## 3 Results and Analysis
### The Impact of Sponge Iron on the Microbial Community Structure
#### 3.3.3 Analysis of Key Functional Bacteria and Their Correlations  
To further explore the impact of SFe addition on the dynamics of the microbial community, a detailed analysis of nitrogen cycle-related functional bacteria in each sample was conducted, and the interrelationships among these functional bacteria were investigated. Figure 8a illustrates the changes in the relative abundances of nitrifying and denittifying bacteria across L1-L4, along with a correlation analysis. The relative abundance of denittifying bacteria is significantly higher than that of nitrifying bacteria in all stages. Notably, during the stable- and final-operation phases of the BSIS system, the relative abundance of denittifying bacteria increased markedly. This enhancement enables a more efficient reduction of nitrates to nitrogen gas, thereby boosting denitrification efficiency. These findings further confirm that the nitrogen removal performance of the reactor with added SFe continues to improve over time. [PERSON] [57] evaluated the effect and mechanism of sponge iron as an inorganic electron donor combined with the WLASI system on  
Figure 7: Microbial analysis at the genus level for each sample (top 20%): (**a**) community structure analysis and (**b**) hierarchical clustering analysis. improving water quality in low C/N water source reservoirs. The study found that this coupled system significantly enhanced the diversity and abundance of aerobic denitrifying microorganisms in the water body, with the relative abundance of key genera such as _Mycobacterium_ and _hgcl_clade_ increasing notably. [PERSON] [3] constructed three biological filters with different fillers--ceramsite (R-C), an iron-carbon mixture (R-Fe-C), and pure iron--carbon (R-Fe)--and found that the addition of iron-carbon material significantly promoted the enrichment of heterotrophic denitrifying bacteria (DNB) and aerobic denitrifying bacteria (HNADB), particularly increasing the relative abundance of HNADB in the R-Fe reactor by 7.3 times, indicating that iron has a significant promoting effect on the growth of HNAD bacteria. The changes in the relative abundance of nitrifying bacteria in L1-L4 are shown in Figure 8b. Across the stages L1-L4, the total relative abundance of nitrifying bacteria was 1.06%, 0.92%, 2.496%, and 1.13%, respectively. In the BSIS system with 90 g/L SFe addition, it exhibited a trend of initial decrease, followed by an increase, and then a subsequent decrease. However, at the end of the operation, the overall relative abundance remained higher than that in the conventional activated sludge system. _Nitrospina_, _Nakamurella_, and  
Figure 8: Changes in relative abundance: (**a**) nitrifying/denitrifying bacteria among samples, (**b**) nitrifying bacteria, and (**c**) denitrifying bacteria. _SMIA02_ are the dominant nitrifying genera. Notably, _Nitrospira_ exhibits the highest relative abundance in all samples, with values of 0.89%, 0.79%, 2.09%, and 1.04%, respectively. _Nitrospira_ is commonly found in various natural environments [58], especially in agricultural soils, lakes, rivers, and underwater sediments, and possesses complete ammonia oxidation capability [45]. _Nakamurella_ is a newly classified taxon under the phylum Actinobacteria, with short, rod-shaped cells and non-pathogenic properties. This genus plays a crucial role in achieving stable denitrification and promoting nitrite oxidation reactions [59]. The relative abundance of nitrifying bacteria in sample L3 is significantly higher than in the other three samples, indicating that even when the abundance of nitrifying bacteria is lower than that of denitrifying bacteria, the addition of SFe can still effectively promote the growth of nitrifying bacteria and accelerate nitrification reactions. However, as the operation process nears its end, the abundance of nitrifying bacteria decreases to the initial level, which may be due to the bio-toxicity of the accumulated iron ions in the reactor, thereby inhibiting their growth.