Abstract: Soil microbial nutrient limitation plays a key role in regulating carbon use efficiency (CUE), but how long-term nutrient addition alters such constraints and their impact on CUE remains unclear. To address that, we conducted a 10-year-long nitrogen (N) and phosphorus (P) addition experiment in a temperate grassland site to clarify how environmental changes under nutrient enrichment can shape microbial nutrient limitation and influence microbial CUE. We found that the long-term N and P addition significantly altered soil nutrient availability, microbial biomass, and enzyme activities. Carbon (C) - and N- acquiring enzyme activities were generally enhanced under fertilized treatments, whereas the activity of P-acquiring enzymes (acid phosphatase, ACP) peaked under P addition. Ecological enzyme vector analysis revealed persistent C and N co-limitation among all treatments.The N treament increased vector length and reduced vector angle, suggesting exacerbated microbial C limitation and stronger relative N limitation, while the P treament increased vector angle, implying alleviation on microbial N limitation. Correspondingly, microbial CUE was higher under fertilized treatments, reaching its maximum under the NP treament.
Integrated analyses using Mantel tests, random forest modeling, and partial least squares path modeling demonstrated that enzyme activities and their stoichiometric ratios played a dominant role in regulating microbial nutrient limitation and CUE, with microbial N limitation acting as a key proximal driver of CUE. Our findings emphasize the importance of microbial nutrient demand and enzyme-mediated nutrient acquisition strategies in mediating soil carbon dynamics under long-term nutrient enrichment in grassland ecosystems under global environmental change.

Key words: microbial metabolic limitation, microbial carbon use efficiency, soil microbial biomass, ecoenzymatic stoichiometry, nutrient addition, temperate grassland