Water was collected via Niskin bottles mounted on a rosette, equipped with a CTD in the Western North Atlantic aboard R/V Atlantic Explorer, cruise AE2413 in May 2024. Sampling was done at the following locations:

stn24: 34.99333, -73.03333
stn25: 34.99685, -68.24842
stn26: 42.17828, -60.0479

At each station, 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, and the activities of polysaccharide hydrolases under varying hydrostatic pressures (0.1, 20, and 40 MPa). A separate glass Duran bottle was filled with seawater from the carboy and sterilized in an autoclave for over 30 minutes to serve as a killed control for various measurements.

For each substrate and time point, three 3 mL Exetainer vials were filled with seawater and one 3 mL Exetainer vial was filled with autoclaved seawater to serve as a killed control. Substrate was added at 3.5 μM monomer-equivalent concentrations, except for fucoidan, which was added at 5 μM concentrations (a higher concentration was necessary for sufficient fluorescence signal). Two 3 mL Exetainer vials – one with seawater and one with autoclaved seawater – with no added substrate served as blank controls. Vials for each substrate were pressurized to either 0.1, 20, or 40 MPa in individual pressure vessels for each time point and stored in the dark at 4ºC for 0, 5, 12, or 22 days.

At each time point, three pressure vessels were depressurized (one at 0.1, 20, and 40 MPa), vials were removed and using a sterile syringe, incubations were filtered through a 0.2 μm pore size syringe filter, and stored frozen until analysis.

Hydrolysis rates were calculated from the change in molecular weight distribution of the substrate over time, as described in detail in Arnosti (2003).