AbstractTwenty‐three strains capable of oxidizing iron were isolated from coal and ore storage sites as well as coal and ore mines, volcanic areas, and hot spring. Four strains were found to have high iron‐oxidizing activity. One strain (T‐4) was selected for this experiment since the strain showed the fastest leaching rate of iron and sulfate from pyrite among the four strains. The T‐4 strain was assigned for Thiobacillus ferrooxidans from its cultural and morphological characteristics.Bacterial treatment was applied to column flotation. An increase of cell density in the microbial column flotation resulted in the increase of pyrite removal from a coal‐pyrite mixture (high sulfur imitated coal) with corresponding decrease of coal recovery. The addition of kerosene into the microbial column flotation increased the recovery of the imitated coal from 55% (without kerosene) to 81% (with 50 μL/L kerosene) with the reduction of pyrite sulfur content from 11% (feed coal) to 3.9% (product coal). The kerosene addition could reduce the pyritic sulfur content by collecting the coal in the recovery. However, the addition could not enhance separation of pyrite from the coal‐pyrite mixture, since pyrite rejection was not affected by the increase of the kerosene addition. An excellent separation was obtained by the microbial flotation using a long column which had a length‐diameter (L/D) ratio of 12.7. The long column flotation reduced the pyritic sulfur content from 11% (feed coal) to 1.8% (product coal) when 80% of the feed coal was recovered without the kerosene addition. The long column flotation not only attained an excellent separation but also reduced the amount of cells for desulfurization to as little as one‐tenth of the reported amount. © 1994 John Wiley & Sons, Inc.