## 4 Discussion
showed that this strain produced a maximum of 83.45% flocculation activity at 12 mg L\({}^{-1}\) flocculant concentration [11]. Another essential parameter for flocculation is the system pH. In the present study, the flocculation efficiency showed pronounced activity in a basic medium, with the maximum activity at pH 11 (Figure 3b). Biofloculants exhibit varying degree of electrical states at varying pH. Alteration of pH alters the charge status on bioflecculant which in turn alters the surface characteristics of suspended materials. This results in a change of the flocculation ability of the flocculants [41; 42]. The present study found conformity with other reports where alkaline pH favored bioflecculation of suspended particles [37; 38; 43]. The present study failed to flocculate the suspended kaolin particles at an acidic pH below 6. The increase in flocculation activity at higher pH than at lower pH could be due to the fact that at higher pH the OH\({}^{-}\) ions might disrupt the formed complexes between kaolin and the bioflecculant, while lower pH might cause the adsorption of H\({}^{+}\) ions by both the bioflecculant and kaolin particles leading to reduced flocculation activity [44]. These results were supported by other reports where bioflecculant produced by _Bacillus megaterium_ strain caused flocculation in the pH range of 7 to 12 with maximum flocculation at pH 9 and acidic pH inhibited flocculation [45]. Another important parameter for flocculation is the presence of cations. Cations neutralize and stabilize negative charges of functional groups of both kaolin particles and the biofleculant [38]. In the present study, the effect of varying concentrations of CaCl\({}_{2}\) on the flocculation activity showed that maximum activity was observed at 2% salt concentration (Figure 3c). Here too, the flocculation activity was better at pH 11 than low pH values. The flocculation activities against various cations are shown in Table 2 indicating that flocculation efficiency was more in the case of divalent cations. The maximum value was for Ca\({}^{2+}\) ions. Divalent cations neutralize negative charges on both the kaolin particles and bioflecculant, which minimizes the repulsive forces, thereby enhancing adsorption of bioflecculant onto the surface of the kaolin clay leading to agglomeration of flocs and sedimentation of the suspended particles [45]. This result was supported by other reports where divalent cations enhanced flocculation while monovalent ions failed to cause effective flocculation [37; 38]. In spite of being a trivalent cation, Fe\({}^{3+}\) failed to increase the bioflecculation activity in the present study. This could be due to the fact that there was an antagonistic effect between Fe\({}^{3+}\) and the bioflecculant. Fe\({}^{3+}\) could have caused an alteration in the charge of kaolin surfaces and at the same time covered the reactive adsorption sites. Consequently, a competition between the positively charged particles and the adsorption sites might have occurred causing an antagonistic effect of Fe\({}^{3+}\) ions and subsequent decrease in flocculation activity [46]. The result was in accordance with other reports [47] where Fe\({}^{3+}\) showed least or no flocculation activity. The experiment on the effect of temperature on the flocculation ability of EPS showed that the EPS was thermally stable (Figure 3d). Flocculation activity was retained even when the EPS was exposed to a temperature of 100 \({}^{\circ}\)C for 30 min. The result was akin to other reports where thermal stability of EPS and retention of flocculation activity were shown [38; 40; 48]. The flocculation activity was higher at pH 11 than at pH 7 even at a varying temperature. Reports suggested that flocculants rich in carbohydrate backbone showed greater thermal stability than those of protein and nucleic acid backbones [38]. The FTIR and NMR analyses of the EPS in our studies showed the presence of carbohydrate in the EPS backbone, thus confirming the thermal stability of the EPS.