Due to the characteristics of simple process, environmental friendliness and low operating costs, biometallurgy has become a popular technology for metals recovering from low-grade ores and tailings. In order to enhance the efficiency of bioleaching functional bacteria acquisition, the 9K agar was optimized by adjusting the ratio of two solutions to achieve better and faster solidification for the functional bacteria growth and isolation. By using the improved 9K agar, six functional stains within genera of Acidithiobacillus ferriphilus, A. ferrooxidans and Leptospirillum ferrooxidans were isolated from the enrichment of acid mine drainage. After the Fe2+ oxidation ability evaluation, three strains of WT1-1, XT2-2, and YT3-1 within the three genera were selected and employed as the individual inoculum for the bioleaching from polymetallic sulfide tailings. Eventually, a maximum leaching efficiency of 58.37% Cu, 53.14% Al, 80.09% Mg, and 76.95% Zn were observed by A. ferriphilus WT1-1 after 28 d. To further improve the bioleaching efficiency, the three strains were mixed proportionally as the inoculum in both one-step and two-step bioleaching processes. Comparing to the pure cultures, the leaching efficiencies of Cu and Mg were significantly enhanced in both one- and two-steps, while no significant change in Zn. By comparing the one- and two-step processes, leaching efficiencies of Al, Mg, and Zn were higher in the one-step process, whereas Cu was observed to be higher in the two-step process. Therefore, the selection on leaching process of one or two steps should be determined based on tailings composition and target metals.
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