## 3 Results and Discussion
### Zeta Potential
\begin{table}
\begin{tabular}{c c c c c c} \hline \hline \multicolumn{3}{c}{**Conditions**} & \multicolumn{3}{c}{**Zeta Potential (mV)**} \\ \hline
**pH** & **Bioflocculant (\% \(\
u\)/\(\
u\))** & **Kaolin** & **Kaolin/CaCl\({}_{2}\)** & **Kaolin/CaCl\({}_{2}\)/Biofloculants** \\ \hline
2.96 & 2 & \(-19.8\) & \(-4.64\) & \(-16.9\) \\
4 & 1 & \(-31.7\) & \(-11.1\) & \(-20.4\) \\
4 & 3 & \(-31.7\) & \(-11.1\) & \(-21.6\) \\
6.5 & 0.59 & & & \(-21.0\) \\
6.5 & 2 & \(-41.2\) & \(-20.3\) & \(-22.6\) \\
6.5 & 3.41 & & & \(-21.4\) \\
9 & 1 & & & \(-21.1\) \\
9 & 3 & \(-44.0\) & \(-17.9\) & \(-20.6\) \\
10.04 & 2 & \(-48.3\) & \(-19.7\) & \(-21.2\) \\ \hline \hline \end{tabular} NB: In kaolin clay samples and Kaolin clay CaCl\({}_{2}\), biofocculants were not added.  
\end{table} 
Table 1: Zeta potential results. \begin{table}
\begin{tabular}{c c c c c c} \hline \hline
**Source** & **Sum of Squares** & **df** & **Mean Square** & **F-Value** & \(p\)**-Value** & \\ \hline Model & 22.97 & 5 & 4.59 & 5.05 & 0.0281 & significant \\ A-pH & 4.18 & 1 & 4.18 & 4.59 & 0.0694 & \\ B-Biofloculant dosage & 0.2002 & 1 & 0.2002 & 0.2200 & 0.6533 & \\ AB & 0.7225 & 1 & 0.7225 & 0.7937 & 0.4026 & \\ A\({}^{2}\) & 17.26 & 1 & 17.26 & 18.96 & 0.0033 & \\ B\({}^{2}\) & 1.74 & 1 & 1.74 & 1.91 & 0.2094 & \\ Residual & 6.37 & 7 & 0.9103 & & \\ Lack of Fit & 6.37 & 3 & 2.12 & & \\ Pure Error & 0.0000 & 4 & 0.0000 & & \\ Corr. Total & 29.34 & 12 & & & \\ \hline \hline \end{tabular} R\({}^{2}\) = 0.7828, adjusted R\({}^{2}\) = 0.6277, predicted R\({}^{2}\) = \(-0.5442\).  
\end{table} 
Table 2: Analysis of variance (ANOVA). to be 0.6277, while the anticipated R\({}^{2}\) value was found to be \(-0.5442\). This discrepancy exceeds the established threshold of 0.2, as [37] (2022) specified, indicating that the model was inadequate. The second order model was established in terms of coded factors expressing the relationship between zeta potential, pH and biofocculant dosage. This is shown by Equation (2), while the depiction of the response is shown in Figure 3.  
\[Zeta\ potential(\mathrm{mV})=-22.60-0.7226A-0.1582B+0.4250 AB+1.57A^{2}+0.5000B^{2} \tag{2}\]  
In addition, the three-dimensional quadratic model surface plot underscores the significance of pH as a determining factor in the modulation of zeta potential, as seen by the asymmetrical distribution observed in the graph. Given the unsatisfactory findings of the zeta potential analysis in establishing the optimal circumstances for maximal flocculation activity, an alternative approach was taken by observing formed flocs under a light microscope, as seen in Figure 4.  
The optical microscope images revealed that the kaolin clay particles had a minimum and dispersed distribution before introducing any other substances. The suspension or dispersion of kaolin clay particles in a solution is due to their negative charge, leading to an electronic double layer [38]. The addition of CaCl\({}_{2}\) resulted in a modest increase in density, which may be attributed to the process of charge neutralization. The occurrence of flocculation is evident in the subsequent images after the introduction of the D2 biofocculant. Larger, more compact aggregates were seen to have developed. The flocs exhibited a discernible shape in contrast to the aggregation of particles induced by CaCl\({}_{2}\), providing more evidence that the flocculation process involves charge neutralization followed by bridging [39]. Figure 3: Graphical illustration of zeta potentials’ relationship with changes in pH and biofocculant dosage. Furthermore, it was apparent from the visual representations that the most favorable aggregation of flocs occurred at a pH level of 6.5, accompanied by a dose of 2% (\(v/v\)). Under such circumstances, the flocs exhibit a moderate size while the dose remains within an optimal range, avoiding excessive or insufficient amounts. Upon examination of the minimum dose, namely 0.59% (\(v/v\)) at pH 6.5, it became evident that the flocs exhibited a diminutive size and poor density.