Table of Contents

• Dataset Description
  • Methods & Sampling
  • Data Processing Description
• Data Files
• Parameters
• Instruments
• Project Information
• Funding

Enzyme assays were conducted to determine the N and O isotope effects of nitrate reduction by 3 forms of nitrate reductase: respiratory nitrate reductase (Nar), periplasmic nitrate reductase (Nap), and eukaryotic assimilatory nitrate reductase (EukNR).

Sources of Nitrate Reductase:
Enzymatic assays were conducted on (a) cell homogenates from the denitrifying bacterial strain Paracoccus denitrificans (American Type Culture Collection [ATCC] 19367) cultured under anaerobic vs. aerobic conditions, on (b) cell homogenates from the photo-heterotrophic bacterial strain Rhodobacter sphaeroides (Deutsche Sammlung von Mikroorganismen [DSM] 158) cultured aerobically, and on (c) purified extracts of recombinant eukaryotic assimilatory nitrate reductases (EukNR) from the flowering plant Arabidopsis thaliana (AtNaR:  E.C. 1.7.1.1) and from the yeast Pichia angusta (YNaR1: E.C. 1.7.1.2), both purchased from NECi (nitrate.com).

Enzymatic assay preparations:
Assays contained 0.5 or 1 mL of cell suspension or of commercially purified EukNR buffered solution, 1 mL of 200 umol L-1 reducing agent – either membrane-permeant benzyl viologen dichloride [Sigma-Aldrich, CAS: 1102-19-8], methyl viologen dichloride hydrate [Sigma-Aldrich, CAS: 75365-73-0], or hydroquinone (for dissimilatory reductases only; [MP Organics]) –  0.2 or 1 mL 10 mmol L-1 KNO3 to a final concentration of 200 or 1000 umol L-1, and the remaining volume of 100 mmol L-1 phosphate buffer [pH 7.9] containing 100 umol L-1 to a final assay volume of 10 mL. After drawing an initial 1 mL sample for quantitation of [NO3-] and [NO2-], the denitrification reaction was commenced by the addition of 1 mL of 57 mmol L-1 sodium dithionite in 29 mmol L-1  sodium bicarbonate, which reduces the electron donor. Initial [NO3-] and [NO2-] values are corrected for this dilution. Serial 1 mL samples were drawn approximately every 90 seconds during room temperature assays and every 3 minutes during assays conducted at 4 degrees C. Samples were mixed vigorously on a vortex mixer for 30 s immediately upon collection to halt the reaction through oxidation of the methyl or benzyl viologen or hydroquinone.  In selected assays, additional ~ 50 uL samples were also drawn throughout the assay reactions for determination of [NO2-] and were measured immediately. In order to ensure complete cessation of enzyme activity, samples placed in an 80 degrees C water bath for 2 to 10 minutes. NO2- was then removed from the samples via the addition of 55 uL 4% (wt/vol) sulfamic acid in 10% vol/vol HCl.

Determination of [NO3-]:
[NO3-] was measured in samples by chemiluminescence detection on a NOx analyzer (model T200 Teledyne Advanced Pollution Instrumentation) following reduction to NO in a heated vanadium solution (Braman and Hendrix 1989). Nitrite had been previously removed from these samples, as vanadium reduces both nitrate and nitrite to NO.

Nitrate N and O isotope ratio analyses:
NO3- δ15N and δ18O were determined with the denitrifier method (Sigman et al. 2001; Casciotti et al. 2002), wherein denitrifying bacteria lacking terminal nitrous oxide reductase (P. chlororaphis f. sp. aureofaciens ATCC 1398) quantitatively convert sample NO3- to N2O gas, which is then extracted, purified and analyzed through a modified Thermo-Scientific Gas Bench II and Delta V Advantage gas chromatograph isotope ratio mass spectrometer.  Samples were standardized through comparison to reference standards IAEA-N3, USGS-34, and USGS-32, which have δ15N (vs air N2) and δ18O (vs V-SMOW) of 4.7‰ and 25.6‰, -1.8‰ and -27.9‰, and 180‰ and 25.6‰ respectively after individually being referenced to pure N2O injections from a common reference gas cylinder.

Nitrate Calculations: delta 15N and delta 18O NO3 were calculated from uncorr_d45_44 and uncorr_d46_44 using equations outlined in the "Nitrate isotope corrections" supplementary file (PDF), followed by reference to IAEA N-3, US34, US32 standards.

Description from NSF award abstract:
This study will test the sensitivity of the amplitude of the denitrification isotope effect to culture conditions pertinent to the ocean environment. The isotope effect amplitude will be explored with respect to electron donor, trace oxygenation, and temperature, in both batch and continuous culture experiments of denitrifiers. The proposed work will also involve measurements of the enzymatic isotope effect of the respiratory nitrate reductase of denitrifiers, measurements of its enzymatic activity among cultures, and examination of cellular nitrate transport kinetics of denitrifying strains. The experiments are designed to reveal the physiological basis of the modulation of the isotope effect amplitude, which will further resolve this manifestation in the environment.

In regards to the broader significance and importance of this study, these new experimental data will provide a basis for integration of nitrogen isotope dynamics in ocean models to test how key environmental parameters can affect the global ocean distribution of nitrogen isotopes.