## 3 Results and Analysis
### The Impact of Sponge Iron on the Microbial Community Structure  
#### 3.3.1 Analysis of Microbial Community Richness and Diversity  
The Simpson [30] index and Shannon [31] index are important indicators for measuring the diversity of microbial species. An increase in these indices means that the types of microorganisms in the sample are more diverse. The Chao1 index [32] and ACE index [33], on the other hand, are used to reflect the species richness of the microbial community. An increase in their values indicates an improvement in the richness of the microorganisms. Generally, the Chao1 index is significantly affected by the number of low-abundance species, while the ACE index reduces this bias by setting the abundance threshold for rare species to 10. The Alpha diversity indices of mixed-liquor sludge samples from each reactor are shown in Table 1.  
Figure 3: Effects of aerobic starvation on nitrogen removal in each reactor: (**a**) NH\({}_{4}\)\({}^{*}\)-N, (**b**) NO\({}_{2}\)\({}^{-}\)-N, (**c**) NO\({}_{3}\)\({}^{-}\)-N, and (**d**) TN. From Table 1, it can be seen that the Shannon indices for each sludge sample are 8.46 (L1), 7.64 (L2), 8.25 (L3), and 7.32 (L4); and the Simpson indices are 0.99 (L1), 0.97 (L2), 0.98 (L3), and 0.96 (L4), respectively. It is found that both the Shannon and Simpson indices in the L2-3 samples are lower than those in the L1 sample, indicating that the addition of 90 g/L SFe reduces the microbial diversity in the reactor, and microorganisms sensitive to iron ion concentration are eliminated. The L3 sample is higher than the L2 and L4 samples, indicating that after a period of operation, iron bacteria and denitrifying bacteria proliferate in the reactor, increasing microbial diversity. However, by the end of the operation, the excessively high accumulated iron ion concentration in the reactor inhibits the growth and reproduction of microorganisms. Compared with L1, the Chao1 and ACE indices of L2, L3, and L4 increased significantly, indicating that the addition of SFe can effectively promote the richness of microorganisms in the reactor. As shown in Figure 4, the microbial diversity analysis of the four mixed-sludge samples identified a total of 13,327 operational taxonomic units (OTUs). Specifically, the number of unique OTUs in the samples from the conventional activated sludge system (L1) and the bio-iron sponge system (BSIS) during the start-up phase (L2), stable-operation phase (L3), and end-of-operation phase (L4) were 2020, 2936, 4646, and 3725, respectively. Notably, the number of OTUs shared among these four systems was only 210. The phenomenon where the number of unique OTUs was significantly higher than that of shared OTUs revealed significant differences in the microbial community structure among the systems. This result clearly indicates that, although there is limited overlap in microbial composition among different samples, the introduction of iron sponge (SFe) significantly promoted the enrichment and succession of new microbial groups, thereby enhancing the diversity level of the entire microbial community at the OTU level. The analysis results indicate that PCoA can explain 75.9% of the variance in the microbial community. By examining Figure 5a, it can be seen that L3 and L4 are closely adjacent on the coordinate axis, while L1 and L2 are more dispersed relative to L4. This result suggests that in the BSIS reactor, the microbial community structure in the mixed-liquor sludge during the middle (L3) and late (L4) stages of operation exhibits a high degree of similarity in the dimensions examined. In contrast, the microbial community composition of the conventional activated sludge system (L1) shows lower similarity to that of the BSIS system. From Figure 5b, it can be observed that, compared to the proximity of sludge samples (L2 to L4) from the BSIS system with added SFe, the control-group sludge sample without SFe (L1) appears relatively isolated. This phenomenon further reveals significant differences in the microbial communities between the two systems.