Elemental analysis of extracellular polymeric substances and granules in chalcopyrite bioleaching microbes

The composition and distribution of elements within extracellular polymeric substances (EPS) and intracellular granules in three species of sulphur and/or iron oxidising microbes were analysed to determine their roles in chalcopyrite oxidation and microbial physiology. Three strains of Acidithiobacillus ferrooxidans, Leptospirillum ferrooxidans, and the archaeon Metallosphaera hakonensis were grown on chalcopyrite (CuFeS2) concentrate as an energy source. Separate cultures of A. ferrooxidans and M. hakonensis were also grown on sulphur and ferrous iron. Whole microbes were examined using energy-filtered transmission electron microscopy (EFTEM), TEM energy dispersive X-ray spectroscopy (EDS) in scanning TEM (STEM) mode, and electron energy-loss spectroscopy (EELS). These methods did not detect iron in the EPS of A. ferrooxidans, as has been previously detected with other methods. However, Perlʹs Blue stain precipitated on the EPS of chalcopyrite grown A. ferrooxidans and L. ferrooxidans indicating the presence of Fe3+. No precipitates were present on Perlʹs Blue-stained A. ferrooxidans grown on sulphur. All three species had intracellular granules containing phosphorous and when iron was present in the culture medium it was also present in granules. EELS indicated that iron in granules was Fe3+, suggesting that it is not stored as an energy source. Microbes slowly lost iron from granules when switched to an iron-free medium. Cells grown in the presence of iron often had a speckled appearance, and STEM EDS indicated that those speckles contained iron and phosphorous. These findings suggest that phosphorous may bind to iron to reduce intracellular concentrations of iron ions. In addition, microbes sometimes contained silica on their EPS and cell wall. Si concentrations have been shown to affect bioleach performance, so element deposition on the microbial catalyst may be a reason for this.