| Candida glycerinogenes, a perfect glycerol production strain whose independent intellectual property belongs to our laboratory, has been successfully used in the industrial production of glycerol because of its high glycerol concentration, yield. However, the glycerol synthesis attenuation exsists in fed-batch fermentation, compared with batch fermentation. In order to reveal the mechanism of glycerol synthesis attenuation of C. glycerinogenes in fed-batch fermentation, metabolic flux analysis, transportation and synthesis of glycerol, product inhibition and energy metabolism were studied under extreme fed-bactch conditions. The results should provide the theoretical basis for relieving glycerol synthesis attenuation of C. glycerinogenes in fed-batch fermentation.At the fermentative level, the glycerol synthesis of C. glycerinogenes in extreme fed-batch fermentation was comprehensively studied. It was found that the glycerol concentration decreased by 13%, the conversion ratio of glycerol decreased by 34%, the glycerol synthesis rate decreased by 67% and the glucose consumption rate decreased by 44% respectively, compared with batch fermentation.At the cellular level, the cell number, plasa membrane composition and membrane permeabilization were also studied. It was found that cell morphology and cell number basically maintained invariable in batch and fed-batch fermentation, which indicated that cell morphology and number have little effect on glycerol synthesis attenuation. As the major part of cell membrane, the membrane protein dropped from 0.040μg/mL to 0.028μg/mL, while the phospholipid content dropped from 0.130 mg/mL to 0.070 mg/mL in fed-batch fermentation. Coefficient of membrane permeability(P'), a parameter used to measure the membrane permeabilization, also dropped from 4.86×10-5 cm/h to 1.11×10-5 cm/h in fed-batch fermentation, which indicated that the glycerol transportation would be impacted.At the enzymatic level, the key enzymes in metabolic pathway were also studied. The activity of ctGPD and G6PDH decreased by 34.9% and 24.4% respectively, while the activity of PYK increased from 131.1 mU to152.5 mU, rising by 16.3%. ctGPD, a key enzyme for glycerol formation, can directly affect the glycerol formation and the canbon flux flowing to glycerol synthesis pathway. G6PDH can influence the carbon flux to HMP, which not only plays an important role in cell growth, but also provides reducing power for glycerol formation. PYK influences the metabolic flux distribution between EMP and glycerol synthesis pathway. On the other hand, the by-product ethanol produced in EMP and glycerol will compete for NADH, which indicates the glycerol production will be affected.At the carbon metabolic level, MFA was used in analyzing the metabolic flux distribution in both batch and extreme fed-batch fermentation. It was found that the metabolic flux distribution changed compared with batch fermentation. The metabolic flux decreased by 48% and 33% respectively in HMP and glycerol synthesis pathway, while the metabolic flux increased in EMP, which led to the increase of by-products. The results suggest that fed-batch fermentation leads to the carbon declination from HMP to EMP.The energy metabolism was also studied. Although the energy metabolism slightly increased after fed-batch, it still kept at a low level, compared with batch fermentation. It simply means that the glucose consumption will remain at a relatively low level. The NADH/NAD+ in product synthesis phase was low and decreased by 40.5%, compared with batch fermentation. The NADH/NAD+ did not increase by the addition of fresh medium, which means that the product synthesis will be impacted.The extreme fed-batch fermentation may be affected because of the high concentration of glycerol at the beginning, and the experiment simulating the real environment after fed-batch and subculture were carried out. It was found that the glycerol existing in the culture broth would reduce the glycerol synthesis rate and the glucose consumption rate. The glycerol synthesis rate dropped from 1.83 g/(L·h) to 0.71 g/(L·h), while the glucose consumption rate dropped from 1.03 g/(L·h) to 0.31 g/(L·h) at the same time. Although the time the yeast took in reaching the stationary phase was extended, the biomss basically maintains constant at last,.Consequently, the decrease in the activity of the key enzymes, NADH/NAD+, the inefficient reducing power for glycerol formation, the carbon flux to glycerol synthesis pathway, the membrane permeabilization and the inhibition of glycerol showed synergy effects on the glycerol synthesis attenuation in fed-batch fermentation. The results should provide the primary data-materials for the regulation by glycerol synthesis in C. glycerinogenes and strain modification. |
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