## 4 Conclusions  
This study analyzed the toxic effects of BPA on the structure and function of activated sludge microbial flora, and the mechanism and reasons for the toxicity of BPA in activated sludge. These effects and mechanisms were analyzed from the perspective of changes in microbial populations, and a recovery strategy was proposed using Req-001 to improve activated sludge functions under BPA stress. The main research conclusions were as follows:  
(1) BPA reduced the concentration and biomass of activated sludge, and the biomass of activated sludge decreased significantly with the increase of BPA concentration. BPA led to an increase in SVI, a decrease in the secretion and accumulation of EPSs, an increase in the ratio of polysaccharide to protein, and a deterioration in the flocculation and settling properties of activated sludge.  
(2) The toxic effects of BPA will reduce the species diversity and richness of microorganisms. In this study, 50 mg/L of BPA increased the abundance of Proteobacteria in activated sludge to 99.7%. Bacteria including _C. koreensis_, _A. bohemicus_, and _B. bullata_ could maintain high abundances under the stress of high concentration of BPA, and were potential BPA-resistant strains. DHA, COD digestion, and the water purification function were all reduced, and the activated sludge functions of the degradation of organic matter, flocculation, and nitrogen and phosphorus removal were all inhibited.  
Figure 8: Bisphenol A (BPA) residues in biofortified sequencing batch reactors (SBR).  
(3) After bioaugmentation by Req-001, the BPA removal rate of activated sludge was increased by 15%, the COD digestion rate was 6.6% higher, and the removal rates of ammonia nitrogen and phosphorus were increased by 8.8% and 22.7%, respectively. In the actual sewage treatment process using bio-augmentation technology, the results indicate that it is necessary to add enhanced strains many times to ensure the long-term persistence of beneficial bacteria in the treatment system.  
The results indicated that the functional recovery ability of the sludge system and the high removal ability of Req-001 make the latter a promising specie for use in BPA bioremediation. These results provide a theoretical basis for a comprehensive understanding of the impact of BPA on sewage systems and activated sludge community structure. Furthermore, the study provides potential biotechnological strategies for the application prospects of strain Req-001 for bioremediation, and has important implications for the bio-augmentation of damaged sludge during wastewater treatment.  
The following supporting information can be downloaded at: [[https://www.mdpi.com/article/10.3390/app12168026/s1](https://www.mdpi.com/article/10.3390/app12168026/s1)]([https://www.mdpi.com/article/10.3390/app12168026/s1](https://www.mdpi.com/article/10.3390/app12168026/s1)). Figure S1: SBR reactor design diagram and flow chart. A: BPA Toxicity Test Reactor; B: Bio-augmented Experimental Reactor; C: SBR Reactor Flow Chart. Figure S2: Each sample OTU-Venn drawn by different stages. The number of overlapping parts between multiple color graphs is the total number of OTUs among multiple samples, and the non-overlapping part is the number of unique OTUs of each sample. Table S1: Simulated sewage reagent content. Table S2: Trace element composition content.  
Conceptualization, K.T.; Data curation, F.M.; Formal analysis, S.L.; Funding acquisition, H.L.; Investigation, Y.Y.; Methodology, Q.Q.; Project administration, M.C.; Writing--original draft, J.S.; Writing--review & editing, H.H. All authors have read and agreed to the published version of the manuscript.  
This research was funded by National Natural Science Foundation of China (No. 51978132).  
Not applicable.  
Informed Consent Statement: Informed consent was obtained from all subjects involved in the study.  
The authors declare no conflict of interest.