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The alterations of surface properties of pyrrhotite after biological conditioning with Leptospirillum ferriphilum and Acidithiobacillus caldus were evaluated by zeta-potential, adsorption and contact angle measurement.Research showed that pyrrhotite isoelectric point (IEP) after interaction with bacterial cells shifted towards cells isoelectric point, indicating the adsorption of cells on chalcopyrite surface.Due to the formation of element sulfur on pyrrhotite surface treated with A.caldus, which showed by SEM and energy spectrum analysis, the surface hydrophobicity of pyrrhotite increase at the late stage of bioleaching, but the surface hydrophobicity of pyrrhotite treated with L.ferriphilum decreased during bioleaching.Although the adsorption density of A.caldus on pyrrhotite was higher than that of L.ferriphilum, L.ferriphilum with strong ability to oxidize ferrous ion showed better leaching efficiency than A.caldus with strong ability to oxidize sulfur for pyrrhotite leaching.The results demonstrated that Fe ion play more important action on microorganisms bioleaching ofpyrrhotite.