One of the main characteristics of the microorganisms used in the leaching process is their capacity to adapt to aggressive environments, characterized by a notable presence of heavy metals. In this study the adaptation of the strains Acidithiobacillus ferrooxidans, Acidithiobacillus thiooxidans and Leptospirillum ferrooxidans was evaluated on a sphalerite concentrate from mining waste. In the adaptation tests, the energy source (ferrous sulphate) was gradually replaced by percentages of mineral pulp, ending with subcultures without the addition of an external energy source. The results show that the strains A. ferrooxidans and A. thiooxidans are more resistant to high concentrations of sphalerite, compared to the strain of L. ferrooxidans, since, in the case of this strain, it was necessary to repeat some tests (8% of pulp), since a deficient development was evident. This was associated with factors such as the decrease of the Fe+2 energy source, the increase of the pulp density, the accumulation of toxic metals and secondary products of the dissolution of minerals and the increase of the pH.