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.
Experiments on (operationally defined) particles were carried out by gravity-filtering water through 3 µm pore size filters. 1/12th sections of the 3 µm pore-size filters were submerged in 15 mL artificial seawater; enzyme activities were measured as described below.
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 – leucine – and four oligopeptides – 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) – 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 timepoints 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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