Bioaugmentation stabilizes high-loading anaerobic N,N-dimethylformamide removal: process stability and microbial...

Sludge-based bioaugmentation is a promising and practical strategy for treating refractory N,N-dimethylformamide (DMF) wastewater, yet the ecological basis underlying its stability under intense DMF loading remains unclear. Here, two parallel anaerobic sequencing-batch reactors were operated for 60 days under progressively increasing DMF concentrations, with R1 receiving periodic residual activated sludge supplementation and R0 serving as the non-bioaugmented control. At the highest influent DMF concentration of 1610 mg/L, R1 maintained an average DMF removal efficiency of 91.2% ± 2.4%, compared with 67.0% ± 11.2% in R0, and exhibited a lower coefficient of variation of effluent DMF concentration (27.3% and 34.0%, respectively). Microbial analysis showed that R1 maintained a larger intermittent-bacteria pool (7.42% compared with 5.55% in R0) and a stronger predicted anaerobic phenotype (9.9% compared with 7.9%). The R1 co-occurrence network contained more associations and exhibited a higher density than R0 (4027 edges and 0.101 density compared with 2982 edges and 0.076, respectively). Notably, representative DMF-related populations, including Paracoccus and Methyloversatilis, were more strongly represented in R0 during the late stage despite its poorer treatment performance. PICRUSt2 further predicted stronger central carbon metabolism, energy conversion, and cellular maintenance potential in R1, whereas R0 showed greater relative representation of DMF-specific functions. Overall, periodic sludge supplementation was associated with repeated microbial immigration followed by selective community reorganization, supporting stable anaerobic DMF removal under increasing loading.