Human activities have greatly influenced the natural nitrogen cycle, causing dramatic degradation of ecosystem function. Net anthropogenic nitrogen input (NANI) is an important factor contributing to the impact of human activities on the regional nitrogen cycle. Here, we analyzed the temporal and spatial variation of NANI in the Pearl River Basin of China between 1986 to 2015, and found that the total amount of NANI significantly increased from 3,362.25 kg N km^-2 yr^-1 to 8,071.15 kg N km^-2 yr^-1. Application of nitrogen fertilizers was the largest component of NANI in the Basin, accounting for 55.53% in the total NANI, followed by food/feed net nitrogen input (21.26%), atmospheric nitrogen deposition (12.95%), and crop nitrogen fixation (10.26%). Over the last three decades, nitrogen inputs from atmospheric nitrogen deposition have become the second largest source of NANI due to rapid industrialization and urbanization in the region. Regression analysis showed that the rapid growth of both GDP and population density were the main contributors to the increase of NANI. In addition, the increase in the number of red tides in the Pearl River Estuary was strongly correlated with NANI discharge (R² = 0.90, p<0.01), suggesting the NANI’s eutrophication effect. In total, this study provides a quantitative understanding of the temporal and spatial variations of NANI in the Pearl River Basin as well as the effects of NANI on estuarine waters, and offered key information for developing an integrated strategy for watershed nitrogen management.

Denitrification is vital to nitrogen removal and N₂O release in ecosystems; in this regard, paddy soils exhibit strong denitrifying ability. However, the underlying mechanism of N₂O emission from denitrification in paddy soils is yet to be elucidated. In this study, the potential N₂O emission rate, enzymatic activity for N₂O production and reduction, gene abundance, and community composition during denitrification were investigated using the ¹⁵N isotope tracer technique combined with slurry incubation, enzymatic activity detection, quantitative polymerase chain reaction (qPCR), and metagenomic sequencing. Results of incubation experiments showed that the average potential N₂O emission rates were 0.51 ± 0.20 μmol⋅N⋅kg^–1⋅h^–1, which constituted 2.16 ± 0.85% of the denitrification end-products. The enzymatic activity for N₂O production was 2.77–8.94 times than that for N₂O reduction, indicating an imbalance between N₂O production and reduction. The gene abundance ratio of nir to nosZ from qPCR results further supported the imbalance. Results of metagenomic analysis showed that, although Proteobacteria was the common phylum for denitrification genes, other dominant community compositions varied for different denitrification genes. Gammaproteobacteria and other phyla containing the norB gene without nosZ genes, including Actinobacteria, Planctomycetes, Desulfobacterota, Cyanobacteria, Acidobacteria, Bacteroidetes, and Myxococcus, may contribute to N₂O emission from paddy soils. Our results suggest that denitrification is highly modular, with different microbial communities collaborating to complete the denitrification process, thus resulting in an emission estimation of 13.67 ± 5.44 g N₂O⋅m^–2⋅yr^–1 in surface paddy soils.