# Assessment of _Bacillus pumilus_ Isolated from Fresh Water Milieu for Bioflocculant Production
## 3 Results and Discussion
### Optimization of Culture Conditions for Biofoculant Production
#### 3.2.5 Effect of Cations on Flocculating Activity of Crude Biofoculant  
The effect of various cations on the activity of the crude biofoculant was investigated and the results are depicted in Figure 5. The stimulating effect of cations in flocculation process is not only dependent on the concentration of the cations added but is also highly dependent on the valence ions [54]; hence, the effect of cations on the flocculating activity was investigated using cations with different valence ions (i.e., monovalent, divalent and trivalent). All the divalent cations tested enhanced the flocculating rate of the biofoculant albeit to varying degrees as follows: Ca\({}^{2+}\) (67.9%), Mg\({}^{2+}\) (74.5%) and Mn\({}^{2+}\) (82.9%) (Figure 5). Among these cations, Mn\({}^{2+}\) gave the highest flocculating activity and was therefore used as a replacement for Ca\({}^{2+}\) in all subsequent experiments. On the other hand, in the previous studies where the flocculating activity was around 80%, the sole carbon source was glucose, whereas, when the flocculating activity was 67.9%, the sole carbon was maltose. This shows that the synergistic effect of cations differs for different biofoculants, which means that the chemical composition of the biofoculant produced from glucose-containing medium differ from the one produced sucrose-containing medium. This might be the reason why a decline in flocculating activity was observed in the biofoculant produced from maltose-containing medium. However, when the biofoculant was produced from optimal culture conditions in the presence of maltose as the carbon source, the flocculating activity was 89.7% with Mn\({}^{2+}\) as the flocculating aid (Figure 5). Furthermore, it was noticed that Na\({}^{+}\), K\({}^{+}\), Fe\({}^{3+}\) completely inhibited the flocculating activity of the biofoculant while Al\({}^{3+}\) stimulated the second highest flocculating activity of 77.2% (Figure 5). The high flocculating activity observed might be due to ionic density of Al\({}^{3+}\) that increases the ionic strength of the suspension, and therefore, the electrostatic repulsive forces among charged groups of the biofoculant chain decrease notably and consequently increases the flocs formation as a result of electrostatic attraction between the biofoculant and the suspended particles. In addition, AlCl\({}_{3}\) is an inorganic flocculant like polyaluminum chloride and ferric chloride (FeCl\({}_{3}\)) that are highly charged and conventionally used in water treatment. The flocculating activity of the biofoculant was enhanced in the presence of Al\({}^{3+}\) and inhibited in the presence of Fe\({}^{3+}\). The enhanced flocculating activity noticed  
Figure 5: Effect of cations on the flocculating activity of the biofoculant. in the presence of Al\({}^{3+}\) might be due to the synergistic effect of Al\({}^{3+}\) with the biofloculant and the poor flocculation observed with Fe\({}^{3+}\) might be due to antagonistic effect of Fe\({}^{3+}\) with the biofloculant thus lead to high flocculating activity for the Al\({}^{3+}\) and poor flocculation in the presence of Fe\({}^{3+}\) when the effects of trivalent cations on the flocculating activity of the biofloculant were compared. Furthermore, it could be safer to say that Fe\({}^{3+}\) might possibly alter the surface charge of kaolin surfaces and cover the adsorb sites [46]. The competition of the positively charged particles and less adsorb sites might induce the antagonist effect of Fe\({}^{3+}\) that resulted into poor flocculation. Moreover, even though both Al\({}^{3+}\) and Fe\({}^{3+}\) are trivalent cations, they belong to different group in the periodic table with different features. For example, Al\({}^{3+}\) has electronegativity of 1.61 and first ionization energies of 577.5 KJ/mol, whereas Fe\({}^{3+}\) has electronegativity of 1.832 and first ionization energies of 762.5 KJ/mol.