## 3 Results and Analysis
### The Impact of Sponge Iron on the Microbial Community Structure
#### 3.3.2 Analysis of the Microbial Community Structure  
The analysis of the microbial structure and hierarchical clustering of the top 10% relative abundance at the phylum level in the conventional activated sludge system and the BSIS system with an SFe dosage of 90 g/L (initial operation L2, stable operation L3, and final operation L4) is shown in Figure 6. The results indicate that there are differences in the microbial community composition between the conventional activated sludge system and the BSIS system, suggesting that the addition of SFe can alter the microbial community structure at the phylum level. The microbial community composition of the BSIS system during the stable-operation phase (L3) and the final-operation phase (L4) is highly similar, which is consistent with the conclusions of the beta diversity analysis. In L1-L4, the top ten phyla by relative abundance include _Proteobacteria_, _Patescibacteria_, _Bacteroidetes_, _Chloroflexi_, _Acidobacteria_, _Actinobacteria_, _Nitrospirae_, and some other phyla with relatively low abundance. Figure 4: Venn diagram of each sample. Figure 5: Beta diversity analysis. (**a**) Principal coordinate analysis (PCoA) and (**b**) non-metric multidimensional scaling (NMDS) analysis. _Proteobacteria_ has the highest relative abundance in all samples, accounting for 44.35% in the stable conventional activated sludge system (L1), and 57.35%, 47.52%, and 43.50% in the BSIS system sludge during the initial-operation phase (L2), stable-operation phase (L3), and final-operation phase (L4), respectively. L2 and L3 increased by 13.00% and 3.17% compared to L1, while L4 was slightly lower by 0.85% compared to L1. _Proteobacteria_ is an important genus in the process of denitrification and nitrite reduction [34], including various nitrogen-fixing, nitrifying, and denitrifying bacteria [35], which can participate in carbon and nitrogen cycles and decompose organic macromolecules such as carbohydrates and proteins [36]. The lipopolysaccharidees on the surface of _Proteobacteria_ are beneficial for microbial attachment and growth and drive activated sludge flocculation [37], playing an important role in biological nitrogen and phosphorus removal and pollutant degradation [38]. In the initial-operation phase of the BSIS system, the relative abundance of _Proteobacteria_ showed an upward trend, which may be due to the fact that iron ions play a vital role in the life cycle of microorganisms, and low concentrations can promote the growth and reproduction of _Proteobacteria_. In the stable-operation phase and final-operation phase of the BSIS system, the relative abundance of _Proteobacteria_ began to decline, indicating that with the increasing concentration of iron ions, it is not conducive to the enrichment of _Proteobacteria_, and there was even an inhibitory trend in the final-operation phase. _Patescobacteria_ is the second dominant bacterium in all samples. This bacterium has a synergistic effect with key genes nifH and amoA in the nitrogen cycle and can participate in the ammonia oxidation process [39] with relative abundances of 15.76%, 11.41%, 21.05%, and 33.29%, respectively. [PERSON] [39] found that _Patescibacteria_ often coexist with denitrifying bacteria in groundwater. [PERSON] [40] found that _Patescibacteria_ were detected in partial nitrification-anammox systems participating in the ammonia oxidation process, and are a typical phylum of the carbon cycle, with a synergistic effect with nitrogen cycle genes nifH and amoA [41]. In the stable-operation phase and final-operation phase of the BSIS system, the relative abundance of _Patescibacteria_ increased, indicating that the addition of SFe is beneficial to the growth and reproduction of _Patescibacteria_. _Bacteroidetes_ is the third dominant bacterium in all samples, with relative abundances of 23.60%, 19.91%, 10.18%, and 7.76%, respectively. _Bacteroidetes_ can participate in protein decomposition and play an important role in organic matter degradation [42].