Objective The aim was to investigate the phased responses and storage characteristics of soil microorganisms in paddy fields to nitrogen (N) fertilization at different times, and to clarify their dynamic roles in the N cycle of paddy fields, and to further improve the calculation system for N balance in paddy fields.

Method The field microplot experiment was conducted using ¹⁵N isotope tracing to systematically investigate the storage characteristics of basal fertilizer, tillering fertilizer, and panicle fertilizer N in paddy periphytic biofilm, together with the underlying microbial mechanisms.

Result The results showed that N application significantly enhanced periphyton development and biomass accumulation, producing consistently higher biomass than the non-fertilized treatment. Periphyton biomass exhibited a distinct phenological pattern, increasing initially and then declining, with the maximum value occurring at the tillering stage. On this basis, periphyton displayed pronounced stage-dependent differences and rapid retention of fertilizer-derived N: Shortly after fertilization, periphyton rapidly accumulated exogenous N and formed a short-term storage peak, with the strongest retention observed at the tillering stage. Further analysis revealed that this retention was not permanent. Across all fertilization stages, the labeled N followed a dynamic pattern of rapid enrichment after fertilization followed by gradual release. Microbially, periphyton N storage was jointly regulated by direct N assimilation by algae and indirect modulation mediated by parasitic fungi.

Conclusion The uptake and release of fertilizer N by paddy periphytic biofilm exhibit pronounced stage-dependent characteristics. Periphytic biofilm acts as a progressive N sink during the basal and tillering fertilizer stages, but shifts from an "N reservoir" to an "N source" during the panicle fertilizer stage, thereby playing a critical regulatory role in N redistribution within rice paddy ecosystems.