Multiscale elucidation of reactive black 5 biodegradation by a synergistic bacterial consortium biofilm: experimental validation and molecular dynamics insights

Abstract

Reactive Black 5 (RB5) is a widely used azo dye in the textile industry that is highly toxic and harms both human health and the environment. The biofilm is a complex matrix comprising microorganisms and extracellular polymeric substances (EPS). Biofilm-based bioremediation is one of the most effective approaches for removing toxic RB5 from water. In this study, a biofilm-forming bacterial consortium comprising Bacillus tequilensis BS007 and Enterobacter mori BS008 was evaluated for treated RB5 dye. The consortium exhibited a specific growth rate (μ_max) of 0.265 h⁻¹ and efficient biofilm formation. The biofilm consortium achieved 98% decolorization of 100 ppm RB5 within 8 h (pH 8, 37 °C). FESEM analysis showed morphological changes in the biofilm, whereas FTIR confirmed the breakdown of azo bonds and changes in other functional groups in the dye after degradation. A biodegradation pathway was proposed based on degradation intermediates analyzed by GC–MS. Molecular modelling and dynamics simulations were performed with two enzymes primarily responsible for its degradation. RMSD and RMSF data up to 100 ns indicated stable docked structures, little structural variation, and a robust protein framework between the azoreductase enzyme and the RB5, which can correlate with the GC–MS data. Phytotoxicity assessment using Cicer arietinum demonstrated that compared to the control, seed germination increased from 40% in the untreated dye to 85% after treatment. These findings highlight the potential of the biofilm consortium as an efficient, environmentally sustainable approach for treating RB5-contaminated textile wastewater.