Sampling

Samples were collected on 6 cruises of the R/V Savannah across the SAB (2-6 April, 15-18 May, 15-19 July, 4-7 Aug, 23-30 Sept and 3-7 Nov of 2014). The stations were divided into four regions: inshore, nearshore, mid-shelf and shelf-break, based on their locations and physical properties (Atkinson et al 1985). Station C8 is on the edge of the shelf-break and is influenced by intrusions of the Gulf Stream; thus, C8 was considered as a shelf-break station in this study. Surface and bottom water samples were collected from all stations, but we also collected samples at depths of 75 (or 100) m and 200 m at shelf-break stations C9 and C10. Seawater samples were collected using 12 L Niskin bottles mounted on a CTD rosette. Profiles of environmental variables (temperature, salinity, dissolved oxygen, relative fluorescence) were taken by sensors mounted on the CTD during hydrocasts to collect water samples. Optical Backscatter measured with a Wetlabs ECO BB Backscatter sensor at a wavelength of 660 nm.

Approximately 1L of water from each station and depth sampled was filtered through 0.22 um pore size Durapore filters (Millipore). The filters were then placed in Whirl-Pak bags, 2 mL of lysis buffer (0.75 M sucrose, 40 mM EDTA, 50 mM Tris; pH=8.3) was added, and the filters were frozen on dry ice and stored at -80C until they were processed. DNA was extracted from filters by first lysing cells with lysozyme and sodium dodecyl sulfate/proteinase K, followed by a phenol-chloroform extraction as described previously (Bano and Hollibaugh 2000, Tolar et al 2013). Approximately 100 mL of the filtrate was frozen at -20C for nutrient analysis (NO2, NO3, NH4, and urea). Ammonium was measured using a fluorometric method described in (Holmes et al 1999). Nitrate plus nitrite (NOx) was analyzed by reducing nitrate to nitrite with cadmium (Jones 1984), and nitrite was determined following (Strickland and Parsons 1972). Urea was measured following (Mulvenna and Savidge 1992) and (Revilla et al 2005). A total of ~1 L of seawater was filtered through 47 mm diameter Whatman GF/C glass fiber filters to collect chlorophyll a during all cruises except May. Two or three drops (~0.1 to 0.2 mL) of magnesium carbonate (0.01 g/mL) were added to the sample at the end of the filtration to avoid degradation to pheophytin. Filters were wrapped in aluminum foil and stored at -20C until processed. Chlorophyll a was extracted in 90% acetone and measured spectrophotometrically as described in (Strickland and Parsons 1972).

Quantitative PCR. All quantitative PCR (qPCR) analyses were performed on a C1000 TouchTM Thermal cycler (Bio-Rad Laboratories, Hercules, CA). qPCR reactions were run in triplicate with a dilution series of standards made from environmental amplicons as described in (Kalanetra et al 2009) and (Tolar et al 2013). We determined the abundance (copies/L) of Thaumarchaeota and Bacteria 16S rRNA genes (rrs); Archaea and betaproteobacteria amoA genes; and Nitrospina 16S rRNA (rrs) genes. Thaumarchaeota and Bacteria rrs were detected using TaqMan probes and iTaq Universal Probes Super Mix (Bio-Rad), while all other genes were quantified using SYBRgreen (iTaq Universal SYBRgreen Supermix, Bio-Rad). The primers and probes used and resulting qPCR efficiencies are given in the table below. Note that Archaea amoA was quantified using the (Wuchter et al 2006) primer set, which is biased against amoA genes from the deep-water clade of AOA (Beman et al 2008, Tolar et al 2013) that we encountered at shelf-break stations. Thaumarchaeota abundance was calculated from gene abundance (copies per liter assuming 100% extraction efficiency, calculated from copies per reaction, volume filtered for each sample, extract volume and the volume of template used in each reaction), assuming one rrs or amoA gene per Thaumarchaeota genome (IMG database). The relative abundance of Thaumarchaeota (percent of prokaryotes) was calculated assuming 1.8 rrs per Bacteria genome (Biers et al 2009) as described in (Kalanetra et al 2009).

Ammonia oxidation (AO) rates. AO rates were measured in 24 h incubations at in situ temperatures in the dark using15N-labeled ammonium (>99 atom-percent 15NH4Cl; Cambridge Isotope Laboratories, Tewksbury, MA) added to a final concentration of 50 nM. Incubations were terminated by freezing at -20 °C. Controls were frozen immediately after the addition of 15NH4Cl. The 15NOx produced was measured using the “denitrifier method” (Sigman et al 2001) as described previously (Beman et al 2012, Dore et al 1998, Popp et al 1995). Briefly, 15N2O produced from NO2- and NO3- by incubation with the denitrifier Pseudomonas aureofaciens cultures was transferred from a reaction vial, cryofocused, separated from other gases using a 0.32-mm i.d. × 25 m capillary column packed with 5 µm CP-PoraBOND Q (Agilent Technologies, Santa Clara, CA) at room temperature, and introduced into a Finnigan MAT-252 isotope ratio mass spectrometer (Thermo Fisher Scientific, Waltham, MA). 15N2O concentrations and d15N were determined to calculate the AO rates with a modified equation described previously (Beman et al 2011, Beman et al 2012, Ward et al 1989).

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