Trace Element Bioavailability Shapes Bacterial Communities and Biological Nitrogen Fixation in Paddy Soils

Biological nitrogen fixation (BNF) is a key nitrogen source in paddy soils, yet its spatial variability remains poorly understood. In this study, we conducted a 90-day 15N2 labeling experiment using soils from seven paddy fields in Zhejiang Province, China, to quantify total nitrogen fixation and fixed-N accumulation in soil layers and plant tissues and to characterize bacterial communities using the 16S rRNA gene sequencing method. Total BNF varied from 3.73 ± 0.39 to 9.95 ± 0.88 kg N ha−1, with ZH4 showing an almost 2.7-fold greater BNF rate than ZH7, the site with the lowest BNF rate. Our results indicated that the subsurface layer (1–15 cm) was the predominant BNF zone, with a 6.5-fold greater soil N fixed rate and the strongest site dependent variation. Available molybdenum and organic matter were strongly correlated with total BNF (r = 0.879, p < 0.009), while bulk density and available manganese showed strong positive associations. Redundancy analysis showed that organic matter and available manganese jointly explained 60.1% of bacterial community variation. In comparison, the second model explained 64.4% of community variation and identified plant-fixed nitrogen as a significant explanatory variable. Overall, these findings indicate that bioavailability of micronutrients and subsurface processes influence BNF more strongly than microbial diversity alone.