The response of Escherichia coli cells to transient exposure (step increase) in substrate concentration and anaerobiosis leading to mixed-acid fermentation metabolism was studied in a two-compartment bioreactor system consisting of a stirred tank reactor (STR) connected to a mini-plug-flow reactor (PFR: BioScope, 3.5 mL volume). Such a system can mimic the situation often encountered in large-scale, fed-batch bioreactors. The STR represented the zones of a large-scale bioreactor that are far from the point of substrate addition and that can be considered as glucose limited, whereas the PFR simulated the region close to the point of substrate addition, where glucose concentration is much higher than in the rest of the bioreactor. In addition, oxygen-poor and glucose-rich regions can occur in large-scale bioreactors. The response of E. coli to these large-scale conditions was simulated by continuously pumping E. coli cells from a well stirred, glucose limited, aerated chemostat (D = 0.1 h−1) into the mini-PFR. A glucose pulse was added at the entrance of the PFR. In the PFR, a total of 11 samples were taken in a time frame of 92 s. In one case aerobicity in the PFR was maintained in order to evaluate the effects of glucose overflow independently of oxygen limitation. Accumulation of acetate and formate was detected after E. coli cells had been exposed for only 2 s to the glucose-rich (aerobic) region in the PFR. In the other case, the glucose pulse was also combined with anaerobiosis in the PFR. Glucose overflow combined with anaerobiosis caused the accumulation of formate, acetate, lactate, ethanol, and succinate, which were also detected as soon as 2 s after of exposure of E. coli cells to the glucose and O2 gradients. This approach (STR-mini-PFR) is useful for a better understanding of the fast dynamic phenomena occurring in large-scale bioreactors and for the design of modified strains with an improved behavior under large-scale conditions. Biotechnol. Bioeng. 2009; 104: 1153–1161. © 2009 Wiley Periodicals, Inc.