The distinct phases of fresh-seawater mixing intricately regulate the nitrogen transformation processes in a high run-off estuary: Insight from multi-isotopes and microbial function analysis

Excessive anthropogenic nitrogen inputs lead to the accumulation of nitrogen, and significantly impact the nitrogen transformation processes in estuaries. However, the governing of nitrogen during its transport from terrestrial to estuary under the influence of diverse human activities and hydrodynamic environments, particularly in the fresh-seawater mixing zone, remains insufficient researched and lack of basis. To address this gap, we employed multi-isotopes, including delta N-15-NO3-, delta O-18-NO3-, delta N-15-NH4+, and delta N-15-PN, as well as microbial function analysis, to investigate the nitrogen transformation processes in the Pearl River Estuary (PRE), a highly anthropogenic and terrestrial estuary. Principle component analysis (PCA) confirmed that the PRE could clearly partitioned into three zone, e.g., terrestrial area (T zone), mixing area (M zone) and seawater area (S zone), in terms of nitrogen transportation and transformation processes. The delta N-15-NO3- (3.38 +/- 0.60%) and delta O-18-NO3- (6.35 +/- 2.45%) results in the inner estuary (T area) indicate that NO3- attributed to the domestic sewage and groundwater discharge in the river outlets lead to a higher nitrification rate in the outlets of the Pearl River than in the reaching and seawater intrusion areas, although nitrate is rapidly diluted by seawater after entering the estuary. The transformation of nitrogen in the T zone was under significant nitrogen fixation (0.61 +/- 0.22 %) and nitrification processes (0.0043 +/- 0.0032 %) (presumably driven by Exiguobacterium sp. (14.1 %) and Cyanobium_ PCC-6307 (8.1 %)). In contrast, relatively low delta N-15-NO3- (6.83 +/- 1.24%) and high delta O-18-NO3- (22.13 +/- 6.01%) imply that atmospheric deposition has increased its contribution to seawater nitrate and denitrification (0.53 +/- 0.13 %) was enhanced by phytoplankton/bacterial (such as Psychrobacter sp. and Rhodococcus) in the S zone. The assimilation of NH4 results from the ammonification of NO3- reduces delta N-15-NH4+ (5.36 +/- 1.49%) and is then absorbed by particulate nitrogen (PN). The retention of nitrogen when fresh-seawater mixing enhances the elevation of delta N-15-NH4+ (8.19 +/- 2.19%) and assimilation of NH4+, leading to an increase in PN and delta N-15-PN (6.91 +/- 1.52%) from biological biomass (mainly Psychrobacter sp. and Rhodococcus). The results of this research demonstrate a clear and comprehensive characterization of the nitrogen transformation process in an anthropogenic dominated estuary, highlighting its importance for regulating the nitrogen dissipation in the freshseawater mixing process in estuarine ecosystems.