Phosphorus limitation regulates the responses of microbial carbon metabolism to long-term combined additions of nitrogen and phosphorus in a cropland

Microorganisms play central roles in the decomposition and retention of soil organic carbon (SOC), but how nutrient addition in intensively managed croplands influences microbial C metabolism remains uncertain. Here, we investigated the effects of coupled phosphorus (P) and nitrogen (N) additions on microbial respiration, growth rate, C use efficiency (CUE), and biomass turnover time in a continuously managed Zea mays cropland, by combining an 18-year field fertilization experiment with the ¹⁸O–H₂O labeling approach. Results showed that adding P at 50 and 100 kg P ha⁻¹, combined with 150 kg N ha⁻¹, increased respiration by 109% and 50.7%, increased growth rate by 207% and 135%, and increased CUE from approximately 0.26 without P addition to around 0.33 and 0.35, respectively. Conversely, adding N at varying rates (0, 100, 150, and 250 kg N ha⁻¹), combined with 50 kg P ha⁻¹, generated variable responses. These findings underscore the significance of P as the primary limiting element for microbial metabolism in this system. Ecoenzyme stoichiometry analysis further revealed that P addition decreased microbial P vs. N limitation, as well as decreased relative C limitation. In total, changes in P vs. N limitation with P and N additions accounted for 39.6% of the variation in microbial respiration, and in conjunction with relative C limitation, co-explained 51.4% of variations in growth rate and 44.0% of variations in CUE. Furthermore, our investigation identified positive associations of CUE with the activities of N and P-acquiring enzymes, but not with SOC. These results demonstrate flexible responses of microbial C metabolism to long-term anthropogenic N and P additions, highlighting their dependence on soil nutrient limitation. Consequently, optimizing the P-to-N fertilization ratio to alleviate relative P and C limitations may maximize microbial C assimilation and SOC retention in agroecosystems.