Impact of operating conditions on N₂O accumulation in Nitrate-DAMO system: Kinetics and microbiological analysis

Nitrate-dependent anaerobic methane oxidation (Nitrate-DAMO) is a novel and sustainable process that removes both nitrogen and methane. Previously, the metabolic pathway of Nitrate-DAMO has been intensively studied with some results. However, the production and consumption of nitrous oxide (N₂O) in the Nitrate-DAMO system were widely disregarded. In this study, a Nitrate-DAMO system was used to investigate the effect of operational parameters (C/N ratio, pH, and temperature) on N₂O accumulation, and the optimal operating conditions were determined (C/N = 3, pH = 6.5, and temperature = 20 °C). In this study, an enzyme kinetic model was used to fit the nitrate nitrogen degradation and the nitrous oxide production and elimination under different operating conditions. The thermodynamic model of N₂O production and elimination in the system also has been constructed. Multiple linear regression analysis found that pH was the most important factor influencing N₂O accumulation. The Metagenomics sequencing results showed that alkaline pH promoted the abundance of Nor genes and denitrifying bacteria, which were significantly and positively correlated with N₂O emissions. And alkaline pH also promoted the production of Mdo genes related to the N₂O-driven AOM reaction, indicating that part of the N₂O was consumed by denitrifying bacteria and the other part was consumed by the N₂O-driven AOM reaction. These findings reveal the mechanism of N₂O production and consumption in DAMO systems and provide a theoretical basis for reducing N₂O production and greenhouse gas emissions in actual operation.