Research On Fermentation Condition Of Polyglutamic Acid Produced By B.Subtilis CGMCC No.1250

The purpose of this research is to optimize the fermentation conditions of polyglutamic acid produced by B. subtilis CGMCC No. 1250.In order to reduce the production cost of polyglutamic acid, the effects of different substitutes of sodium glutamate, carbon source, nitrogen source and their concerntrations, volume, temperature on γ-PGA synthesis were studied. The best carbon source and the best nitrogen source were sucrose and tryptone, the best substitutes' of glutamic acid Na salt was refined (glutamic acid) raw material. When 70 g/L of sucrose, 50 g/L of tryptone, 30 g/L of NaCl and 266ml of refined (glutamic acid) raw material were added to the media(pH7.0) and B. subtilis 508-13 was cultivated at 37℃ , 220rpm, for 24 h, the concentration of γ-PGA reached 41.7g/L.The metal ions in cultivation media were optimized on the basis of previous study. Three factors were selected among 9 factors by the aid of Plackett-Burman method. Then those 3 factors were further investigated to actualize the best productivity of γ-PGA, which was found to be as follows: calcium ion 3.27mmol/l, magnesium ion 2.35mmol/l, kalium ion 4.13mmol/l. The γ-PGA concentration of B. subtilis CGMCC No. 1250 was increased from 21.5g/L to 51.4g/ L after optimizing the fermentation conditions.A novel method for the quantitative determination of sucrose concentration in broth by high-performance capillary electrophoresis with indirect absorbance detection was also described. The best separation condition was obtained with an uncoated capillary column (75μm×68cm), a running buffer of 30mM 4-aminosalicylic acid (pH 11.0), and a separation voltage of 15kV at 25℃, the separation was closed to 6.661 min, linear calibration curve of peak area versus sucrose concentration was between 0.078g/L—20g/L, the correlation coefficient was 0.99985, the detection limit was 0.035g/L. The influence of buffer concentration, pH and separation voltage, temperature on the value of peak-area and retention time in this method was developed. The time curves of sucrose concentration and Bacillus subtilis cell density in γ-PGA zymotic broth and the correlation between sucrose concentration and Bacillus subtilis cell density were reported.