Members of Leptospirillum genus have emerged not only as one of the most representative bacteria in the Río Tinto ecosystem, but also in other acidic environments (AMD), and in biohydrometallurgical operations. The main objective of this work was to study the role of chemolithoautotrophic bacteria of the genus Leptospirillum in the Río Tinto iron cycle (an extreme acidic environment, characterized by its constant low pH) to better understand and control industrial biohydrometallurgical processes. Different strains of Leptospirillum were isolated from the Río Tinto basin and physiologically and genetically characterized. Certain metabolic capabilities, such as pyrite leaching, iron oxidation and nitrogen fixation, were determined for each strain. Complementary molecular ecology techniques (FISH, CARD-FISH and cloning) were used to study the microbial diversity and the distribution of leptospirilli along the iron gradient in the different phases of Río Tinto: water column, anaerobic sediments and biofilms.