Optimizing control of Escherichia coli cultures depends on the availability of appropriate on-line sensors for the main culture components. An efficient and practical way to maintain E. coli cultures in the neighborhood of the optimal operating conditions is to regulate the acetate concentration at a constant low level. Unfortunately, reliable acetate probes are currently not available on the market. A way to overcome this lack of information is to design software sensors. In this work, observability conditions are first examined, and Extended Kalman Filters (EKF) are developed for various hardware sensor configurations, taking account of their reliability and cost, and including a possible kinetic parameter estimation. The filters are validated using experimental data collected on a lab-scale bioreactor. (C) 2012 Elsevier Ltd. All rights reserved.