Seawater was transferred to 20 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 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.
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Two substrates, -glucose and -glucose linked to a 4-methylumbelliferyl (MUF) fluorophore, were used to measure glucosidase activities. Five substrates linked to a 7-amido-4-methyl coumarin (MCA) fluorophore, one amino acid \u2013 leucine \u2013 and four oligopeptides \u2013 the chymotrypsin substrates alanine-alanine-phenylalanine (AAF) and alanine-alanine-proline-phenylalanine (AAPF), and the trypsin substrates glutamine-alanine-arginine (QAR) and phenylalanine-serine-arginine (FSR) \u2013 were used to measure exo- and endo-acting peptidase activities, respectively. Hydrolysis rates of the substrates were measured as an increase in fluorescence as the fluorophore was hydrolyzed from the substrate over time [as in Hoppe, 1993; Obayashi and Suzuki, 2005]. Incubations with the seven low molecular weight substrates were set up in a 96-well plate. For each substrate, triplicate wells were filled with a total volume of 200 L seawater for experimental incubations; triplicate wells were filled with 200 L autoclaved seawater for killed control incubations. Substrate was added at saturating concentrations. A saturation curve was determined with surface water from each station to determine saturating concentrations of substrate. The saturating concentration was identified as the lowest tested concentration of substrate at which additional substrate did not yield higher rates of hydrolysis. Fluorescence was measured over 24-48 hours incubation time with a plate reader (TECAN spectrafluor plus; 360 nm excitation, 460 emission), with time points taken every 4-6 hours. Hydrolysis rates were calculated from the rate of increase of fluorescence in the incubation over time relative to a set of standards of known concentration of fluorophore. Scripts to calculate hydrolysis rates and produce the figures shown here are available in the associated Github repository [Hoarfrost, 2017].
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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 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 \u03bcM monomer-equivalent concentrations, except for fucoidan, which was added at 5 \u03bcM concentrations (a higher concentration was necessary for sufficient fluorescence signal). Two 50 mL falcon tubes \u2013 one with seawater and one with autoclaved seawater \u2013 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 six subsequent time points (t1-t6): 2 days, 5 days, 10 days, 17 days, 30 days, and 42 days. 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 \u03bcm pore size syringe filter, and stored frozen until processing.
BCO-DMO Processing Notes:
\n- added conventional header with dataset name, PI name, version date
\n- modified parameter names to conform with BCO-DMO naming conventions
\n- removed 'cast00' and 'stn0' from data records for the cast and station columns