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doi:10.2204/iodp.proc.329.109.2011

Microbiology

Sediment samples for microbiological studies were obtained by APC coring, primarily from Holes U1371E and U1371F. PFT was continuously injected into the drilling fluid for quantification of sample contamination. Samples for cell and virus-like particle (VLP) abundance were taken from the cut cores that face interstitial water whole-round samples and fixed for subsequent microscopic studies. After core recovery, core sections were transferred to the core refrigerator on the Hold Deck, where microbiological whole-round cores were sampled. The temperature of the core refrigerator during subsampling ranged from 7°–10°C. Microbiological whole-round cores were taken at a high depth resolution from the first core (1H) and the bottom core (14H) of Hole U1371F. All whole-round cores for cultivation were immediately transferred into N₂-flushed foil packs to prevent excess oxidation of fresh microbiological samples from the air and stored at 4°C before subsampling.

Cell abundance

Microbial cells were enumerated by direct counting using epifluorescence microscopy (Table T15; see “Microbiology” in the “Methods” chapter [Expedition 329 Scientists, 2011a]). Sediment subcores (2 cm³) for shipboard analysis were taken using tip-cut syringes from Holes U1371E and U1371F. In addition, eighteen 10 cm whole-round cores were taken for postcruise analysis from Holes U1371E and U1371F and stored at –80°C.

Six blanks were prepared and counted during processing of the samples from Site U1371. As at the previous site, a slurry of heat-sterilized (4 h at 450°C) sediment was used as a blank instead of Tris-EDTA buffer. The mean blank value of the heat-sterilized blanks was 3.2 × 10² cells/cm³ with a standard deviation of 2.5 × 10² cells/cm³, resulting in minimum detection limit (MDL; blank plus three times standard deviation) of 1.1 × 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-U1371E-1H-1, 35–43 cm) is ~6 × 10⁶ cells/cm³ and gradually decreases to 28.55 mbsf. Below this depth, cell abundance is below the MDL or even below the blank, with two exceptions at 50 and 95 mbsf (Fig. F37). Because of the limited time before the end of the expedition, no samples from below 114 mbsf were counted and no samples without cell extraction steps were examined. These will be quantified postcruise. Only two samples were recounted by another shipboard microbiologist. These counts were in good agreement with the original counts, suggesting that differences in cell recognition among observers are small.

Virus abundance

Eighty samples for VLP enumeration were taken at a high depth resolution from Holes U1371E and U1371F (Table T16). All of these samples were preserved at –80°C for shore-based analyses.

Cultivation

Sediment samples

Sediment whole-round cores were subsampled aseptically with sterile, tip-cut syringes to make slurries for inoculation of a variety of media (Table T17). After aseptic slurry inoculation in the Microbiology Laboratory, the headspace in the vials containing culture medium for anaerobic microbes was flushed with N₂ and sealed with an autoclaved rubber stopper. Additional samples (referred to as SLURRY in Table T17) 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 U1371 with a sterile 500 mL glass bottle immediately after the R/V JOIDES Resolution arrived at the site. Aerobic heterotrophic bacteria were cultured on marine agar and marine R2A plates (see Table T8 in the “Methods” chapter [Expedition 329 Scientists, 2011a]) at 25°C. No visible colonies were observed on either marine agar or R2A plates after 5 days of incubation.

Bottom seawater was collected from the mudline of Core 1H in Holes U1371B and U1371G, placed in a sterile plastic bag and stored at 4°C. The water was filtered through 0.2 µm pore size polycarbonate filters into sterile 50 mL serum bottles and sparged 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 abundances of cultivable aerobic heterotrophic bacteria on marine agar and R2A plates were ~2.5 × 10³ and ~2.3 × 10³ colony-forming units (cfu)/mL, respectively, which are 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 ~2.5 × 10³ cfu/mL.

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 for shore-based molecular ecological studies (see “Microbiology” in the “Methods” chapter [Expedition 329 Scientists, 2011a]). Sixteen 10 cm whole-round core samples were taken from Hole U1371E as routine microbiology samples (curatorial code MBIO) and stored at –80°C. These samples will be stored at –80°C in the Gulf Coast Core Repository at Texas A&M University (USA) to accommodate future sample requests.

