Samples for quantification of B-vitamins B1, B7, CB12 and Methionine were collected at a six of depths within the euphotic zone (5-250m). Seawater was collected from each CTD depth using Niskin bottles and immediately filtered. Particulate samples for chlorophyll quantifications were collected using in-line 0.2um, 3µm and 10um pore-size filters and a peristaltic pump (flow rate < 50 ml per minute), transferred into sterile cryovials and were immediately stored at -80 degrees C until analysis. Pigments were extracted from the filters in 3 mL of methanol, BHT(butylated hydroxytoluene) was added and placed in a -20 degrees C freezer overnight. For chlorophyll-a measurements, 100 microliters of the pigment extraction were diluted in acetone (50x dilution) and analyzed using a Turner 10AU fluorometer.
B-vitamins and Methionine samples were analyzed as in Suffridge et al. 2017. Two liters of seawater were filtered through um pore-size filters and then preconcentrated using a C18 resin (HF Bondesil (Agilent Technologies) and analyzed by liquid chromatography/triple mass spectrometry (LC/MS/MS/MS). The LCMS system consists of a ThermoTSQ Quantum Access electro-spray ionization triple quadrupole mass spectrometer, coupled to a Thermo Accela High Speed Liquid Chromatography system. The LC system used a stable- bond C18 reversed-phase column (DiscoveryHSC18 10cm × 2.1mm,5 μm column, Supelco Analytical) with a100 uL sample loop. In order to increase the sensitivity and precision, theLC/MS was run in full-loop mode (100 ul/injection).
Bacterial cell counts were heterotrophic prokaryotes were enumerated by flow-cytometry (Becton–Dickinson FACScalibur) (Gasol & del Giorgio, 2000).