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doi:10.2204/iodp.proc.336.103.2012

Microbiology

This section documents samples taken on 23 September 2011 from the old Hole 395A CORK ROV platform, thermistor cable, and CORK wellhead elements. This CORK observatory was installed in 1997 during ODP Leg 174B (Becker and Davis, 1998; Becker et al., 1998; Becker, Malone, et al., 1998).

The old Hole 395A CORK and ROV platform were picked up from the seafloor by a recovery tool and transported through the water column for ~9 h during the pipe trip before recovery in the moonpool. In the moonpool, rust deposits/mats on the ROV platform were sampled using autoclaved metal tools and sterile 50 mL conical vials, sterile Whirl-Pak bags, and sterile deoxyribonuclease (DNase)/ribonuclease (RNase)–free 1.5 mL centrifuge tubes. Immediately after collection, the samples were stored on ice until processing in the laboratory a few hours later.

As shown in Figure F12, several different areas of the platform were sampled, with multiple samples originating from the same spot in some cases. Samples were labeled according to the area where they were collected. Table T5 lists the different samples that were collected and how they were processed.

After the ROV platform was sampled and cut off of the CORK body, the thermistor cable was pulled out of the top of the CORK wellhead on the rig floor using Yale grips and instrument string lifting tools. Note that the thermistor cable was exposed to the water column during the pipe trip and ROV platform sampling. Each Yale gripped section was pulled up into the derrick and suspended in mid-air for as long as 10 min before being pulled down on the rig floor for cutting out the thermistors and adjacent cable sections. Rig floor workers wore fresh nitrile gloves, and handling of the cable around the sampling locations surrounding the thermistors was minimized when possible to avoid contamination. A ~1 m section immediately below each thermistor section was secured, and then this section (along with the adjacent thermistor) was cut out with rig floor loppers (not sterilized). The thermistor was immediately cut off, and the subsequent thermistor cable cut between the locations where it had been handled into ~10–15 cm pieces and placed directly into sterile Whirl-Pak bags or conical vials, all of which were stored on ice until processing a few hours later. Note that samples were labeled 1–10, with 1 equaling the shallowest thermistor, although this numbering sequence is in reverse of the thermistor labeling (i.e., microbiology Thermistor 1 samples equate with Becker’s Thermistor 10 samples; Table T6). Scrapings of mineral deposits were also collected into 1.5 mL eppitubes from the cable sinker bar (N = 8), the packer inflation element (N = 11; Fig. F13), and the stinger (N = 3; Table T7).

Enrichments were initiated on board for some of the samples to select for iron-oxidizing bacteria. Prior to inoculation, six 150 mL serum bottles were filled with 60 mL autoclaved and filter sterilized seawater. Each was stoppered and then brought to and maintained at a boil for 5 min with a 22.5 gauge needle as a pressure outlet. After 5 min, another needle was inserted, from which flowed nitrogen gas at 2–4 psi. The bottle was flushed with nitrogen while boiling for 5 min. At this point, both needles were removed and the bottles were allowed to cool on the bench top. In an anaerobic chamber, a “tip of spatula” amount of material from the CORK ROV platform Area 9D and Packer 1 samples (Tables T5, T7) were added into two bottles each. Additionally, the ThermStr-5 sample, representing a mid-depth string sample, was shaken in a Whirl-Pak bag with 50 mL autoclaved seawater. The bag was then vortexed for 90 s to shake loose any colonizing microbes on the external polyethylene rope. One milliliter of this water was used as inoculum in one bottle. After inoculation, 0.25 g of steel wool was added to all bottles, and 15% of headspace was replaced with pure O₂. One bottle was treated as a negative control (no inoculum).

Enrichments were also established for anaerobic heterotrophs. Media consisted of 1 L of GYPS (see “Microbiology” in the “Methods” chapter [Expedition 336 Scientists, 2012a]) that was autoclaved, dispensed into 60 mL serum vials through a 0.2 µm mesh filter, stoppered, and then boiled for 5 min with a needle vent, followed by boiling while purging with nitrogen gas. Cultures were inoculated in an anaerobic chamber, as described above, and incubated in the dark at 10°C.

Enrichments were also established to isolate high pressure–adapted heterotrophs from Hole 395A CORK rust and the thermistor cable. Rust samples (~100 µL) were mixed with 25 mL of Marine Broth 2216 or 1:10 diluted Marine Broth 2216 (for oligotrophic heterotrophs) in glass bottles. Samples were stored at 4°C for shore-based experiments. Thermistor cable samples were also stored at 4°C in sterile Whirl-Pak bags for shore-based experiments. In the shore-based laboratory, high-pressure incubation will be utilized to enrich and select for high pressure–adapted microorganisms (see “Microbiology” in the “Methods” chapter [Expedition 336 Scientists, 2012a]). Isolated high pressure–adapted microorganisms will then be characterized and compared with other deep-sea high-pressure microorganisms isolated from different ocean provinces.

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