740 often hindered by geopolitical and social constraints [7,15]. However, conventional hydrometallurgical recovery remains limited by high operational costs and secondary effluent generation [5,15]. Microbial bioleaching offers a sustainable alternative for metal recovery from low-grade residues [5,12]. This process utilizes acidophilic chemolithotrophic microorganisms to catalyze the oxidation of reduced iron and sulfur, generating reactive lixiviant agents that release target metals [3,11]. Bioleaching systems are increasingly recognized for their low carbon footprint, reduced energy consumption, and compatibility with in situ or column-based processing [12,15,16]. Despite these advantages, tailings bioleaching is often constrained by slow initial kinetics, low indigenous microbial populations, and suboptimal physicochemical conditions [9,10,11,14]. Consequently, recent research has focused on optimized microbial inoculation strategies using pre-adapted consortia to reduce lag phases and enhance sulfide oxidation rates [9–11,13]. Successful inoculation depends on timing, microbial composition, and the hydrodynamic conditions within the system [11,14,18] among main factors. Furthermore, integrated approaches such as acid curing are employed to neutralize acid consumption and facilitate microbial establishment [8,15]. Within this context, the present study evaluates various inoculation strategies for the bioleaching of cobalt-bearing sulfidic tailings. The primary objective is to determine how specific inoculation approaches influence metal solubilization efficiency, particularly regarding cobalt recovery. Through column bioleaching experiments and systematic monitoring, this work aims to develop optimized biotechnological strategies for tailings valorization, supporting sustainable mining practices and environmental stewardship [12,15,16,18]. 2. MATERIALS AND METHODS 2.1 Microbial consortium acquisition and selection To identify microbial consortia suitable for bioleaching, microbiological scouting was conducted at the San José de Pucobre flotation plant (Copiapó, Chile). Samples collected from various stages of the flotation process served as initial inoculum for batch cultures. These cultures were incubated at 30, 45, and 55 °C in KMD media (0,99 g/l (NH4)2SO4, 0,15 g/l NaH2PO4*2H2O, 0,1 MgSO4*7H2O, 0,021 g/l CaCl2*2H2O, 0,05 g/l KHPO4*2H2O, supplemented with 4 g/l FeSO4*7H2O or/and 0,1% S°) to evaluate microbial growth alongside iron and sulfur-oxidizing activities. Growth and metabolic activity were observed exclusively at 30 and 45 °C, while no microbial activity was detected at 55 °C. Out of 11 samples analyzed, six exhibited metabolic activity compatible with biomining processes. Based on these results, two consortia were selected for further biohydrometallurgical experimentation: Kobold A (30 °C) and Kobold B (S) DSM 35389 (45 °C), (henceforth referred to as Kobold B). Additionally, cell viability assays were performed for these consortia under optimal growth conditions and in the presence of "Cola Scavenger" mineral residues. 2.2 Taxonomic characterization Taxonomic characterization of both consortia was performed by 16S rRNA gene sequencing, using the 16S Barcoding Kit 1-24 (SQK-16S024) protocol and the Oxford Nanopore Technologies (ONT) platform. 2.3 Sampling and characterization of mineral residue Two independent sampling campaigns were conducted to obtain the "Cola Scavenger" fraction from the cleaning circuit (CS-CL) at the San José de Pucobre flotation plant. In the first campaign, 60 kg of material were collected and processed via quartering, primary and secondary crushing, and subsequent dry homogenization. For the second campaign, 150 kg of fresh sample were obtained and subjected to the same
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