Sampling and analytical procedures:

 1. Analysis of NO3- N and O isotope ratios with the denitrifier method

The denitrifying bacteria strains Pseudomonas chlororaphis f. sp. aureofaciens (ATCC 13985, Manassas, VA, USA) and Pseudomonas. chlororaphis (ATCC 43928, Manassas, VA, USA) were used. Cultures were inoculated from cryo-preserved aliquots (Weigand et al., 2011) into sterile growth media prepared as originally described (Sigman et al., 2001; Casciotti et al., 2002) in 700 mL glass bottles containing 600 mL of medium, then sealed with gas-tight lids. Cells were cultured for 7-10 days at 20˚C on a rotary shaker table. Cultures were harvested by centrifugation and resuspended into 220 mL of fresh medium without potassium nitrate addition, achieving ca. 3-fold concentration of the bacteria. Two mL of the cell concentrates were added to respective 20-mL headspace glass vials, capped with pre-rinsed butyl rubber septa and crimp-seals (Mcllvin and Casciotti, 2011). Vials were sparged with a water-scrubbed N2 gas stream for 6 hours to remove any N2O produced from the residual NO3- in the medium. NO3- samples were then injected into each vial to achieve a final sample size of 10 nmoles of N. Vials were incubated inverted in order to prevent potential N2O leakage. Following overnight incubation in the dark, ca. 0.1 ml of 10 mol L-1 NaOH was injected into each vial to kill the cultures and sequester CO2 into carbonate species. The N2O gas in the vials was extracted, purified and analyzed with a Delta V Advantage continuous flow gas chromatograph isotope ratio mass spectrometer (Thermo Fisher Scientific, Waltham, MA, USA) interfaced with a modified Thermo Fisher Scientific Gas Bench sample preparation device fronted by dual cold traps (Casciotti et al., 2002) and a GC Pal autosampler (CTC Analytics, Zwingen, Switzerland). Samples were referenced to pure N2O injections from a common reference gas cylinder.

2. Demonstration of volume effects in analyzes of NO3- reference materials

The reference solutions were prepared from salts into primary stocks at 200 µmol L-1 in deionized water (DIW) from a Milli‐QTM water purification system (EMD Millipore, Burlington, MA, USA), and stored frozen. Primary stocks of NO3- reference materials (IAEA-NO3 and USGS-34) were diluted in NO3--deplete surface Sargasso seawater or in aged DIW to concentrations of 1, 5 and 20 µmol L-1, corresponding to respective injection volumes of 10, 2 and 0.5 mL, in order to aliquot 10 nmoles of N analyte. The NO3- aliquots were injected into the sparged bacterial concentrates of either P. chlororaphis or P. aureofaciens. Following bacterial conversion, the resulting N2O in the reaction vials was extracted, purified and analyzed on the isotope ratio mass spectrometer.

res1.csv