To develop an automated feeding strategy for ε-poly-L-lysine(ε-PL) production during fed-batch fermentation and resolve the problem of ε-PL synthesis rate decreased in the middle phase of fermentation derived from the decrease or cease of Streptomyces sp.M-Z18 cell growth,we have investigated DO-stat feeding glycerol approach and constant feeding different organic nitrogen sources during fermentation in this study.Based on the optimization of initial glycerol concentrations(60 g / L),different ranges of glycerol concentrations(0 ~ 2,0 ~ 10,0 ~ 20,0 ~ 30 g / L) have been achieved by DO-stat feeding strategy during fed-batch fermentation,and determined by 0 ~ 10 g / L as the optimal concentration for ε-PL production and cell growth by Streptomyces sp.M-Z18.The experiments of feeding beef and yeast extracts during fed-batch fermentation from 96 h showed that yeast extract had significantly enhanced by 28.7% in ε-PL production when pH was kept at 3.8.When DO-stat feeding glycerol approach and constant feeding yeast extract together with the two-stage pH control strategy(3.5→3.8) was simultaneously performed in ε-PL fedbatch fermentation by Streptomyces sp.M-Z18,ε-PL production and productivity were finally attained by 38.77 g / L and 4.85 g /(L·d),respectively,after 196 h fermentation.