Nanoscale Characterization of Fungal-Induced CaCO₃ Precipitation: Implications for Self-Healing Concrete

Cracks in concrete compromise structural integrity by exposing steel reinforcement to corrosion agents, shortening its service life. Fungal-induced calcium carbonate (CaCO₃) precipitation via urea hydrolysis offers a fast and robust self-healing mechanism to seal the cracks, extending the lifespan while reducing the carbon (C) footprint of concrete infrastructure. However, current studies rely on bulk-scale analytical methods, which lack the spatial resolution and chemical sensitivity to distinguish and map CaCO₃ polymorphs at the nanoscale. This study combined scanning electron microscopy (SEM) and synchrotron-based scanning transmission X-ray microscopy (STXM) with near-edge X-ray absorption fine structure (NEXAFS) spectroscopy to characterize fungal CaCO₃ polymorphs at the nanoscale. CaCO₃ biominerals precipitated by three urease-positive fungi were sectioned into 75-200 nm thin layers. STXM data were collected from at least two spots per section, focusing on Ca (L-edge) and C (K-edge) chemical speciation and elemental quantitative mapping. Calcite, the thermodynamically most stable polymorph, was identified as the predominant mineral phase precipitated by all fungi species, while aragonite and non-CO₃-Ca species (CaCl₂ or Ca adsorbed onto extracellular polymeric substances (EPS)) occurred as minor components. In fungal species 2, we observed nanoscale heterogeneity in Ca phases across five analyzed spots, three dominated by calcite with minor contributions of other Ca species, while the others showed mixed CaCO₃/non-CO₃ phases, as confirmed by NEXAFS spectra. These findings suggest that biomineralization in the fungal micro and nanoenvironment is influenced by localized physicochemical and metabolic conditions that shape mineral phases. C NEXAFS spectra further supported the Ca data, showing C-specific spectral features in the calcite-rich regions across all samples. This underscores STXM's capability to resolve complexities and mechanisms of fungal CaCO₃ formation (e.g., mineral phase composition, fungal organic-mineral interactions, and spatial heterogeneity). Overall, this study provides critical nanoscale insights into fungal CaCO₃ precipitation, thus providing valuable guidance in optimizing fungal systems in self-healing concrete applications.