Abstract In this article, the theory of fractional control and state estimation applied to biological science is studied, particularly in hybrid wastewater treatment. For nonlinear systems with stable and known states, an interconnected fractional robust control design with high gain state estimation is proposed to generate a control insensitive to nutritional perturbations originated by an advanced oxidation process in a microalgae culture. An online study is proposed for the mineralization of glyphosate and its feedback in a microalgae cultivation process where through the designed control the light dynamics is manipulated to robustly and automatically regulate the biomass signal provided by an analog sensor and nutrient estimation via state observers. In the literature, there are few results developed with real-time results. This work is a multidisciplinary study with online results where the performance and improvement of the proposed complex process are concluded.