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doi:10.2204/iodp.proc.329.107.2011

Microbiology

Sediment samples for microbiological studies were obtained by APC coring, primarily in Holes U1369C and U1369E. Sample contamination was monitored by PFT injection into the drilling fluid. Samples for cell counting 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 microbiological whole-round cores were sampled. The temperature of the core refrigerator was 7°–10°C. Microbiological whole-round cores were generally taken at a high depth resolution from the first core (1H), as well as the core above the sediment/basalt interface (Core 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 using tip-cut syringes from Hole U1369C for shipboard analysis. For shore-based analysis, 10 cm whole-round cores were taken from Hole U1369E and frozen at –80°C. Thirty-two 2 cm³ syringe samples (Table T10) and six whole-round cores (Sections 329-U1369E-1H-1, 1H-2, 1H-4, 2H-2, 2H-4, and 2H-5) were taken from Site U1369.

Six blanks were prepared and counted during processing of the samples from Site U1369. Instead of Tris-EDTA (TE) buffer, a slurry of heat-sterilized sediment (4 h at 450°C) was used as a blank. The mean values of the heat sterilized blanks was 6.1 × 10² cells/cm³ with a standard deviation of 2.9 × 10² cells/cm³, resulting in a minimum detection limit (MDL; blank plus three times standard deviation) of 1.5 × 10³ cells/cm³. As the blanks did not vary much between sites, they were pooled from all sites. 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-U1369C-1H-1, 15–20 cm) is ~8 × 10⁴ cells/cm³. Numbers decreased sharply to ~10³ cells/cm³ at 2 mbsf (Sample 1H-2, 50–60 cm) and remained below the MDL downhole to the basement (Fig. F31). Because of the limited transit time from Site U1369 to U1370, only two samples (329-U1369C-1H-1, 15–20 cm, and 135–140 cm) were recounted by another shipboard microbiologist for cross-comparison. These counts were in good agreement with each other, suggesting that differences in cell recognition among microscope observers is small (Table T10). Cell counts on samples without cell extraction were made on the three uppermost samples (329-U1369C-1H-1, 15–20 cm, 50–60 cm, and 100–110 cm). Cells were only detected in the topmost nonextracted sample, where abundance was ~4 × 10⁵ cells/cm³ (slightly higher than in the extracted samples). Both deeper samples were below the blank.

Virus abundance

Samples for counting VLPs at a high depth resolution were taken from Holes U1369C and U1369E (Table T11). A subset of these samples was used for shipboard counting of VLPs, and other syringe samples were preserved at –80°C for shore-based analyses. Enumeration of VLPs followed the protocol described in “Microbiology” in the “Methods” chapter (Expedition 329 Scientists, 2011a).

For the uppermost sample (329-U1369C-1H-1, 15–20 cm), 1.7 × 10⁶ VLP/cm³ were observed. VLP abundance decreased rapidly within the uppermost 2 m of the sediment column, and the numbers of VLPs are generally higher than cell numbers estimated by direct microscopic count (Fig. F31). The number of VLPs is ~10⁵ VLP/cm³ (Sample 329-U1369C-1H-2, 135–140 cm) at 2 mbsf and decreases slightly with depth. Samples from deeper horizons (Sections 329-U1369C-2H-3 and 2H-6 and 329-U1369E-2H-5 and 2H-6) were not counted onboard due to ship movement during transit.

Cultivation

Sediment samples

Sediment whole-round cores were subsampled aseptically with sterilized tip-cut syringes to make slurries for inoculation of a variety of media (Table T12). Additional samples (referred to as SLURRY in Table T12) were stored in N₂-flushed serum bottles or in syringes packed in sterile foil packs and stored at 4°C for shore-based cultivation experiments.

Seawater control sample

A surface seawater sample was collected from Site U1369 with a sterile 500 mL glass bottle immediately after the R/V JOIDES Resolution arrived at the site. Cultivation of aerobic heterotrophic bacteria in the seawater sample was performed on marine agar and marine R2A plates (see Table T8 in the "Methods" chapter [Expedition 329 Scientists, 2011a]) at 25°C. The abundance of cultivable aerobic heterotrophic bacteria on marine R2A plates was ~1 colony-forming unit (cfu)/mL. No visible colonies were observed on marine agar after three days of incubation.

