## 2 Materials and Methods
### Biologically Produced Acid-Leading Medium  
A stable acidophilic microbial culture that is capable of producing an acid-leading medium was necessary. This biologically produced medium will be employed as an analytic in electrokinetic studies. In order to stimulate the native microbial population, the soil (tailings) was first incubated in a modified TK medium containing 1 M H\({}_{2}\)SO\({}_{4}\) at a fixed pH of 2 and consisting of (NH\({}_{4}\))\({}_{2}\)SO\({}_{4}\) 0.625 g L\({}^{-1}\), MgSO\({}_{4}\) 7H\({}_{2}\)O 0.625 g L\({}^{-1}\), KH\({}_{2}\)PO\({}_{4}\) 0.625 g L\({}^{-1}\), and FeSO\({}_{4}\)\(\cdot\)7H\({}_{2}\)O 12.45 g L\({}^{-1}\). The flasks were open to air oxygen and maintained at 150 rpm and 30 \({}^{\circ}\)C during the incubation period. For obtaining an acid-leading medium (called a BL medium), aliquots were regularly taken and inoculated into fresh medium until a stable microbial culture capable of oxidizing the iron and sulfur present was formed. Once the stable microbial autochthonous culture was obtained, the acid-leading medium was produced thanks to its biological activity for obtaining a leaching medium capable of oxidizing metal sulfides.  
Metagenomics (Darwin Bioprospecting, Paterna, Spain) was used to characterize the mixed microbial culture. The results showed that _Acidithiobacillus ferriphilus_ (82.1%) and _Acidipililium multivorum_ (11.3%) were the predominant bacterial species, with _Acidibrevivacterium_ sp. (1.5%) and _Acidibacillus ferrooxidants_ (0.7%) being less common.  
Several considerations can be made regarding the use of a leaching medium (referred to as the BL medium in this paper) generated in an external bioreactor to conduct bioleading of the mine tailings. Using the already generated BL medium in an agitated slurry-phase reactor can avoid some drawbacks related to microbial metabolism, such as microbial inhibition caused by increased metal concentrations [21] and potential damage to biofilms attached to solid particles due to mechanical stirring. For in situ bioleaching combined with electrokinetics, previously reported operational problems include challenges in maintaining pH and providing oxygen, as well as temperature increases due to ohmic heating [15; 22].  
\begin{table}
\begin{tabular}{c c} \hline \hline \multicolumn{3}{c}{**Total Metal Concentration (mg kg\({}^{-1}\))**} \\ \hline Fe & 32,045 \\ Cd & 15.3 \\ Zn & 1613 \\ Mn & 555.8 \\ Ni & 41.2 \\ Cr & 45.6 \\ Co & 14.9 \\ \hline \multicolumn{3}{c}{**Other parameters**} \\ \hline pH & 6.03 \\ EC (mS cm\({}^{-1}\)) & 487 \\ Sand (\%) & 5.5 \\ Silt (\%) & 80.0 \\ Clay (\%) & 14.5 \\ Particle size (mm) & 2 \\ \hline \hline \end{tabular}
\end{table}
Table 1: Chemical properties and characterization of the mine tailings.  
These issues can be mitigated by using the previously generated BL medium. Additionally, it should be considered that this leaching medium can be produced through a low-cost biological process. For these reasons, this study selected the use of a previously generated BL medium, which will chemically leach metals without the need to maintain conditions compatible with microbiological activity.