Identification of bacteria recovered from acid mine environments by reverse sample genome probing (T. acidophilus, T. thiooxidans, Leptospirillum ferrooxidans, Thiobacillus ferrooxidans)

Acid mine drainage is an extreme environment. However, observations of the microbiota indicate that a variety of microorganisms can exist in such an environment. To eliminate acid mine drainage, and also to improve commercial bioleaching, it is imperative to identify the species of bacteria present in such environments. Reverse sample genome probing (RSGP) is a technique recently described for the identification of bacteria by DNA hybridization. The purpose of this investigation was (1) to isolate environmental strains of Thiobacillus ferrooxidans, T. acidophilus, T. thioaxidans and Leptospirillum ferrooxidans and (2) microbiotato apply RSGP to their identification in acid mine drainage samples. Environmental strains of T. ferrooxidans and T. acidophilus were isolated and purified by repeated plating and single-colony isolation on iron salts and tetrathionate media, respectively. Different media were used to isolate T. thiooxidans, namely, thiosulfate, tetrathionate and sulfur media. For the isolation of Leptospirillum ferrooxidans , iron salts and pyrite media were inoculated with environmental samples. However, L. ferrooxidans was never recovered on solid media. Type cultures and environmental strains of T. ferrooxidans, T. acidophilus, T. thiooxidans, and L. ferrooxidans were used as standards and it was shown that RSGP was able to distinguish them. Environmental samples were analyzed by RSGP after enrichment in different liquid media. Ferrous sulfate medium supported the growth of T. ferrooxidans only, whereas all thiobacilli grew in sulfur medium, T. thiooxidans being dominant. Growth in glucose followed by growth in tetrathionate resulted in the selection of T. acidophilus. The selected microorganisms were identifiable by RSGP. DNA was also extracted directly (without enrichment) from cells recovered in acid mine drainage and sediments and analyzed by RSGP to describe the communities present in such environments. A better hybridization pattern was obtained when the environmental sample was prefiltered.