NADH-Driven Bioreductive Degradation of Azo Dyes: Mechanisms of High NADH Production, Electron Transfer, and Microbial Responses

Bioreductive co-metabolic degradation of azo dyes represents a promising green technology for addressing the environmental pollution caused by azo dyes. This study investigated the impact of co-metabolized substances on NADH production in microbial systems, focusing on the production of reducing power, electron transfer, and the synergistic effects of microbial communities and associated mechanisms during azo dye degradation. A culture system was developed to maximize NADH production at 3   g/L yeast extract, and it was observed that the system exhibited a significant increase in reducing power, with NADH concentration reaching 909.48   pg/mL (60   h). The electron transfer process in this system primarily depended on factors such as redox mediators, azoreductase, and formic acid. Azo dye reductive degradation and decolorization occurred through an indirect electron transfer pathway. Flavin-based redox mediators (riboflavin and flavin mononucleotide) played a key role in the system, with the application of riboflavin and flavin mononucleotide increasing the system's dye reduction ratio by 14.45% and 14.40%, respectively. They were endogenously expressed by the system and facilitated efficient electron transfer by synergizing with specific reductases, particularly when the electron transport chain was inhibited, and alternative pathways ensured the continuation of the reduction reaction. NADH production primarily occurred through glycolysis, the TCA cycle, and fatty acid β-oxidation, with glycolysis contributing the most. Microorganisms such as Enterococcus , Burkholderia , and Escherichia within the microbial community played a crucial role in NADH production while regulating community behavior through a quorum sensing system, thereby enhancing the stability and efficiency of dye degradation. This study investigated the bioreduction of azo dyes in terms of reducing power, offering a theoretical foundation and practical guidance for optimizing the microbial system and enhancing the biodegradation efficiency of azo dyes.