Dataset: Sediment Core Incubations - Sylt 2013

Sediments were collected from three intertidal sites near Königshafen on the island of Sylt in the North Sea, Germany. The ‘Schlickwatt (CL)’ and ‘Mischwatt (SLT)’ sites were located inside a small lagoon, while the ‘Sandwatt (SD)’ site was more openly exposed to wind and waves. Thirty intact push cores (30cm length, 10cm OD) were taken using polycarbonate core liners having vertical lines of silicone sealed holes (ø 3mm) at 1-cm intervals to allow porewater collection. Cores were retrieved leaving ~10 cm of overlying water and sealed with double o-ring caps to minimize gas exchange during transport, and brought immediately back to the laboratory. The gas-tight sealed sediment cores were incubated in the dark at in situ temperatures (19˚C) while being continuously supplied with filtered seawater at a flow rate of 1.8  0.06 ml/min for ~8 days. For experimental manipulations, four different inflow seawater compositions were used: “Low nitrate” (air sparged; ~20uM; LN), “Low oxygen, low nitrate” (sparged with N2 to 30-35% O2 saturation; ~20uM; LOLN), “High nitrate” (amended with NaNO3 to ~120uM (above background nitrate); HN) and “low oxygen, high nitrate” (combined treatments; LOHN). Samples of each sediment core effluent were taken twice per day.

Concentrations of NO3- + NO2- were measured by chemiluminescence after reduction in a hot acidic vanadyl sulfate solution on a NOx analyzer (Braman and Hendrix, 1989). Concentrations of NO2- were quantified by using the Griess-Ilosvay method followed by measuring absorption 540nm, and NO3- was quantified by difference (Grasshoff et al., 1999). Concentrations of NH4+ were measured by fluorescence using the OPA method (Holmes et al., 1999). Concentrations of N2O were made using the integrated peak area of the m/z 44 beam on the IRMS, standardizing to analyses of known amounts of N2O (injected into N2 sparged seawater in 160ml serum bottles) and normalizing to sample volume (158ml).

