Study On Molecular Breeding Of Lactobacillus Rhamnosus And L-lactic Acid Fermentation

Lactic acid is extensively used in food and pharmaceutical industries. It isincreasingly used as feedstocks for potential biodegradable plastics fromlow-cost and renewable carbohydrates. L-lactic acid is more important forindustrial uses. Microbial fermentation technology can make desired opticallypure lactic acid isomers. Molecular breeding of L-lactic acid production strainbecomes a focus that is paid attention by many research members.Lactobacillus strains have been particularly useful due to their high yield, highgrowth rate and their ability to be genetically engineered for selectiveproduction of D or L optical isomers. The desirable characteristics of industrialmicroorganisms are their ability to rapidly and completely ferment with cheapraw materials, requiring minimal amount of nitrogenous substances, providinghigh yields of preferred stereo specific lactic acid under conditions of low pHand high temperature, production of low amounts of cell mass and negligibleamounts of other byproducts. Development of L. rhamnosus that is more tolerant lowpH and higher volumetric productivity may decrease the requirement for neutralizingagents and lower the cost of downstream processing and reducing the possibility forcontamination, and fermentation at low pH may be combined with efficient in situ productremoval. For an industrial strain, improvement in productivity of microbialmetabolite by the organisms is done not only by strain improvement but alsoby manipulating the nutritional parameters and physical parameters.Experimental design and data analysis using appropriate software makes theanalysis easier as observed in the present study and more attentions were paidon it. The optimization of fermentation conditions, particularly physical andchemical parameters are of primary importance in the development of anyfermentation process owing to their impact on the economy and practicabilityof the process. New low-cost media for L-lactic acid fermentation are desiredto enhance the economics of the L-lactic acid production by lactic acidbacteria.In this study, we aim to simultaneously improve acid tolerance andvolumetric productivity of the L. rhamnosus by using genome shuffling. Themutant strains were obtained with subtle improvements than L. rhamnosuswild-type strain by ultraviolet (UV) irradiation and nitrosoguanidine (NTG)mutagenesis, and then they were subjected for recursive protoplast fusing. Allthe mutant strains were screening on low pH YE plates and then proceed with2% CaCO3 YE plates screening and shake-flask test. The behavior of shuffledstains was investigated in shake-flask and bioreactor. Here we improved theacid tolerance and volumetric productivity of an industrial strain L. rhamnosusby genome shuffling. Five strains with subtle improvements in pH toleranceand volumetric productivity were obtained from the populations generated byultraviolet irradiation and nitrosoguanidine mutagenesis, and then they weresubjected for recursive protoplast fusing. A Library that was more likely toyield positive results was created using fusing the lethal protoplasts obtainedfrom both ultraviolet irradiation and heat treatments. After three rounds ofgenome shuffling, four strains that could grow at pH 3.6 were obtained. Weobserved 3.1-fold and 2.6-fold increases in lactic acid production and cellgrowth of the best performing at pH 3.8, respectively. The maxium volumetricproductivities were of 5.8 g/liter/h when fermented 10% glucose underneutralizing condition with CaCO3, which was 27% higher than the wild type.The L-lactic acid production of Lc-WT and Lc-F34 at pH 4.5 is compared. AtpH 4.5, the lactate concentration of Lc-F34 obtained was 83.8 g/liter withaverage volumetric productivity of 0.998 g/liter/h at 84 hours and themaximum volumetric productivity was 3.59 g/liter/h. While the lactateconcentration of Lc-WT obtained was 52.17 g/liter with average volumetricproductivity of 0.623 g/liter/h at 84 hours and the maximum volumetricproductivity was 3.02 g/liter/h. The average volumetric productivity of L-F 34improved 60% more than Lc-WT. The maximum volumetric productivity andaverage volumetric productivity of Lc-F34 were both markedly improved thanLc-WT. The cell dry weight of Lc-F34 was 4.54 g/liter and markedly higherthan that of the Lc-WT, 3.58 g/liter. The ratio of L-and D-lactic acid infermentation broth were analyzed using Boehringer Mannheim's kit. Theoptically pure L-lactic acid was 98% optical purity of total lactic acid.In order to realize large-scale L-lactic acid production by genome shuffledstrain, an attempt was made to optimize medium components and culturalconditions for maximizing the production of the strain using statisticalapproaches. The optimal condition based on the single factor experiment wasmade