741 preparation protocol. Cobalt concentrations were determined using atomic absorption spectroscopy (AAS). 2.4 Mineralogical and granulometric characterization Mineralogical characterization of both CS-CL sample batches was conducted using TESCAN Integrated Mineral Analyzer (TIMA) software, supplemented by X-ray Diffraction (XRD) analysis for the initial batch. These analyses were performed to quantify the abundance of sulfide phases and gangue minerals, as well as to determine the degree of target mineral liberation. The granulometric profile of both batches was established by quantifying the distribution of valuable species within individual mineral particles. Particles were binned according to their valuable species content into 10% class intervals, spanning from <10% to ≥90%, and these classes were subsequently used to evaluate the particle size distribution. 2.5 Biohydrometallurgical tests in flasks As a preliminary stage, bioleaching tests were performed in flasks, using 0,5X KMD medium supplemented with Fe(II) and a pulp concentration of 20% (w/w) of Cola Scavenger. A total of six flasks were evaluated under conditions with and without microbial inoculation: M1 (uninoculated, 30 °C), M2–M3 (inoculated with Kobold A, 30 °C), M4 (uninoculated, 45 °C), and M5–M6 (inoculated with Kobold B, 45 °C). The experiments were conducted under agitation and temperature control, pH, Eh (redox potential with Ag/AgCl electrode) and Cobalt recovery was systematically monitored throughout the duration of the test. 2.6 Bioleaching tests in minicolumns Following the batch assays, five bioleaching campaigns were conducted using 30-cm-high minicolumns to evaluate cobalt recovery under varying operational parameters. This experimental phase aimed to simulate percolation conditions while assessing the influence of key variables on leaching performance. The following parameters were systematically evaluated: - Microbial Consortia: Performance comparison between the Kobold A and Kobold B consortia. - Operating pH and Temperature: Systems were maintained at pH levels of 1.0 or 1.5, and temperatures of 30 °C or 45 °C. - Acid Curing: The mineral was subjected to chemical pre-treatment at 60% and 80% acid curing levels to neutralize gangue and promote initial metal mobilization. - Inoculation Dynamics: Inoculum concentrations ranging from 1×10⁶ to 1×10⁸ cells·g⁻¹ of total solids were tested. - Inoculation Methodology: Two inoculation modes were compared: delivery via irrigation and application immediately following the acid-curing resting period. Physicochemical variables, including pH, Eh, and dissolved metal concentrations (cobalt, nickel, copper, and iron), were monitored periodically to characterize the kinetic behavior and overall recovery efficiency of the system. 3. RESULTS 3.1 Microbial consortia To isolate an optimal microbial consortium for the bioleaching process, a comprehensive scouting was performed at the San José de Pucobre flotation plant. Samples were retrieved from diverse operational points within the flotation circuit and subsequently cultivated in batch systems at 30 °C, 45 °C, and 55 °C. Microbial growth was confirmed at 30 °C and 45 °C (Table 1), whereas no growth was observed at 55 °C. Of the 11 samples evaluated, six exhibited robust iron and/or sulfur oxidation activity. Based on these results, two distinct cultures were established: Kobold A (30 °C) and Kobold B (45 °C).
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