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Mutation Breeding and Process Optimization of Lactic Acid High Yield Strain Preparation Using Xylose

Author: CaiCong
Tutor: OuYangJia
School: Nanjing Forestry University
Course: Microorganism
Keywords: Bacillus coagulans L-lactic acid plasma injection xylose fermentation
CLC: Q939.97
Type: Master's thesis
Year: 2013
Downloads: 71
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Lactic acid, which is one of three organic acids, is an important multi-purpose organicacid, especially the L-lactic acid. It has a broad application prospect due to the distinctconfiguration advantages. In this thesis, xylose and Bacillus coagulans NL01which wasthermophilic strain were selected as raw material and fermentation strains, respectively.Plasma implantation mutagenesis techniques were used to improve the xylose utilization ofsuch strain and to study how to utilize xylose and a steam exploded corn stoverprehydrolyzates to produce L-lactic acid efficiently. The main contents and results are asfollows.Firstly, the fermentation experiment was conducted with regard to steam exploded cornstover prehydrolyzates full of xylose in order to determine the fermentation feasibility of Bcoagulans using the prehydrolyzates. Simultaneously, the influence of different inhibitors onthe fermentation was developed through the analysis of components of the wash water. Theresults showed:1) there was a significant promotion at the formic acid concentration of2g/L,2) acetic acid which had the highest concentration of wash water ingredients could tolerate nomore than10g/L,3) the hydroxymethylfurfural had a significant role in promotingfermentation at the concentration of1g/L,4) when the concentration of levulinic acid andfurfural acid production were not greater than0.5g/L and0.25g/L, respectively, thesignificant promotion was found. The test has proven that the main inhibitors of the washwater did not have significant impact on Bacillus coagulans fermentation.Due to the defects regarding xylose utilization of the majority of the current L-lactic acidproduction strains, the wild-type B coagulans NL01was used as the starting strain. They wereselectively bred, and then rapidly screened by plasma mutation breeding, and high sugar andacid rapid screening tablet, respectively, thereby simplifying the screening process andeffectively finding a strain N01-17with excellent characteristics of xylose fermentation andhigh L-lactic acid yield. The mutagenic conditions:1) the power of ion implanter wascontrolled around100W;2) the irradiation distance was2.5mm;3) the helium flux was10slpm;4) the optimal irradiation time was60s. In terms of the fermentation outcomes of strainN01-17, the maximum yield of L-lactic acid was83.20g/L, increasing21.5%compared withthe starting strain. Meanwhile, the sugar acid conversion rate also improved16.0%comparedto the starting strain.The main factors of culture conditions and medium regarding the mutant strain N01-17producing L-lactic acid were optimized and screened through the single-factor experiment andPlackett-Burman experimental methods. The optimal fermentation conditions:1) rubberstopper was used as fermentation sealing;2) fermentation temperature was50℃;3) culture shaker speed was150rpm;4) initial pH was7.2. The best combination of fermentationmedium ingredients optimized by response surface design were (g/L): xylose100, yeastextract powder9.3, corn steep liquor powder4.5, FeSO47H2O0.03, MnSO4H2O0.03,CaCO350. Xylose was approximately exhausted after60h fermentation, and the L-lactic acidyield and sugar acid conversion rate were85.12g/L and86.12%, respectively. After120hfermentation in3L fermenter, both the L-lactic acid yield and sugar acid conversion ratereached the highest ones:90.29g/L and94.37%, respectively.

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