| Poly(γ-glutamic acid) (γ-PGA) is a water-soluble polymer that consists of D- and L-glutamic acid and is mainly produced by several Bacillus species outside the cells. Potential and unique applications of γ-PGA and its derivatives have been of interest in the past few years in a broad range of industrial fields such as agriculture, food, medicine, cosmetics and water treatment, which can be used as highly water absorbable materials, thickener, drug carrier, biopolymer flocculants and heavy metal absorber. Therefore, the objective of this dissertation is to carry out a fermentation bioprocess with a high γ-PGA yield. The contents mainly include: (1) selection and identification of the strain producing y-PGA; (2) fermentation process optimization and mechanism of y-PGA synthesis; (3) breeding of the high yield strain; (4) the purification and the effects of physical chemistry factors on γ-PGA molecule structure; (5) potential application of y-PGA and the strain. The main results of this paper are as follows:[1] Bacillus sp. B53 was selected from 231 strains that were isolated from 76 specimen and shown to produce poly( γ -glutamic acid). The polymer had an absorption peak at 212nm and its molecular weight was between 570 kD and 669 kD. With the following analysis of colony morphology, physiological and biochemistry experiments, G+C content (mol%) and 16S rRNA gene sequence as well, the strain was identified as Bacillus subtilis.[2] The yield of γ-PGA by B. subtilis B53 was different on various carbon sources and nitrogen sources, and among those, citric acid, glycerol, L-glutamic acid and ammonium sulfate were much better than others; Inorganic salts such as K2HPO4, CaCl2 and FeCl3 facilitated γ-PGA synthesis clearly. Using orthogonal experimental design and regression analysis, the optimum fermentation medium was obtained as follows(g/L): L-Glutamic acid 20, Citric acid 9.86, Glycerol 80.36, (NH4)2SO4 7, MgSO4 7H2O 0.5, FeCl3 6H2O 0.02, K2HPO4 0.89, CaCl2 2H2O 0.03, MnSO4 H2O 0.3; pH 6.5 with NaOH. Under these conditions, γ-PGA 26.67 g/L was achieved in shake flask culture with temperature of 37℃, seed age 24h, inoculation amount 4%(V/V), adding 50mL medium each 300 mL flask and 150 r/min for 84 h. Batch fermentation of γ-PGA in 5 L bioreactor was carried out. The organism cell and the polymer structure changed evidently in the course. Compared with the consumption of citric acid and glutamic acid, the metabolism of glycerol was faster; and γ-PGA synthesis accelerated in the term of glycerol consumption increasing fast; the γ-PGA could be discovered after 12 h and its yield was achieved peak at 84 h, then reduced a few; the molecular weight of γ-PGA was basically no change before 54 h, but its range became wide later; γ-PGA 18.61 g/L was achieved in the batch fermentation. The results of fed-batch fermentation showed adding a few glycerol could increase γ-PGA yields in the middle of the course; and adding penicillin 20~100 U/mL or biotin 10 μ g/L could improve the γ-PGA synthesis in the medium, but adding surfactants (Tween20 or SDS) would be disadvantageous to γ-PGA high-yield.[3] Using protoplasts fusion technique, Bacillus subtilis B53 and Corynebacterium glutamicum B9 were successfully fused. A stable fusion hybrid R162 was obtained, which was clearly different with the parents, its γ-PGA yield was doubled in the medium of glucose and (NH4)2SO4 as carbon and nitrogensources and a few polysaccharide were also formed.Bacillus subtilis B53 was irradiated by 60Co γ-rays; a high-yield mutant was selected and named F2-28, its γ-PGA yield was improved 82.31% by contrast with B53 in the same condition. Cell morphology and forming spore capability of the mutant were researched; the results showed the capsules lays of F2-28 were thicker clearly than before and its forming spore capability changed weakly.[4] γ-PGA was separated by ethanol precipitation. A good separating result was obtained adding 2.5 times 95% ethanol of culture broth after the organisms were removed when γ-PGA concentration of the bro...
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