Intermittent Perturbation with High-Frequency Aerobic-Anoxic Cycling Enhances N ₂ O Reductase Activity during Wastewater Treatment

Nitrous oxide (N ₂ O) is a potent greenhouse gas, with a global warming potential nearly 300 times that of carbon dioxide, and its emission from wastewater treatment plants is a growing environmental concern. In this study, we investigated the effects of intermittent perturbation with high-frequency aerobic-anoxic cycling on N ₂ O reduction in activated sludge systems. The study aimed to enhance N ₂ O reductase (N ₂ OR) activity and N ₂ O reduction potential by optimizing redox state cycling. Six bioreactors were operated under different aeration patterns—constant aerobic (CA), continuous perturbation (CP), and intermittent perturbation (IP)—at two dissolved oxygen levels (2 mg/L and 8 mg/L). Results indicated that IP conditions significantly increased N ₂ OR activity, resulting in higher N ₂ O consumption rates compared to the other aeration patterns. Metagenomic and metaproteomic analyses revealed Hyphomicrobium as a key microorganism contributing to N ₂ O reduction, with increased enzyme abundance and activity under IP conditions. The frequent aerobic-anoxic transitions were found to enhance electron availability for N ₂ OR, facilitating more efficient N ₂ O reduction. These findings suggest that intermittent aeration, particularly with high-frequency cycling, can be an effective strategy for mitigating N ₂ O emissions in wastewater treatment systems. This approach has the potential to improve the environmental sustainability of wastewater treatment by leveraging microbial dynamics and enzyme activity to reduce greenhouse gas emissions.