Mesocosm incubations:

For mesocosm (large volume) incubation experiments (referred to as “LV” incubations), 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. Four carboys were filled from bottom water and deep chlorophyll maximum (DCM) water each, according to the CTD. Triplicate 20L carboys (a total of 9 carboys) were amended with high molecular weight material isolated from the diatom Thalassiosira, or the polysaccharide fucodian, or the polysaccharide arabinogalactan; unamended single carboys were used for controls. 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 the activities of peptidases, and glucosidases. A separate glass Duran bottle was filled with seawater from the carboy and sterilized in an autoclave to serve as a killed control for microbial activity measurements. All mesocosms were incubated in the dark at near in-situ temperatures. Mesocosms were sub-sampled for peptidase and glucosidase activity measurements at the start of incubation (0 days), and then after at approximately 5d, 10d, 15d, and 25d. To measure gluosidase and peptidase activities at each timepoint, water was collected from the mesocosms as described above.

For these measurements, seven glucosidase and peptidase substrates were set up in a 96-well plate. The substrates used included alpha-glucose and beta-glucose linked to a 4-methylumbelliferyl (MUF) fluorophore to measure exo-acting glucosidase activities. Five substrates linked to a 7-amido-4-methyl coumarin (MCA) fluorophore, including 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. For each substrate, triplicate wells were filled with a total volume of 200 uL seawater for experimental incubations; triplicate wells were filled with 200 uL autoclaved seawater for killed control incubations. Substrate was added at saturating concentrations. A saturation curve was determined with surface water from each station to identify 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 infiniteF200; 360 nm excitation, 460 emission), with timepoints taken every 4-6 hours. Bottom water measurements were made in a cold van/cold room at 4 C; water from the deep chlorophyll maximum was incubated at room temperature.

Bulk water incubations:

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 the activities of 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.Two substrates, alpha-glucose and beta-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. 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 uL seawater for experimental incubations; triplicate wells were filled with 200 uL autoclaved seawater for killed control incubations. Substrate was added at saturating concentrations. A saturation curve was determined with surface water from each station to identify 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 0-72 hours incubation time with a plate reader (TECAN infiniteF200; 360 nm excitation, 460 emission), with timepoints taken every 4-6 hours.