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This study is prompted by the high leaching efficiency of Zijinshan copper bio-heap leaching industrial plant. Bioleaching columns with 100 mm diameter and 1 m height were used to investigate copper bioleaching at different operating conditions. Elevated temperature, high total iron concentration and high acidity significantly increased copper leaching rate as determined by solution and residue assays. At 60 °C with 50 g/L iron (initial Fe3+/Fe2+ gram ratio 2.5), pH 1.0 and no aeration, copper extraction was achieved 90% after 60 days. However, at 30°C, 5 g/L total Fe, pH 1.5 and no aeration, copper extraction reached 80% and 85% after 90 and 200 days, respectively. Real-time PCR assay showed that only 105 cells/ml and 2×105 cells/g are in solution and on the ore surface at the condition of 60 °C 50 g/L iron and pH 1.0, respectively. In addition, a similar leaching rate was observed in the tests with and without inoculation. The column without inoculation was directly irrigated with acid mine drainage (AMD). Our results indicate high copper leaching efficiency at extreme conditions for mineral oxidizing bacteria. Inoculation and aeration are not necessary in Zijinshan copper mine bio-heap leaching process.