Adaptation of Penicillium simplicissimum with different heavy metals present in a spent hydrocracking catalyst, as well as one-step, two-step, and spent medium bioleaching of the spent catalyst by the adapted fungus, was examined in batch cultures. Adaptation experiments with the single metal ions Ni, Mo, Fe, and W showed that the fungus could tolerate up to 1500 mg/L Ni, 8000 mg/L Mo, 3000 mg/L Fe, and 8000 mg/L W. In the presence of multi-metals, the fungus was able to tolerate up to 300 mg/L Ni, 200 mg/L Mo, 150 mg/L Fe and 2500 mg/L W. A total of 3% (w/v) spent catalyst generally gave the maximum extraction yields in the two-step bioleaching process (100% of W, 100% of Fe, 92.7% of Mo, 66.43% of Ni, and 25% of Al). The main lixiviant in the bioleaching was shown to be gluconic acid. The red pigment produced by the fungus could also possibly act as an agent in Al leaching. (C) 2010 Elsevier Ltd. All rights reserved.