Ammonia monooxygenase-mediated cometabolic biotransformation of volatile 4-chlorophenol in nitrifying counter-diffused biofilms: A combined molecular dynamics simulation, DFT calculation and experimental study.

Ammonia monooxygenase (AMO)-mediated cometabolism of organic pollutants has been widely observed in biological nitrogen removal process. However, its molecular mechanism remains unclear, hindering its practical application. Furthermore, conventional nitrification systems encounter significant challenges such as air pollution and the loss of ammonia-oxidizing bacteria, when dealing with wastewater containing volatile organic pollutants. This study developed a nitrifying membrane-aerated biofilm reactor (MABR) to enhance the biodegradation of volatile 4-chlorophenol (4-CP). Results showed that 4-CP was primarily removed via Nitrosomonas nitrosa-mediated cometabolism in the presence of NH4+-N, supported by the increased nicotinamide adenine dinucleotide (NADH) and adenosine triphosphate (ATP) content, AMO activity and the related genes abundance. Hydroquinone, detected for the first time and produced via oxidative dechlorination, as well as 4-chlorocatechol was primary transformation products of 4-CP. Nitrosomonas nitrosa AMO structural model was constructed for the first time using homology modeling. Molecular dynamics simulation suggested that the ortho-carbon in the benzene ring of 4-CP was more prone to metabolismcompared to the ipso-carbon. Density functional theory calculation revealed that 4-CP was metabolized by AMO via H-abstraction-OH-rebound reaction, with a significantly higher rebound barrier at the ipso-carbon (16.37 kcal·mol-1) as compared to the ortho-carbon (6.7 kcal·mol-1). This study fills the knowledge gap on the molecular mechanism of AMO-mediated cometabolism of organic pollutants, providing practical and theoretical foundations for improving volatile organic pollutants removal through nitrifying MABR.