in YE medium. We aimed to research effect of the ingredient of medium,inoculum, temperature, pH and alkali and so on factor on growth andproduction of Lc-F34. The optimal carbon was glucose, and the optimal initialconcentration of glucose was 90~110 g/liter. The optimal nitrogen was yeastextract, and the second one was corn steep liquor. The optimal concentrationof Mg2+ and Mn2+ were 0.3% and 0.05%, respectively. The optimal inoculumvolume was 8% (v/v), and the optimal inoculum age was 10 h. Statisticalexperimental designs were used for the optimization of L-lactic acidproduction by the mutant Lc-F34, the best performative strain improved bygenome shuffling. Using Plackett–Burman design, glucose, corn steep liquorand yeast extract were identified as significant factors for L-lactic acidproduction. The quantitative effects of the selected medium components andthe process variables were investigated by central composite design. Theexperimental data obtained were fitted to a second-order polynomial equationusing multiple regression analysis. By solving the regression equation andanalyzing the response surface contour plots: the optimum variables thosesupported maximum L-lactic acid production were 104.5 g/liter glucose, 2.6g/liter yeast extract and 33.6 g/liter corn steep liquor for the mutant strainLc-F34. It has been possible to achieve 95 % conversion of glucose to L-lacticacid to give a concentration of 99.1 ± 2.4 g/liter in 17 h in optimal mediumslightly better than that of the YE media. Corn steep liquor is an inexpensiveand abundantly available raw material in comparison with yeast extract, andthe cost of yeast extract is many times higher than that of corn steep liquor. Ithas further scope for improving L-lactic acid production by optimizing themost significant variables and culture conditions from the present analysis. Itwas verified that corn steep liquor supplemented with yeast extract appeared tobe an economical alternative to yeast extract alone. Temperature and pH playimportant roles in the batch fermentation process. The effects of pH andtemperature on L-lactic acid production were studied using CCD. Accordingto the regression model, the maximum L-lactic acid concentration was reachedat pH 5.5 and a temperature of 40.2°C. L-lactic acid Fed-batch fermentationproduction by Lc-F34 was operated in corn steep liquor medium containing10% CaCO3 under the optimum condition. Batch fermentation was carried outin 30-L fermenter containing 20-L medium. 1200 g glucose was added after 24h. The concentration of lactic aicd in fermentation broth was 154.6 g/L, andyield was 91%.The kinetics model was developed to describe cell growth, substrateutilization and lactic acid production by Lc-F34 that was fermentated in 5-Lfermenter containing 3-L YE medium under controlling pH with 10% CaCO3or NH4OH throughout the process.The kinetics model of bath fermentation when pH was controlled withNH4OH:Cell growth rate model: ddXt = 0 .579×??? 1?7.3X20???XLactic acid production rate model: ddpt = 1 .435×???ddXt???+1.345XGlucose unilization rate model: ? ddSt =4 .246×???ddXt???+1.541×???ddPt???The kinetics model of bath fermentation when pH was controlled with CaCO3:Cell growth rate model: ddXt = 0 .425×??? 1?6.8X22???XLactic acid production rate model: ddpt = 1 .833×???ddXt???+2.237XGlucose unilization rate model: ? ddSt =4 .037×???ddXt???+1.691×???ddPt???Genome shuffling is an efficient approach for the rapid improvement ofindustrially important microbial phenotypes, and it is easy to operate byinactivated parental protoplasts fusing. This improved genome shuffling couldreduce time for screening fusants and improve work efficiency. Our resultssuggest that it is possible for the compatibility of the optimal genomic profilesfor the two phenotypes. As a result, this approach has great potential tofacilitate industrial stains to the rapid improvement of many phenotypes. Fourimproved strains in productivety and acid tolerant were breeded and screenedby genome shuffling. The medium components and cultural conditions formaximizing the production of the shuffed strain were optimized using thesingle factor experiment, Plackett-Burman design response surfacemethodology. The shuffled strain as biocatalysts for the production of opticallypure L-lactic acid has several advantages including high volumetricproductivity, acid tolerance and no aeration was needed. As using this strainL-lactic acid should reduce costs associated with purification, containment,biological oxygen demand, and waste treatment. The kinetics model wasdeveloped under controlling pH with 10% CaCO3 or NH4OH throughout thebatch fermentation process. It can offer theory base for transition from bacthfermentation to fed-bactch and continuous fermentation, mathematic base forfermentation on line control and important information for opitimizingbioreactor design.