Regulate Mechanism Of Dissolved Oxygen (DO) On The Glycerol Fermentation By Candida Glycerinogenes

Candida glycerinogenes, used commercially for glycerol production as an osmotolerant yeast, was used as a model to demonstrate the regulation mechanism of dissolved oxygen (DO) on the glycerol fermentation and thus guide the efficient production of glycerol. Based on the well understanding of relationship between dissolved oxygen and glycerol fermentation, the kinetic characteristics, intracellular metabolic flux distribution, enzyme levels, energy metabolism and redox balance were investigated. The main results were described as follows: The relationship between dissolved oxygen and glycerol fermentation by C.glycerinogenes with shake-?ask experiments is close, and ethanol is the primary by-product of the fermentation process (maximum of 19.7 g/L); the carbon flow of EMP pathway is the mainly competition of glycerol production by C. glycerinogenes; intracellular enzyme of cytoplasmic 3-phosphoglycerate dehydrogenase (ctGPD), 6-phosphate glucose dehydrogenase (G6PDH) and pyruvate kinase (PYK) have a direct regulation to the critical path of glycerol fermentation by C. glycerinogenes.The regulation mechanism of dissolved oxygen on the glycerol fermentation by C. glycerinogenes with the 5-L bioreactor was demonstrated. The highest glycerol production was 113.3 g/L at DO 30%. Analysis of the kinetic characteristics showed that biomass synthesized fast at initial of logarithmic phase with DO 60%, glycerol synthesized fast at the fermentation process with DO 30%, while glucose consumed fast at the fermentation process with DO 10%. The highest biomiss yield on glucose appeared at logarithmic phase of DO 60%. The highest glycerol yield on glucose appeared at initial of logarithmic phase with DO 60%, and a higher level at other fermentation process with DO 30%. Intracellular metabolic flux distribution showed that synthesis of ethanol through EMP pathway was the mainly competition of carbon flux for glycerol production by C. glycerinogenes.Analysis of energy metabolism and redox balance showed that the regulation mechanism of DO on glycerol fermentation by C. glycerinogenes was in fact the regulation of intracellular energy levels, thereby affecting the intracellular NADH reoxidation pathway. The intracellular energy and coenzyme impacted the biological processes, and led to the formation of different production with the regulation of enzyme, thus affecting the synthesis of glycerol. Adequate ATP was supplied for cell metabolism at high DO (60%), but the oxidative phosphorylation will allocate a lot of NADH, so NADH can't reach a higher level for glycerol synthesis. Insufficient ATP was supplied at low DO (1% ~ 10%), cell will gather sufficient ATP through substrate level phosphorlation, and the pressure of pyruvate overflow will be released by synthesize of ethanol and other by-products. Cell does not require excessive substrate level phosphorlation, nor pyruvate overflow pressure at relatively suitable DO (30%), while the NADH gathered by EMP will be reoxidation by oxidative phosphorylation a certain extent, the ethanol synthesis is no longer the key competitive of NADH reoxidation, so there is plenty of NADH to glycerol synthesis.Based on the regulation mechanism of dissolved oxygen on the glycerol fermentation by C. glycerinogenes, the two stage DO control strategy for glycerol fermentation by C. glycerinogenes was proposed to improve glycerol production on 5-L fermenter. Through control DO 60% and change to DO 30% at 36 h get a higher glycerol production (120.2 g/L), and 6.1 % higher than the 113.3 g/L (DO 30%). The time of glucose consumed completely was advance from 132 h (30%) to 120 h, which improved the efficiency of the glycerol synthesis. Above results further confirmed the regulation mechanism of dissolved oxygen on the glycerol fermentation by C. glycerinogenes.