Zhou Xiang-Shan, Fan Wei-Min, Zhang Yuan-Xing
State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, Shanghai 200237, China.
Sheng Wu Gong Cheng Xue Bao. 2003 Sep;19(5):618-22.
Chemostat culture was performed to characterize the growth, substrate consumption and the hirudin production, and to disclose their interrelations in the fermentation of recombinant Pichia pastoris. The Andrew substrate-inhibited growth model is more suitable than Monod model to simulate the growth of Pichia pastoris on methanol. Therefore, two stationary states can be obtained in the continuous culture at a certain dilution rate because of the substrate inhibition on cell growth. The stationary state could be obtained if only the dilution rate not more than 0.048 h(-1) in the continuous fermentation. The concentrations of cell, methanol and hirudin were constant after 50 h continuous culture with dilution rate at 0.04 h(-1). However, it could not be obtained when the dilution rate more than 0.048 h(-1) because the other stationary point at S > 0.048 h(-1) is unstable. Therefore, it was found that the cell concentration declined and the methanol concentration increased from 2.9 g/L to 18.1 g/L within 18h at dilution rate 0.06 h(-1). Thus, the fed-batch culture with a constant specific growth rate was carried out to disclose the fermentation behavior at high and constant methanol concentration in aid of a methanol sensor. The theoretical maximum specific growth rate, microm = 0.0464 h(-1), was found under critical methanol concentration, Scrit = 3.1 g/L. The growth of P. pastoris was typically methanol-limited at the methanol concentration S < Scrit. It was, however, inhibited at S > Scrit. The maximum specific Hir65 production rate qp was obtained at 0.2 mg/(g x h) when methanol concentration and mu were 0.5 g/L and 0.02 h(-1), respectively. The specific Hir65 production rate qp increased with the increase of mu and S at mu < 0.02 h(-1), and decreased at mu > 0.02 h(-1). The specific methanol consumption rate increased with the increase of S when S < 5 g/L, but decreased when S > 5 g/L. At last, the high Hir65 production rate 0.2 mg/(g x h) was obtained in the fermentation conducted under methanol-limited concentration and mu controlled at 0.5 g/L and 0.02 h(-1), respectively, while the specific methanol consumption rate is low only at 0.04 g/(g x h), showing the potential for the strategy of getting high Hir65 production rate at the low consumption of methanol.
进行恒化器培养以表征重组巴斯德毕赤酵母发酵过程中的生长、底物消耗和水蛭素生产，并揭示它们之间的相互关系。与莫诺德模型相比，安德鲁底物抑制生长模型更适合模拟巴斯德毕赤酵母在甲醇上的生长。因此，由于底物对细胞生长的抑制作用，在连续培养中以一定稀释率可获得两个稳态。在连续发酵中，只有当稀释率不超过0.048 h⁻¹时才能获得稳态。以0.04 h⁻¹的稀释率连续培养50 h后，细胞、甲醇和水蛭素的浓度保持恒定。然而，当稀释率超过0.048 h⁻¹时无法获得稳态，因为在S > 0.048 h⁻¹时的另一个稳态点是不稳定的。因此，发现在0.06 h⁻¹的稀释率下，细胞浓度在18 h内下降，甲醇浓度从2.9 g/L增加到18.1 g/L。因此，进行了具有恒定比生长速率的补料分批培养，以借助甲醇传感器揭示在高且恒定的甲醇浓度下的发酵行为。在临界甲醇浓度Scrit = 3.1 g/L下，发现理论最大比生长速率μm = 0.0464 h⁻¹。当甲醇浓度S < Scrit时，巴斯德毕赤酵母的生长通常受甲醇限制。然而，当S > Scrit时，生长受到抑制。当甲醇浓度和μ分别为0.5 g/L和0.02 h⁻¹时，最大比水蛭素Hir65生产率qp为0.2 mg/(g·h)。当μ < 0.02 h⁻¹时，比水蛭素Hir65生产率qp随μ和S的增加而增加，当μ > 0.02 h⁻¹时则下降。当S < 5 g/L时，比甲醇消耗率随S的增加而增加，但当S > 5 g/L时则下降。最后，在甲醇限制浓度下进行发酵，μ分别控制在0.5 g/L和0.02 h⁻¹，获得了0.2 mg/(g·h)的高水蛭素Hir65生产率，而比甲醇消耗率仅为0.04 g/(g·h)，显示了在低甲醇消耗下获得高水蛭素Hir65生产率策略的潜力。
