Recombinant mixed feed bioprocesses are characterized by the controlled feeding of multiple defined carbon sources aiming at increased productivities. However, mixed feed process design is challenging due to physiological constraints such as adaptation times and catabolite repression. A novel soft-sensor assisted dynamic method that allows the science-based process design with respect to co-utilization of primary and secondary substrate was developed. The method is based on the control of the specific uptake rates of primary and secondary substrate via a combination of a rate-based soft sensor and in-line infrared spectroscopy. Maximum secondary substrate specific uptake rates and adaptation times are determined by a combination of dynamic pulse and ramp experimentation. The power of the presented method was demonstrated on a recombinant Escherichia coli pBAD mixed feed process with D-glucose as primary and L-arabinose as secondary carbon source. Onset of catabolite repression was observed once a total specific substrate uptake rate of 1.0 g/gh was exceeded. Adaptation times to L-arabinose were determined as similar to 10 min. The presented method can be considered generically applicable for the physiological investigation of mixed feed systems. Furthermore, metabolic capabilities of the promising but yet unexplored E. coli pBAD mixed feed system were explored for the first time. (C) 2013 Elsevier Ltd. All rights reserved.