Water-nitrogen coupling optimizes soil microbial carbon use efficiency by alleviating metabolic limitations in cropland soils

Water and nitrogen management are two major agronomic practices that play a crucial role in the cropland carbon cycle. However, the mechanisms by which their interactions regulate microbial carbon use efficiency (CUE) through extracellular enzyme allocation strategies remain poorly understood. To address this, we conducted a field experiment on water-nitrogen regulation in the wheat-maize rotation system on the Loess Plateau. By combining 18 O-H 2 O stable isotope tracing with ecoenzymatic and microbial biomass stoichiometry, we developed a framework illustrating how water–nitrogen inputs reshape soil and microbial stoichiometry, alleviate metabolic limitation, and enhance the microbial CUE. The results showed that microbial CUE increased by approximately 40.31% under moderate water and nitrogen supply (70–85% field capacity, 180/200 kg N ha −1 for wheat/maize) but plateaued at higher water-nitrogen coupling levels, indicating a nonlinear response. The moderate water–nitrogen interaction demonstrated the minimum relative carbon limitation and phosphorus vs . nitrogen imbalance, thereby optimizing microbial resource allocation. These changes were reflected in microbial biomass stoichiometry: the C:N ratio decreased by 33.35%, and the C:P and N:P ratios increased by 14.23% and 71.63%, respectively. Random forest models consistently identified the enzyme C:N ratio as an important driver of the microbial respiration and CUE, whereas enzyme C:P and N:P ratios primarily regulated microbial growth. Partial least squares structural equation modeling revealed that water-nitrogen inputs indirectly altered vector length (negative effect) and vector angle (positive effect) by affecting microbial stoichiometry, together explaining 58% of the variation in CUE. Overall, this study revealed the nonlinear regulation of water–nitrogen interactions on microbial carbon metabolism. The optimized water–nitrogen management regime (70–85% field capacity, 180/200 kg N ha −1 for wheat/maize) shifted microbial strategies from high enzymatic investment for multiple resource acquisition pathways toward metabolism characterized by lower respiratory costs and higher CUE. These findings provide a process-based framework and practical guidance for optimizing water and nitrogen supply to simultaneously enhance soil fertility and nutrient availability, promoting higher carbon use efficiency and carbon sequestration in agroecosystems.