Bioleaching of coal's pyrite is a promising option to reduce sulphur content of high sulphur coals from an economical, technical and environmental point of view. In this research, a mixed culture of acidophilic iron- and sulphur-oxidizing mesophilic microorganisms including Acidithiobacillus ferrooxidans, Acidithiobacillus thiooxidans and Leptospirillum ferrooxidans was used to remove pyritic sulphur from the high sulphur coal of Mehr Azin, Tabas, Iran. The influence of various parameters, namely nutrient medium type (Norris and 9 K), initial pH (1, 1.5 and 2) and the addition of ferrous sulphate on the pyritic sulphur removal and ash content of coal was investigated using an orthogonal array L-18 Taguchi design. Shake flasks experiments were carried out in pulp density of 5% (w/w), particle size of smaller than 500 mu m, and a stirring rate of 150 rpm at 35 degrees C for 30 days. Analysis of variance (ANOVA) was used to determine the effects of the variables on sulphur and ash reduction from the coal. The maximum sulphur removal (50.3% total sulphur) was obtained at the initial pH of 1, the ferrous sulphate addition of 0.02 M and Norris nutrient medium. Total sulphur and ash content values were decreased from 3.87% to 1.92% and 25.72% to 11.6%, respectively. SEM/EDS and XRD analyses showed that a high level of sulphate and jarosite precipitations could be occurred at the initial pH of 2 when 0.05 M ferrous iron addition and 9 K nutrient medium were used. (C) 2014 Elsevier Ltd. All rights reserved.