## 3 Results
### Bio-Augmented Activated Sludge
#### 3.2.2 Enhancement of Req-001 to Improve Effluent Quality  
The COD effluent concentration of the control group was 25.3-50.25 mg/L, and the removal rate was 86-92.9% (Figure 7A,D). The COD effluent of the non-biofortified group was 81-172.9 mg/L, and the removal rate was 51.9-77.4%; the COD effluent of the Req-001 group was 50.9-151.2 mg/L, and the removal rate was 57.9-85.8%. The COD removal rate of the Req-001 reactor was 15.3% higher than that of the BPA reactor. In 3-24 h, the activated sludge adapted to the presence of BPA, the removal ability of organic matter recovered, and the COD concentration of the effluent of the BPA group reactor decreased. The COD removal rate of the Req-001 reactor was 6.6% higher than that of the BPA reactor. The results showed that Req-001 had good bio-enhancement ability at the beginning of the experiment, which could effectively improve the organic matter degradation ability of the reactor.  
The control group showed good ammonia nitrogen removal performance, with an effluent concentration between 0.63 mg/L and 1.59 mg/L, and a removal rate above 92.9%. At 1-24 h, the ammonia nitrogen removal efficiency of the effluent from the biofortified reactor was 85.9%, which was 40% higher than that of the non-biofortified reactor (45.9%)  
Figure 6: Abundance of biofortified Req-001.  
Figure 7: Req-001 bioaugmentation reactor effluent indicators. (**A**) Chemical oxygen demand; (**B**) Ammonia nitrogen; (**C**) Total phosphorus (TP); (**D**) Chemical oxygen demand removal rate; (**E**) Ammonia nitrogen removal rate; (**F**) Total phosphorus removal rate. Control: Activated sludge; Not Biofortified: Addition of bisphenol A (BPA) sludge; Biofortified: Addition of BPA + Req-001 sludge.  
(Figure 7B,E). On the second day, the Req-001 group of reactors exhibited better ammonia nitrogen removal capacities, with removal efficiencies of 18.3-40.4%. On the third day, the removal rate of ammonia nitrogen in the Req-001 group of reactors increased by 8.8% compared with the BPA reactor. The results showed that the addition of bio-enhanced strain Req-001 reduced the ammonia nitrogen content in the effluent of the bio-enhanced reactor, and the toxic effect of BPA on activated sludge was weakened. Req-001 could improve the ammonia nitrogen removal ability of activated sludge under BPA stress, and could achieve good ammonia nitrogen strengthening ability in a short time (24 h). The TP concentration in the effluent of the control group was between 0.63-1.58 mg/L. In the non-biofortified group reactor, the TP removal rate on the first day was 41.2% (Figure 7C), while the biofortified group reactor in the same period showed good phosphorus removal performance at the beginning (61.9%), and reached 70.5% after 24 h. Phosphorus removal from the biofortified reactor remained stable on the second day, ranging from 65.1-74%. At 24 h on the third day, the phosphorus removal rate of the biofortified reactor was 84.7%, which was lower than the phosphorus removal ability of the activated sludge of 92%. Compared with the non-biofortified group of reactors, Req-001 weakened the functional effect of BPA on activated sludge and improved the phosphorus removal capacity of the activated sludge system by 22.6%. The results showed that Req-001 could restore the removal of ammonia nitrogen and phosphorus from sludge under BPA stress.  
This effect was due to the multi-pathway metabolism of _Rhodococcus_ and the existence of gene homologues. Req-001 has a strong metabolic capacity and can effectively degrade BPA [69]. Req-001 has a good bio-enhancement ability in that the bio-enhanced bacteria improve the toxicity tolerance of activated sludge to BPA. In addition, because of the removal of BPA in a short time, it contributes to the bioremediation of BPA activated sludge damage. Req-001 improved the removal capacity of the reactor organic substrate, weakened the influence of BPA on the activated sludge, and thus restored the denitrification and phosphorus removal function of the activated sludge in the reactor.