Detection of Acidithiobacillus ferrooxidans in acid mine drainage environments using fluorescent in situ hybridization (FISH)

An important microorganism of acid mine drainage (AMD) and bioleaching environments is Acidithiobacillus ferrooxidans which oxidizes ferrous iron and generates ferric iron, an oxidant. Most investigations to understand microbial aspects of sulfide mineral dissolution have focused on understanding physiological, metabolic, and genetic characteristics of A. ferrooxidans. In this study, a 16S rRNA oligonucleotide probe designated S-S-T.ferr-0584-a-A-18, and labeled at the 5'-end with indocarbocyanine dye (CY3), was used in a fluorescent in situ hybridization (FISH) procedure on pure cultures of nine isolates of A. ferrooxidans. These isolates were recovered from acid mine drainage and mining environments. The probe was also used to detect cells of A. ferrooxidans, recovered from AMD samples, growing on FeTSB and FeSo solid media in a FISH procedure. In addition, the presence of cells of A. ferrooxidans in an environmental water sample from an AMD site in Copper Cliff, Ontario, Canada was analyzed using the FISH technique. Probe specificity was first confirmed with A. ferrooxidans ATCC 19859 (positive control) and Acidithiobacillus thiooxidans ATCC 19377, Acidiphilium acidophilum ATCC 27807, and Lactobacillus plantarum ATCC 8014 (negative controls). Positive and negative control cells were also used to determine optimal stringency conditions for hybridizations with the probe. Cells of the nine isolates of A. ferrooxidans stained positive, although the fluorescent signal varied in intensity from isolate to isolate. Colonies of A. ferrooxidans from the environmental water sample of the AMD site were recovered only on FeTSB solid medium after 22 days of incubation. The probe was able to detect cells of A. ferrooxidans in a FISH procedure. However, no cells of A. ferrooxidans were detected in the AMD water sample without cultivation. Thus, probe S-S-T.ferr-0584-a-A-18 hybridized effectively with cells of A. ferrooxidans recovered from pure cultures but failed to directly detect cells of A. ferrooxidans in the AMD site.