Deep seawater control sample

For shore-based molecular ecological studies, the bottom seawater was collected from the top part of mudline cores (1H) in Holes U1371B and U1371G. Approximately 300 mL of seawater was collected in a sterilized plastic bag and stored at 4°C in the Microbiology Laboratory until further processing. The sample was then filtered through 0.2 µm pore size polycarbonate membrane filters under aseptic conditions and the filters stored at –80°C.

Fluorescence in situ hybridization analysis

Duplicate 10 cm³ subcores of sediment from Sections 329-U1371E-3H-3, 5H-3, 7H-2, 9H-3, 11H-2, and 14H-3 and 329-U1371F-1H-1 through 1H-3, 14H-3, and 14H-4 were collected using sterile tip-cut syringes and 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 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]). Six whole-round cores from Sections 329-U1371E-5H-2, 8H-2, and 11H-2 and 329-U1371F-1H-2, 2H-2, and 14H-4 were processed for stable isotope tracer incubation experiments, as described in “Microbiology” in the “Site U1365” chapter (Expedition 329 Scientists, 2011b).

Whole-round interval 329-U1371E-1H-2, 110–120 cm (2.8 mbsf), was used for slurry experiments on metabolic activities using radioactive and stable isotopes. The whole-round core section had been stored in the core refrigerator on the Hold Deck until processing in the Microbiology/Chemistry Laboratory cold room. Incubation experiments were prepared in the Isotope Isolation Van using radioactive and stable isotope or a combination according to the methods described in detail (see “Microbiology” in the “Methods” chapter [Expedition 329 Scientists, 2011a]).

Seven sediment whole-round cores were collected from Hole U1371E (Samples 329-U1371E-1H-1, 20–30 cm; 2H-2, 50–60 cm; 5H-4, 90–100 cm; 7H-5, 20–30 cm; 9H-5, 10–20 cm; 11H-4, 0–10 cm; and 14H-4, 60–70 cm) for stable isotope probing and nuclear magnetic resonance biomass experiments. Subsamples were taken with 5 cm³ tip-cut syringes and amended with stable isotope tracers dissolved in low-nutrient growth media (¹³C-labeled sodium acetate, sodium benzoate, methanol, and methane sulfonic acid) and incubated at 4°C (see “Microbiology” in the “Methods” chapter [Expedition 329 Scientists, 2011a]). Four whole-round cores collected from Hole U1371E (Samples 329-U1371E-1H-1, 25–35 cm; 2H-4, 20–40 cm; 5H-4, 20–40 cm; and 9H-1, 40–60 cm) were simultaneously processed in a similar manner.

For sulfate reduction rate measurements, 24 whole-round cores were collected from Hole U1371E (Samples 329-U1371E-1H-4, 50–60 cm; 1H-5, 50–60 cm; 1H-6, 20–30 cm; 2H-1, 100–110 cm; 2H-2, 20–30 cm; 2H-3, 50–60 cm; 2H-4, 50–60 cm; 2H-5, 50–60 cm; 3H-2, 100–110 cm, 3H-3, 50–60 cm; 3H-4, 50–60 cm; 3H-5, 50–60 cm; 3H-6, 10–20 cm; 4H-1, 50–60 cm; 4H-2, 50–60 cm; 4H-3, 50–60 cm; 4H-4, 50–60 cm; 4H-5, 50–60 cm; 4H-6, 10–20 cm; 5H-2, 60–70 cm, 5H-4, 60–70 cm; 6H-4, 50–60 cm; and 6H-6, 50–60 cm). Five to seven subsamples (~2.5 cm³) were collected directly from each whole-round core for the shore-based distillation analysis of ³⁵S-labeled reduced sulfur compounds (see “Microbiology” in the “Methods” chapter [Expedition 329 Scientists, 2011a]).

Contamination assessment

We used perfluoromethylcyclohexane tracer as PFT to monitor drilling fluid contamination in sediment cores. PFT was continuously injected into drilling fluids during APC coring in Hole U1371E. Subcore samples (3 cm³) were taken from whole-round cores on the catwalk and stored in vials with 2 mL of water prior for postexpedition gas chromatography measurement (see “Microbiology” in the “Methods” chapter [Expedition 329 Scientists, 2011a]).

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