Integrated biodegradation experiments and DFT calculations reveal the structure-dependent promotion of azo dye biodegradation by an ethanol cosubstrate

The role of ethanol as a cosubstrate in enhancing microbial degradation of azo dyes is recognized, yet the structural basis for its variable efficacy remains unclear. This study systematically investigated the effect of ethanol on the biodegradation of seven structurally diverse carbocyclic azo dyes by Providencia rettgeri FZB001. Decolorization efficiency varied significantly among dyes; ethanol boosted the 1-day decolorization by up to 60.64% for Direct Red 5B, while having a marginal effect (3.17%) on Congo Red. Density functional theory (DFT) calculations linked this disparity to molecular descriptors, revealing that dyes with higher polarity, vertical electron affinity, and lower LUMO energy—indicative of a higher reduction potential—were significantly more susceptible to ethanol-facilitated decolorization (|r| > 0.72). We hypothesize that ethanol promotes the regeneration of the intracellular NADH pool, thereby accelerating the rate-limiting electron transfer step catalyzed by key reductases. Consequently, ethanol stimulated a greater diversity of degradation pathways (e.g., enabling asymmetric cleavages) and increased reaction rates, as tentatively identified by HPLC-MS/MS and Fukui function analysis. These findings elucidate a structure-dependent co-metabolic mechanism and provide preliminary structure-activity relationships for applying ethanol to enhance the biological treatment of azo dye wastewater.