Unravelling the Methyl Orange induced acclimation strategies using morphological, physiological, molecular and proteomic analyses and dye degradation in Anoxybacillus rupiensis TPH1 coupled with its mechanism, pathway and metabolites toxicity assessment

The present study investigated the biodegradation of MO by the thermophilic bacterium Anoxybacillus rupiensis TPH1, highlighting its acclimation responses and underlying mechanisms. The biodegradation kinetics followed the modified Gompertz model, indicating that methyl orange degradation by A. rupiensis TPH1 is a growth-associated process supported by cellular acclimation by the bacterium. Azo bond reduction leads to formation of metabolites such as N,N-dimethyl-p-phenylenediamine and 4-aminobenzenesulfonic acid. Phytotoxicity and cytotoxicity assays confirmed these metabolites were non-toxic. Increased activities of antioxidative enzymes (SOD, CAT, TrxR, POX, GPx, GR) and a decreased reduced-to-oxidized ratio of non-enzymatic antioxidants (ascorbate, glutathione) enabled effective mitigation of MO-induced stress, preserving cellular function. SEM analysis showed MO-exposed cells embedded in an exopolysaccharide-rich matrix, likely enhancing dye degradation. Elevated azoreductase expression and activity under MO treatment confirmed their key role in azo bond reduction. Upregulation of carbon and energy metabolism proteins (acyl-CoA synthetase, acyl-CoA dehydrogenase, succinate CoA ligase, cytochrome oxidase) indicated enhanced energy generation supporting degradation. Simultaneously, proteins related to oxidative stress response (thiol peroxidase, catalase, alkyl hydroperoxide reductase) and xenobiotic degradation (azoreductases, alcohol dehydrogenase) were upregulated, reflecting coordinated metabolic adjustments to manage redox status and sustain degradation. Overall, A. rupiensis TPH1 emerges as a robust, sustainable candidate for eco-friendly remediation and management of dye-contaminated effluents.