裂殖壶菌(Schizochytrium sp.S31)作为典型好氧产二十二碳六烯酸(docosahexaenoic acid, DHA)微生物,其生产过程中同时维持葡萄糖和溶解氧浓度(dissolved oxygen concentration, DO)于充足水平有利于细胞快速生长和DHA的大量积累。然而,裂殖壶菌细胞若长期处于DO充足状态时,存在胞内活性氧(reactive oxygen species, ROS)易发生积累,损害裂殖壶菌生物量和DHA产量的难题。为此,该文提出一种新型的“葡萄糖充足-DO周期”组合控制策略,即通过脉冲流加葡萄糖方式将其质量浓度维持于40 g/L左右的高浓度水平,与此同时,DO控制模式则是将培养阶段等分为10个10 h阶段,每个阶段的前8 h维持DO于充足水平(>10%),后2 h维持DO于受限水平(接近于零)。结果表明,采用该组合控制策略时:(1)最大胞内ROS浓度从对照的209.37 FI/g DCW降至146.38 FI/g DCW的低水平;(2)维持代谢系数从0.026 6 1/h降至0.021 2 1/h的较低水平;(3)DHA合成所需的关键前体物质乙酰辅酶A和辅因子NADPH合成途径中相关关键酶的活性、细胞比生长速度和葡萄糖消耗速率均得到增强;(4)DHA产量达到了13.69 g/L的最高水平,相比于对照两批次的3.92 g/L(葡萄糖充足-DO受限)和9.76 g/L(葡萄糖充足-DO充足),分别提高了256.1%和40.3%。
Controlling concentrations of glucose and dissolved oxygen (DO) at high and sufficient level could be considered as an important factor for effective production of docosahexaenoic acid (DHA) by Schizochytrium sp.S31. However, the structure and skeleton of cells would be obviously damaged with severe accumulations of reactive oxygen species (ROS) when cells were subjected to the environment of sufficient DO for a long time, which eventually lead to a limited DHA production. To solve these questions, a combinational control strategy of “glucose sufficient-periodic dissolved oxygen concentration operation” was proposed. When using this strategy, glucose concentration was maintained at about 40 g/L throughout the entire cultivation (feeding) phase. At the same time, in the DO control scheme, typical cultivation (feeding) phase was divided into ten 10 h subintervals, during which alternate DO supplement was applied, namely sufficient DO for the first 8 h and limited DO for the last 2 h. When using this combinational strategy, the maximum concentration of intracellular ROS could be decreased from 209.37 fluorescence intensity(FI)/g DCW to 146.38 FI/g DCW; the distribution of glucose to cell maintenance was decreased from 0.026 6 1/h to 0.021 2 1/h; the activities of key enzymes involved in DHA biosynthesis metabolism, cell growth rate and glucose consumption rate were apparently improved; DHA concentration reached the highest level of 13.69 g/L, which was increased by 40.3% and 256.1%, respectively, compared to that of the traditional control strategies of “glucose sufficient-DO limited” (3.92 g/L) and “glucose sufficient-DO sufficient” (9.76 g/L).
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