## 3 Results and Discussion
### Influence of Fe Application on the Quantity of 16S rRNA and nifD Genes in Fractionated DNA  
In the soil incubated with \({}^{15}\)N\({}_{2}\), nitrogen-fixing microorganisms incorporate \({}^{15}\)N, and their \({}^{15}\)N-labeled DNA becomes heavier than \({}^{14}\)N-(\({}^{15}\)N-unlabeled) DNA. CsCl gradient ultracentrifugation was used to separate \({}^{15}\)N-labeled heavy DNA from \({}^{14}\)N-labeled light DNA based on their different densities.  
In the analysis of Con plot soil DNA, the relative abundance of the 16S rRNA gene for the \({}^{14}\)N\({}_{2}\)-treated samples reached its maximum value at a density of around 1.71, while for the \({}^{15}\)N\({}_{2}\)-treated samples, apparent peak shifts toward heavier densities were observed at all incubation periods (Figure 2a-c), indicating that atmospheric nitrogen was incorporated by bacteria. To confirm whether the genus _Anaeromyxobacter_ and family _Geobacteraceae_ (A&G) were involved in \({}^{15}\)N incorporation, qPCR was performed targeting the nitrogen fixation genes (_nifD_) derived from these bacteria in each fraction. Due to the large amount of pseudo-_nifH_ present in soil environments [35], the _nifD_ gene, a more reliable marker than the _nifH_ gene, was used in this study. The relative abundances of the A&G _nifD_ showed  
Figure 1: The amount of \({}^{15}\)N incorporated in iron-applied (Fe) and non-applied native (Con) plot paddy soils. Data are presented as means \(\pm\) standard deviations (SD) from triplicate tests. A different letter above the bar indicates a significant difference.  
a peak shift similar to that of the 16S rRNA gene in all samples (Figure 2d-f), suggesting the incorporation of \({}^{15}\)N by bacteria belonging to the genus _Anaeromyxobacter_ and family _Geobacteraceae_ in native (Con plot) paddy soils.  
In the analysis of Fe plot soil DNA, peak shifts of the relative 16S rRNA gene (Figure 2g-i) and A&G _nifD_ (Figure 2j-l) abundance toward heavier fractions were also observed in all \({}^{15}\)N\({}_{2}\)-treated samples compared to corresponding \({}^{14}\)N\({}_{2}\)-treated samples. These results indicate atmospheric nitrogen fixation by bacteria, including those in the genus _Anaeromyxobacter_ and the family _Geobacteraceae_, in the Fe plot soil as well. Peak shifts in both the 16S rRNA gene and A&G _nifD_ qPCR were greater in Fe plot soil DNA analysis than in Con plot soil DNA analysis, suggesting increased nitrogen fixation by bacteria,  
Figure 2: Relative abundance of the 16S rRNA gene (**a–c,g–i**) and A&G _(Anaeromyxobacter_ and _Geobacteraceae_) _nifD_ (**d–f,j–l**) in fractionated DNA derived from non-applied native (Con) plot paddy soils (\({}^{14}\)N-Con and \({}^{15}\)N-Con) on day 2 (**a,d**), day 4 (**b,e**), and day 6 (**c,f**) and iron-applied (Fe) plot paddy soils (\({}^{14}\)N-Fe and \({}^{15}\)N-Fe) on day 2 (**g,j**), day 4 (**h,k**), and day 6 (**i,l**) of soil incubation. The open and closed circles show \({}^{14}\)N-treated and \({}^{15}\)N-treated samples, respectively. L1, L2, H1, and H2 indicate the samples chosen for amplicon sequencing. Data are shown in triplicate.  
particularly by the genus _Anaeromyxobacter_ and the family _Geobacteraceae_ in iron-applied (Fe plot) paddy soil.