Abstract:The replacement of depressants used in sulfide mineral beneficiation, with bacteria and their metabolites, promises to reduce the environmental impact left by the mining industry. In this study, the attachment of Leptospirillum ferrooxidans, L.f, to chalcopyrite and pyrite was investigated through Scanning Electron Microscopy (SEM). The impact of selective attachment, bacterial growth conditions, and extracellular polymeric substances (EPS) was investigated through bio-flotation. L.f exhibits selective attachment to pyrite between 0 h and 168 h exposure via an indirect contact mechanism. Separation of chalcopyrite from pyrite was achieved through exposing the minerals for 72 h with an L.f culture grown on either HH media, chalcopyrite, or pyrite. The results produced 80.4, 43.4, and 47.4% recovery of chalcopyrite, respectively. However, EPS supernatant extracted from L.f grown on chalcopyrite, conditioned for 48 h, provided the best separation efficiency by the selective depression of pyrite resulting in 95.8% Cu recovery. Polysaccharide-rich EPS selectively attaches to pyrite within 48 h, depressing its floatability and ensuring successful separation with a PIPX collector.