to 5 cells per 300 fields of microscopic view), this count is below our MDL.

Virus abundance

Sediment samples from Sections 329-U1366F-1H-1, 1H-2, 2H-3, 3H-4, and 4H-3 were used for viral extraction (see “Microbiology” in the “Methods” chapter [Expedition 329 Scientists, 2011a]). The extracted virus-like particles (VLPs) were stained with SYBR Gold and counted using epifluorescence microscopy. Additional samples were taken and preserved at –80°C for shore-based analysis of viral abundance.

Abundance of VLPs in Hole U1366F is ~8 × 105 VLP/cm3 for the uppermost sample (0.4 mbsf; Fig. F34). The abundance of VLPs decreases with depth to ~2 × 104 VLP/cm3 at 9.5 mbsf.

During the estimation of VLP abundance from Sections 329-U1366F-2H-3, 3H-4, and 4H-3, the numbers of VLPs appeared to be very low (Table T11) and showed weaker fluorescence than samples from shallower depths. Additionally, ship movement during transit prohibited confident detection of VLPs. Thus, data on VLP counts in deeper sediment samples are considered to be preliminary. All samples will be reanalyzed postcruise.


Multiple cultivations were initiated onboard using a variety of media for heterotrophic (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 T12). 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 T12). For future cultivation efforts, 0.2 µm 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.

Molecular analyses

Sediment samples

Whole-round cores for shore-based molecular analyses were taken throughout the entire sediment column of Hole U1366F and transferred to –80°C freezers for storage. These samples will be used to determine microbial diversity, community composition, and the presence or absence of selected functional genes. Eight 10 cm whole-round core samples (MBIO) were taken as routine microbiology samples (RMS) for storage at –80°C at the Gulf Coast Core Repository (Texas A&M, College Station, Texas) for future sample requests (Sections 329-U1366F-1H-2, 2H-3, 3H-1, 4H-1, 4H-2, and 4H-5).

Deep seawater control sample

As a control sample for shore-based molecular analysis, 300 mL of bottom seawater was collected from the mudline Core 329-U1366B-1H 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 pore sized polycarbonate membrane filters under aseptic conditions, and the filters were stored at –80°C for shore-based microbiological analyses.

Basalt samples

Samples from the sediment/basalt interface and alteration material from Section 329-U1366F-4H-5 were stored at –80°C. Before being frozen, the basalt piece was separated from surrounding soft materials, washed three times with 3% NaCl solution, briefly flamed, and crushed into powder. Some portions of these interface samples were incubated at 4°C in RNAlater solution (Qiagen) overnight and then frozen at –80°C.

Fluorescence in situ hybridization analysis

Duplicate 10 cm3 subcores of sediment from Sections 329-U1366F-1H-1 through 1H-3, 2H-3, 3H-4, 4H-2, and 4H-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 vial, 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 U1366, five whole-round cores (Sections 329-U1366D-1H-2 and 2H-3 and 329-U1366F-2H-3, 3H-5, and 4H-5) 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 U1366 were used for sediment slurry experiments on potential metabolic activities (i.e., assimilation and dissimilative respiration) using radio and stable isotopes: Samples 329-U1366D-1H-2, 120–130 cm; 3H-2, 110–120 cm; and 4H-3, 120–130 cm (see “Microbiology” in the “Methods” chapter [Expedition 329 Scientists, 2011a]). These incubation slurry samples from Site U1366 were processed 19–21 November 2010, at the same time as other samples from Sites U1365, U1367, and U1368, in the Isotope Isolation Van.

Samples of sediment, basalt, and alteration material from the sediment/basalt interface (Section 329-U1366F-4H-5) were stored at 4°C. Incubations of the sediment/basalt interface samples with 15N-labeled NO3 and 13C-labeled HCO3 or acetate were initiated onboard.

Contamination assessment

We used perfluoromethylcyclohexane as PFT to monitor the level of drilling fluid contamination in sediment cores. The PFT was constantly injected into drilling fluids during APC coring in Holes U1366D and U1366DF. Three cubic centimeters subcores of sediments were taken from whole-round cores in the cold room and stored in glass vials with 2 mL of water for shore-based analyses (see “Microbiology” in Expedition 329 Scientists, 2011a, 2011b).