Grab samples were taken using Niskin bottles that collected seawater at the same depth and location of the Environmental Sample Processor deployed at Station M0 (36.835 N, 121.901W). Water was transferred to a low-density polyethylene cubitainer and maintained at ambient temperature until return to lab within 30 min.

Chlorophyll a: 150 ml of seawater was filtered through a 25 mm GF/F filter in triplicate using a vacuum pump and <5 in Hg pressure. The filter was placed in a glass scintillation vial and 10 ml of 90% acetone was added and placed in -20 freezer for at least 24 hours to extract the pigment. Extracted chlorophyll a was quantified using fluorometry (Pennington and Chavez, 2000).

Flow Cytometry: Cubitainer seawater was transferred to a 50 ml Falcon tube using laminar flow. 1.8 ml was then aliquoted to triplicate cryovials and preserved with 200 ul of 5% glutaraldehyde and stored at -80 degrees C. Analysis was run on a Beckman Coulter Altra flow cytometer for detection of DNA, pigments, and forward and side light scatter (Monger and Landry, 1993).

Akashiwo Microscopy Counts: 7 - 14 ml of seawater was preserved to 1% final concentration electron microscopy grade glutaraldehyde and stored at 4 degrees C. Slides were made by filtering the full volume onto a 0.22 um black polycarbonate filter (GE Water & Process Technologies) using a vacuum pump (<5 in Hg), and cells were counted under epifluorescence microscopy.

DMSP concentrations: Immediately upon return to the deck, duplicate samples were collected from the Niskin bottle for in situ dissolved DMSP (DMSPd) (see details below) before seawater transfer to the cubitainer. Upon return to the laboratory, the cubitainer of water was gently mixed by inversion and three replicate 10 ml sub-samples were removed by pipette into individual 15 ml centrifuge tubes (Corning, polypropylene). The samples were immediately acidified with 0.3 ml of 50% concentrated HCl (1.5% final concentration of concentrated HCl) to preserve total DMSP (dissolved plus particulate). These DMSPt samples were closed tightly and stored until analysis (described below) which took place within three months of collection. 

DMSPd consumption: To measure the consumption rate of dissolved DMSP, we used the glycine betaine (GBT) inhibition technique (Kiene & Gerard, 1995; Li et al., 2016). Immediately upon return to the laboratory, six 500 ml glass bottles were filled with seawater from the gently-mixed cubitainer. Three of the bottles were treated with 25 ul of a 100 mM GBT anhydrous reagent (Sigma) solution (10 uM final GBT concentration), and three were left untreated as controls. Bottles were incubated in seawater maintained within 1 degree C of the in situ temperature. Immediately after GBT addition, the first time point was collected by simultaneously filtering ~50 ml sub-samples from each bottle through 47 mm Whatman GF/F filters using the small volume gravity drip filtration protocol of Kiene and Slezak (2006). The first 3.5 ml of filtrate from each sample was collected into 15 ml centrifuge tubes (Corning, polypropylene) that contained 100 ul of 50% HCl to immediately preserve any DMSP passing through the GF/F filter, which is defined as dissolved DMSP (DMSPd). Additional time points from each bottle were collected at 3 and 6 h. The rate of change of DMSPd in no-treatment bottles was subtracted from the rate of change in the +GBT bottles to obtain an estimate of DMSPd consumption rate (Kiene and Gerard, 1995).

< 5 µm DMSPd: The DMSP that was less than 5 µm was measured in water from the cubitainer in the lab, using the drip filtration protocol, as described above for DMSPd, except that a 5.0 µm pore size, track-etched polycarbonate filter was used for the filtration.

DMSP Analysis: DMSP was quantified by proxy as the amount of DMS released from samples after alkaline cleavage (White, 1982). For DMSPt, 0.05 to 0.5 ml of each preserved sample was pipetted into a 14 ml glass serum vial, with the volume being adjusted based on the concentration of DMSPt in the sample. For DMSPd, the volume pipetted was 1.0 to 3.0 ml. Each serum vial was treated with 1 ml of 5 M NaOH and capped with a Teflon-faced serum stopper (Wheaton). After 1 h, the amount of DMS in each vial was quantified by purge and trap gas chromatography with flame photometric detection. Briefly, each vial was attached to the purge system and a flow of helium (90-100 ml per minute) allowed bubbling of the solution. An excurrent needle led to a Nafion dryer and six-port valve (Valco). The DMS in the samples was cryotrapped in a Teflon tubing loop immersed in liquid nitrogen. After a 4 min sparge, during which >99% of the DMS in the samples was removed, hot water replaced the liquid nitrogen to introduce the DMS into the Shimadzu GC-2014 gas chromatograph. Separation of the sulfur gases was achieved with a Chromosil 330 column (Supelco; Sigma) maintained at 60 degrees C with a helium carrier flow of 25 ml per minute. The flame photometric detector was operated in sulfur mode and maintained at 175 degrees C. Minimum detection limits during this study were 0.5 to 1 pmol DMS per sample with minimum detectable concentrations ranging from 0.17 to 10 nM, depending on the volume analyzed. The GC-FPD system was calibrated with a gas stream containing known amounts of DMS from a permeation system.

Problem report: For November chlorophyll a samples, fluorescence after acid addition not measured but estimated from samples with similar total fluorescence (Pennington and Chavez, 2000).