## 4 Discussion
Utilizing EPS produced by bacteria in metal adsorption is one of the most profoundly driven approaches in metal remediation [18]. One of the important parameters for metal adsorption by bacterial EPS is the initial pH of the solution. The pH not only affects the speciation of metal ions in the solution, but also influences the surface charges and dissociation of binding sites of the biosorbent [15]. In the present study, the adsorption was low for both the metal ions at pH 5, while the adsorption increased with an increase of pH values from 5 to 8 (Figure 4a). A low pH is responsible for raising the concentration of hydrogen ions in the solution, which increases the positive charges of functional groups present on the biosorbent surfaces. As a result, an electrostatic repulsion can occur between the positively charged functional groups and the positively charged metal ions. Additionally, a competition between metal ions and hydrogen ions for binding sites on EPS surfaces can also arise, which would result in fewer binding sites available on the surface of EPS to bind the metal ions. At high pH on the other hand, the biosorbent surface becomes negatively charged due to deprotonation of metal binding sites, and consequently the metal ions get electrostatically attracted and adsorbed on the surface [28; 36]. Carboxylic groups play an important role in the pH-responsive protonation-deprotonation reactions as discussed above and participate in the complexation of metal ions resulting in the best adsorption performance at pH 7 [49]. The EPS isolated in the present study showed the presence of carboxyl groups [4], which might be the reason behind the better adsorption of metal ions at pH 7 by the isolated EPS. There are reports showing a constant adsorption of Cu(II) from pH 1-9, while for Zn(II) there was an initial increase in adsorption at initial increase in pH from 1-3 after which up to pH 9 there was a constant adsorption without any change [36]. Similarly, reports showed that there was a constant increase in the amount of Cu(II) adsorbed by EPS from 0.09 mmol g\({}^{-1}\) EPS at pH 3.0 to 0.37 mmol g\({}^{-1}\) EPS at pH 7.0 [28]. Another important parameter for metal sorption by bacterial EPS is the initial concentration of the EPS used. The capacity of sorption of metal ions by EPS in this study reached a constant value when the concentration of EPS was 50 mg L\({}^{-1}\) and it remained constant even at 100 mg L\({}^{-1}\). This can be due to the fact that, an increase of the biosorbent dosage resulted in an increase in the number of sites available for adsorption at lower EPS concentrations. A higher EPS concentration, on the other hand, led to a saturation of binding sites on the biosorbent surface. Higher concentration of EPS could have resulted in interference between the binding sites themselves. Another reason which led to the formation of the plateau region as seen in Figure 4b could be due to the fact that there was an insufficient number of metal ions in the solution with respect to the number of available binding sites [28; 29; 50]. A previous study [28] showed that an increase in EPS concentration from 26 to 135 mg L\({}^{-1}\) resulted in the increase in adsorption of Cu(II), while further increase in EPS concentration failed to increase the adsorption of Cu(II). Similarly, reports showed an increase in adsorption of metal ions with initial increase in EPS concentration while further increase failed to increase the adsorption of metal ions [29; 36]. Optimization of adsorption systems and determination of the efficiency of adsorption is accomplished by equilibrium adsorption isotherms. The Langmuir isotherm postulates the monolayer adsorption on homogenous surfaces, and Freundlich isotherm assumes the multilayer formation on heterogenous surfaces [28]. The model parameter values obtained (Table 3) indicated that the adsorption of metal ions onto the EPS synthesized by _Bacillus licheniformis_ is isolated from the earthworm gut fitted to the Langmuir model better than the Freundlich model.