Denitrification-enhanced anaerobic digestion for stable synergy of hydrolysis and methanogenesis of N, N-dimethylformamide by nitrate addition: Delayed stimulation effect for successful enrichment of functional microorganisms

Methanogenic degradation of N, N-dimethylformamide (DMF) is still a critical technical bottleneck, as DMF-hydrolyzing bacteria (DHB) lack the ability of autonomous proliferation in strict anaerobic environments. In this work, a novel denitrification-enhanced anaerobic digestion (DEAD) strategy was developed, where nitrate was introduced as an exogenous stimulant to realize efficient treatment of high-strength industrial wastewater containing approximately 2000 mg/L of DMF. A lab-scale up-flow anaerobic sludge blanket (UASB) reactor was operated continuously for 350 days to validate the feasibility of this strategy. The results indicated that DHB isolated from aerobic activated sludge could not maintain efficient and stable DMF degradation with DMF as the sole carbon source under anaerobic conditions, resulting in a gradual decline of reactor treatment performance. Nitrate supplementation induced a functional transformation of DHB from specialized DMF-degrading bacteria to denitrifying bacteria, which allowed DHB to achieve effective proliferation in anaerobic systems and further construct a stable DEAD system based on denitrification-driven hydrolysis (DDH). This established DEAD system successfully realized the synergistic metabolism of denitrification and methanogenesis. Microbial analysis and functional gene prediction verified that nitrate addition significantly promoted the enrichment of core DHB like Paracoccus and Hyphomicrobium, and these DHB are also denitrifying bacteria. Functional gene reads for DMFase, dimethylamine dehydrogenase, co-methyltransferase and methyltransferase which are closely related to DMF hydrolysis, were also elevated by the DEAD system. This research provides a new technical approach and theoretical basis for the anaerobic biodegradation of refractory nitrogenous organic pollutants in industrial wastewater. © 2026 Elsevier Inc.