Bioleaching of metal sulfides is an interfacial process comprising the interactions of attached bacterial cells and bacterial extracellular polymeric substances with the surface of a mineral sulfide. Such processes and the associated biofilms can be investigated at high spatial resolution using atomic force microscopy (AFM). Therefore, we visualized biofilms of the meso-acidophilic leaching bacterium Acidithiobacillus ferrooxidans strain A2 on the metal sulfide pyrite with a newly developed combination of AFM with epifluorescence microscopy (EFM). This novel system allowed the imaging of the same sample location with both instruments. The pyrite sample, as fixed on a shuttle stage, was transferred between AFM and EFM devices. By staining the bacterial DNA with a specific fluorescence dye, bacterial cells were labeled and could easily be distinguished from other topographic features occurring in the AFM image. AFM scanning in liquid caused deformation and detachment of cells, but scanning in air had no effect on cell integrity. In summary, we successfully demonstrate that the new microscopic system was applicable for visualizing bioleaching samples. Moreover, the combination of AFM and EFM in general seems to be a powerful tool for investigations of biofilms on opaque materials and will help to advance our knowledge of biological interfacial processes. In principle, the shuttle stage can be transferred to additional instruments, and combinations of AFM and EFM with other surface-analyzing devices can be proposed.
Extracellular polymeric substances (EPS) of Acidithiobacillus ferrooxidans and other
leaching microorganisms mediate the attachment of cells to pyrite and other minerals. They also
play a pivotal role in indirect leaching of base and precious metals via the contact mechanism. The
aim of this study is to get more insight on the influence of the growth substrates iron(II) ions, pyrite,
chalcopyrite and elemental sulfur on EPS formation, attachment and biofilm formation. The
synthesis of EPS by cells of A. ferrooxidans strain A2 is strongely influenced by the growth
substrate or attachment substratum of the cells. Cells grown with soluble iron(II) ions generally
generate less EPS than cells grown with solid pyrite, chalcopyrite or elemental sulfur. Planktonic
cells grown in the presence of solid substrate produce two to four times more EPS than iron(II) ion
grown cells. With sessile cells, this factor is further increased to 50 to 240 depending on the specific
substrate. The EPS of all the differently grown planktonic and sessile cells of A. ferrooxidans strain
A2 contained neutral sugars, fatty acids, uronic acids, proteins and metal ions. The composition of
these compounds varied with the growth substrate and type (planktonic or sessile). The attachment
behavior of cells of A. ferrooxidans strain A2 also differed with the substrate of the pre-culture.
Cells grown on iron(II) ions, pyrite or chalcopyrite attached rapidly to pyrite and chalcopyrite,
while attachment to elemental sulfur was poor. On the contrary, sulfur grown cells attached well to
elemental sulfur but weakly to pyrite and chalcopyrite. Attachment of EPS-free cells to all
substrates was also diminished. Cells of A. ferrooxidans strain A2 cover mineral surfaces with a
dense biofilm after a few days of cultivation, as visualized by fluorescence microscopy and AFM.
Large amounts of EPS are formed, which eventually cover the cells and the mineral surface. Even
after a few weeks of cultivation the biofilm remained monolayered on all substrates.
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