Arachidonic acid (ARA) yield and productivity of Mortierella alpina mutant D20 were enhanced by integrating a fed-batch culture combined with a two-stage pH control strategy. Following a kinetic analysis of the whole fermentation process, a two-stage pH control strategy was developed in which the pH was maintained at 5.5 for the first 48 h and then shifted to 6.5 till the end of fermentation. Using this strategy, a maximum ARA production of 8.12 g/L was achieved. On the basis of pH control, the effects of fed-batch cultures on ARA productivity were further investigated. A maximum ARA productivity of 1.40 g/L/d was obtained with a two-stage constant-speed glucose feeding strategy, starting with a glucose concentration of 50 g/L. This strategy was simple and economical to operate, and it may be possible to apply this approach for large-scale industrial production of ARA. (C) 2015 Elsevier Ltd. All rights reserved.