Extracellular Matrix of Biofilm Regulates the Stress Tolerance Mechanism in Aspergillus terreus HLP5 Against Lead and Phenanthrene Co‐Pollutants

Lead [Pb(II)] and phenanthrene are major environmental co‐pollutants that pose a serious threat to all life forms. The present study highlights the biofilm‐associated stress responses in the marine filamentous fungus Aspergillus terreus HLP5 to various concentrations of Pb(II) and phenanthrene (25–500 ppm, 1:1 ratio). Highest biofilm formation shifted from 48 h in control to 72 h in presence of co‐pollutants. A reduced surface‐to‐volume ratio in biofilm formed at 25–50 ppm Pb(II) + Phe indicated increased mycelial aggregation and compact architecture. The hyphal diameter increased with co‐pollutant concentration. Gene expression analysis revealed downregulation of ags gene, whereas somA , medA , uge5 , uge3 , and gtb3 expression peaked at 72–96 h in 25–100 ppm of Pb(II) + Phe. Extracellular matrix (ECM) exhibited compositional and conformational modifications in polysaccharide and protein content. Major shifts were observed in functional groups corresponding to hydroxyl, Amide I‐II, carboxyl, and phosphate moieties. Distinct peak at 8.36 ppm in 1 H NMR revealed phenanthrene‐ECM interaction. A decrease in fluorescence intensity of tryptophan‐like residues (290/355 nm) indicated ECM‐co‐pollutant binding. Upregulation of stress response genes ( gst , acot2 ) and antioxidants like glutathione S‐transferase, superoxide dismutase, and proline contributed to reduced ROS accumulation and redox homeostasis. These findings illustrated enhanced stress resilience of ECM‐covered biofilm under co‐pollutant stress.