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In the present study, we applied a scanning transmission X-ray microscopy (STXM) technique to investigate the spatial distribution of biomolecules at the microbe–mineral boundary during bioleaching process of pyrite (FeS₂). STXM-based carbon and oxygen near-edge X-ray absorption fine structure (NEXAFS) directly showed that polysaccharides were localized within 150 nm of the bacteria–pyrite boundary and that sessile bioleaching bacteria produced polysaccharides-abundant extracellular biomolecules on the pyrite grain. The STXM-based NEXAFS technique has multiple advantages: (i) a high spatial resolution less than 50 nm, (ii) a nondestructive analytical technique with a high element of specificity, (iii) X-ray spectroscopic chemical speciation useful for the analysis of hydrated biomolecules. Thus, the STXM-based NEXAFS technique is a powerful tool to investigate the mechanism of biological …