Study On Improvement Of 1, 3-Propanediol-producing Strains By Mutation And Its Fermentation

1,3-propanediol is acknowledged as one of six new petrochemical products currently in the world. Its main function is as an important monomer to synthesize a new type of polyester,polyether,polyurethane. 1,3-Propanediol is produced by two ways:chemical synthesis and microbial conversion. Producing 1,3-propanediol by microbial fermentation have many obvious advantages and become the focuses of research.Klebsiella sp. was used as the original strain for further research. A high 1,3-Propanediol-producing strain was obtained by UV and (0.5mg/mL)NTG-ultrasound (200W, 50kHz, 20min) inducing mutagenesis. In batch fermentation of mutant,the production of 1,3- propanediol was increased to 23.31 g/L (by 36.72%). The mutant strain showed genetic stability after 10 generations. The by-product, acetate, was increased, and ethanol was decreased, respectively. The amount of enzyme was not found changed significantly, but the activity of GDHt rises by 21% and the activity of PDOR also rise in a certain extent.The fermentation condition for Klebsiella sp. mutant producing 1,3-propanediol was preliminary optimized by single factor experiment. Different concentration of glycerol, yeast extract, KH2PO4 and other factors influencing the glycerol conversing to 1,3-propanediol was studied respectively. A further study on the fermentation medium composition was by orthogonal test. The optimum medium for mutant was as follows: glycerol 60g/L, yeast extract 6g/L, KH2PO4 3g/L,MgSO4·7H2O 1g/L. In flask fermentation, the production of 1,3- propanediol was increased to 27.03 g/L(by 9.92%).Effects on 1,3-propanediol production of Klebsiella sp. mutant by addition of reductant(VC, GSH, Cys, DTT)were studied.The yield of 1,3-propanediol can reach 29.77 g/L, 29.32 g/L, 29.87 g/L by adding 0.35 mmol/L DTT, 0.35 mmol/L Cys, 0.45mmol/L GSH singlely. They were all exceeded the value of control(27.01 g/L). The yield of 1,3-propanediol is 30.01 g/L(rise by 11.11%)by adding 0.25 mmol/L VC. Studying on the concentration variation of side products by adding VC, the results showed that the final concention of acetic acid, ethanol is increased by 3.77%, 3.35% accordingly. Experimental results also showed that addition of VC had an inhibitory effect on cell growth, but increased 1,3-propanediol production per cell considerably.Effects on 1,3-propanediol production of Klebsiella sp. by addition of ATP were studied. The result showed that addition of ATP had an inhibitory effect on cell growth. Adding 0.04 g/L ATP had a remarkable inhibitory effect on cell growth, the value of OD600 decreased by 26.5%. The yield of 1,3-propanediol is 30.05 g/L(rise by 11.26%)by adding 0.06 g/L ATP. Addition of ATP increased 1,3-propanediol production per cell and the conversion of side product acetate.Comparing to the control, the yield of acetic acid rised by 8.7% by adding 0.06 g/L ATP. Adding 0.06 g/L ATP to flasks at different fermentation stage(0h,4h,18h), the highest yield of 1,3-propanediol is 30.13 g/L(by 11.26%) by adding 0.06 g/L ATP at 4h. The value of 1,3-PD/OD is maximum (rise by 25.84%)by adding 0.06 g/L ATP at 0h, respectively.