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doi:10.2204/iodp.proc.329.108.2011

Microbiology

Sediment samples for microbiological studies were obtained by APC coring, primarily from Holes U1370E and U1370F. PFT was continuously injected into drilling fluid for quantification of sample contamination with gas chromatography. Samples for cell and virus-like particle (VLP) abundance were taken from the cut cores facing interstitial water whole-round samples and preserved for subsequent microscopic studies. After core recovery, 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 during subsampling ranged from 7°–10°C. Microbiological whole-round cores were taken at a high depth resolution from the first core (1H) as well as the penultimate core (7H) because the bottom core (8H), which contained the sediment/basalt interface, was highly disturbed. All the whole-round cores for cultivation from Cores 329-U1370F-4H through 8H 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 (see “Microbiology” in the “Methods” chapter [Expedition 329 Scientists, 2011a). Sediment subcores (2 cm³) were taken using tip-cut syringes from Holes U1370E and U1370F for shipboard analysis. For shore-based analysis, 10 cm whole-round cores were taken from Hole U1370F and frozen at –80°C. Fifty-nine 2 cm³ syringe samples (Table T13) and 11 whole-round cores were taken at Site U1370.

Two blanks were prepared and counted during processing of the samples from Site U1370. 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 1 × 10³ cells/cm³ with a standard deviation of 2.7 × 10² cells/cm³, resulting in an minimum detection limit (MDL; blank plus three times standard deviation) of 1.8 × 10³ cells/cm³. As the blanks showed little variation 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-U1370E-1H-1, 35–43 cm) is ~3 × 10⁴ cells/cm³. With one exception at 7.55 mbsf (Sample 2H-1, 135–140 cm), cell abundances were above the detection limit to 36 mbsf, which is much deeper than most of the previous sites drilled during this expedition. Below this depth, cell abundances were below the MDL (with a few below the blank) (Fig. F35).

Six samples were recounted by another shipboard microbiologist for cross-comparison. These counts were generally in good agreement with the original counts, suggesting that differences in cell recognition among observers are very small (Table T13). Cell counts on samples without cell extraction steps were made on three samples (329-U1370E-1H-1, 135–140 cm; 1H-2, 135–140 cm; and 1H-3, 135–140 cm). Cells in the topmost sample were above the nonextracted detection limit, ranging from 9 × 10⁴ to 2 × 10⁵ cells/cm³, slightly higher than in the extracted samples. In the two deeper samples, nonextracted counts were below the blank.

The sediment/basalt interface sample was collected in the core catcher from Core 329-U1370E-9H, which included a relatively large piece of basalt. Microbial cell abundance on the basaltic rock sample was 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]). No cells were observed in 300 fields of microscopic view.

Virus abundance

Sixty three samples for VLP enumeration were taken at a high depth resolution from Holes U1370E and U1370F (Table T14). All samples from Site U1370 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. Subsamples from Cores 329-U1370F-4H through 8H were taken under anoxic conditions in the anaerobic chamber deployed in the core refrigerator on the Hold Deck (Table T15). Hydrogen was added as a reductant to remove oxygen in the anaerobic chamber, H₂ concentration was maintained at 2%–3%. Oxygen concentration was consistently 0 ppm. After inoculation, vial headspace for heterotrophic anaerobic cultivation was flushed with N₂, if necessary, to remove hydrogen in the headspace. Additional samples (referred to as SLURRY in Table T15) 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 U1370 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 (Table T8 in the “Methods” chapter [Expedition 329 Scientists, 2011a]) at 25°C for 5 days. The abundances of cultivable aerobic heterotrophic bacteria on marine agar and R2A plates were ~39 and ~22 colony-forming units (cfu)/mL, respectively.

Bottom seawater was collected from the mudline of Core 1H of Holes U1370A–U1370F, 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 ~5.7 × 10³ and ~4.0 × 10³ 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 cfu/mL. Contrary to the seawater samples, we rarely observed colony formation from sediment slurries prepared from Site U1370; only for Core 329-U1370-3H did we observe heterotrophic growth of colonies on marine agar and R2A plates (137 and 112 cfu/cm³, respectively). It is not clear if the colonies were derived from an indigenous microbial community in the sediment or from contamination. This will be addressed by both PFT analysis and postcruise 16S rRNA sequence analysis of the 19 isolates from the core.

Molecular analyses

Sediment samples

Nine 10 cm whole-round cores were taken throughout the entire sediment column (Sections 329-U1370E-CC and 329-U1370F-1H-3, 2H-3, 3H-5, 4H-5, 5H-5, 6H-5, 7H-4, and 7H-5) as routine microbiology samples (curatorial code MBIO) and transferred to –80°C freezers for storage. These samples will be used for shore-based molecular studies (See “Microbiology” in the “Methods” chapter [Expedition 329 Scientists, 2011a]).

Deep seawater control sample

Bottom seawater overlaying the seafloor was collected from the top part of mudline cores (1H) in Holes U1370A–U1370F for shore-based molecular studies. Approximately 300 mL seawater was collected in a sterile plastic bag and stored at 4°C in the Microbiology Laboratory until further processing. The sample was then filtered through 0.2 µm polycarbonate membrane filters under aseptic conditions and stored at –80°C.

Basalt samples

Sediment and basalt samples at the sediment/basalt interface were collected from Section 329-U1370E-9H-CC on the catwalk immediately after core recovery. The basalt piece was separated from surrounding soft materials, washed three times with 3% NaCl solution, briefly flamed, and crushed into powder (see “Microbiology” in the “Methods” chapter [Expedition 329 Scientists, 2011a]). The samples were stored at –80°C for shore-based microbiological and (bio)mineralogical analyses.

Fluorescence in situ hybridization analysis

Duplicate 10 cm³ subcores of sediment from Sections 329-U1370F-1H-1 through 1H-3, 3H-2, 4H-3, 5H-2, 7H-2, 7H-4 through 7H-6, and 8H-1 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 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]). Six whole-round cores from Sections 329-U1370F-1H-2, 2H-3, 4H-3, 6H-3, 7H-5, and 7H-6 were processed for stable isotope tracer incubation experiments as described in “Microbiology” in the “Site U1365” chapter (Expedition 329 Scientists, 2011b). Subcore samples (15 cm³) were taken from the inner part of sedimentary whole-round cores.

Whole-round interval 329-U1370C-1H-2, 120–130 cm (2.7 mbsf), from Site U1370 was used for slurry experiments on metabolic activities using radioactive and stable isotopes. The whole-round core section had been stored in the Hold Deck core refrigerator until processing in the Microbiology/Chemistry Laboratory cold room. Samples from Sites U1369 and U1370 were processed together on board. Incubation experiments were prepared to test for autotrophic and heterotrophic activities using radio and stable isotope or a combination thereof according the methods described in “Microbiology” in the “Methods” chapter (Expedition 329 Scientists, 2011a).

For sulfate reduction rate measurements, 13 whole-round cores were collected from Hole U1370E (Samples 329-U1370E-1H-2, 10–20 cm; 1H-4, 50–60 cm; 2H-2, 50–60 cm; 2H-5, 50–60 cm; 3H-2, 50–60 cm; 3H-5, 50–60 cm; 4H-2, 50–60 cm; 4H-5, 50–60 cm; 5H-2, 50–60 cm; 5H-5, 50–60 cm; 6H-2, 30–40 cm; 6H-5, 40–50 cm; and 9H-2, 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

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 U1370C and U1370F. Sediment subcores (3 cm³) were taken from core sections on the catwalk 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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