A glucose-glycerol mixed carbon source led to higher production of both epsilon-poly-L-lysine and biomass, whether in the batch or fed-batch fermentation. However, as the major benefit of the mixed carbon source, the high specific epsilon-poly-L-lysine formation rate in the batch fermentation sharply declined when the feeding strategy was initiated. In this study, we examined the development of cell physiology in epsilon-poly-L-lysine production to obtain an insight into this undesirable phenomenon. The simultaneous consumption of glucose and glycerol generated abundant reactive oxygen species in the batch fermentation stage. Transcriptome analysis further confirmed the existence of excessive oxidative stress with the glucose-glycerol mixed carbon source. Serious oxidative damage in the mixed carbon source resulted in deterioration in cell viability, intracellular levels of ATP and L-lysine in the following fed-batch fermentation. Moreover, the stress situation was worsened by a low antioxidant capacity with the mixed carbon source. As a result, these damaged cells showed decreased specific epsilon-poly-L-lysine formation rate. This study provided information for the understanding of the underlying change of cell physiology in epsilon-poly-L-lysine fed-batch fermentation, which was greatly needed for a further enhancement of epsilon-poly-L-lysine production in culture with an intensive cell respiration. (C) 2015 Elsevier Ltd. All rights reserved.