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doi:10.2204/iodp.proc.323.102.2011

Microbiology

The microbiology objectives of Expedition 323 are (1) to constrain global models of subseafloor biomass by quantifying subseafloor cell abundance in an extremely high productivity region of the ocean and (2) to determine how subseafloor community composition is influenced by extremely high productivity in the overlying ocean. To meet these objectives, our analyses included quantification of total cell abundance and assessment of the diversity and structure of the microbial community by the use of nucleic acid–based techniques and fluorescent in situ hybridization (FISH).

Core handling and sampling

Cores were sectioned on the catwalk, and the sections were immediately run through the SHMSL. To minimize temperature effects on microbial community composition, all further handling took place in the Cold Laboratory (7°C). All materials, including core cutters, were kept cold so that the samples did not warm. In addition, because core liners are not sterile and the outer surfaces of cores are contaminated during drilling (Smith et al., 2000a, 2000b), subsampling excluded the sediment next to the core liner. In the Cold Laboratory, core sections were cut into multiple whole rounds for interstitial water analyses. Immediately after cutting a whole round, the edge of the remainder section was cleaned with a sterile blade. Samples for prokaryotic cell counts, contamination tests, and nucleic acid analyses were then taken with sterile 5–60 cm³ cut-off syringes.

The above procedures were followed only for cores retrieved from microbiology-dedicated holes. In addition, a limited number of samples were collected from nondedicated holes. These samples were taken directly on the catwalk adjacent to whole rounds collected for interstitial water analyses.

Prokaryotic cell counts and contamination tests

Cell counts (cells per cubic centimeter) serve as the basis for subseafloor biomass estimates (e.g., Whitman et al., 1998; Parkes et al., 2000; Kallmeyer et al., 2008). Samples for prokaryotic cell counts were taken with sterile 3 cm³ cut-off syringes, placed in 15 mL vials containing 8 mL of 2% formalin, and stored at 4°C for postcruise analyses (Kallmeyer et al., 2008).

Contamination during drilling and handling was evaluated by tests using micrometer-sized fluorescent beads and perfluorocarbon tracer (PFT). These tests have previously shown that core samples can be obtained without introducing prokaryotic cell contamination, which is essential for all microbiological analyses that follow core retrieval (Smith et al., 2000a, 2000b).

A suspension of submicrometer-sized fluorescent microspheres was introduced into the drill fluid in all microbiology-dedicated holes. A plastic bag positioned within the core catcher released a suspension of beads inside the core barrel as it hit the sediment, allowing for the maximum effectiveness of the beads as tracers of potential bacterial contamination. Particle contamination in the samples will be evaluated during postcruise analyses of subsamples taken for total cell counts.

PFT was continuously fed into the seawater drill fluid at a tracer concentration of 1 mg/L seawater drill fluid. The concentration of PFT was measured in all sections used for microbiological studies. For this purpose, 5 cm³ subsamples were routinely taken, as described by Smith et al. (2000b), and placed in headspace vials. PFT was analyzed on a GC equipped with an electron capture detector and an HP-AL/S 15 m × 0.25 mm ID, 5.00 µm film thickness (Agilent P/N 19091P-531) column. Additionally, air samples were occasionally taken to monitor the ambient concentration of PFT in the Cold Laboratory air. The concentration of PFT at the outer periphery of the drill cores was measured to verify delivery of the PFT.

Diversity and structure of the microbial community

Microbial molecular analyses

Samples for microbial molecular analyses were collected in 5 and 60 cm³ sterile cut-off syringes, which were frozen at –80°C in Mylar or whirlpack bags.

Molecular analysis of the 16S ribosomal ribonucleic acid (rRNA) will be performed during postcruise investigations. 16S ribosomal deoxyribonucleic acid (rDNA) sequence libraries for prokaryote diversity will be established and compared downhole and between sites. Functional genes will be analyzed through application of specific polymerase chain reaction (PCR) primers. This will enable the characterization of the diversity of specific functional guilds such as sulfate-reducing bacteria.

Fluorescence in situ hybridization

Samples for FISH analysis were collected in 2 cm³ sterile syringes and transferred to Eppendorf tubes. Sediment was fixed following the protocol by Pernthaler et al. (2001) in buffered paraformaldehyde solution (gram-negative bacteria) or ethanol (Archaea and gram-positive bacteria). Fixed samples were washed several times, and ethanol was added. Samples were then stored at –20°C until processing in onshore laboratories.

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