Since the first isolation of the well-known iron oxidizer Acidithiobacillus ferrooxidans various media and techniques have been developed to isolate new species of acidophilic iron-oxidizing bacteria. A successful strategy in many cases was the use of iFeo medium in double-layer plates with a heterotrophic strain in the underlayer. However, even with samples which had been shown by molecular techniques to be dominated by "Ferrovum myxofaciens" and Gallionella-related bacteria, these bacteria were isolated considerably less frequently than Acidithiobacillus spp. on iFeo. Therefore, a new medium was designed which corresponded largely to the chemical composition of the mine water in a treatment plant dominated by the bacterial groups mentioned and was called artificial pilot-plant water (APPW). The analyses of approximately 500 colonies obtained from mine waters of two different sampling sites by PCR with primers specific for Acidithiobacillus spp., "Ferrovum" spp., Gallionella relatives, and Acidiphilium spp. revealed higher abundances of "Ferrovum" spp. and Gallionella relatives on the newly designed APPW medium than on iFeo which favored Acidithiobacillus spp. Molecular analysis of the colonies obtained indicated the occurrence of at least two species of iron-oxidizing bacteria and/or the heterotrophic Acidiphilium spp. in most of the colonies. Furthermore, the influence on the isolation of the concentrations of iron, phosphate, and ammonium of APPW, in levels of the iFeo medium previously described was studied.
Keywords: Acid mine water; Gallionella relatives; Iron-oxidizing bacteria; Isolation; Overlay plates; “Ferrovum”.