Sediment samples were obtained from 23 core sections between 1.4 and 171.2 mbsf of Hole 1149A. Immediately after a core was cut into 1.5-m sections on the outside catwalk, a thin layer of sediment was removed from the section end using a sterile scalpel to expose an uncontaminated surface. A 1-cm3 sample was then taken with a sterile (autoclaved) 5-mL syringe from which the luer end had been removed. The sample was aseptically ejected directly into a serum vial containing 9 mL of filter-sterilized (0.2 µm) 4% formaldehyde in artificial seawater for preservation and storage. This was the first ODP leg where contamination checks were conducted for microbiology, and these checks demonstrated that the inner portion of cores, where the microbiological samples were taken, were free from potential sampling contamination (Smith et al., 2000).
Acridine orange staining and microscopic observations were based on the general recommendations of Fry (1988) with minor modifications. Fixed samples were vortex mixed and a subsample of between 15 and 40 µL was added to 10 mL of 2% filter-sterilized (0.1 µm) formaldehyde in artificial seawater. Acridine orange (50 µL) was added to give a final concentration of 5 mg/dm3. After 3 min the solution was filtered through a 25-mm Nucleopore black polycarbonate membrane (Osmonics Inc. of Minnetonka, Minnesota) of 0.2-µm pore size. The filter was rinsed further with 10 mL of 2% filter-sterilized formaldehyde and mounted in a minimum amount of paraffin oil under a coverslip.
Mounted membrane filters were viewed under incident illumination with a Zeiss Axioskop microscope fitted with a 50-W mercury vapor lamp, a wideband interference set for blue excitation, a 100x (numerical aperture = 1.3) Plan Neofluar objective lens, and 10x eyepieces. Green fluorescing bacterial cells were counted. Cells attached or unattached to particles were counted separately, and the numbers of those attached to particles were doubled in the final calculations to account for cells hidden from view by particles (Goulder, 1977).
We prepared three replicate membranes for each sample; however, where calculated 95% confidence limits of the mean population size were greater than ±0.5 log10 units, additional membranes were prepared and enumerated until the confidence limits were reduced below ±0.5 log10 units. All 23 samples were satisfactorily enumerated with three membranes, and the mean confidence limit for all 23 samples was ±0.33 log10 units.
The concentrations of ammonium, sulfate, and methane, plus the measurement of pH, were obtained directly from the database generated during Leg 185 (Shipboard Scientific Party, 2000). The methods are described in detail therein. Dissolved manganese and acetate concentrations were subsequently obtained from shore-based laboratory analysis, and methods are described below.
Dissolved Mn was measured by inductively coupled plasma-atomic emission spectrometry (ICP-AES) at Boston University, following the general analytical procedures described by Murray et al. (2000), using a Jobin-Yvon 170C ICP spectrometer. Calibration standards were constructed using Mn-spiked seawater (of Scituate, Massachusetts) to provide an appropriate matrix match. Replicate analyses of separate natural samples indicated that the data were precise to 2% of the measured values. Accuracy was checked by comparison to a Mn-spiked aliquot of International Association for the Physical Sciences of the Oceans standard seawater.
Pore water acetate concentrations were measured at Bristol University following the enzymatic method of King (1991), which measures bioavailable acetate, using high-performance liquid chromatography (Dionex [UK] Ltd.) separation. Samples were injected onto an analytical column (Supelco LC-18-T, 25 cm x 4.6 mm) with a mobile phase of 0.1-M KH2PO4 (pH 6.0) at 30°C at 0.8 mL/min (Wellsbury and Parkes, 1995). Detection was by UV/VIS at 254 nm and quantification by peak-area integration (Spectra-Physics, United Kingdom).