## 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
Additionally, according to the analysis in Figure 9a, there is a positive correlation between _Nitrospira_ and _SMIA02_. In contrast, _Nakamurella_ shows a significant negative correlation with both _Nitrospira_ and _SMIA02_, which may be attributed to the inhibitory effect of SFe. The changes in the relative abundance of denitrifying bacteria in L1-L4 are shown in Figure 8c. In L1-L4, the overall relative abundances of denitrifying bacteria are 32.84%, 24.42%, 39.47%, and 43.90%, respectively. As the reactor continues to operate, the abundance of denitrifying bacteria within the BSIS system shows an increasing trend, indicating that SFe has a significant promoting effect on the aggregation of denitrifying bacteria. The increase in iron ion concentration is positively correlated with the enrichment degree of denitrifying bacteria. During this process, the relative abundances of _Saccharimonadales_, _Arenimonas_, and _Acinetobacter_ continue to rise. In particular, _Saccharimonadales_, a typical denitrifying bacterium within the Patescibacteria phylum, shows an abundance proportional to that of denitrification-related genes [50], reaching a relative abundance of 35.07% in the BSIS system at the end of the operation. _Arenimonas_, an autotrophic denitrifying bacterium capable of utilizing iron and reducing nitrates, has a relative abundance of 2.90% in the BSIS system at the end of the operation. _Acinetobacter_, an autotrophic denitrifying bacterium that plays a key role in degrading complex organic matter and removing COD\({}_{\text{Cr}}\), reaches a relative abundance of 0.41% in the BSIS system at the end of the operation. The enrichment of these denitrifying bacteria contributes to the removal of nitrogen in the system. In contrast, _Ferruginibacter_, _Rhodoferax_, _Rhodobacter_, _Defluviimonas_, and _Thauera_ have  
Figure 9: Functional bacteria correlation analysis (**a**) functional bacteria and (**b**) denitrifying bacteria. * _p_\(<=\) 0.05.  
significantly lower relative abundances in the BSIS system compared to the conventional activated sludge system, indicating that these species have poor tolerance to iron ions. _Ferruginibacter_ plays a significant role in denitrification and exhibits strong tolerance to compounds such as Zn(II) [60]. Additionally, _Rhodoferax_ is a typical denitrifying bacterium, widely distributed in both biological nitrogen removal and natural systems, and an increase in its relative abundance facilitates the transformation of NO3-N [61]. _Defluvimonas_ possesses the capability to reduce nitrate to nitrogen gas under aerobic conditions, thereby promoting the occurrence of simultaneous nitrification and denitrification [62]. _Thauera_ represents a denitrifying bacterium frequently encountered within wastewater treatment facilities [63]. The low abundance of these bacteria in the BSIS system also confirms that the iron ion environment may exert a certain selection pressure or inhibitory effect on these species. By analyzing Figure 9a,b, complex correlations among different denitrifying bacteria can be observed. For example, there is a strong positive correlation between _Saccharomyces-adas_ and _Acetobacteraceae_, indicating that these two genera may be interdependent or co-occur under specific environmental conditions. Similarly, a positive correlation exists between _Theromonas_ and _Flavobacterium_, suggesting they may share similar niche strategies or competitive relationships. On the other hand, _Rhodoftermus_ and _Pseudoxanthomonas_ show a significant negative correlation, indicating that they may compete for resources in different environments or inhibit each other in the same environment. The negative correlation between _Hydrogenophaga_ and _Inhella_ may reflect their competitive relationship under specific environmental conditions.