Seawater was transferred to 20 liter (L) carboys that were rinsed three times with water from the sampling depth and then filled with seawater from a single Niskin bottle, using silicone tubing that had been acid washed and then rinsed with distilled water prior to use. From each carboy, water was dispensed into smaller glass containers that were cleaned and pre-rinsed three times with water from the carboy prior to dispensing. This water was used to measure cell counts, bacterial productivity, and the activities of polysaccharide hydrolases, peptidases, and glucosidases. A separate glass Duran bottle was filled with seawater from the carboy and sterilized in an autoclave for 20-30 minutes to serve as a killed control for microbial activity measurements.
The potential of the seawater microbial community to hydrolyze six high-molecular-weight polysaccharides (arabinogalactan, chondroitin sulfate, fucoidan, laminarin, pullulan, and xylan) was investigated in surface and bottom water. For each substrate, three 50 milliliter (mL) falcon tubes were filled with seawater and one 50 mL falcon tube was filled with autoclaved seawater to serve as a killed control. Substrate was added at 3.5 micromolar (μM) monomer-equivalent concentrations, except for fucoidan, which was added at 5 μM concentrations (a higher concentration was necessary for sufficient fluorescence signal). Two 50 mL falcon tubes – one with seawater and one with autoclaved seawater – with no added substrate served as blank controls. Incubations were stored in the dark at as close to in situ temperature as possible. Subsamples of the incubations were collected at time zero, and at a sequence of subsequent time points. At each time point, 2 mL of seawater was collected from the 50 mL falcon tube using a sterile syringe, filtered through a 0.2 micrometer (μm) pore size syringe filter, and stored frozen until processing.
The hydrolysis of high molecular weight substrate to lower molecular weight hydrolysis products was measured using gel permeation chromatography with fluorescence detection, after the method of Arnosti [1996, 2003]. In short, the subsample was injected onto a series of columns consisting of a 21-centimeter (cm) column of G50 and a 19-cm column of G75 Sephadex gel. The fluorescence of the column effluent was measured at excitation and emission wavelengths of 490 and 530 nanometers (nm), respectively. Hydrolysis rates were calculated from the change in molecular weight distribution of the substrate over time, as described in detail in Arnosti (2003).
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