Denitrification fractionates N and O isotopes of nitrate following a ratio independent of carbon sources in freshwaters.

The stable isotope technique has been used in tracking nitrogen cycling processes, but the isotopic characteristics are influenced by environmental conditions. To better understand the variability of nitrate isotopes in nature, we investigated the influence of organic carbon sources on isotope fractionation characteristics during microbial denitrification. Denitrifying cultures were inoculated with freshwater samples and enriched with five forms of organic compounds, that is, acetate, citrate, glucose, cellobiose, and leucine. Though the isotope enrichment factors of nitrogen and oxygen ( 15 ε and 18 ε) changed with carbon sources, 18 ε/ 15 ε always followed a proportionality near 1. Genome-centred metagenomics revealed the enrichment of a few populations, such as Pseudomonas, Enterobacter, and Atlantibacter, most of which contained both NapA- and NarG-type nitrate reductases. Metatranscriptome showed that both NapA and NarG were expressed but to different extents in the enrichments. Furthermore, isotopic data collected from a deep reservoir was analysed. The results showed δ 18 O- and δ 15 N-nitrate did not correlate in the surface water where nitrification was active, but 18 ε/ 15 ε followed a proportionality of 1.05 ± 011 in deeper waters (≥ 12 m) where denitrification controlled the nitrate isotope. The independence of 18 ε/ 15 ε from carbon sources provides an opportunity to determine heterotrophic denitrification and helps the interpretation of nitrate isotopes in freshwaters.