# A Simple Analysis Method of Specific Anammox Activity Using a Respirometer
## 2 Materials and Methods
### Batch Test  
A batch test was performed with a working volume of 36 L and was conducted in two steps. In step 1, the granules that were not classified by size were used. Additionally, gas production was measured until all of the reactions were completed to determine the optimal reaction time for SAA analysis using the respirometer (BRS-100, EETech Co., Chuncheon, Korea) (Figure 1). In step 2, the experiments were conducted according to the granule size (\(<\)1, 1-2, and \(>\)2 mm) and temperature (30, 35, and 40 \({}^{\circ}\)C). Moreover, the granules were separated using a sieve for each size.  
Herein, N\({}_{2}\) gas production was calculated using two methods based on the actual measured gas (\(\Delta_{n}N_{2}\)) using a respirometer (Equation (2)). In addition, theoretical gas production (\(\Delta_{m}N_{2}\)) was calculated from the actual consumed ammonium and nitrite concentrations based on the following stoichiometry equation (Equations (3) and (4)):  
\[\Delta_{r}N_{2}(molN_{2}/min)=\frac{P\cdot\Delta N}{R\cdot T\cdot\Delta t} \tag{2}\]  
\[\Delta_{m}N_{2}(molN_{2}/min)=\frac{1.02\cdot(n\Delta A)\cdot V_{L}}{M\cdot \Delta t}\text{ at }\frac{the\text{(1 atm); \(\rm V_{L}\) is the volume of liquid (0.1 L); M is the molecular weight of nitrogen gas (28 g/mol); and \(n\) is the \(\rm N_{2}\) gas modifying factor based on the stoichiometry equation (Equation (1)), which is one when the ratio of removed \(NO_{2}\)\({}^{-}\)-\(N\) divided by the removed \(NH_{4}\)\({}^{+}\)-\(N\) is \(\geq\)1.32, and 1.32 when the ratio of removed \(NO_{2}\)\({}^{-}\)-\(N\) divided by the removed \(NH_{4}\)\({}^{+}\)-\(N\) is \(<\)1.32.  
SAA was calculated from the \(\rm N_{2}\) gas production rate divided by the biomass concentrations (gVSS/L) in the vial (Equations (5) and (6)):  
\[\rm rSAA\ (gN_{2}/gVSS/day)=\frac{\Delta rN_{2}\cdot M\cdot f}{\rm{XV_{L}}} \tag{5}\]  
\[\rm mSAA\ (gN_{2}/gVSS/day)=\frac{\Delta mN_{2}\cdot M\cdot f}{\rm{XV_{L}}} \tag{6}\]  
where rSAA is the SAA calculated based on the \(N_{2}\) gas production rate using a respirometer; mSAA is the SAA calculated based on the \(N_{2}\) gas production rate from the actual removed ammonium and nitrite; \(\rm X\) is the biomass concentration (gVSS/L); M is the molecular weight of nitrogen gas (28 g/mol); \(f\) is the time modifying factor (1440 min/day); and \(\rm V_{L}\) is the volume of liquid (0.1 L).  
All of the batch experiments (3 gVSS/L) were performed in duplicate under a thermostatic chamber maintained at 35 \({}^{\circ}\)C, except for step 2 (On-lab Co., Busan, Korea). The reactors were carried out under a complete mixing condition using a stirrer at 120 rpm. Before the experiments, each reactor was purged with argon gas for 10 min to remove the oxygen and ensure anaerobic conditions in the reactor. The composition of the medium was as follows: 100 mg/L \(NH_{4}\)\({}^{+}\)-\(N\), 100 mg/L \(NO_{2}\)\({}^{-}\)-\(N\), 1.010 g/L NaHCO\({}_{3}\), 0.055 g/L KH\({}_{2}\)PO4, 0.005 g/L CaCl\({}_{2}\):5H\({}_{2}\)O, 0.5 g/L MgSO\({}_{4}\):7H\({}_{2}\)O, 0.010 g/L FeSO\({}_{4}\).7H\({}_{2}\)O, 0.005 g/L EDTA.