L-Lactic Acid Fermentation Process Optimization And Central Carbon Metabolic Network Construction Of Lactobacillus Paracasei

Lactobacillus paracasei is a potential strain for industrial lactic acid production which can produce lactic acid with high productivity, optical purity and concentration and tolerate high osmotic pressure. But high nutritional requirements become a major obstacle on the road to its industrial production. On the other hand, the usage of calcium hydroxide as neutralizing agent in the traditional lactic acid production process led to the production of large amounts of calcium sulfate as a byproduct which had a significant negative impact on environment. High chemical and optical pure L-lactic acid production based on NH3·H2O as neutralizing agent in fermentation processes and chromatography- or electrodialysis-based separation methods mark the beginning of a new way forward for industrial-scale lactic acid production.Based on the above two issues, this paper studied the following aspects.C/N ratio of the fermentation medium of L. paracasei was optimized firstly with the initial glucose concentration maintained at 135 g/L.When the C/N ratio (mass ratio) of the fermentation medium was 28.97, lactic acid produced by per gram cell dry weight reached its maximum.Then the influences of culture temperature (37,39 and 41℃)and pH (5.5,6.0,6.5, 7.0,7.5) on growth, lactic acid production, consumption of sugar and formation of by-products were investigated.The results showed that 37-39 is the optimum growth temperature of I. paracasei; pH6.0-7.0 is the optimum growth pH range.The most optimum temperature and pH for lactic acid production is 37℃ and 6.0, respectively. At higher temperature condition, the amount of acetic acid in fermentation broth increased, and when the pH was higher than 6.5, a significant increase of acetoin content in the fermentation broth was displayed.Secondly, the original analytical pure reagents (Yeast Extract and Tryptone) were alternatively substituted by domestic and industrial grade reagents, and yeast extract FM802 and peptone FP101 exhibited their best performance on replacement. At batch fermentation process with 135 g/L glucose in 5L fermenter, L-lactic acid concentration, yield and the maximum productivity were 110 g/L,87.1% and 8.75 g/L/h respectively, which reached the same level as the control; the cost for feedstock of nitrogen sources was reduced by 71.0% after substitution, while the fermentation time only delayed by 1 h. When the glucose concentration of the batch fermentation process was increased to 180 g/L, the fermentation time was more than 60 h and the productivity of L-lactic acid decreased to 2.71 g/L/h. To shorten the fermentation time and enhance the productivity, a pH-based fed-batch fermentation method was set up using a mixture of glucose and ammonia as feeding solution, in which the ratio of glucose concentration to ammonia was decided by the quantitative relationship among glucose consumption, lactic acid production and the consumption of ammonia. When the concentration of residual glucose was maintained around 9 g/L, the fermentation process was finished within 36 h. With the establishment of the cleaner production processes of lactic acid by L. paracasei using ammonia as a neutralizing agent, 180 g/L glucose was consumed at the consumption of 12 g/L industrial grade organic nitrogen source and a higher productivity of 4.44 g/L/h were obtained than that in batch fermentation processes.However, the above results do not solve the problem of the high nutritional requirements of L. paracasei fundamentally. The construction of the global metabolic network and metabolic flux analysis will help us to find the nutritional requirements of the cell exactly, which can provide the bases for low-cost nitrogen sources substitution and genetic modification. For this purpose, central carbon metabolic network of L. paracasei was established by the method of 13C-labeling experiment with [1-13C] glucose as substrate. The results showed that the network included EMP, PPP, TCA cycle, pyruvate metabolic pathway and anaplerotic pathway of oxaloacetate, no Entner-Doudoroff pathway and glyoxylate cycle involved. This work laid the foundation for the further metabolic regulation of L. paracasei.