All N and O isotopic composition measurements (d15N and d18O (or d17O); where d15N = [(15Rsample/15RAir)-1)*1000 in units of ‰, and 15R = 15N/14N and where d18O = [(18Rsample/18RVSMOW)-1)*1000 in units of ‰, and 18R = 18O/16O (or 17O/16O) were made after conversion of analytes to nitrous oxide, followed by purification with a customized purge and trap system similar to that previously described (McIlvin and Casciotti, 2010) and analysis on a continuous flow IsoPrime 100 isotope ratio mass spectrometer (IRMS). D17O refers to the excess 17O beyond that defined by the terrestrial fractionation line for the oxygen isotope system and is defined as D17O = d17O*0.52 - d18O. Nitrate was converted to N2O using the denitrifier method (Casciotti et al., 2002; Sigman et al., 2001) after removal of nitrite by addition of sulfamic acid (Granger and Sigman, 2009). Corrections for drift, size and fractionation of O isotopes during bacterial conversion were carried out as previously described using NO3- standards USGS 32, USGS 34 and USGS 35 (Casciotti et al., 2002; McIlvin and Casciotti, 2011), with a typical reproducibility of 0.2‰ and 0.4‰ for d15N and d18O, respectively. Nitrate D17O measurements were made on separate aliquots by routing denitrifier-produced N2O through a gold tube (1/16” OD) held at 780˚C, thermally decomposing the N2O into N2 and O2, which were chromatographically separated using a 2m column (1/16” OD) packed with molecular sieve (5Å) before being sent into the IRMS (Kaiser et al., 2007; Komatsu et al., 2008). Nitrate standards USGS 35 and USGS 34 were used to normalize any scale contraction during conversion, with typical reproducibility of D17O measurements of 0.8‰. All samples for nitrite N and O isotope measurements were converted to N2O within 2 hours of collection using the azide method (McIlvin and Altabet, 2005). Internal nitrite isotope standards (WILIS 10, 11 and 20) were run in parallel at 3 different sizes to correct for any variations in sample size and instrumental drift, with a typical reproducibility for both d15N and d18O is 0.2‰. Based on calibrations against isotope standards USGS 32, 34 and 35 for d15N (Böhlke et al., 2003) and N23, N7373, and N10129 for d18O (Casciotti et al., 2007), the values of internal standards WILIS 10, 11, and 20 are reported here as -1.7, +57.1, and -7.8‰ for d15N and +13.2, +8.6 and +47.6‰ for d18O, respectively. Nitrite D17O measurements were made after conversion to N2O using the azide method and normalized using a combination of NO2- and NO3- isotopic standards. D17O values of NO2- isotope standards WILIS 10 and WILIS 11 were calibrated previously against USGS 34 and USGS 35 using the denitrifier method followed by thermal decomposition of N2O to N2 and O2 as described above – yielding D17O values of 0‰ for both. For sample NO2-, raw d17O and d18O values were first normalized for oxygen isotopic exchange with water during the azide reaction (McIlvin and Altabet, 2005) using the calibrated d17O and d18O values of WILIS 10 and WILIS 11. During the same IRMS run, N2O produced from USGS 34 and USGS 35 via the denitrifier method was also thermally converted and analyzed as N2 and O2. Because any isotope fractionation occurring during these reactions is mass dependent (e.g., D17O is unaffected), the D17O of NO2- can be calculated by normalizing to D17O values of these NO3- standards. We disregard the small amount of oxygen isotope exchange occurring during the denitrifier method, as this would have only a small impact on the calculated D17O values. Total reduced nitrogen (TRN, e.g., DON + NH4+) was measured in a subset of incubation cores by oxidation of the total dissolved nitrogen (TDN) pool via persulfate digest – followed by d15N analysis using the denitrifier method, similar to that previously described (Knapp et al., 2005). The d15N of the TRN pool was then calculated by mass balance by subtracting the molar contribution of the measured d15N of NO3- and NO2- pools to the TDN pool. Based on the measurement of NH4+ concentrations, the DON flux was generally of the same magnitude as the NH4+ flux (not shown). For dissolved N2O, samples were extracted from the 160ml serum bottles using a purge and trap approach similar to that previously described (McIlvin and Casciotti, 2010). Liquid samples were quantitatively transferred from the sample bottle into a purging flask using a 20psi He stream, followed by He-sparging (~45 min) and cryogenic trapping using the same system described above for nitrate and nitrite derived N2O. Isotopic composition of the dissolved N2O was measured by direct comparison against the N2O reference tank. The composition of this tank (d15N bulk = -0.7‰; d18O = +39.1‰; site preference (SP) = -5.3‰, where SP = d15N alpha – d15N beta, and alpha and beta refer to the central and outer N atoms in the linear N2O molecule, respectively) was calibrated directly against aliquots of two previously calibrated N2O tanks from the Ostrom Lab at Michigan State University, having been calibrated by Tokyo Tech. Several sample analyses of tropospheric N2O from the study site using this system yielded isotope values of +6.8  0.7‰ for d15N bulk, +44.1  1.7‰ for d18O and +17.4  2.2‰ for SP. Reported values have been corrected for any size linearity of isotopic ratios (31/30, 45/44 and 46/44) by using a series of reference tank subsamples injected into 20ml headspace vials using a gastight syringe. Precision for replicate analyses of our reference gas analyzed as samples for d15N is  0.3‰, for d18O is 0.4‰ and for SP is 0.8‰. The D17O of N2O was calculated similar to that described above for NO2-. After extraction and cryotrapping, the N2O sample is thermally decomposed to N2 and O2 and chromatographically separated before measurement on the IRMS. Regular analyses of N2O converted from NO3- isotope standards (USGS 35 and USGS 34) via the denitrifier method were made to normalize D17O values.