# A Simple Analysis Method of Specific Anammox Activity Using a Respirometer
## 3 Results and Discussion
### Comparison of rSAA and mSAA According to Temperature and Granule Size  
The reliability and accuracy of the SAA analysis method using a respirometer were examined. The SAA was analyzed using two methods (rSAA and mSAA) according to granule size and temperature. \begin{table}
\begin{tabular}{c c} \hline \hline
**Time (min)** & **SAA (gN\({}_{2}\)/gVSS/Day)** \\ \hline
60 & 0.449 \(\pm\) 0.133 \\ \hline
120 & 0.384 \(\pm\) 0.083 \\ \hline
360 & 0.197 \(\pm\) 0.033 \\ \hline
600 & 0.111 \(\pm\) 0.022 \\ \hline \hline \end{tabular}
\end{table} 
Table 1: Changes in the rSAA value over the reaction time. Figure 2: Changes in nitrogen gas amount (bar) as a function of time (min) and accumulated nitrogen gas amount (line) during the total reaction time. The grey area indicates the standard deviation for accumulated N\({}_{2}\) gas production. Herein, the two methods showed a similar trend according to granule size and temperature. Both rSAA and mSAA showed the highest SAA values at 35 \({}^{\circ}\)C regardless of the granule size, followed by 40 and 30 \({}^{\circ}\)C (Figure 3). The temperature range, in which the presence and activity of anammox bacteria were detected, was very wide (\(-\)5\(-\)80 \({}^{\circ}\)C). However, the optimal temperature for most of the anammox species used in the wastewater treatment was between 30 and 40 \({}^{\circ}\)C [22]. Therefore, the optimal temperature for anammox bacteria is 35 \({}^{\circ}\)C.  
Figure 3: The rSAA and mSAA changes according to the temperature by granular size. (**a**) Granule size \(<\)1 mm, (**b**) granule size 1-2 mm, and (**c**) granule size \(>\)2 mm. The SAA decreased with the increasing granule size and showed a strong negative linear relationship, according to the granule size under each temperature condition (Figure 4). A granular size of <1 mm showed the highest SAA regardless of the temperature, followed by 1-2 and >2 mm. Anammox bacteria with a small granular sludge are highly active due to the fact that the nutrients are quickly and actively supplied to the center of the granules. However, a granular sludge larger than 2 mm had a low SAA. The larger granule size causes extensive tunnels and empty spaces inside the granule, which lead to the low activity and reduction of granule stability [23]. The \(t\)-test and Wilcoxon rank-sum test were performed to determine if the SAA values using the two methods are significantly different. Prior to this step, a Shapiro-Wilk normality test was performed for the SAA value under each condition (Table 2). All of the data appeared to follow a normal distribution since the \(p\)-values resulting from the Shapiro-Wilk normality test were >0.05. All of the datasets showed a normal distribution. However, the \(t\)-test and Wilcoxon rank-sum test were performed together since the number of samples was too small (N = 6). At each condition, the \(p\)-values from the \(t\)-test and Wilcoxon rank-sum test for rSAA and mSAA were <0.05, which indicates that the SAA values using the two methods were similar. Therefore, a respirometer is well-suited for SAA analysis. However, since there is a limit for the evaluation of the process state, it is necessary to use this value as an indirect indicator, along with various factors that can confirm the process state.