Isolation and characterization of acidophilic heterotrophic iron-oxidizing bacterium from enrichment culture obtained from acid mine drainage treatment plant

An acidophilic heterotrophic bacterium, designated as HIB4, having the ability to oxidize ferrous ion was newly isolated from a sample of an enrichment culture for iron-oxidizing bacteria, using the modified washed agarose/yeast extract (WAYE) medium with ferrous sulphate. The isolate HIB4 was an acidophilic, heterotrophic, mesophilic and gram-positive bacterium. Phylogenetically, it was classified under the genus Alicyclobacillus and was the closest to Alicyclobacillus disulfidooxidans SD-11 with 99.7% 16S rDNA homology. It grew and oxidized ferrous ion in the medium containing 0.02% (w/v) yeast extract. Yeast extract was an essential substrate for this bacterium because it could not grow or oxidize ferrous ion without yeast extract. However, a higher concentration of yeast extract inhibited the growth of HIB4, so that the optimum concentration of yeast extract for this bacterium to grow was 0.02% (w/v) at 0.08 mol/l of ferrous ion. On the other hand, ferrous ion oxidation occurred almost at the end of the bacterium's logarithmic growth phase and the isolate was able to grow without ferrous ion. These results denote that HIB4 did not obtain any energy from the ferrous ion oxidation and that HIB4 is an obligate heterotrophic and aerobic bacterium even though it oxidized ferrous ion. Also, HIB4 could not utilize any organic compounds, among the several organic chemicals used in this study, as a carbon source except yeast extract. These characteristics were completely different from these of A. disulfidooxidans SD-11 so that HIB4 might be a different species.