## 3 Results and Discussion  
### Overall Performance of PN1 and PN2  
PN2 achieved a stable nitritation reaction with an NAE above 90% at an influent NH4\({}^{+}\)-N concentration of 50 mg/L, compared to the PN1 reactor (Figure 1). PN1 was operated with a mainstream wastewater-level influent NH4\({}^{+}\)-N concentration, maintaining an FA concentration of 10 mg/L. The oxygen inflow was adjusted to keep the DO concentration below 0.5 mg/L to induce nitrification over the initial 5 days. During this period, the effluent NH4\({}^{+}\)-N, NO2\({}^{-}\)-N, and NO3\({}^{-}\)-N concentrations were 11.5 mg/L, 1 mg/L, and 39.9 mg/L, respectively. These values indicate that most NH4\({}^{+}\) was oxidized to NO3\({}^{-}\) rather than accumulating as NO2\({}^{-}\). Following this initial period, PN1 continued to operate with an ARE of 77.1% and an NAE below 5%. This demonstrates that NOB was not sufficiently suppressed, preventing stable nitritation.  
PN2 was started with an influent NH4\({}^{+}\)-N concentration of 300 mg/L, influenced by an FA at 50 mg/L. This concentration was gradually reduced to the mainstream wastewater-level (NH4\({}^{+}\)-N 50 mg/L, FA 10 mg/L). After a 5-day period of starting up nitrification, PN2 showed an effluent NH4\({}^{+}\)-N concentration of 23.4 mg/L, NO2\({}^{-}\)-N of 279.7 mg/L, and NO3\({}^{-}\)-N of 7.6 mg/L, achieving an NAE of 98.3%. As the influent NH4\({}^{+}\)-N was gradually reduced to 200 mg/L and 100 mg/L, the NAE remained high, at 98.8% and 96.5%, respectively. When the influent NH4\({}^{+}\)-N was further reduced to 50 mg/L, the effluent concentrations were as follows: NH4\({}^{+}\)-N at 3.0 mg/L, NO2\({}^{-}\)-N at 47.3 mg/L, and NO3\({}^{-}\)-N at 3.9 mg/L; at this state, the ARE was 96.1% and an NAE was 95.1%. The inhibition of NOB  
\begin{table}
\begin{tabular}{c c c c} \hline \hline
**Primer** & **Sequence (5\({}^{\prime}\)-3\({}^{\prime}\))** & \multicolumn{2}{c}{**PCR Conditions**} \\ \hline  & & \(\bullet\) & Initial denaturation step: 3 min at 95 \({}^{\circ}\)C. \\ V3F & CCTACCGGAGGCAGC & \(\bullet\) & PCR steps (25 cycles) \\  & & \(\bigcirc\) & denaturation: 30 s at 95 \({}^{\circ}\)C; \\  & & \(\bigcirc\) & annealing: 30 s at 55 \({}^{\circ}\)C; \\ V4R & GGACTACHVGGGWTCTAAT & \(\bigcirc\) & extension: 30 s at 72 \({}^{\circ}\)C. \\  & & \(\bullet\) & Final extension step: 5 min at 72 \({}^{\circ}\)C. \\ \hline \hline \end{tabular}
\end{table}
Table 2: PCR parameters for amplifying the V3-V4 regions of the bacterial 165 rRNA gene sequence.  
due to the high FA levels in PN2 effectively promoted stable accumulation of NO2-, which maintained the nitritation reaction even as the influent NH4+-N concentration decreased. In comparison, a previous study reported that carriers subjected to an FA shock of 1068 mg/L in a moving bed biofilm reactor (MBBR) achieved stable nitritation after 60 days with an influent NH4+-N concentration of 63 mg/L [29]. This resulted in effluent concentrations of 12 mg/L for NH4+-N, 49 mg/L for NO2--N, and 0.2 mg/L for NO3-N. This study confirms that a high FA strategy is advantageous for suppressing NOB, as evidenced by the stable nitritation observed in PN2 following an initial shock of high FA.  
After 50 days of operation, the activities of AOB and NOB in reactors PN1 and PN2 were evaluated through batch tests (Figure 2). The activity of AOB was similar in both reactors, with PN1 at 0.43 \(\pm\) 0.042 gN/gVSS/d and PN2 at 0.40 \(\pm\) 0.005 gN/gVSS/d. In contrast, the activity of NOB was significantly higher in PN1 at 1.32 \(\pm\) 0.059 gN/gVSS/d, compared to 0.01 \(\pm\) 0.018 gN/gVSS/d in PN2. The PN2 reactor, influenced by 50 mg/L of FA, was more favorable for stably enriching AOB that accumulate NO2-.  
Figure 1: Variation in concentrations of nitrogen compounds, ammonium removal efficiency (ARE), and nitrite accumulation efficiency (NAE) during nitritation enrichment: (**a**,**c**) PN1 reactor; (**b**,**d**) PN2 reactor.  
Figure 2: Activity of AOB and NOB on day 50 under influent NH4+-N of 50 mg/L.