| As an important C3 platform chemical, propionic acid (PA) and its derivatives are widely used in organic synthesis, food, feed, spices, paint, dye, medicine and so on. Currently, PA is mainly produced by petrochemical process. With the increase of fossil fuel price and the desire of human for the green, healthy bio-based chemicals, the biotechnological production of PA attracts increasing attention.In this thesis, Propionibacterium acidipropionici, a strain could accumulate high concentration of PA is used for PA production. It is very important to optimize the cultivation process of P. acidipropionici to achieve high concentration, high yield and high productivity of PA. The obtained main results are as follows.(1) The propionic acid tolerance of P. acidipropionici was improved from 2.5 g/L to 20 g/L via a passage transfer approach, in which the propionic acid concentration was improved gradually. After one year of such repeated passage, an evolved strain was obtained. Compared to the parental strain, the fermentation time of evolved strain was reduced by 14.3%, and DCW was increased by 23.1%, and PA concentration was increased by 41.1% and the productivity was increased by 65% with 20 g/L of initial glycerol in batch culture. The analysis of the intracellular micro-environment of the parental strain showed that the energy charge of the evolved strain was increased by 100%, and the redox potential was increased by 16.5% and the H+-ATPase activity was increased by 52.7%.(2) The optimal nutrition and environment conditions in flask level were investigated. The optimal conditions were:glycerol 20 g/L, yeast extract 10 g/L, poptone 5 g/L, CoCl2 10 mg/L with inoculum ratio of 10% and culture temperature at 30℃.(3) The culture mode was investigated in 7 L fermentor. Four initial glycerol concentrations were investingated to determine their effects on microbial growth and substrate consumption. It was concluded that high concentration, high yield and high productivity of both cell and PA could not be consolidated in a batch process. The effects of different cultivation modes such as pulse feeding and constant-rate feeding on PA production were also studied. High production of cell and PA were all realized in these cultivation modes of glycerol feeding. The fed-batch culture with constant feeding rate at 0.01 L/h of 400 g/L glycerol solutions from 72 h to 120 h was the most suitable approach to attain not only high concentrations but also high yields and high productivities of cell. The PA in the cultivation of P. acidipropionici under which the maximum dry cell weight and PA reached 4.46 g/L and 44.62 g/L at 220 h, respectively.(4) The optimal culture mode was carried out in 10 m3 fermentor, and the maximum PA concentration of 47.28 g/L was obtained at 240 h, and PA production was increased by 6% than in 7 L fermentor, indicating that this culture mode has broad applicability on the industrial scale production of PA on the fundamental realization of high production, high yield and high productivity.(5) The extraction process of calcium propionate was studied. The viscosity of fermentation broth was reduced and the efficiency of filtration was increased when the broth was keeped at 70℃for 10 min. The multiple application of diatomaceous earth showed the best effect and the method was as follows:1 g of coarse diatomaceous earth was firstly used to make filter cake in Buchner funnel and then 1 g of thin diatomaceous earth was added into 70℃of culture broth. After vacuum filtration, the microbial cells were eliminated and the powder calcium propionate was obtained by simple spray dried method. The specific spray drying conditions:inlet temperature of 180℃～200℃and the outlet temperature>85℃. |
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