Benzo(b)fluoranthene degradation pathway in Bacillus subtilis BMT4i (MTCC 9447)

Benzo(b)fluoranthene (BbF) is a high-molecular-weight polycyclic aromatic hydrocarbon (HMW-PAH) known for its persistence, toxicity, and probable carcinogenicity. Its hydrophobic and structurally stable nature limits natural degradation, making its accumulation in soil and aquatic environments a major ecological concern. Although fungal and algal degradation pathways have been reported, bacterial mechanisms remain poorly understood. This study evaluates the ability of Bacillus subtilis BMT4i, previously known to degrade benzo(a)pyrene (BaP), to utilize BbF as its sole carbon and energy source. The strain was grown in basal salt medium (BSM) containing 50 µg/ml BbF, and degradation was monitored through high-performance liquid chromatography (HPLC). Metabolites formed at 7, 15, and 40 days were analyzed using Ultra-High-Performance Liquid Chromatography coupled with Tandem Mass Spectrometry (UHPLC-MS/MS) to determine structural transformations based on molecular ions and fragmentation patterns. B. subtilis BMT4i achieved approximately 75% BbF degradation within 42 days, showing a strong correlation between bacterial growth and BbF utilization. UHPLC-MS/MS analysis revealed 10 structurally characterized metabolites out of 23 chromatographic peaks, indicating active biotransformation. These intermediates reflected key steps such as dioxygenation, dehydrogenation, aromatic ring cleavage, and side-chain oxidation. Based on the detected metabolites, a detailed multi-step degradation pathway for BbF was proposed, the first comprehensive bacterial report of its kind highlighting the strong bioremediation potential of B. subtilis BMT4i for HMW-PAH-contaminated environments.