Overflow metabolism characterizes cell strains that are likely to produce inhibiting metabolites as a result of an excess of substrate feeding and a saturated respiratory capacity. In this study. extremum seeking is used to assess the actual critical substrate level, which is a function of the cell respiratory capacity. The latter is modelled as a function representing the existence of two different metabolic modes, namely the respirative mode and the respiro-fermentative mode, as well as modulation factors taking account of inhibiting metabolites. A model-free approach is taken due to the high uncertainty in modelling bioprocesses, and two alternative gradient estimation procedures are considered and compared, e.g., a bank of filters and a recursive least-squares formulation. The RLS technique is simple to implement and provides quite satisfactory results in the presence of model uncertainties and measurement noise. (C) 2011 Elsevier Ltd. All rights reserved.