Evidence for Assimilatory Nitrate Reduction as a Previously Overlooked Pathway of Reactive Nitrogen Transformation in Estuarine Suspended Particulate Matter

Suspended particulate matter (SPM) contributes to the loss of reactive nitrogen (Nr) in estuarine ecosystems. Although denitrification and anaerobic ammonium oxidation in SPM compensate for the current imbalance of global nitrogen (N) inputs and sinks, it is largely unclear whether other pathways for Nr transformation exist in SPM. Here, we combined stable isotope measurements with metagenomics and metatranscriptomics to verify the occurrence of dissimilatory nitrate reduction to ammonium (DNRA) in the SPM of the Pearl River Estuary (PRE). Surprisingly, the conventional functional genes of DNRA (nirBD) were abundant and highly expressed in SPM, which was inconsistent with a low potential rate. Through taxonomic and comparative genomic analyses, we demonstrated that nitrite reductase (NirBD) in conjunction with assimilatory nitrate reductase (NasA) performed assimilatory nitrate reduction (ANR) in SPM, and diverse alpha- and gamma-proteobacterial lineages were identified as key active heterotrophic ANR bacteria. Moreover, ANR was predicted to have a relative higher occurrence than denitrification and DNRA in a survey of Nr transformation pathways in SPM across the PRE spanning 65 km. Collectively, this study characterizes a previously overlooked pathway of Nr transformation mediated by heterotrophic ANR bacteria in SPM and has important implications for our understanding of N cycling in estuaries.