Selection Of High Production L-lactic Acid Bacteria And Optimization Of Fermentation Medium

Lactic acid is extensively used in food and pharmaceutical industries. It isincreasingly used as feedstocks for potential biodegradable plastics and the greaterdemand is for the L isomer. For an industrial strain, improvement in productivity ofmicrobial metabolite by the organisms is done not only by strain improvement butalso by manipulating the nutritional parameters and physical parameters. Bothobjectives were obtained by using genome shuffling and experimental design anddata analysis with appropriate software in the present study.In this work, six 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.All the mutant strains were screening on 15%Glucose YE plates, low pH YE platesand 2% CaCO₃ YE plates. Then they were subjected for recursive protoplast fusing.A Library that was more likely to yield positive results was created using fusing thelethal protoplasts obtained from both ultraviolet irradiation and heat treatments.After two rounds of genome shuffling, four strains that could grow at pH 3.6 wereobtained. The maxium lactic acid production was of 125.2g/L by Lc-F22 , which isthe best performative strain improved by genome shuffling. When Lc-F22 fermentedafter 36 hour under neutralizing condition of 15% glucose with 12%CaCO₃, its lacticacid production was 61% higher than the wild type. The volumetric productivity ofLc-F22 was 3.48 g/L/h and the 83.4% conversion of glucose to L-lactic acid wasobtained, all of them were markedly improved than Lc-WT. So we got the improvedstrain of high volumetric productivity, acid tolerance and relative high sugarcontent,they also got genetic stability.In order to realize large-scale L-lactic acid production by genome shuffledstrain, an attempt was made to optimize medium components and cultural conditionsfor maximizing the production of the strain using statistical approaches. The optimalcondition based on the single factor experiment was made in YE medium. We aimedto research effect of the ingredient of medium, inoculum and factors on growth andproduction of Lc-F22. The optimal carbon was glucose;the optimal nitrogen wasyeast extract, and the second one was corn steep liquor. The optimal inoculumvolume was 9% (v/v), and the optimal CaCO3 was 12%. Statistical experimentaldesigns were used for the optimization of L-lactic acid production by the mutantLc-F22, Using Plackett–Burman design, glucose,molasses, corn steep liquor andyeast extract, Tween,MnSO4 were identified as significant factors for L-lactic acidproduction. The quantitative effects of the selected medium components wereinvestigated by central composite design. The experimental data obtained were fittedto a second-order polynomial equation using multiple regression analysis. Bysolving the regression equation and analyzing the response surface contour plots: themaximum L-lactic acid production was 111.5 g liter-1, yield was 93% andproductivity was 4.65 g/L/h. Corn steep liquor is an inexpensive and abundantlyavailable raw material in comparison with yeast extract. It was verified that cornsteep liquor could be an economical alternative to yeast extract alone.In short, the shuffled strain Lc-F22 which we screened in this study has severaladvantages for the production of L-lactic acid including high volumetric productivity,acid tolerance and relative high sugar content.