Irrational nitrogen (N) fertilizer management and application practices have led to a range of ecological and environmental problems that seriously threaten food security. In this study, an effective N fertilizer management strategy was established for improving N fertilizer utilization efficiency (NUE). Biochar, N2-fixing bacteria (Enterobacter cloacae), and a nitrification inhibitor (dicyandiamide, DCD) were simultaneously added to the soil during maize cultivation. The goal was to increase soil ammonium nitrogen content and NUE by regulating the relative abundance, enzyme activity, and functional gene expression of N conversion-related soil microbes. Biochar combined with E. cloacae and DCD significantly increased soil N content, and the NUE reached 46.69 %. The relative abundance of Burkholderia and Bradyrhizobium and the activity of nitrogenase increased significantly during biological N2 fixation. Further, the abundance of the nifH gene was significantly up-regulated. The relative abundance of Sphingomonas, Pseudomonas, Nitrospira, and Castellaniella and the activities of ammonia monooxygenase and nitrate reductase decreased significantly during nitrification and denitrification. Moreover, the abundance of the genes amoA and narG was significantly down-regulated. Correlation analyses showed that the increase in soil N2 fixation and the suppression of nitrification and denitrification reactions were the key contributors to the increase in soil N content and NUE. Biochar combined with E. cloacae and DCD synergistically enabled the supplementation, slow release, and retention of N, thus providing adequate N for maize growth. Thus, the combination of biochar, E. cloacae, and DCD is effective for mitigating the irrational application of N fertilizers and reducing N pollution.
