## 3 Results and Discussions
### Effect of Divalent Cations on EPS Production and Compositions  
The EPS can be generally divided into the tightly bound EPS (B-EPS) and the outer-layer soluble EPS (S-EPS) [29]. In this study, the EPS amount and content of the anammox granules were varied by the salinity stress and following the augmentation of divalent cations (Figure 4). Under salinity stress conditions (5 g NaCl/L), the anammox granules (C2) produced more EPS than the normal granules (C1) under the non-salinity stress conditions. The amount of the EPS from the granules under the salinity stress was increased further by adding 200 mg/L of Ca\({}^{2+}\) (C3) and was also increased by 70 mg/L of Mg\({}^{2+}\) (C4). This suggests that effect of divalent cations might be associated with changes in the amount and composition of the EPS. Mg\({}^{2+}\) augmentation increased the production of the EPS more than Ca\({}^{2+}\) augmentation for both the B-EPS and S-EPS. In the anammox granules under all conditions, the amount of the B-EPS was approximately 8-10 times higher than those of the S-EPS. Previous studies have shown similar trends. The amount of the B-EPS was approximately 4-20 times higher than those of the S-EPS in the anammox UASB reactors under saline conditions (2.5-15 mg NaCl/L) with Ca\({}^{2+}\)[30]. Figure 4: PS and PN concentrations, and PN/PS ratio in soluble EPS (**a**) and bound EPS (**b**) anammox granules at C1 (no salt and no cation), C2 (5 g NaCl/L and no cation), C3 (5 g NaCl/L and 200 mg/L Ca\({}^{2+}\)), and C4 (5 g NaCl/L and 70 mg/L Mg\({}^{2+}\)). Both EPSs are composed mainly of PN and PS [31]. Usually, the PS are reported to contribute to the hydrophilicity of the EPS, whereas the PN is the hydrophobic component [25; 32; 33]. The decrease in the PN/PS ratio in the EPS led to poor cell surface hydrophobicity, thereby decreasing the flocculating ability of the granule. The PN/PS ratios in the B-EPS and S-EPS in this study were varied depending on the augmented divalent cations. Those changes in the EPS affect the properties and the aggregation of the anammox granules based on the variation in the PN/PS ratios, which may vary depending on the environmental conditions [34; 35]. In all conditions, the PN/PS ratio of the B-EPS was relatively high, but it was difficult to find a correlation with the SAA. However, in the case of the S-EPS, although the amount of the S-EPS was small, C1 and C3 showed a relatively high PN/PS ratio, which showed the same trend as the variations of the SAA at each condition. As a result of the ANOVA test, it was confirmed that there was a statistically significant difference because the \(p\)-value (0.0086) for the PN/PS ratio of the S-EPS was less than 0.05. Principally, there is a statistically significant difference because the \(p\)-values for the PN/PS ratio between C1 and C2 (\(p\)-value = 0.0277), C2 and C3 (\(p\)-value = 0.0082), and C3 and C4 (\(p\)-value = 0.03072) were less than 0.05. However, the p-value (0.0086) for the PN/PS ratio of the B-EPS was greater than 0.05, indicating that there was no statistically significant difference. Ca\({}^{2+}\) and Mg\({}^{2+}\) would have a difference in the salinity stress alleviation because Ca\({}^{2+}\) and Mg\({}^{2+}\) showed different PN and PS functional groups in the S-EPS. This suggests that the S-EPS plays a more important role in maintaining anammox activity than the B-EPS under saline conditions. In addition, the EPS in microbial aggregates have many functional groups (carboxyl), proteins, carbohydrates, and others). FTIR spectra were analyzed to investigate the detailed variations in the PN and PS of the EPS. The functional groups of the EPS were varied by the condition of the salinity stress and following the divalent cation augmentation (Figure 5). In the case of the B-EPS, the anammox granules under the non-salinity stress (C1) and the granules under the salinity stress augmented by divalent cations (C3 and C4) showed similar patterns of FTIR spectra, while the granules under the salinity stress (C2) showed differences in FTIR spectra.