## 3 Results and Discussions
### Effect of divalent Cations on Alleviation of Salinity Stress  
The augmentation of divalent cations (Ca\({}^{2+}\) and Mg\({}^{2+}\)) relieved the salinity stress of the anammox caused by the IC\({}_{50}\) (5 g NaCl/L) but the mitigation effect differed according to the ion (Figure 2). The rSAA by the augmentation of Ca\({}^{2+}\) increased to 77.9% (0.105 gN\({}_{2}\)/gVSS/day for the SAA at 200 mg Ca\({}^{2+}\)/L) with increasing the Ca\({}^{2+}\) from 30 to 200 mg/L but decreased again to 66.4% (0.084 gN\({}_{2}\)/gVSS/day for the SAA) at 300 mg Ca\({}^{2+}\)/L (Figure 2a). In the case of Mg\({}^{2+}\) augmentation, the rSAA increased slightly to 71% (0.084 gN\({}_{2}\)/gVSS/day for the SAA at 70 mg M\({}^{2+}\)/L) with increasing the Mg\({}^{2+}\) from 20 mg/L to 70 mg/L but decreased to 55% (0.067 gN\({}_{2}\)/gVSS/day for the SAA) at 100 mg Mg\({}^{2+}\)/L. It is known that Ca\({}^{2+}\) above an appropriate level inhibits the energy and the substrate metabolism of anammox bacteria [20], and Mg\({}^{2+}\) addition causes the breakdown of the granules [21]. However, in this study, the saline stress alleviation effect by the Ca\({}^{2+}\) addition was higher than that by Mg\({}^{2+}\). Moreover, the optimal Ca\({}^{2+}\) and Mg\({}^{2+}\) concentrations for alleviating salinity stress were determined to be 200 mg/L and 70 mg/L, respectively.  
Figure 1: Effect of the salt concentration (g NaCl/L) on the specific anammox activity (rSAA, gN\({}_{2}\)-N/gVSS-day) (**a**) and relative specific anammox activity (rSAA, %) (**b**).  
The SEM showed that the salinity stress shrank the anammox granule size and roughed its surface (compare Figure 3a,b). When the cations were added, fewer changes in the granule shape and surface evenness were observed (Figure 3c,d). When the divalent cations were added, the granule size and the surface properties became similar with that of the control without the NaCl treatment (Figure 3e,f). Although both the divalent cations contributed to the alleviation of the salinity stress, a denser granular structure was observed in the granules augmented by Ca\({}^{2+}\) than by Mg\({}^{2+}\). In previous studies, the addition of Ca\({}^{2+}\) (100 mg/L) promoted the secretion of the EPS from the activated sludge [22], and the sludge granulation was enhanced by Ca\({}^{2+}\) (\(>\)150 mg/L) [23]. In addition, Ca\({}^{2+}\) was observed to play a role in a cross-bridge connecting bacteria and the EPS to protect the aggregates of the granules [20]. It is widely known that EPS plays an important role in granulation and substrate transfer, as well as acts as the first barrier against adverse stress [24; 25]. Mg\({}^{2+}\) was known to be essential for phosphodiesterase synthesis to produce bis-(3'-5')-cyclic dimeric guanosine monophosphate, which acted as an important regulator for the anammox activity under unfavorable conditions [26; 27; 28].  
Figure 2: Effect of the Ca\({}^{2+}\) (**a**) and Mg\({}^{2+}\) (**b**) concentrations on relative specific anammox activity (rSAA, %) at 5 g/L of salt concentration.  
Figure 3: SEM images of particles and surface for anammox granules according to cation additions at 5 g NaCl/L: (**a**) The particles of an anammox granule without salt; (**b**) The surface of an anammox granule without salt; (**c**) The particles of an anammox granule at 5 g NaCl; (**d**) The surface of an anammox granule at 5 g NaCl/L; (**e**) The particles of an anammox granule with 200 mg/L Ca\({}^{2+}\) at 5 g NaCl/L; (**f**) The surface of an anammox granule with 200 mg/L Ca\({}^{2+}\) at 5 g NaCl; (**g**) The particles of an anammox granule with 70 mg/L Mg\({}^{2+}\) at 5 g NaCl/L; (**h**) The surface of an anammox granule with 70 mg/L Mg\({}^{2+}\) at 5 g NaCl/L.