Removal of aniline by biochar-immobilized fungal pellets is size-dependent: A spatial-temporal analysis at the microscale

Biochar-based fungal immobilization is widely used to degrade organic contaminants, yet microscale biochemical dynamics governing size-dependent performance of filamentous fungal pellets remain unclear. Here, the microscale distribution of enzyme activity and pH during aniline removal by Phanerochaete chrysosporium immobilized pellets was quantified using in situ zymography and planar optode mapping. After acclimation, conditions of aniline removal by immobilized fungal pellets were optimized using response surface methodology. Among 12 biochars, straw-derived magnetic biochar pyrolyzed at 700°C showed the highest aniline adsorption ( q e = 16.36 mg·g −1 ) and thus was selected as the carrier. Under optimized conditions (24 mL inoculum, 1.2 g biochar, 10 h pre-adsorption, 30°C, pH 6.0), magnetic biochar-immobilized fungal pellets (MB-IFPs) removed 98.99% of aniline in 8 days and retained 74.10% efficiency after five cycles. Comparison of three pellet sizes (1–2, 3–4, and 5–6 mm) revealed size-dependent microenvironmental heterogeneity: the maximum β-glucosidase activity influence radius reaches 26–34 mm after 48 h’ incubation compared to free fungi (about 5 mm). Enzyme activity and pH gradients were the strongest near the pellet core and weakened toward the periphery, with immobilized fungal pellets inducing stronger core acidification than free fungi. Medium-sized pellets (3–4 mm) showed the most favorable early-stage microscale performance, reaching a peak β-glucosidase activity of 2.27 μmol cm −2 h −1 , being 61% higher than free fungi. These findings reveal an inside-out attenuation pattern of biochemical activity in immobilized fungal pellets and identify pellet size as a critical design parameter for optimizing fungal remediation systems.