Measurements of peptidase and glucosidase activities in large volume mesocosm incubations on RV/Endeavor EN584, July 2016 (Patterns of activities project) (NCEI Accession 0291535)

Acquisition Description:
For mesocosm (large volume) incubation experiments (referred to as “LV” incubations), a 30L Niskin bottle rosette was used to collect the water. Separate casts were used to collect surface water, bottom water, and water from the depth at which oxygen showed a minimum, according to the CTD. From each depth, 20L seawater from single Niskin bottles was dispensed using cleaned silicon tubing into a single carboy. Prior to filling, carboys were rinsed 3x with water from the same Niskin bottle used to fill the carboy. Four carboys were filled at each depth. Triplicate 20L carboys were amended with ca. 500 mg (exact mass was recorded for each addition) of HMW Thalassiosira; unamended single carboys were used for controls. All mesocosms were incubated in the dark at near in-situ temperatures. Mesocosms were sub-sampled at the start of incubation (0 days), and then after 2 d, 7d, and 16d for the following assays: bacterial production using 3H-Leucine, dissolved organic carbon (DOC), nutrients, bacterial cell counts, peptidase and glucosidase activity measurements.

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]. 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].