Molecular Breeding Of Lactobacillus Rhanmosus By Genome Shuffling And L-lactic Acid Fermentation

Lactic acid and its derivatives have been wildly applied in the field of food, agriculture, environment, pharmacy, forage, commodity and chemical industry as industrial chemicals and fine chemicals. Because humans and animals can only metabolize L-lactic acid, L-lactic acid has attracted increasing attention in food, feed and pharmaceutical industries in recent years. L-polylactic acid with high intensity rapidly develops in the field of industrial packaging and biocompatible and biodegradable materials. Also, the L-lactic acid has the tremendous impact on the physical nature, productivity and biodegradable rate of polylactic acid. Thus breeding of L-lactic acid bacteria with high optical purity, high production, high yield, and excellent properties suitable for the fermentation has become the focus of researchers. Compared with the enzymatic and chemical methods for producing L-lactic acid, fermentation has obvious advantages in raw materials, production costs and product quality Otherwise, the homofermentative lactic acid bacteria had higher yield, lower production cost, less chance of contaminating hybrid than Rhizopus. Genome shuffling has the great potential in improving the Lactobacillus without the need of knowing genomesequencing data. Therefore, it is feasible to use genome shuffling to breed the lactic acid bacteria with good capacity of fermenting glucose to L-lactic acid which then used for polylactic acid. In this paper, Lactobacillus rhamnosus MEE539 used as the original strain to generate the shuffled strain with glucose tolerant, high lactic acid production, high yield, and high productivity in the laboratory-scale fermentation. To achieve this goal, a systematic study was carried out on the L-lactic acid bacteria breeding and the fermentation process in fermentor and shake-flask.Substrate inhibition is one of the conventional characteristics in lactic acid batch fermentation. Breeding glucose-resistant mutant and then studying its fermentation characteristics will play an important role in industrial production. Application of genome shuffling for strain improvement will increase the frequency of positive mutation and obtain positive mutant strains in a short time without the detailed genetic background of parent strains. In this study, we aim to simultaneously improve two phenotypes (glucose tolerance and high production) of the L. rhamnosus by using genome shuffling. Eight mutant strains were obtained with subtle improvements than L. rhamnosus wild-type strain by ultraviolet (UV) irradiation and nitrosoguanidine (NTG) mutagenesis, and then they were subjected for recursive protoplast fusing. Plate method with high glucose concentration and 2% CaCO3 was proved to be an effective means of initial screening. The formation and regeneration of protoplasts were studied. When bacteria were cultured with 1.2% glycine in advance and treated with lysozyme (10 mg/ml), mutanolysin (30μg/ml) at pH 7.0, the protoplast formation rate was 99.6%.The protoplast regeneration was 16.1% using sugar as the stabilizer in RM. After two rounds of genome shuffling, four strains that can grow on 40% glucose YE solid medium were obtained. The best performed shuffled strain, F2-2, produced 140g/l L-lactic acid, about 1.8 fold over the parent strain, with 150g/l initial glucose in the 16L bioreactor. The cell growth and rate of glucose consumption of F2-2 were 50.7% and 62.2% higher than those of the wild-type strain, respectively. The higher lactic acid concentrations were obtained when the initial glucose concentrations up to 160 and 200g/l in batch fermentation. The research here demonstrated that genome shuffling could greatly accelerate the improvement of important phenotypes of microorganism.The optimizations of medium and fermentation condition are very important for the improvement of lactic acid production Among the nutritional parameters affecting the fermentative lactic acid production, yeast extract (YE) leads to the highest lactic acid concentrations in a variety of nitrogen sources. However, the high cost of YE impairs the economics of lactic acid fermentation because YE was estimated to account for about 38% of the total production cost. Hence, the low cost corn steep liquor was tested in this paper as an effective alternative nitrogen source with the other components. Firstly, the inoculum age (18h), inoculum size (6%), neutralizer (NH4OH) and CaCO3 concentration (8%) were determined.Then glucose, molasses, YE, CSL, and soy hydrolysate were selected by one factor a time method. They were used for the Plackett-Burman design with minerals and surfactant.The glucose,molasses, YE, CSL, MnSO4 and Tween80 were most significant factors. Response surface methodology involving central composite design was successfully applied to evaluate the effect of corn steep liquor along with glucose, molasses, Tween80 and MnSO4 on L-lactic acid fermentation. Corn seep liquor was investigated as a sole and low cost nitrogen source in cooperation with other components to substitute yeast extract for the economical production of L-lactic acid. Statistical analysis of the results showed that the linear and quadric terms of these five variables had significant effects. The interactions between the five variables were found to contribute to the response at a significant level. A second-order polynomial regression model estimated that the maximum lactic acid production of 113.05g/l was obtained when the optimum concentrations of glucose, molasses, corn steep liquor, Tween80 and MnSO4 were 118.20g/l, 37.27ml/l, 42.54g/l, 1.52ml/l and 0.30g/l, respectively. Verification of the optimization showed that L-(+)-lactic acid production of 115.12g/l was achieved in the shake-flask experiment, while the concentration of lactic acid in fermenter were 110.00 g/l. However, the fermentation time using fermenter was shorter than the corresponding fermentation time obtained from the shake-flask cultivation by 12 hours. Comparing to the lactic acid production in the medium with yeast extract as the only nitrogen source, lactic acid production in the optimized medium was increased by 30.4%. Moreover, the yield and the average volumetric productivity of lactic acid were as high as 96% and 4.58g/l.h, respectively. At last, the optimal temperature was set at 40℃and pH was 6.3.This paper also discusses the cell growth, lactic acid production and substrate consumption kinetics model of F2-2 in batch cultivation. A simple model was proposed using Logistic equation for growth, the Luedeking-Piret equation for lactic acid production and carbon balance equation for glucose consumption. The software of Matlab and Origin were used for solving equations and the optimal values of parameter estimated were achieved through nonlinear least-squares fitting method. The model was as follows:Fed-batch fermentation may effectively reduce substrate and product inhibition and toxic substances in the medium. According to the parameters of batch fermentation, two kinds of fed-batch fermentation were carried out. The results showed that exponential fed-batch fermentation of L-lactic acid by F2-2 was better than the fed-batch fermentation with glucose concentration controlling in respect of lactic acid production, yield and productivity. When the total sugar was180g/l, the lactic acid concentration was 162.62g/l, yield was 94.5% and productivity was 3.68g/lh.In short, this paper using genome shuffling technology to obtain the glucose tolerant Lactobacillus rhamnosus F2-2 with high lactic aid production. The Plackett-Burman design and Central Composite Design were applied to optimize the medium. The low-cost corn steep liquor with small amount of molasses, manganese sulfate and Tween80 completely replaced the expensive yeast extract in the medium. The optimized medium also contributed to the improvement of lactic acid production. In addition, the cell growth, lactic acid production and substrate consumption kinetics model were established as the batch fermentation of F2-2 was performed in 30L fermentor. At last, both the exponential fed-batch fermentation and the fed-batch fermentation with glucose concentration controlling were developed for L-lactic acid production.