Long-term nitrogen addition stabilizes microbial carbon use efficiency through antagonistic biotic and abiotic regulation in cropland soils

Chronic nitrogen fertilization, as a common anthropogenic practice associated with global change, has important implications for soil carbon cycling. Microbial carbon use efficiency (CUE), which determines the partitioning of assimilated carbon between biomass production and respiratory loss, is therefore expected to respond to prolonged nitrogen addition. However, its long-term response in cropland soils remains unresolved. Here, we combined a meta-analysis with six long-term fertilization experiments across China’s croplands and integrated substrate-independent 18O-H2O measurements with metagenomic analyses to test whether, and why, microbial CUE changes under chronic nitrogen addition. The meta-analysis showed that the initially positive response of microbial CUE weakened with fertilization duration and converged toward zero after approximately 30 years. Consistently, long-term nitrogen addition did not significantly alter microbial CUE in field experiments, despite substantial shifts in soil chemistry and microbial functional potential. Nitrogen addition decreased soil pH, the DOC:DON ratio, and the abundances of genes involved in assimilatory nitrate reduction (narB and nirB) and organic nitrogen metabolism (ureB), which together tended to increase microbial CUE. These positive effects were offset by nitrate accumulation, alleviated relative carbon limitation, and increased abundance of the dissimilatory nitrate reduction gene narI, which together suppressed microbial CUE. Our results show that the long-term stability of microbial CUE is not due to microbial insensitivity, but emerges from antagonistic biotic and abiotic regulation, highlighting a compensatory mechanism that may buffer microbial carbon processing under chronic global change.