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Optimization and control strategies are necessary to keep wastewater treatment plants (WWTPs) operating in the best possible conditions, maximizing effluent quality with the minimum consumption of energy. In this work, a benchmarking of different hierarchical control structures for WWTPs that combines static and dynamic real time optimization (RTO) and nonlinear model predictive control (NMPC) is presented. The objective is to evaluate the enhancement of the operation in terms of economics and effluent quality that can be achieved when introducing NMPC technologies in the distinct levels of the multilayer structure. Three multilayer hierarchical structures are evaluated and compared for the N-Removal process considering the short term and long term operation in a rain weather scenario. A reduction in the operation costs of approximately 20% with a satisfactory compromise to effluent quality is achieved with …