Using a small boat, samples were collected in 20L carboys in Tylerfjord-Young Sound. Three rivers that feed into Tyrolerfjord-Young Sound (Tyroler River, Lerbugten River and Zackenberg River) were sampled; surface and subsurface water samples were also collected at transition sites where the rivers feed into the fjord (Tyro_01, Zac_30, Ler_30, altogether referred to as ‘river transition sites’). Enzyme activities were measured in unfiltered water. In addition, water was size-fractionated using gravity filtration through a GF/A filter to capture ≥1.6 µm particles.

Two substrates, a-glucose and b-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].  All substrates were used to measure enzyme activities in unfiltered water, as well as particle-associated (≥1.6 µm) enzymatic activities.

In unfiltered water, enzyme activities were measured by adding 4 mL of water to triplicate cuvettes. One incubation containing autoclaved water served as the killed control. This procedure was applied to each of the 7 substrates and one live blank and autoclave blank (no substrate addition). Each cuvette containing either live or autoclaved water was amended with one substrate to a concentration of 100 µM. Fluroescence was measured using a Promega Quantifluor solid-state single-cuvette fluorimeter; excitation and emission maxima were 365 nm and 410–450 nm, respectively,

To measure particle-associated enzyme assays, 1/12th piece of a GF/A filter through which water had been gravity filtered was put into a cuvette containing 4 mL of cooled, autoclaved water from the same station/depth as the live samples. In addition, killed controls were set up using sterile GF/A filters cut into 1/12th pieces. Bulk water and particle-associated enzyme assays were incubated for up to 24 and 16 hours, respectively; timepoints were taken at specific intervals. Incubations were kept in the dark either at 0°C, 5°C, or 8°C, depending on in situ water temperature at the time of sampling.

Activities of polysaccharide hydrolases were measured using fluorescently labeled polysaccharides (Arnosti 2003). Activities of enzymes that hydrolyze pullulan, laminarin, xylan, fucoidan, arabinogalactan, and chondroitin sulfate were measured in unfiltered water, and using GF/A filters through which water had been gravity-filtered. For these measurements, substrate was added (of 3.5 µM monomer equivalent) to 15 mL of water; autoclaved ambient water served as the killed control. Particle-associated activities were measured by submerging 1/12th of a GF/A filter in 15 ml autoclaved seawater. Samples were incubated in the dark at near in situ temperature (0°C, 5°C, or 8°C), and sub-sampled at specific time intervals—t0 (0h, upon substrate addition), t1 (120 h), t2 (240 h), t3 (360 h) and t4 (600 h). Sub-samples from each timepoint were filtered using 0.2 µM pore size SFCA (surfactant-free cellulose acetate) syringe filters, and the filtrate was collected in tubes and frozen at -20°C until processing in the lab. Sub-samples were processed using gel permeation chromatography (Arnosti, 2003).