Chemical formation of hybrid di-nitrogen calls fungal codenitrification into question

Removal of excess nitrogen (N) can best be achieved through denitrification processes that transform N in water and terrestrial ecosystems to di-nitrogen (N2) gas. The greenhouse gas nitrous oxide (N2O) is considered an intermediate or end-product in denitrification pathways. Both abiotic and biotic denitrification processes use a single N source to form N2O. However, N2 can be formed from two distinct N sources (known as hybrid N2) through biologically mediated processes of anammox and codenitrification. We questioned if hybrid N2 produced during fungal incubation at neutral pH could be attributed to abiotic nitrosation and if N2O was consumed during N2 formation. Experiments with gas chromatography indicated N2 was formed in the presence of live and dead fungi and in the absence of fungi, while N2O steadily increased. We used isotope pairing techniques and confirmed abiotic production of hybrid N2 under both anoxic and 20% O2 atmosphere conditions. Our findings question the assumptions that (1) N2O is an intermediate required for N2 formation, (2) production of N2 and N2O requires anaerobiosis, and (3) hybrid N2 is evidence of codenitrification and/or anammox. The N cycle framework should include abiotic production of N2.

Technical Note: Simultaneous measurement of sedimentary N₂ and N₂O production and a modified ¹⁵N isotope pairing technique

Dinitrogen (N2) and/or nitrous oxide (N2O) are produced through denitrification, anaerobic ammonium oxidation (anammox) or nitrification in sediments, of which entangled processes complicate the absolute rate estimations of gaseous nitrogen production from individual pathways. The classical isotope pairing technique (IPT), the most common 15N nitrate enrichment method to quantify denitrification, has recently been modified by different researchers to (1) discriminate between the N2 produced by denitrification and anammox or to (2) provide a more accurate denitrification rate under considering production of both N2O and N2. In case 1, the revised IPT focused on N2 production being suitable for the environments of a low N2O-to-N2 production ratio, while in case 2, anammox was neglected. This paper develops a modified method to refine previous versions of IPT. Cryogenic traps were installed to separately preconcentrate N2 and N2O, thus allowing for subsequent measurement of the two gases generated in one sample vial. The precision is better than 2% for N2 (m/z 28, m/z 29 and m/z 30), and 1.5% for N2O (m/z 44, m/z 45 and m/z 46). Based on the six m/z peaks of the two gases, the 15N nitrate traceable processes including N2 and N2O from denitrification and N2 from anammox were estimated. Meanwhile, N2O produced by nitrification was estimated via the production rate of unlabeled 44N2O. To validate the applicability of our modified method, incubation experiments were conducted using sediment cores taken from the Danshuei Estuary in Taiwan. Rates of the aforementioned nitrogen removal processes were successfully determined. Moreover, N2O yield was as high as 66%, which would significantly bias previous IPT approaches if N2O was not considered. Our modified method not only complements previous versions of IPT but also provides more comprehensive information to advance our understanding of nitrogen dynamics of the water–sediment interface.