Unveiling mechanistic insights of inactivated Aspergillus niger spore by fishery-waste-derived chitosan via synchrotron radiation tomography.

Aspergillus niger (A. niger) is a pathogenic fungus responsible for severe and potentially fatal invasive pulmonary aspergillosis. For the first time, this study elucidates the antifungal mechanism of fishery-waste-derived chitosan (CTS) against A. niger using synchrotron-based X-ray microscopy integrated with conventional assays, enabling 3D visualization of the inactivation process. CTS (2.0 g L-1) exhibited potent efficacy, achieving a 6.05-log reduction within 6 h. Crucially, transmission X-ray microscopy revealed a transition from spherical spores to centrally collapsed structures, while cryo-soft X-ray microscopy showed pronounced organelle shrinkage and plasma-membrane detachment within 2 h, followed by severe structural rupture at 6 h, with cell volume decreasing from 34.9 to 6.5 μm3. Atomic force microscopy further demonstrated reduced cell height (4.55 to 1.90 nm) and increased surface adhesion (0.72 to 2.21 nN) and roughness (3.51 to 13.03 nm). Fluorescence imaging and potassium-leakage assays confirmed significant ion efflux that disrupted osmotic balance and cellular homeostasis. Collectively, the results indicate that CTS compromises cell integrity through electrostatic interactions between protonated amino groups and the fungal surface, leading to cytoplasmic leakage and irreversible intracellular collapse. This work identifies CTS as an effective carbohydrate-based antifungal agent and highlights the power of synchrotron imaging for resolving subcellular antimicrobial mechanisms under near-native conditions.