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Eicosapentaenoic acid (EPA), which is one kind of important polyunsaturated fatty acid, has many kinds of physiological functions, such as decreasing blood viscosity, preventing atherosclerosis, preventing cardiovascular disease, anti-cancer, anti-inflammatory, etc. And EPA has been widely applied in medicine, food and chemical industry. In this paper, the characteristics of EPA fermentation process with Pythium D283 were studied, and the controls of EPA fermentation process and conditions production were optimized to improve EPA yield. The results were as follows:(1) The characteristics of the kinetic parameters of cell growth rate and EPA synthesis rate were studied. Then, the phase control strategy, in which glucose concentration, dissolved oxygen and pH were main control parameters, was proposed and optimized. Through the way of the phase control, the biomass and EPA yield of Pythium D283 respectively reached 14.32 g/L and 0.554 g/L and were increased by 8% and 15.4% comparing to traditional fermentation of Pythium D283.(2) The studies of EPA high-density fermentation of Pythium D283 in 250-ml Erlenmeyer flask containing 80 ml liquid medium showed that supplementing with soybean-oil, increasing the ventilation volume and flowing carbon source improved the biomass, EPA content and yield, respectively. Supplementing with 2.5% (W/V) soybean oil could promote the accumulation of cell, and improving ventilation could increase EPA content in the latter fermentation. 300 ml of 30% (W/V) glucose solution was flowed with the rate of 120 ml/h into the fermentation broth of Pythium D283, when being at the largest growth rate, and the results showed that the biomass and EPA yield were 24.4 g/L and 0.78 g/L, respectively, which increased by 38.65% and 16.4% comparing to not flowing glucose solution. (3) Based on the GC-MS analysis of microbial oil, the synthesis pathway of EPA in Pythium D283 was at the first time proposed, and two key enzymes (Δ12-desaturase,D12 andΔ15-desaturase,D15) and one rare enzymeΔ8-desaturase(D8) involving in EPA synthesis pathway were diagnosed and determined.(4) The studies of low-temperature EPA fermentation showed that the fermentation broth of Pythium D283 was cultured at 8℃with 150 rpm for 4 days at the end of the fermentation process, and the EPA yield reached 0.702 g/L and was increased 18.4% comparing to non-low-temperature-step EPA fermentation. The fermentation broth was supplemented with 20.3 g/L ethanol and 3.9 g/L sodium nitrate and cultured at 9.0℃with 150 rpm for 4 days at the end of the fermentation process, EPA yield reached 0.856 g/L and was increased 25.52% comparing to non-low-temperature fermentation.
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