# Assessment of _Bacillus pumilus_ Isolated from Fresh Water Milieu for Bioflocculant Production
## 3 Results and Discussion
### Optimization of Flocculating Activity of Purified Biofleculant
#### 3.7.4 Effect of Cations on the Flocculating Activity  
The effect of cations on the flocculating activity of the purified bioflocculant was investigated and the results are depicted in Figure 12.  
Figure 11: Effect of pH on the flocculating activity of the purified bioflocculant.  
It was found that all tested cations stimulated the biofoculant activity, albeit to varying degrees except for Fe\({}^{3+}\), which only showed a flocculating activity of 21%. The flocculating activities observed with all the other cations were above 70% (Figure 12). The divalent cation Mn\({}^{2+}\) demonstrated the highest flocculating activity (96.5%) for the purified biofoculant when compared with all the other cations tested (Figure 12). The synergistic effect of cations was optimal with Mn\({}^{2+}\), which showed that it helps to neutralize negative charges on the biofoculant and the suspended kaolin particles, shortening the distance between them, increasing the initial adsorption of the biofoculant onto the kaolin particle and thus leading to floc formation and sedimentation [57; 71]. Increasing the number of valence ions in suspension increases the ionic strength, which in turn decreases the electrostatic repulsive forces among biofoculant charged groups. In the event of the above phenomenon, the conformation of the biofoculant becomes altered into a more closely-packed structure which is not able to form bridges [69]. This might account for the reduced flocculating ability of the biofoculant in the presence of Fe\({}^{3+}\) and Al\({}^{3+}\) compared to monovalent and divalent cations. However, when comparing the two trivalent cations, it was evident that the synergistic effect of Al\({}^{3+}\) led to a high flocculating activity compared to the antagonistic effect of Fe\({}^{3+}\). In addition, aluminum salts have, on their own, been used in industries as flocculants [3].  
From our results for the effect of cations on the flocculating activity of both crude and purified biofoculant, we observed that monovalent cations completely inhibited the flocculating activity of the crude biofoculant. The experiment for the effect of cations on the flocculating activity of crude biofoculant was carried out at pH 7 (pH of the kaolin suspension was adjusted to neural). On the other hand, the effect of cations on the flocculating activity of purified biofoculant was carried out at the optimal pH 10 (Figure 11).  
The pH of the reaction mixture in a flocculation process is one of the key factors that determine flocs formation and the stability of suspended particles [72]. Furthermore, this shows that the ionization of the functional groups in the molecular chain of the biofoculant is pH-dependent [73]. It will be safe to assume that the spatial charge arrangements for flocculation were not advantageous for the crude biofoculant with the monovalent cations at pH 7 and that was why the flocculating rates were completely inhibited. Conversely, for the purified biofoculant, the synergistic effect of the monovalent cations was highly enhanced with the monovalent cations at alkaline condition pH 10 (Figure 11). Thus, this is an indication that the biofoculant exhibits different electric states at different pH conditions and consequently affected the flocculating efficiency of both crude and purified biofoculant for kaolin particles.  
Similarly to our findings, [PERSON] et al. [44] also reported that Mn\({}^{2+}\) was the most preferred metal ion for the flocculating activity of the biofoculant produced by _Cobeta_ spp. The biofoculant activity of MBF-3 produced by _Bacillus_ sp. was stimulated by Al\({}^{3+}\), Mg\({}^{2+}\), Ca\({}^{2+}\), K\({}^{+}\), and Na\({}^{+}\), with Al\({}^{3+}\) being the most effective cation [74].  
Figure 12: Effect of cations on the flocculating activity of the purified biofoculant.