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To clarify the role and mechanism of Acidithiobacillus ferrooxidans (A. ferrooxidans) in bio-electro-generative-leaching (BEGL), an experiment was made on the electro-generative leaching of chalcopyrite-MnO2 in the presence of the bacteria which grew respectively in Fe(Ⅱ) and S0 media. A dual cell system with chalcopyrite anode and MnO2 cathode was used to study the relationship between time and both of electric quantity and dissolved rate of the two minerals in BEGL. The results show that the dissolved rates for Cu2+ and Fe2+ under the action of the bacteria cultivated by S0 medium are almost 2 times faster than those by Fe(Ⅱ). And the leaching ratio for Mn2+ and the electric output increase by near 3 times. The oxidation residue of chalcopyrite was characterized by SEM and XRD, whose patterns are similar to those of raw ore in BEGL. The mechanism of anodic reaction for CuFeS2-MnO2 leaching in the presence of A. ferrooxidans cultivated by S0 medium is proposed as a successive reaction of two independent sub-processes. The first stage is the dissolution of chalcopyrite to produce Cu2+, Fe2+ and sulfur, and the second stage is bio-oxidation of sulfur, which is the control step of the process. However, dissolution of MnO2 lasts until the reaction of chalcopyrite stops or the ores exhaust in two types of leaching.