Stable carbon degradation strategies drive microbial carbon use efficiency after afforestation

Microbial carbon use efficiency (CUE) is a critical metric to assess soil microbial participation in the carbon cycle. However, the mechanistic links between microbial functional traits and CUE under land-use change remain poorly understood. By selecting soil samples from a 45-year Robinia pseudoacacia (RP) afforestation chronosequence and combining an 18O-H₂O labeling method with metagenomic techniques to explore how afforestation alters soil substrate conditions and microbial metabolism. We found that CUE increased due to a relatively high increase in microbial biomass and a relatively low increase in microbial respiration after afforestation. This outcome is jointly determined by soil stoichiometry and carbon substrate characteristics. Nutrient imbalances, especially phosphorus limitation, stimulated microbial communities to mine stable organic matter for essential elements. Since the breakdown of stable substrates requires higher activation energy, microbes must enhance their metabolic efficiency, thereby increasing CUE. Functional gene analysis further revealed that forest soils with higher abundances of stable carbon-degrading genes exhibited enhanced CUE, while farmland soils with fewer such genes showed low CUE. These findings provide empirical evidence for microbial mediation of soil substrate changes and emphasize that the influence of the stable carbon pool on microbial metabolism is not negligible in soil carbon cycle.