Analytical improvements and assessment of long-term performance of the oxidation-denitrifier method

The analysis of the nitrogen (N) isotopic composition of organic matter bound to fossil biomineral structures (BB-δ¹⁵ N) using the oxidation-denitrifier (O-D) method provides a novel tool to study past changes in N cycling processes.

We report a set of methodological improvements to the O-D method, including (a) a method for sealing the reaction vials in which the oxidation of organic N to NO₃ ^- takes place, (b) a recipe for bypassing the pH adjustment step before the bacterial conversion of NO₃ ^- to N₂ O, and (c) a method for storing recrystallized dipotassium peroxodisulfate (K₂ S₂ O₈ ) under Ar atmosphere.

The new sealing method eliminates the occasional contamination and vial breakage that occurred previously while increasing sample throughput. The protocol for bypassing pH adjustment does not affect BB-δ¹⁵ N, and it significantly reduces the processing time. Storage of K₂ S₂ O₈ reagent under Ar atmosphere produces stable oxidation blanks over more than 3.5 years. We report analytical blanks, accuracy, and precision for this methodology from eight users over the course of ~3.5 years of analyses at the Max Planck Institute for Chemistry. Our method produces analytical blanks characterized by low N content (0.30 ± 0.13 nmol N, 1σ, n = 195) and stable δ¹⁵ N (-2.20 ± 3.13‰, n = 195). The analysis of reference amino acid standards USGS 40 and USGS 65 indicates an overall accuracy of -0.23 ± 0.35‰ (1σ, n = 891). The analysis of in-house fossil standards gives similar analytical precision (1σ) across a range of BB-δ¹⁵ N values and biominerals: zooxanthellate coral standard PO-1 (6.08 ± 0.21‰, n = 267), azooxanthellate coral standard LO-1 (10.20 ± 0.28‰, n = 258), foraminifera standard MF-1 (5.92 ± 0.28‰, n = 243), and tooth enamel AG-Lox (4.06 ± 0.49‰, n = 78).

The methodological improvements significantly increase sample throughput without compromising analytical precision or accuracy down to 1 nmol of N.