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Ore samples from sulfidic mines in Romania were analyzed quantitatively for the occurrence of the ferrous-ion-oxidizing bacteria Thiobacillus ferrooxidans and Leptospirillum ferrooxidans. Thiobacillus ferrooxidans was detected regularly, whereas L. ferrooxidans occurred only at sites with a temperature above 20°C and with sufficient humidity. Accordingly, L. ferrooxidans survived preservation experiments only in nutrient solution with and without pyrite for 56 days at 17°C. Thiobacillus ferrooxidans also survived this period of time in liquid nitrogen, frozen at -70°C with and without protective buffer, and after freeze-drying, although the cell numbers and activity were reduced. Thiobacillus thiooxidans survived all techniques. After growth on ferrous sulfate, the ferrous-ion-oxidizing activity of L. ferrooxidans was about 40% of the activity of T. ferrooxidans. After growth on sulfidic ore, both species exhibited a lower but similar activity. Cells ofL. ferrooxidans exhibited 35% activity and cells of T. ferrooxidans 15%. Laboratory experiments with pure cultures of these lithotrophs and mixed with strains of Acidiphilium sp. demonstrated that chemoorganotrophic microorganisms enhanced only the metabolic activity of L. ferrooxidans. Furthermore, in mixed culture with Acidiphilium sp. cells of L. ferrooxidans formed microcolonies or flocks. Flocks did not appear in pure cultures nor in mixed cultures of T. ferrooxidans and Acidiphilium strains. The significance of this finding is discussed.Considering the porous system of an ore particle in a humid environment, high ferric ion concentration, and a low pH value, the results indicate that L. ferrooxidans is as important as T. ferrooxidans for metal solubilization and thus for microbial leaching.