Recombinant protein production in Escherichia coli can be significantly reduced by acetate accumulation. It is demonstrated that acetate production can be detected on-line with a standard dissolved oxygen sensor by superimposing short pulses to the substrate feed rate. Assuming that acetate formation is linked to a respiratory limitation, a model for dissolved oxygen responses to transients in substrate feed rate is derived. The model predicts a clear change in the character of the transient response when acetate formation starts. The predicted effect was verified in fed-batch cultivations of E. coli TOPP1 and E. coli BL21(DE3), both before and after induction of recombinant protein production. It was also observed that the critical specific glucose uptake rate, at which acetate formation starts, was significantly decreased after induction. On-line detection of acetate formation with a standard sensor opens up new possibilities for feedback control of substrate feeding.