Bottom seawater was collected from the mudline of the upper core (1H) of Holes U1369A–U1369D, placed in a sterile plastic bag, and stored at 4°C. The water was filtered through 0.2 µm pore sized polycarbonate filters into sterile 50 mL serum bottles and flushed with N₂ for 5 min. The bottles were capped with rubber stoppers and aluminum crimp caps and stored at 4°C for future preparation of liquid media on shore. Aerobic heterotrophic bacteria were cultured from the unfiltered sample at 25°C for 3 days. The abundance of cultivable aerobic heterotrophic bacteria on marine agar and R2A plates were ~5.7 × 10³ and ~5.2 × 10³ cfu/mL, respectively, which is in marked contrast to numbers obtained from surface seawater as described above. The estimated abundance of Vibrio-like species, based on selective enrichment for this genus on thiosulfate citrate bile salts sucrose agar, was ~15 cfu/mL.

Molecular analyses

Sediment samples

Three whole-core samples were taken from Hole U1369E (Sections 329-U1369E-1H-3, 2H-2, and 2H-4) as routine microbiology samples (curatorial code MBIO) and transferred to –80°C freezers for storage. These samples will be available for shore-based molecular studies (See “Microbiology” in the “Methods” chapter [Expedition 329 Scientists, 2011a]).

Deep seawater control samples

As a control sample for shore-based molecular analyses, ~300 mL of bottom seawater was collected from the mudline of the uppermost cores (1H) of Holes U1369A–U1369D. The water was collected in a sterile plastic bag and stored at 4°C in the Microbiology Laboratory until further processing. The sample was filtered through 0.2 µm polycarbonate membrane filters under aseptic conditions and stored at –80°C for shore-based microbiological analyses.

Fluorescence in situ hybridization analysis

Duplicate 10 cm³ subcores of sediment from Sections 329-U1369E-1H-1, 1H-2, 2H-1, 2H-3, and 2H-6 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 U1369, four whole-round cores (Sections 329-U1369E-1H-2, 2H-2, 2H-5, and 2H-6) were processed for the stable isotope tracer incubation experiments, as described in the U1365 site report (see “Microbiology” in the “Site U1365” chapter [Expedition 329 Scientists, 2011b]).

Four whole-round intervals from Site U1369 were collected for slurry experiments on potential metabolic activities (i.e., assimilation and dissimilative respiration) using radio- and stable isotopes (intervals 329-U1369C-1H-2, 110–130 cm; 1H-4, 100–123 cm; 2H-3, 120–140 cm; and 2H-6, 110–130 cm) (see “Microbiology” in the “Methods” chapter [Expedition 329 Scientists, 2011a]) and stored in the core refrigerator of the Hold Deck prior to the isotopic sample processing.

For stable isotope probing and nuclear magnetic resonance biomass experiments, two sediment whole-round cores were collected from Hole U1369C (Samples 329-U1369C-1H-1, 120–130 cm, and 2H-5, 120–130 cm). Subsamples were taken with 5 cm³ tip-cut syringes and amended with stable isotope tracers dissolved in low-nutrient growth media (¹³C-labeled acetate, benzoate, methanol, and methane sulfonic acid) and incubated at 4˚C (see “Microbiology” in the “Methods” chapter [Expedition 329 Scientists, 2011a]) in the Isotope Isolation Van.

For sulfate reduction rate measurements, four whole-round cores were collected from Hole U1369C (Samples 329-U1369C-1H-2, 60–65 cm; 2H-1, 60–65 cm; 2H-4, 60–65 cm; and 2H-7, 54–59 cm). Five to seven subsamples (~2.5 cm³) were collected from each whole-round core for the shore-based distillation analysis of ³⁵S-labeled reduced sulfur (see “Microbiology” in the “Methods” chapter [Expedition 329 Scientists, 2011a]).

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 U1369C and U1369E. Sediment subcores (3 cm³) were taken from whole-round cores on the catwalk and in the core 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]).

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