| Ploy-?-glutamic(?-PGA) is a type of capsule were amino acid polymer mainly produced by Bacillus subtilis. Because of good water solubility, biodegradability,biocompatibility, strong binding, and no toxic effect on the environment, it has a good prospect of agricultural application. The purpose of this study is to provide theoretical support for the large-scale production of agricultural poly glutamic acid. In laboratory,the primary screening of the culture conditions of Bacillus subtilis yt-102 was carried out with the constant temperature oscillator. Then the simulation of mass production is carried out by using 5, 10 and 50 L Fermentation tank. The method of quantitative determination of ?-PGA was discussed. The effect of application of?-PGA was verified by pot-experiment and hydroponic-experiment. In summary, through the research, we get the conclusion as follows:At first, remake the original culture medium of Bacillus subtilis. yt-102 to be cheap and contaminated in the laboratory. The best production medium was obtained(g/L): Molasses 100, Soybean hydrolysate 30, Monosodium glutamate 50, Corn steep liquor 8,(NH4)2SO4 7,MgSO4 0.5, KH2PO4 15. The best culture condition is temperature 37?, inoculation volume 8%, the shaking speed 220r/min, and culture22~24 hours. The highest yield could be achieved is 2.97%, ethanol precipitation obtained 5.77%.Secondly, correction parameters of ?-PGA were carried out in 5, 10 and 50 L tank of optimized fermentation experiments in the LAB. The effects of initial carbon source concentration on the growth and yield of Bacillus subtilis yt-102 was studied by using single-factor experiment. The program of fed batch fermentation was determined:Molasses concentration were 6% in the beginning, after 12 hours later, add the other.The highest yield could be achieved is 3.27%. The best culture condition of the tank is temperature 37 ?, inoculation volume 8%, the shaking speed 400r/min, and pH6.5~7.5.Contrast the accuracy and advantages and disadvantages of ethanol precipitation,Ultraviolet spectrophotometric method, HPLC assay, viscosity and hydrolysis method.The parameter conditions of the hydrolysis method were determined: removal of impurities by centrifugation of samples, determinate the free glutamic acid content,add 5 times the volume of 18.5% concentrated hydrochloric acid, 110 oil bath hydrolysis 4 hours. Then measure the glutamic acid content again. The latter minus the former is the content of?-PGA.The application effect of PGA was validated by pot experiment. Poly glutamic acid can increase the absorption of fertilizer and promote plant growth. Long term application of poly glutamic acid can effectively improve the soil. When using different fertilization methods of potted plants, microbial biomass had a significant difference. The experimental results show that the weak acid type poly glutamic acid fermentation liquid used in experiments in the soil have a certain ease, the content of organic matter compared to other treatment has greatly improved. The nitrogen and phosphorus content in soil decreased, which has greatly improved the ability of plants to absorb inorganic elements. |
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