Microorganisms were enumerated and isolated on selective solid media from a pilot-scale stirred-tank bioleaching operation in which a polymetallic sulfide concentrate was subjected to biologically accelerated oxidation at 45°C. Four distinct prokaryotes were isolated: three bacteria (an Acidithiobacillus caldus -like organism, a thermophilic Leptospirillum sp., and a Sulfobacillus sp.) and one archaeon (a Ferroplasma -like isolate). The relative numbers of these prokaryotes changed in the three reactors sampled, and the Ferroplasma isolate became increasingly dominant as mineral oxidation progressed, eventually accounting for >99% of plate isolates in the third of three in-line reactors. The identities of the isolates were confirmed by analyses of their 16S rRNA genes, and some key physiological traits (e.g., oxidation of iron and/or sulfur and autotrophy or heterotrophy) were examined. More detailed studies were carried out with the Leptospirillum and Ferroplasma isolates. The data presented here represent the first quantitative study of the microorganisms in a metal leaching situation and confirm that mixed cultures of iron- and sulfur-oxidizing prokaryotic acidophiles catalyze the accelerated dissolution of sulfidic minerals in industrial tank bioleaching operations. The results show that indigenous acidophilic microbial populations change as mineral dissolution becomes more extensive.

Two novel extremely acidophilic, iron-oxidizing actinobacteria were isolated, one from a mine site in North Wales, UK (isolate T23^T), and the other from a geothermal site in Yellowstone National Park, Wyoming, USA (Y005^T). These new actinobacteria belong to the subclass Acidimicrobidae, and in contrast to the only other classified member of the subclass (Acidimicrobium ferrooxidans), both isolates were obligate heterotrophs. The mine site isolate was mesophilic and grew as small rods, while the Yellowstone isolate was a moderate thermophile and grew as long filaments, forming macroscopic flocs in liquid media. Both isolates accelerated the oxidative dissolution of pyrite in yeast extract-amended cultures, but neither was able to oxidize reduced forms of sulfur. Ferrous iron oxidation enhanced growth yields of the novel mesophilic actinobacterium T23^T, though this was not confirmed for the Yellowstone isolate. Both isolates catalysed the dissimilatory reduction of ferric iron, using glycerol as electron donor, in oxygen-free medium. Based on comparative analyses of base compositions of their chromosomal DNA and of their 16S rRNA gene sequences, the isolates are both distinct from each other and from Acidimicrobium ferrooxidans, and are representatives of two novel genera. The names Ferrimicrobium acidiphilum gen. nov., sp. nov. and Ferrithrix thermotolerans gen. nov., sp. nov. are proposed for the mesophilic and moderately thermophilic isolates, respectively, with the respective type strains T23^T (=DSM 19497^T=ATCC BAA-1647^T) and Y005^T (=DSM 19514^T=ATCC BAA-1645^T).