## 3 Results and Discussion
### Zeta Potential  
Zeta potential measures the force of repulsion between suspended particles and the distance that must be covered/overlapped for agglomeration [31]. The zeta potential measurements were used to determine if charge neutralization was the responsible mechanism and to determine the optimal conditions for flocculation using central composite design (CCD). Table 1 shows the zeta potential results under different physicochemical conditions. The zeta potential of kaolin clay decreased with an increase in pH values, i.e., it was \(-19.8\) mV at pH 2.96 and decreased to \(-48.3\) mV at pH 10.04. This resulted from increased electrostatic repulsion force on the negatively charged flocs due to raised charged densities from pH increases. However, the zeta potential increased after the addition of CaCl\({}_{2}\) for all pH conditions because Ca\({}^{2+}\), which is a divalent cation, enhances flocculation activity through charge neutralization and stabilization of negative charges [34]. It was further  
Figure 2: A graphical illustration of changes in flocculation activity of biofoculant, _Bacillus_ spp. D2 (from _Bacillus megaterium_), stored in various conditions over twenty days. noted that adding biofocculant D2 to a suspension of kaolin clay and CaCl\({}_{2}\) decreased the zeta potential's negative charge. This observation confirms that the biofocculant has a negative charge, namely an anionic charge, which, therefore, leads to the observed drop, hypothetically reducing the interfacial double layer (DL). The negative zeta potential of the biofocculant may also be attributed to the presence of functional groups possessing negative charges, as shown by the FTIR analysis. The reduction observed may be attributed to the electrical repulsion resulting from the presence of similarly charged layers between the biofocculants and kaolin clay [35]. This implied that the primary mechanism of flocculation is not charged neutralization since it was shown to occur only after the introduction of a cation. This indicates the involvement of a bridging mechanism, therefore necessitating further investigation by a bonding-type test. Similarly, some biofocculants, such as poly(\(\gamma\)-glutamic acid) (\(\gamma\)-PGA) produced by _Bacillus subtilis_ with a high flocculating activity stimulated by 0.325 mM of Mg\({}^{2+}\), provide an alternative source of biodegradable, eco-friendly Bioflocculant [36]  
Additionally, it should be noted that the zeta potential results do not provide a direct means of determining the optimal conditions for achieving maximal flocculation activity. The results underwent Analysis of Variance (ANOVA) to statistically assess the suitability and significance of the model generated to describe the association between zeta potential, pH and biofocculant dosage. However, it was determined that the model needed to be improved. The results from the ANOVA are shown in Table 2. According to [8] (2019), a \(p\)-value of less than 0.05 indicates statistical significance for the analyzed model or component. The current model had a significance level of 0.0281, indicating its significance. Nevertheless, when evaluating the model's dependability using the coefficient of determination (R\({}^{2}\)), it was determined that there was an insufficient correlation between the projected R\({}^{2}\) and the experimental response. The corrected R\({}^{2}\) value was determined