Field measurements and bottle collections: Seawater was collected on the research vessel Fay Slover using 5 L Niskin bottles along a gradient from the open ocean near the Chesapeake light tower into the Chesapeake Bay and the James River in June 2019. Conductivity, temperature, and depth were measured using a SBE 32 CTD equipped with a Wetlabs fluorometer and a Wetlabs transmissometer (650 nm).
Field collection of ATP: ATP samples were processed using the hot-water extraction method described by Bochdansky et al. (2021). Three types of ATP samples were collected: 1) pATP in 5 ml seawater filtered through 0.2 mm pore size polycarbonate filters (Isopore GTTP, 25 mm diam.), 2) dissolved ATP (ATP in the filtrate of 0.2 mm filtration), and 3) 0.5 ml ATP in whole water without filtration. Polycarbonate filters were chosen over GF/F filters because of their higher retention efficiency (Taguchi and Laws, 1988; Lee et al., 1995) and because the volumes filtered were small, to ensure filtration times were kept to a minimum even with the use of the smaller pore size. Five milliliters of each depth were immediately and simultaneously filtered using a filtration manifold (25 mm-diameter filters, stainless steel screen supports) preloaded with the polycarbonate filters. The filters were placed in 15 ml polypropylene centrifuge tubes (FalconTM) within seconds after the water passed. The dissolved ATP was captured in 15 ml polypropylene centrifuge vials placed underneath the filtration manifold and inside the vacuum flasks. The dissolved ATP samples (i.e., the 0.2 mm filtrates) were immersed in a boiling water bath for ~15 minutes immediately after filtration to sterilize the water and inactivate ATPases and then frozen at -80 °C until analysis.
In June 2019, instead of boiling on board, filters, filtrates, and whole water samples were collected as above but immediately frozen in liquid nitrogen, transported to the laboratory at -20 °C, and stored at -80 °C in the lab.
Hot-water extraction was then performed on the still frozen samples (i.e., without prior thawing) in the laboratory (see Bochdansky et al. 2021 for details). The shock freezing-boiling treatment breaks up cells more efficiently than boiling alone, which results in higher extraction efficiencies (Bochdansky et al. 2021).
In 2019, 500 microliter of water from each bottle was placed in 15 ml centrifuge tubes and shock-frozen in liquid nitrogen. These unfiltered samples thus contained both particulate and dissolved ATP and were labeled total ATP (tATP). All samples were brought to the laboratory in a -20 °C freezer and subsequently stored at -80 °C in the laboratory.
For analysis of the 500 microliter shock-frozen samples, boiling hot water was added to the samples and extracted for ~15 minutes in a boiling-water bath.
Particulate and total ATP samples were topped up to 5 ml with ultrapure water using the gradations on the centrifuge tubes and mixed with a vortex mixer. The 500 microliter whole-water samples were also diluted to 5 ml with ultrapure water to reduce salt effects that strongly decrease the luminescence yield. It should be noted that the whole-water extraction method used here (for total ATP) requires sufficiently high ATP levels to produce a signal. This was possible because samples were taken in the mesotrophic coastal ocean and in a eutrophic estuary. Such a small amount of water (500 microliter) would be insufficient in oligotrophic or deep-sea environments. Hot-water extraction is only one of two methods proposed by Bochdansky et al. (2021). Many of our subsequent collections were based on chemical extraction using phosphorus benzalkonium chloride (P-BAC) instead. Both methods give highly consistent results, with the values from the chemical extraction method exceeding that of the hot water extraction by 20% (Bochdansky et al. 2021). The hot-water method used here has the advantage that measurements of dissolved ATP can be added easily to the protocol as hot water for the inactivation of ATPases is already at hand.
Particulate carbon and nitrogen: During the June 2019 cruise, between 75 ml and 250 ml of seawater (less in the inshore and more in the offshore stations) was filtered onto pre-combusted (450 °C, 4 hours) GF/F filters. The filters were stored frozen (-20 °C) and later dried at 50 °C for approximately 2 days. Filters were then rolled in a tin wrap and pressed into pellets to be analyzed in a Europa 20‐20 isotope ratio mass spectrometer (IRMS) equipped with an automated N and C analyzer.
