Studies On The Process Of Glycerol Anaerobic Fermentation For 1,3-propanediol Production By Klebsiella Pneumoniae

1,3-propanediol has become one of the most interesting feedstocks in recent years because of its wide industrial applications, such as synthesis of polytrimethylene terephthalate and other polyester fibers. Current commercial production is based on the chemical route, which uses acrolein or ethylene as the starting material. Compared with the chemical synthesis method, biotransformation has the advantage of utilizing inexpensive, renewable resources such as glycerol or glucose, and becomes increasingly attractive.To make the price more attractive, the yield, productivity and concentration of 1,3-propanediol need to be enhanced. In this paper, we had a study on culture medium optimization, aeration conditions and dynamics of fed-batch culture. Based on this, 1,3-propanediol production was enhanced by repeated fed-batch culture with organic acids addition, and the results of experiments were as follows.The composition of medium for seeds culture and fermentation were optimized separately by orthogonal experiments. Based on this, the cell growth and glycerol consumption increased significantly in anaerobic bottle and the concentration and yield of 1,3-propanediol reached 6.06 g-L-'and 0.41 mol-mol-1, respectively,62% and 7.9% higher than that of the control, respectively. At the same time, the 1,3-propanediol production in 5-L bioreactor was also enhanced significantly, similar to that in anaerobic bottle. In addition, the concentrations of yeast extract and ammonium chloride both decreased sharply compared with that of the control, which could decrease the cost of the 1,3-propanediol production.Under initial conditions, the 1,3-propanediol production would not be enhanced under the strategy of two-stage aeration and nutrient addition in late phase of fermentation. At the same time, experiments carried out in 250-mL with aerobic condition indicated that the increased volume of culture medium would be benefitial to PDO production. Based on this, effects of strategies including two-stagy aerating, aeration air and anaerobic aeration on the 1,3-propanediol production was studied separately as the glycerol was used as the solely carbon source, the results of experiments indicated that aerating N2 to maintain anaerobic condition would be benefitial to 1,3-propanediol production than that of others.The specific growth rate of cells in fed-batch culture decreased as the products increasingly accumulated in culture medium, which further would not be benefitial to glycerol consumption and 1,3-propanediol production. Based on this, the specific growth rate was controlled by glycerol feeding rate. The experimental results indicated the specific growth rate affected significantly yields of productions. The yields of ethanol and formic acid on glycerol were high as the specific growth rate was maintained at low level. Increased the specific growth rate, the yields of ethanol and formic acid on glycerol decreased sharply while the yield of 1,3-propanediol on glycerol increased significantly. At the same time, the glycerol specific consumption rate and PDO formation rate both rapidly increased. As the cell growth and 1,3-propanediol specific formation rate declined sharply in late phase of fermentation, the glycerol concentration at the range of 15-26 g-L-1 was testified to be beneficial to the yield of 1,3-propanediol increase. Combined the specific growth rate controlled at higher level and glycerol concentration maintained range of 15-26 g-L-1 could effectively decreased the yields of lactic acid and ethanol on glycerol while increased the yield of 1,3-propanediol on glycerol.The 1,3-propanediol production could be enhanced significantly while lactic acid and ethanol production decreased sharply with the parent strain under the strategy of organic acids mixture addition in the process of fermentation. Based on this, the effects of concentration of organic acids mixture addtion and time of organic acids mixture addition was studied separately and the results indicated that 3.80 mM organic acids mixture was added at the biomass (OD650) of 3.0 would be benefitial to 1,3-propanediol production and the concentration of 1,3-propanediol reached above 70 g-L"1at 30 h of culture.The accumulation of products in medium of fed-batch culture would strongly inhibit cell growth. The strain used for 1,3-propanediol production could maintain the ability of 1,3-propanediol production in long-time cultivation in repeated batch culture in anaerobic bottle. However, when the cells used as inoculums which was maintained for a long-time in late phase of culture would not be benefitial to cell growth in next cycle of repeated fed-batch culture. The 1,3-propanediol productivity and concentration reached 2.86 g-L"--h-1and 44.60 g·LL1, respectively, in repeated fed-batch culture under initial condition. Based on this, the 1,3-propanediol production could be further enhanced in repeated fed-batch culture with organic acids mixture addition. At the same time, multiple-cycle of repeated fed-batch culture with industry-grade yeast extract replacing regent-grade yeast extract, the concentration, yield and productivity of 1,3-propanediol reached 66 g-L-,0.61 mol-mol1 and 3.40 g-L--h-, respectively, which was higher than that of the reported.