Abstract:[Background] Efficient bioleaching is closely related to the active iron and sulfur metabolism mediated by microorganisms. Thus, the lack of iron metabolism will restrict low-grade chalcocite bioleaching. [Objective] To improve the bioleaching of low-grade chalcocite by enhancing iron and sulfur metabolism and the "contact". [Methods] The iron-oxidizing Leptospirillum ferriphilum (screened with the double-plate method), iron-reducing Acidiphilium sp., sulfur-oxidizing Acidithiobacillus caldus, and the Fe2+/Fe3+-pyrite-fibrous waste acid hydrolysate system (waste utilization) were combined to improve low-grade chalcocite bioleaching. [Results] A large number of micropores and pits were observed on the slag surface under the scanning electron microscope (SEM), suggesting the active action of the bacteria. Fourier transform infrared (FTIR) spectroscopy revealed that bonds such as N-H, C=O, and O-H were closely related to extracellular polymer substance (EPS), and significantly enhanced absorption peaks of S=O and C-O-S testified more intense sulfur metabolism. Confocal laser scanning microscope (CLSM) manifested that more attached cells and EPS existed in the optimized system, laying the foundation for the "contact" mechanism. After 40 days of leaching, the concentration of planktonic and attached cells increased by 2.51 and 5.73 times, respectively. The maximum specific growth rate (μmax) appeared 1.5-5.3 days earlier, and the highest cupric ion leaching rate reached 67.6%. [Conclusion] The intervention of iron-oxidizing bacteria, iron-reducing bacteria, and exogenous iron-containing substances strengthens the iron and sulfur metabolism of the leaching system and accelerates the dissolution of minerals. The acid hydrolysate promotes iron circulation and the growth of bacteria. The increase in the number of attached cells and secreted EPS enhances the "contact", thus effectively improving the bioleaching microenvironment and efficiency.