In the present work, key parameters in copper bioleaching from chalcopyrite have been investigated at long-term operation. In detail, the type of mixed microbial consortium (origin and adaptation); the composition of two mineral media (the growth medium and the modified 9K medium); its buffer capacity by the buffers HCl/KCl and Na2HPO4/KH2PO4; and the influence of different ore grades in relation with the potential alkalinity associated have been investigated. For the first time, a mixed microbial consortium, obtained from a gas-phase biotrickling filter treating high loads of H2S, was employed revealing significant copper extraction by biological leaching. Results reveal that a single adaptation step of this biomass improved both kinetics and process efficiency, nearly doubling the amount of copper obtained compared with the non-adapted consortium. Nevertheless, the growth medium also influences the efficiency of the bioleaching process, enhancing copper extraction at higher sulfate concentration. The ore containing the metal is also a determining factor, obtaining same copper extraction for biotic and abiotic in one case, and enhancing up to 50 times from the abiotic in the other. Thus, this becomes a relevant limitation for the applicability of bioleaching for some ores, mainly due to the composition of the matrix.