In biotechnological processes such as fed-batch reactors the lack of reliable and robust on-line sensors and the limited number of actuators make the task of operating at optimal conditions very difficult. We present a feedback controller that aims at regulating the substrate concentration at an optimum value such that biomass production is enhanced while by-product formation is not favored. We use a virtual output that is estimated using a bank of weighted super-twisting observers to drive an output-feedback extremum-seeking controller. The only online measurements needed are the biomass concentration and the oxygen and carbon dioxide mass transfer rates. Simulations on a fed-batch bioreactor model show its applicability. (C) 2015 Elsevier Ltd. All rights reserved.