## 3 Results and Discussion
### Influence of Fe Application on the Diazotrophic Communities in Soil  
Amplicon sequencing of the 16S rRNA gene in the L1,2 and H1,2 fractions was performed to clarify the active nitrogen fixing bacteria in native (Con plot) and iron-applied (Fe plot) paddy soils, particularly to confirm whether the bacteria belonging to the genus _Anaeromyxobacter_ and the family _Geobacteraceae_ were the most active diazotrophs. Soil samples collected on days 2, 4, and 6 were subjected to this analysis to cover a wide variety of diazotrophic bacterial communities containing bacteria with different growth rates. In some cases of qPCR targeting A&G _nifD_ (Figure 2d,k,l), \({}^{15}\)N-incorporated _Anaeromyxobacter_/_Geobacteraceae_ DNA was present in heavier fractions than top-peak fractions of the 16S rRNA gene. Therefore, in addition to using the highest peaks of the 16S rRNA gene (L1 and H1), we performed amplicon sequencing of the L2 and H2 fractions (Figure 2). Bacteria with increased relative abundance in the \({}^{15}\)N treatments compared to the \({}^{14}\)N treatments are shown as a heatmap (Figure 3). In the 16S rRNA gene sequence analysis of Con plot soil, _Geomonas_, _Anaeromyxobacter_, and _Geobacter_, all of which are iron-reducing bacteria, were most frequently detected in \({}^{15}\)N treatments with higher abundance than in \({}^{14}\)N treatments (Figure 3a-c). This result suggests that _Geobacteraceae_ and _Anaeromyxobacter_ are the primary drivers of nitrogen fixation in the native (Con plot) paddy soil. _Anaerolinea_ (days 2, 4, and 6), _Cyanobacteria_ (days 2 and 4), and _Rhodoplanes_ (days 2 and 4) were also frequently detected, suggesting that they are active nitrogen fixers as well. _Cyanobacteria_ are well-known nitrogen-fixing bacteria in paddy soils [6]. Within the genus _Rhodoplanes_, some strains, e.g., _R. roseus_, _R. piscinae_, and _R. elegans_, possess a nitrogenase gene cluster in their genome, while _R._ sp. Z2-YC6860 does not (NPEX01000005.1, NPEW01000030.1, NPEU01000442.1, and NZ_CP007440.1). Thus, some bacteria belonging to _Rhodoplanes_ likely have nitrogen-fixing abilities. On the  
Figure 3: Bacterial community composition based on 16S rRNA gene sequencing at the genera with higher relative abundances in \({}^{15}\)N-labelled DNA(\({}^{15}\)N-Con and \({}^{15}\)N-Fe) than in \({}^{14}\)N-labelled DNA (\({}^{14}\)N -Con and \({}^{14}\)N-Fe) of non-applied native (Con; **a–c**) and iron-applied (Fe; **d–f**) plot samples on day 2 (**a,d**), day 4 (**b,e**) and day 6 (**c,f**) of soil incubation. (U) indicates unclassified bacteria. Data are shown as the average of triplicates. other hand, bacteria belonging to the genus _Anaerolinea_ have rarely been isolated, and only genomic information on _Anaerolinea thermophila_, which does not possess nitrogenase genes, is available in the NCBI database \"[[https://www.ncbi.nlm.nih.gov](https://www.ncbi.nlm.nih.gov)]([https://www.ncbi.nlm.nih.gov](https://www.ncbi.nlm.nih.gov)) (accessed on 14 June 2023)\". Given the presence of a partial nitrogenase gene in the genome of _Anaerolineae_ bacterium isolate ANRL2 (CAG0970598.1), _Anaerolinea_ harboring nitrogenase may be found as the number of isolates increases. In the 165 rRNA gene analysis of Fe plot soil, _Geomonas_, _Anaeromyxobacter_, and _Geobacter_ were most frequently detected, indicating that these iron-reducing bacteria are primary drivers of nitrogen fixation in the iron-applied (Fe plot) paddy soil as well (Figure 3d-f). In addition to these bacteria, _Bacteroides_ spp., which were not detected in Con plot soil, were frequently detected, indicating that they are another primary driver of nitrogen fixation in iron-applied paddy soil. Although the nitrogen-fixing ability of _Bacteroides_ spp. has not been verified, nitrogenase gene clusters are present on the genomes of _Bacteroides graminisolvens_ (BAJS01000002.1), _Bacteroides reticulormitis_ (NZ_BAIV01000007.1), and _B. luti_ (RQTV01000005.1), suggesting diazotrophic ability in some _Bacteroides_ spp.