Laboratory analysis of ATP : Fifty microliters of each sample (in triplicates) were transferred to 6 ml pony scintillation vials (Research Products International), and received 3 ml of ultrapure water, and 50 µL of CellTiter-Glo 2.0 (Promega Corporation). Internal standards were used by spiking a fourth vial with 50 µL of samples with 50 µL of 0.0164 µM ATP standard. Using internal standards instead of separate calibration curves corrects for matrix effects that change the luminescence signal caused by the presence of ions, acids, and organic material (Bochdansky et al. 2021). Luminescence was analyzed in a PerkinElmer Liquid Scintillation Analyzer with a single photon counting protocol of 1 minute each. The counter was programmed to cycle samples five times in sequence. We determined that values from the second cycle were the most consistent and were thus subsequently used for all analyses.

See supplemental files for formula to calculate ATP, also available in Bochdansky et al. 2021.

Microscopic analysis of biomass, 2018 samples: For bacteria abundance, 20 ml of the Niskin bottle sample was added to a 50 ml Falcon tube and preserved with 2% (fin. conc.) formaldehyde. Within 24 hours, 5 ml of the formaldehyde sample (in duplicates) was filtered onto a 25 mm diameter, 0.2 mm pore size, black polycarbonate filter (Isopore type GTBP, Millipore Corp.). The filter was put onto a slide and one drop of Vectashield Antifade Mounting Medium with DAPI (H1200, Vector Laboratories) was applied. A cover slip was added and the slide was stored at -20 °C.

The slides were brought to room temperature in a desiccator to remove condensation before immersion oil was added to the cover slips. Bacteria were enumerated under an Olympus BX61 epifluorescence microscope (100 x oil immersion objective, 2 x loupe, 10 x ocular magnification). ToupView (Toup Tek Photonics) imaging software was used to take images of the bacteria slides. Images were analyzed using a custom macro developed for ImageJ software (National Institutes of Health). This macro inverted the image, converted it into an 8 bit image, applied a FFT Bandpass Filter, binarized the image, and finally applied a watershed feature that outlined each bacterium. The algorithm automatically counted the number of bacteria and the area of each bacterium on each picture. A fixed value of 20 femtogram (fg) carbon per prokaryotic cell was applied (Ducklow, 2018). We preferred the higher value as cell carbon is typically higher in eutrophic systems than in open and deep ocean environments (Ducklow, 2018; Herndl et al., 2023).

For the enumeration of eukaryotes and measurements of size, two types of analysis were performed: epifluorescence and inverted microscopy. The same slides used for the bacteria counts were also used to count eukaryote microbes directly. The eukaryotes were counted if the bright blue spots were at least twice the size (linear dimension) of the bacteria in the image. If a large red fluorescence due to chlorophyll was noticed but there was no noticeable blue nucleus, it was still counted as a eukaryote. Organisms that were completely inside the picture were counted; if an organism landed on the top or left side of the picture and at least ¾ of the organism was visible, then it was counted too. If the organism landed on the right or the bottom of the image and was cut off, then it was not counted. The total number of eukaryotes was taken for each station and depth and a fixed value of 2,200 fg was applied, assuming that the overwhelming majority of these eukaryotes were nanoplankton (Pomeroy 1974, Fukuda et al., 2007; Sohrin et al., 2010). Given the wide size range in eukaryotes, this step represents a majr simplification.

For inverted microscopy, 20 ml sample vials were filled with seawater to the vial shoulder and 6 drops of Lugol’s solution were added to produce a tea-colored solution. Once in the lab, samples were mixed by inversion and 10 ml of the sample was measured into a graduated cylinder. Another 4 drops of Lugol’s solution were added and then topped off with equal-salinity 0.2 mm-filtered artificial seawater. Once the chamber was topped off, a glass plate was placed on the top to make it air-tight and to hold the water column in the settling chamber. After 24–48 hours of settling, the chamber was drained and replaced with a cover slip.

The slide was then processed under an Olympus CK7 inverted microscope equipped with a digital camera (see above). Organisms on the entire slide were imaged and all of those with the following criteria were analyzed. Organisms that were over 9,000 pixels in area (~ 100 mm2) and had a smooth shape to the structure (indicating a cell membrane) were included. For ciliated cells, only the area inside the cell membrane was measured. Diatom cell chains were measured as one composite organism. Each organism on the image was outlined and the area in pixels was recorded. If two or more organisms were identical, then only one would be measured and that measurement would be extrapolated to all identical organisms on the image. Any empty-shelled organisms were excluded. Due to time constraints, only approximately every tenth image was examined, and the results extrapolated to the entire sample. The area in pixels was converted to mm2 then the volume was calculated according to Bochdansky et al. (2017a). Volumes were converted into carbon values using equation 2 (Menden-Deuer and Lessard, 2000):

Log10(carbon (pg)) = -0.665+0.939*Log10(volume (mm3)