The water column in Monterey Bay (coastal California, USA) was sampled near-monthly from May 2014-February 2016 at stations M1 (36.747 ºN, 122.022 ºW) and M2 (36.697 ºN, 122.378 ºW), on board the RV Western Flyer or RV Rachel Carson using a CTD Rosette sampler (Sea-Bird Scientific, Bellevue, WA). For each hydrocast, the CTD collected data on conductivity, temperature, depth, dissolved oxygen (DO), total CO2, and transmissivity (turbidity). Additional samples were collected from 11-12 depths from the cast (0, 5, 10, 20, 30, 40, 60, 80, 100, 150, 200 m; 500 m included for 2015-2016) to measure nutrients (ammonia, nitrite, nitrate, silicate, phosphate), chlorophyll a and phaeopigment concentrations. These were processed using established methods as part of the Monterey Bay Time Series (http://www3.mbari.org/bog/Projects/CentralCal/summary/ts_methods_and_materials.htm; Pennington and Chavez 2000). Light penetration depth (LPD; 0.1-50 % of surface light) was estimated by secchi disk.

Approximately 1 L sample seawater was filtered using a peristaltic pump onto duplicate filters – 10 µm polycarbonate (PCTE, Sterlitech; pre-filter), 0.2 µm GVWP (Millipore; final filter) – for molecular analysis from 6-10 depths per site per month (0-500 m depth). Samples were immediately frozen on liquid N2 and stored at -80°C upon return to laboratory until processing.

DNA was co-extracted with RNA using previously described methods (Smith et al. 2014a), with slight modification – both 0.1 and 0.5 mm sterile glass beads (BioSpec) were used for bead beating on the FastPrep (Thermo) and fresh -mercaptoethanol was added to Lysis/Binding buffer (10 µL per mL) immediately before extraction. Concentration of DNA was measured using a Qubit fluorometer (Invitrogen). Gene abundance was determined using published methods for total archaeal amoA (Francis et al. 2005), water column group A (WCA) and water column group B (WCB) amoA (Beman et al. 2008); modified to TaqMan assay, (Mosier and Francis 2011), and two archaeal nirK groups (AnirKa and AnirKb; Lund et al. 2012). 

Water samples were collected from 6-10 depths for nitrification rate measurements using 15NH4Cl as a tracer. Sample seawater was spiked with 15NH4Cl, and placed in ship-board seawater flow-through incubators for 24 h. Incubations were carried out in the dark or at estimated in situ light using stainless steel tubes with pre-drilled evenly spaced and sized holes (Pennington and Chavez 2000; Smith et al. 2014). At the end of incubations, samples were filtered (0.2 µm) and frozen at -20 ºC. δ15N values were measured from NOx in each sample, converted to N2O via the bacterial denitrification assay (Sigman et al. 2001) using a ThermoFinnigan Gas Bench and PreCon trace gas concentration system interfaced with the Delta VPLUS isotope-ratio mass spectrometer (Bremen, Germany) at the UC Davis Stable Isotope Facility.