<p>Biotechnology translation from laboratory to industrial applications requires scale-up through piloting. This paper describes the fabrication and optimization of a pilot bioreactor system comprising a cascade of three tanks (50 L each) connected in series for continuous bioleaching of sulfidic minerals. Tests were conducted to optimize suspension of solids and gas–liquid mass transfer in the bioreactor vessel, investigating combinations of different agitation speeds, aeration rates, temperatures, and pulp densities. The combination of 400 rpm agitation speed and 2&#xa0;L&#xa0;min⁻¹ air supply achieved optimal solids suspension within the reactor vessel, as well as a gas dissolution rate exceeding oxygen requirement by commonly used bioleaching prokaryotes. However, importantly, the findings indicate that a greater aeration rate (4 to 6&#xa0;L&#xa0;min⁻¹) would be required to maintain sufficient dissolved oxygen levels during biooxidation of sulfides at high pulp densities (&gt; 20%) and temperatures (&gt; 60&#xa0;°C), without adjusting the agitation speed. The current study provides the characterization of a pilot reactor system that will be used for scaling up improved and novel biohydrometallurgical technologies prior to commercialization, addressing major biotechnological and engineering challenges commonly encountered in biomining operations.</p> Graphical Abstract <p></p>