Breeding Of L-lactic Acid High-producing Strain By Genome-shuffling And Research Of The Ferment Technology

Lactic acid (2-hydroxy propionic acid) is generally recognized as one of the three organic acids. The molecular structure of lactic acid has a asymmetric carbon atom, so there are three kinds of optical isomers: L-lactic acid, D-lactic acid and DL-lactic acid, a racemic mixture. Lactic acid, lactate and its derivatives are widely used in food, pharmaceutical, and textile industries. Moreover, an enormous potential is the use of L-lactic acid as a precursor to produce ploy-lactic acid for synthesis of biodegradable plastic. At present, the research effort is focused on breeding of L-lactic acid high-producing strain and selecting inexpensive ferment raw materials to reduce the product cost.During the batch fermentation, the accumulation of lactic acid will inhibit the microorganism growth and productivity. To maintain the pH of the fermentation broth between 5.5 and 6.5, conventional method is to neutralize with calcium carbonate. However, the method complicates downstream process and reduces the lactic acid yield. In addition, it produces large amounts of gypsum as a by-product which is normally dumped into the environment as waster. Hence, it is significant commotional importance to increase acid tolerance of the producing strain, which will make the fermentation with continuous lactic acid removal by advanced extract technology. It will increase lactic acid concentration of the broth and predigest the producing process.In this paper, a strain of Lactobacillus rhamnosus that can produce high optical isomer L-lactic acid was studied. Multiple mutagenesis treatments with ultraviolet irradiation and nitrosoguanidine were applied and the mutants with a slight increase in lactic acid production were selected as the parental strains of genome shuffling. All mutants were screened on low pH YE plates and on calcium carbonate plates and shake-flask test. We improved the recombinant screening efficiency by inactivated parental strains protoplast fusion technique with ultraviolet irradiation and heat treatment. Because it is different of sites damaged by the two treatments, it is propitious to regeneration of the parental protoplast complementary fusion. Using one-factor-at-a-time approach, we optimized the conditions of the protoplast generation. The result is that the strain is cultivated in MRS culture medium with 1.2% glycin for four hours, concentrations of bacteriolysin and mutanolysin are 10mg/ml and 10μg/ml respectively, the time of cell fission is seventy minutes, the time of ultraviolet irradiation is thirty minutes, and the time of heat treatment is two hours. After three cycles of genome shuffling, we obtained five mutants that could grow at pH 3.6 plates, and have higher productivity, namely Lc-F31-4. We observed 3.4-fold and 2.6-fold increases in lactic acid production and cell growth at pH 3.8 YE culture medium, respectively. The maximum volumetric productivity was of 5.8 g/L/h at YE culture medium with 10% glucose under neutralizing with CaCO3, which was 27% higher than the wild type. The experimental results prove that the mutants have characters of increased acid tolerance and improved lactic acid productivity by genome shuffling technology. Lactic acid bacteria are generally recognized as nutritionally fastidious. Lactobacillus rhamnosus can synthesize B-vitamins and amino acids limitedly itself. Yeast extract is the most appropriate nitrogen sources in lactic acid fermentation, but it is too expensive to large-scale industrial production. As an inexpensive source of essential microbial nutrients, corn steep liquor (CSL) containing abundant vitamins, amino acids and metal ions can substitute yeast entirely or partially. In this work, we replaced yeast extract with corn steep liquor mostly, and optimized fermentation medium components and the conditions of lactic acid fermentation by Plackett-Bruman design, steepest ascent and central composite design. The results revealed that there was an considerable interaction between yeast extract and CSL, the optimum medium composition for lactic acid production was found to be: glucose 126.5g/L, yeast extract 3.2g/L, and corn steep liquor 29.3g/L, the optimum fermentation conditions was: pH5.5, 40.2℃,inoculation volume 8%, and neutralization in 25% ammonia.We had lucubrated lactic acid fermentation technology. Batch fermentation of lactic acid was carried out in 30L fermenter containing 15L medium with 120g/L glucose. Maintaining the pH by calcium carbonate, the fermentation periods was 32 hours; terminal lactic acid concentration was 116Lg/L; the maximum volumetric productivity was 6.25g/L/h; and the conversion ratio was 98.3%. Maintaining the pH by 25% ammonia, the fermentation periods was 32 hours too; terminal lactic acid concentration was 100Lg/L; the maximum volumetric productivity was 7.00g/L/h; and the conversion ratio was over 95%. In this paper, the L-lactic acid production by Lactobacillus rhamnosus fermentation using different fed-batch strategies was studied. The effects of different fed-batch methods such as pulse fed-batch, constant glucose concentration fed-batch and exponential fed-batch on the fermentation of lactic acid were determined. According to the results of experiments, exponential fed-batch culture is an effective method for the fermentation of L-lactic acid. It is propitious to the growth of bacterium and the shortening of fermentation time. The maximum L-lactic acid concentration (162.62g/L) was obtained, the fermentation time, average productivity, and conversion ratio were 44 hours, 3.63g/L/h, and 95.4%, respectively.As our research showed, we have obtained acid tolerance strains by using genome shuffling technology. The inexpensive medium components and the fermentation conditions were achieved by biology optimization design. We further study the ferment technology systemically, and obtained the best fed-batch method. These provide integrated theoretic foundation for the industrial production of L-lactic acid.