In rice paddy soil, biological nitrogen fixation is important for sustaining soil nitrogen fertility and rice growth. Anaeromyxobacter and Geobacteriaceae, iron-reducing bacteria belonging to Deltaproteobacteria, are newly discovered nitrogen-fixing bacteria dominant in paddy soils. They utilize acetate, a straw-derived major carbon compound in paddy soil, as a carbon and energy source, and ferric iron compounds as electron acceptors for anaerobic respiration. In our previous paddy field experiments, a significant increase in soil nitrogen-fixing activity was observed after the application of iron powder to straw-returned paddy field soil. In addition, combining iron application with 60–80% of the conventional nitrogen fertilizer rate could maintain rice yields similar to those with the conventional nitrogen fertilization rate. It was thus suggested that iron application to paddy soil increased the amount of nitrogen fixed in the soil by enhancing nitrogen fixation by diazotrophic iron-reducing bacteria. The present study was conducted to directly verify this suggestion by ¹⁵N-IRMS analysis combined with ¹⁵N-DNA-stable isotope probing of iron-applied and no-iron-applied plot soils in an experimental paddy field. In no-iron-applied native paddy soil, atmospheric ¹⁵N₂ was incorporated into the soil by biological nitrogen fixation, in which diazotrophic iron-reducing bacteria were the most active drivers of nitrogen fixation. In iron-applied paddy soil, the amount of ¹⁵N incorporated into the soil was significantly higher due to enhanced biological nitrogen fixation, especially via diazotrophic iron-reducing bacteria, the most active drivers of nitrogen fixation in the soil. Thus, our previous suggestion was verified. This study provided a novel picture of active nitrogen-fixing microorganisms dominated by diazotrophic iron-reducing bacteria in paddy soil, and directly proved the effectiveness of iron application to enhance their nitrogen fixation and increase the incorporation of atmospheric nitrogen into soil. The enhancement of biological nitrogen fixation in paddy fields by iron application may lead to novel and unique paddy soil management strategies to increase soil nitrogen fertility and ensure rice yields with reduced nitrogen fertilizer input and lower environmental nitrogen burdens.

The application of iron powder stimulated the growth of iron-reducing bacteria as a respiratory substrate and enhanced their nitrogen (N)-fixing activity in flooded paddy soils. High N fertilization (urea) in the flooded paddy soils has caused adverse environmental impacts such as ammonia (NH₃) volatilization, nitrous oxide (N₂O) emissions, and nitrate (NO₃⁻) leaching. This study aims to investigate the effects of N fertilization rates in combination with an iron amendment on rice yields and N losses from flooded paddy fields. We performed a 2-year field plot experiment with traditional rice–wheat rotation in China’s Yangtze River Delta. The investigation consisted of seven treatments, including 100%, 80%, 60%, and 0% of the conventional N (urea and commercial organic manure) fertilization rate, and 80%, 60%, and 0% of the conventional N with the iron powder (≥99% purity) amendment. The rice yields decreased with a reduction in the conventional N fertilization rate, whereas they were comparable after the iron application under the 80% and 60% conventional N rate. The critical N losses, including NH₃ volatilization, N₂O emissions, and NO₃⁻ and NH₄⁺ leaching, generally decreased with a reduction in the conventional N fertilization rate. These N losses were significantly greater after the iron amendment compared with the non-amended treatments under the 80% and 60% conventional N fertilization rate in the first rice-growing season. However, it was comparable between the iron-amended and the non-amended treatments in the second season. Furthermore, NO₃⁻ leaching was the most significant N loss throughout the two rice seasons, followed by NH₃ volatilization. The iron amendment significantly increased soil Fe²⁺ content compared with the non-amended treatments irrespective of N fertilization, suggesting the reduction of amended iron by iron-reducing bacteria and their simultaneous N fixation. A combination of the iron application with 60–80% of the conventional N fertilization rate could maintain rice yields similar to the conventional N fertilization rate while reducing the critical N losses in the flooded paddy field tested in this study. Our study leads to the establishment of novel and practical rice cultivation, which is a step towards the development of green agriculture.