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Elective culture of Acidithiobacillus ferrooxidans and Acidithiobacillus thiooxidans in 9K medium modified with pyrrhotite was studied. Bioleaching of flotation concentrate of sphalerite by the selected bacteria was carried out. The results show that the microorganisms cultured by pyrrhotite are a mixture of Acidithiobacillus ferrooxidans and Acidithiobacillus thiooxidans, of which the capability to oxidize ferrous to ferric irons is enhanced by the high mass ratio of Fe to S in pyrrhotite. Three pyrrhotite samples were separated into various parts with corresponding S/Fe ratios by magnetic separation and were used to culture the elective bacteria as the substrate. The association of the cultures could provide a more rapid and complete oxidation of sphalerite than that of bacteria cultivated by conventional methods.