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doi:10.2204/iodp.proc.302.103.2006

Microbiology

One finding of great significance from ODP was the discovery of microorganisms living in deeply buried marine sediments (see Parkes et al., 2000, for review). To date, microorganisms have been observed in samples collected at depths as great as ~880 mbsf. Extrapolation of these data to total global ocean sediments suggests that the marine subsurface contains a significant fraction of the biomass on Earth (Whitman et al., 1998). However, the global coverage of sites examined for microorganisms is still quite sparse. Arctic sediments are completely unexplored with respect to microbiological parameters. Therefore, the results of this expedition will allow us to determine whether our current working models of global life in deeply buried marine sediments apply to this region as well. In addition to the total biomass, the structure, diversity, and function of subsurface microbial communities remain poorly understood.

Core handling and sampling

In order to better evaluate microbiological data in the geochemical setting, microbiological samples were taken adjacent to whole-round samples collected for IW chemistry. All microbiological analyses will be conducted at shore-based laboratories. Therefore, it was imperative to collect clean samples and to prepare them properly for storage and transport. Because the samples were retrieved from a very stable sedimentary environment, the microorganisms are presumed to be sensitive to chemical and physical change, particularly oxygen, temperature, and pressure. Consequently, microbiological samples were taken on the Vidar Viking as soon as the core passed through the MSCL (~20 min after sectioning). It is essential to handle these sediment samples aseptically to prevent introduction of extraneous bacteria. As the sediments are predominantly anoxic, anaerobic handling methods must be used to prevent the introduction of oxygen. In addition, as the core liner is not sterile and the outer surface of the core is contaminated during drilling, subsamples for microbiological analyses were taken from the uncontaminated interior of the core (Smith et al., 2000). After the whole round was cut and removed, a sterile spatula was used to scrape a small section of the freshly exposed sediment surface. Sterile polypropylene syringes (3, 10, or 60 cm3) with the luer-lock end cut off were pushed into the core to collect subcores aseptically. The syringes containing the sediment subcore were then treated as follows depending on the intended downstream analysis.

Nucleic acid analyses

Syringes with samples for deoxyribonucleic acid (DNA) analyses were sealed with parafilm on the exposed end and placed in a trilaminate foil bag (Schoelle) and heat-sealed. The samples were then transferred to an ultra-low-temperature freezer (–57°C) for transport to shore-based laboratories. Nucleic acid analyses (e.g., small subunit ribosomal gene sequencing) will be performed in shore-based laboratories. These analyses will be used to determine the community structure of the microorganisms inhabiting the deep-buried marine sediments of the Lomonosov Ridge.

Total cell counts

The most immediate method to visualize and quantify the deep microbial biosphere is total bacterial cell counts using the nucleic acid stain acridine orange. These counts have been made on a wide range of ODP (Parkes et al., 1994) and IODP sediment cores. In general, these counts have demonstrated an exponential decrease of microbial cells with depth below seafloor. Yet, minimum cell densities of 104–105 cells/cm3 have been consistently detected even in the deepest sediments. The method detects sediment layers of increased cell density that often coincide with particular geochemical conditions that are conducive to bacterial growth (Parkes et al., 2000). Subcores (1 cm3) collected in cutoff syringes were extruded into sterile polypropylene tubes containing filter-sterilized (0.2 µm) formalin in 3.5% NaCl. The tubes were stored at 4°C for transport to a shore-based laboratory. Microorganisms in the samples will be enumerated using epifluorescence microscopy after staining the DNA fluorochrome acridine orange (Fry et al., 1988).

Biomarker analysis

Syringes with samples for DNA analyses were sealed with parafilm on the exposed end and placed in a trilaminate foil bag (Schoelle) and heat-sealed. The samples were then transferred to an ultra-low-temperature freezer (–57°C) for transport to shore-based laboratories. Biomarker analyses will be conducted in shore-based laboratories. These analyses will be used to determine the community structure of the subsurface microbial community.

Cultivation techniques

Syringes with samples for cultivation experiments were placed in a plastic bag containing oxygen scrubber (Anaerocult) that was previously wetted. The bag was heat-sealed and then placed inside of a trilaminate bag (Schoelle) that was then heat-sealed. The bags were stored at 4°C for transport. This method of storage has been shown to maintain anaerobic conditions for months (Cragg et al., 1992). Attempts at cultivating microorganisms from these sediment samples will be conducted onshore.