Endophytic bacteria (Bacillus licheniformis) mediated degradation and detoxification of Direct Red 20: an in silico and in vitro insights

Synthetic azo dyes released from textile industries represent a major source of water pollution because of their chemical stability, toxicity, and resistance to conventional wastewater treatment processes. Among these dyes, Direct Red 20 is widely used in textile dyeing and is highly recalcitrant, making it a persistent environmental pollutant. The present study evaluated the biodegradation and detoxification potential of Bacillus licheniformis toward Direct Red 20 through an integrated experimental and computational approach. Maximum degradation efficiency of 79.31% was achieved after 120 h under optimized conditions (37 °C, 0.1 mg/mL dye concentration, 200 rpm, and the natural pH of nutrient broth), with 65% degradation after 72 h. Enzyme assays demonstrated the involvement of oxidative and reductive enzymes during biodegradation, with peroxidase exhibiting the highest activity (0.377 U/mL), followed by azoreductase (0.358 U/mL) and laccase (0.0067 U/mL), indicating their role in azo dye degradation. Molecular docking further supported these findings, with peroxidase showing the strongest interaction with Direct Red 20, exhibiting a binding affinity of − 10.69 kcal/mol. FTIR and GC–MS analyses confirmed structural transformation of the dye into simpler compounds, while zebrafish embryo toxicity assays demonstrated significant detoxification, with embryo survival increasing from 13.33 to 80.00% in untreated dye samples to 66.67–93.33% following biodegradation. To evaluate its potential for industrial application, B. licheniformis cells were immobilized in calcium alginate beads, achieving 39.78% degradation after 72 h. Although the degradation efficiency was lower than that of free cells, immobilization provided practical advantages including cell protection, easy recovery, reusability, and suitability for continuous wastewater treatment. The novelty of this study lies in integrating biodegradation, enzyme activity profiling, molecular docking, immobilized-cell technology, structural characterization and in vivo toxicity assessment to systematically evaluate Direct Red 20 detoxification. These findings demonstrate that B. licheniformis is a promising eco-friendly biocatalyst for the remediation of azo dye-contaminated wastewater. Future studies should focus on optimizing immobilization conditions and evaluating the process using real textile wastewater for large-scale applications.