Biodegradation of Toxic Dyes using a Multi-Microbial System with Pseudomonas putida and Lysinibacillus sphaericus Consortium and Pathway Elucidation for Acid Yellow 42, Reactive Red 198 and Reactive Black 5

Abstract

Azo dyes, including Acid Yellow 42 (AY), Reactive Red 198 (RR), and Reactive Black 5 (RB), are persistent environmental contaminants known for their toxicity and resistance to conventional treatment methods. In this study, a co-culture of Pseudomonas putida and Lysinibacillus sphaericus was employed to investigate the biodegradation potential of these structurally diverse azo dyes. The microbial system was cultivated in minimal salt medium (MSM) under optimized conditions, demonstrating complete degradation of 50 ppm dye concentrations within 24 hours and 100 ppm within 48 hours at pH 7.2 and 34 ± 0.3 °C under continuous shaking at 180 rpm. Enzymatic profiling confirmed the active involvement of azoreductase, laccase, and NADH-DCIP reductase, suggesting a multi-enzyme facilitated degradation mechanism. To precisely map the transformation of dye molecules, electrospray ionization mass spectrometry (ESI-MS) was employed as the primary analytical tool. ESI-MS enabled the identification of intermediate metabolites formed during the degradation process, which were subsequently used to construct detailed biochemical degradation pathways for AY, RR, and RB. The strategic use of mass spectrometry-based metabolite tracking presents a powerful alternative to traditional analysis methods, offering direct insights into the molecular breakdown of complex pollutants. This work highlights a sustainable, enzyme-driven, and analytically streamlined route for azo dye degradation, with potential application in industrial wastewater bioremediation.