Metabolite profiling reveals the degradation pathways of Methylene Blue by Co-cultures of Pseudomonas aeruginosa and Daedalea dickinsii

• Mixed cultures of P. aeruginosa and D. dickinsii enhanced MB biodecolorization • MB biodecolorization reached 62.3% after 7 days • MB was transformed into several degradation metabolites • Three possible MB biodegradation pathways were proposed Methylene Blue (MB) is a thiazine-based synthetic dye widely used in industrial applications and is recognized for its high stability and potential toxicity to human health. Biological treatment using fungi has attracted attention as an environmentally friendly approach for dye removal; however, single-microorganism systems often exhibit limited biodecolorization efficiency. Previous studies have shown that the brown-rot fungus (BRF) Daedalea dickinsii is capable of MB biodecolorization and biodegradation, although its performance still requires improvement. In this study, the effect of Pseudomonas aeruginosa addition on MB biodecolorization by D. dickinsii was investigated in a Potato Dextrose Broth (PDB) medium under static conditions at 30°C for 7 days. The results demonstrated that the addition of P. aeruginosa significantly enhanced MB biodecolorization, with the highest removal efficiency of 62.3% obtained at 10 mL bacterial inoculum, compared to only 16.5% achieved by the single fungal culture. Furthermore, several MB degradation metabolites were identified, including C₈H₁₂N₂ (m/z = 137), Azure B (m/z = 270), C₈H₁₁N (m/z = 120), C₂₀H₂₅N₃O₅S₂⁺ (m/z = 418), C₆H₈N₂O₃S (m/z = 188), and C₈H₁₄N₂O₄S (m/z = 217), enabling the proposal of a possible MB biodegradation pathway. The novelty of this study lies in the integration of a fungal–bacterial co-culture system with metabolite-based analysis to provide mechanistic insight into MB biodegradation beyond color removal. These findings highlight the potential of fungal–bacterial synergism as an environmentally friendly approach for dye-contaminated wastewater treatment.