Long-term manure amendment enhances N2O emissions from acidic soil by alleviating acidification and increasing nitrogen mineralization

• Manure amendment, especially combined with inorganic fertilizer, increased soil N 2 O emissions. • Increased N 2 O emissions resulted from alleviated acidification and enhanced gross N mineralization. • Manure amendment favored N 2 O-producing over N 2 O-consuming denitrifiers. • Higher soil aggregate stability closely correlated with increased N 2 O emissions. Long-term manure application has the potential to alleviate soil acidification, and increase carbon sequestration and nutrient availability, thus improving cropland fertility. However, the mechanisms behind greenhouse gas N 2 O emissions from acidic soil mediated by long-term manure application remain poorly understood. Herein, we investigated N 2 O emission and its linkage with gross N mineralization and nitrification rates, as well as nitrifying and denitrifying microbes in an acidic upland soil subjected to 36-year fertilization treatments, including an unfertilized control (CK), inorganic fertilizer (F), 2 x rate of inorganic fertilizer (2F), manure (M), and the combination of inorganic fertilizer and manure (FM) treatments. Compared to the CK treatment (1.34 μg N kg –1 d –1 ), fertilization strongly increased N 2 O emissions by 34-fold on average, with more pronounced increases in the manure-amendment (10.6–169 μg N kg –1 d –1 ) than those in the inorganic fertilizer treatments (3.26–5.51 μg N kg –1 d –1 ). The manure amendment-stimulated N 2 O emissions were highly associated with increased soil pH, mean weight diameter of soil aggregates, substrate availability (e.g., particulate organic carbon, NO 3 − and available phosphorus), gross N mineralization rates, denitrifier abundances and the ( nirK + nirS )/ nosZ ratio. These findings suggest that the increased N 2 O emissions primarily resulted from alleviated acidification, increased substrate availability and improved soil structure, thus enhancing microbial N mineralization and favoring N 2 O-producing denitrifiers over N 2 O consumers. Moreover, AOB rather than AOA positively correlated with soil NO 3 − concentration and N 2 O emissions, indicating that nitrification indirectly contributed to N 2 O production by supplying NO 3 − for denitrification. Collectively, manure amendment potentially stimulates N 2 O emissions, primarily resulting from alleviated soil acidification and increased substrate availability, thus enhancing N mineralization and denitrifier-mediated N 2 O production. Our findings suggest that consideration should be given to the greenhouse gas budgets of agricultural ecosystems when applying manure for managing the pH and fertility of acidic soils.