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doi:10.2204/iodp.proc.336.110.2012

Methods

All described data were logged using a laboratory notebook and Microsoft Excel spreadsheet; all equipment was mounted on top of the bridge on the port side of the ship to minimize potential ship-borne contamination of the samples.

Atmospheric and ship parameters

Each morning when the R/V JOIDES Resolution was on site, atmospheric parameters including temperature, barometric pressure, humidity, wind speed, and wind direction were logged using a Kessler 4500 anemometer and ship compass. When the JOIDES Resolution was under way, measurements were taken at the end of the low-volume filtration A sample period. Between ~1100 and 1300 h (local time; Universal Time Coordinated [UTC] – 3 h) each day, ultraviolet-C (UVC)-254 and ultraviolet (UV)-40 measurements were taken using UV meters. Ship data were taken each morning and while under way at the end of each sample period and include GPS latitude and longitude, ship heading, and ship speed.

Particle count data

A ParticleScan Pro was used to collect particle count data every 10 min, daily. These data were logged into an Excel spreadsheet using a laptop running Microsoft Windows. Daily spreadsheets of these data were saved using the following naming convention: (month/day/year)iodp336. These data were used to determine daily and per sample period aerosol loads.

Low-volume membrane filtration samples

One short-term and one long-term low-volume sample were collected daily. Short-term samples were collected over ~4 h and long-term samples were collected over ~12 h using a portable apparatus as previously described (flow rate = ~1.75–3.5 L/min, as logged). These samples were plated on R2A media and incubated at room temperature in the dark. Bacterial and fungal colony forming unit (CFU) growth was monitored over a 10 day period and logged. CFUs were picked and stored in cryogenic storage for shore-based sequence identification. A maximum of 5 CFUs were picked from those plates where the CFUs appear to be identical in morphology and pigmentation.

High-volume membrane filtration samples

High-volume membrane filtration samples were collected over a 24 h period and started daily at ~0700 h. The high-volume membrane filtration unit was a Staplex TF1A filtration unit, and TFAGF41 glass fiber filters were employed for this study. Each filter was weighed daily, and the filter housing was assembled with the filter in place, wrapped in aluminum foil, and autoclaved prior to use. The unit flow rate is 20 ft³/min. After filtration, the filter was stored in a labeled zip-top bag at –80°C.

Liquid impinger samples

Liquid impinger samples were collected over a 4 h period daily between 0500 and 1200 h (time is Eastern Standard Time [EST] unless otherwise stated; up to 6 November UTC = EST + 5 h, starting 6 November UTC = EST + 4 h). For onboard analyses, 100 µL aliquots were spread-plate with R2A and TCBS agar. R2A media was used to determine cultivable bacteria and fungi, and TCBS agar was used to determine cultivable Vibrio spp. Plates were incubated at room temperature in the dark and enumerated at 5 and 10 days. After spread-plating was completed, the samples were stored at –80°C. These samples will be screened for bacteria and fungi using universal qPCR analyses postexpedition.

Surface water samples

Surface water grabs (~45–50 mL) were collected daily between 1045 and 1200 h from a port/bow location (under the bridge) at a maximum depth of ~0.25 m using a 50 mL tube and weight attached to a string. Salinity, temperature, and pH were recorded using a thermometer, handheld pH meter, and refractometer. Two 100 µL samples were used for spread-plate Vibrio analyses. Samples were incubated for 10 days at room temperature in the dark, and CFUs were enumerated at 5 and 10 days. CFU pigmentation (yellow or green) was recorded for each colony. All colonies were picked and subcultured in ~500 µL of TSB media. These isolates were incubated for a minimum of 3 days at room temperature, at which time ~200 µL of glycerol was added to each tube. The tubes were then vortexed and stored at –80°C for postexpedition 16S sequence identification. For each sample, two 1 mL subsamples were centrifuged for 20 min at 14,500 rotations per minute (rpm); after centrifugation, the supernatant was removed and discarded using a micropipette. These samples were stored at –80°C for postexpedition qPCR analyses for total bacteria and Vibrio spp. Two 10 mL subsamples were used for bacteria and viruslike particle direct counts. The protocol used for these assays follows Griffin et al. (2001). The remaining aliquot of surface water for each sample date was stored at –80°C for postexpedition cataloging.

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