Studies On Breeding Of 1,3-propanediol Hyperproduing Strain And Fermentation From Klebsiella Pneumoniae

Poly-Trimethylene Terephthalate (PTT), a newfashioned polyester, has numerous promising properties and applications. 1,3-propanediol (1,3-PDO) as a pivotal monomer to the synthesis of PTT, it has attracted particular attention of many famous corporation in the world in these years. Compared with chemical synthesis, microbial fermentation processes to 1,3-PDO have many obvious advantages, the environmental friendship and price advantage make it more promising, and become the focuses of research.The inhibition of products on microorganism cells will limit the product titer, the yield of 1,3-PDO on glycerol, and lead the product recovery and purification to a troublesome task. These problems may be effectively solved if mutants with tolerance to high concentration of 1,3-PDO and less byproducts were obtained by selective breeding.1,3-PDO,lactate, acetate and succinate were added to fermention media, and their effects were investigated on the aerobic fermentation of Klebsiella pneumoniae. As a result, 1,3-PDO at a concentration of 90 g/L would inhibit the growth of cells completely. Acetate was favorable to cells growth, but repressive to 1,3-PDO accumulation. Cells grow was repressive by lactate. At a certainty concentration, succinate would increase the producion of 1,3-PDO and lactate.Forty-four mutants were obtained after the mini-Tn5 transposon insertion or NTG mutagenesis of K. pneumoniae and selected on seed medium containing 100 g/L 1,3-PDO and 0.17M NaBr-NaBrO₃ (proton suicide method). Mutants were investigated by shaking flasks, and a mutant named No.9 with high 1,3-propanediol productivity was selected. Its lactate dehydrogenase (LDH) activity was only 162U (about half of the parent strain).In the fed-batch cultures of No.9, a 1,3-propanediol titers of 59.85 g/L was obtained (at the moment, the productivity of 1,3-PDO is 0.88 g/L/h), the mole conversion efficiency of glycerol was 53.94%. In the batch fermentation with a initial glycerol concentration at 43.9 g/L, No.9 finally accumulated 21.2 g/L of 1,3-PDO (80% higher than wild type strain), its productivity of 1,3-PDO was 0.96 g/L/h (65% higher than wild type strain). Furthermore, only two products, 1,3-PDO and acetate, were found in the culture.