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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 (15ε and 18ε) changed with carbon sources, 18ε/15ε 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 δ18O‐ and δ15N‐nitrate did not correlate in the surface water where nitrification was active, but 18ε/15ε followed a proportionality of 1.05 ± 011 in deeper waters (≥ 12 m) where denitrification controlled the nitrate isotope. The independence of 18ε/15ε from carbon sources provides an opportunity to determine heterotrophic denitrification and helps the interpretation of nitrate isotopes in freshwaters. [ABSTRACT FROM AUTHOR]

Microbial nitrate reduction can be driven by organic carbon oxidation, as well as by inorganic electron donors, such as reduced forms of sulfur and iron. An apparent inverse oxygen isotope fractionation effect was observed during nitrate reduction in sediment incubations from five sampling sites of a freshwater lake, Hongze Lake, China. Incubations with organic and inorganic electron donor additions were performed. Especially, the inverse oxygen isotope effect was intensified after glucose addition, whereas the incubations with sulfide and Fe2+ showed normal fractionation factors. Nitrate reductase encoding genes, napA and narG, were analysed with metagenomics. Higher napA/narG ratios were associated with higher oxygen fractionation factors. The most abundant clade (59%) of NapA in the incubation with glucose was affiliated with Rhodocyclales. In contrast, it only accounted for 8%–9% of NapA in the incubations with sulfide and Fe2+. Differences in nitrate reductases might explain different oxygen isotope effects. Our findings also suggested that large variance of O‐nitrate isotope fractionations might have to be considered in the interpretation of natural isotope records. [ABSTRACT FROM AUTHOR]