We report the application of confocal laser scanning microscopy CLSM and Raman spectroscopy on the (bio)chemical oxidation of pyrite and chalcopyrite, in order to understand how surface sulfur species ( ) affects biofilm evolution during mineral colonization by Acidithiobacillus thiooxidans. We found that cells attachment occurs as cells clusters and monolayered biofilms within the first 12？h. Longer times resulted in the formation of micro- and macrocolonies with variable cell density and higher epifluorescence signal of the extracellular polymeric substances (EPS), indicating double dynamic activity of A. thiooxidans: sulfur biooxidation and biofilm formation. Raman spectra indicated consumption modification during biofilm evolution. Hence, cell density increase was primarily associated with the presence of ; the presence of refractory sulfur species on the mineral surfaces does not to affect biofilm evolution. The EPS of the biofilms was mainly composed of extracellular hydrophobic compounds (vr. gr. lipids) and a minor content of hydrophilic exopolysaccharides, suggesting a hydrophobic interaction between attached cells and the altered pyrite and chalcopyrite. 1. Introduction Sulfide minerals (SMs) are the main source of base metals (e.g., Fe, Ni, Cu, Zn, and Pb) in the world. Most of SMs are semiconductors, and, in their crystalline phase, the orbitals of the atoms (sulfur and metal) form electronic bands with different energy levels; the highest fully occupied electron energy levels form the valence band. For SM as pyrite (FeS2), the valence bands are orbitals from the metal atoms, while the valence bands of other SMs as chalcopyrite (CuFeS2) are derived from both metal and sulfur orbitals [1]. In chalcopyrite, the Fe3+ ion and the protons (H+) can remove electrons from the valence band; thus, chalcopyrite is soluble in acid. In contrast, pyrite is acid insoluble, as Fe3+ is its main oxidizing agent at high and low pH, as it has been described by Sand et al. [1] and Schippers and Sand [2]. These authors concluded in these reports that the mechanism and chemistry of SM oxidation are determined by such electronic structure as well as the acid solubility. In both reports, the same authors also proposed the corresponding dissolution mechanisms for acid soluble and acid insoluble SMs, defined as polysulfide and thiosulfate (bio)oxidative pathways, respectively. Accordingly, the oxidation of an acid insoluble SM proceeds via the thiosulfate mechanism by means of electron extraction, by the indirect attack of hydrated Fe(III) ions. The main reduced
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