Long-term nitrogen fertilization accelerates labile biomolecules decomposition and retains recalcitrant compounds in a temperate agroecosystem

The influence of long-term nitrogen (N) fertilization on the quantity, molecular constituents, and origin of soil organic matter (SOM) within the soil profile remains an area of ongoing investigation. We employed a comprehensive set of biomarkers (including free lipids, bound lipids, lignin phenols, neutral sugars, and amino sugars), ¹³C NMR techniques, and soil extracellular enzyme activities, to evaluate the SOM characteristics in response to 18-year N fertilizer gradients (i.e., 0, 57.5, 97.5, 137.5 kg N ha⁻¹ yr⁻¹) to a depth of 60 cm in Northeast China. The results showed that N fertilization distinctly modified the molecular signature of SOM (e.g., biomolecules, degradation, and source) within the soil profile, particularly within the top 20 cm layer. N additions led to a 13.6–17.3 % increase in microbial-derived free lipids (predominantly short-chain lipids, <C₂₀) and accelerated the degradation of aliphatic lipids in the topsoil, as indicated by an increased average chain length of aliphatic lipids. The response of lignin phenols and their degradation to varying levels of N addition differed across soil layers. Compared to the control, N enrichment increased cutin compounds by 26.7–93.7 %, whereas the impact on cutin and suberin-derived lipids was negligible. N fertilization reduced plant-derived neutral sugars by 5.8–26.5 % and microbial-derived neutral sugars by 10.2–15.9 % relative to ambient. Changes in bacterial and fungal necromass C, total microbial necromass C, and their contributions to soil organic C varied across treatments within specific soil layer. Furthermore, N enrichment was associated with microbial C limitation (as indicated by increased vector length) and a decrease in soil microbial C use efficiency, with a bias towards the decomposition of more labile biomolecules such as short-chain lipids, O-alkyl C, while more recalcitrant compounds (e.g., alkyl C, aromatic + phenolic C) were preserved. Collectively, our study offers mechanistic insights into the consequences of N enrichment on SOM compositions and stability at the molecular level, critical for understanding its persistence and broader functionality under impending global change paradigms.