Study On The Fermentation Conditions For Endophytic Bacillus Amyloliquefaciens CC09 Producing Iturin A And The Purification Process Of Iturin A

Iturin A is one of the cyclic lipopeptides (CLPs) antibiotics produced by Bacillus genus and has broad antimicrobial spectra. However, due to the limited production of Bacillus, it has not been industrialized yet. In this study, we have optimized the fermentation conditions of an endophytic Bacillus amyloliquefaciens CC09 so as to increase the production of Iturin A, and the purification process that could enhance the recovery of Iturin A from the fermentation broth, which could provide basic references for Iturin A industrialization. The main results include:1. Based on single factor test and multiple factor orthogonal experiments, we have investigated the effect of medium composition and cultural conditions on Iturin A production of B. amyloliquefaciens CC09 under flask-shaking batch fermentation. Result indicated that the optimal fermentation condition is:starch concentration (5 g/L), tryptone:yeast powder (3:1) 15 g/L, NaCl 1 g/L, pH 6.0, shaking speed 120 r/min, medium volume 20%, temperature 28℃. In this condition, the strain CC09 could produce up to 690 mg/L of Iturin A, which was about 5 fold as high as it was fermented in LB medium (138 mg/L). The average cost of Iturin A is 6.96 RMB/g.2. In order to reduce the fermentation cost and increase of Iturin A production (basic requirement for Iturin A industrialization), we have optimized the culture media and condition of strain CC09 based on literature survey of the media that were used for B. amyloliquefaciens fermentation by using Plackett-Burman method, the Central Composite Design and Response Surface Technique. Results showed that the strain CC09 could produce up to 501 mg/L Iturin A under the optimized fermentation condition:wheat powder 14 g/L, soybean meal 25 g/L, corn flour 8 g/L, MgSO4 0.1 g/L, MnSO4 2.5 mg/L, FeSO4 0.8 mg/L, liquid volume 60 mL/250 mL, pH6,28℃, 125 r/min, which was 4.2 times as high as the strain CC09 fermented in LB medium. The average cost of Iturin A is 0.30 RMB/g, which was reduced by 95.7% compared to the cost under the above mentioned fermentation condition.3. According to optimized fermentation condition in flask-shaking batch based on Response Surface method, we have determined the optimal fermentation condition of B. amyloliquefaciens CC09 to produce Iturin A in a 25L fermenter. Result indicated that the production of Iturin A by strain CC09 was up to 512 mg/L under the optimal fermentation condition (inoculum size 5%, ventilation 1.2 vvm and 150 r/min) for 48h, which was a little bit higher than that under the flask-shaking batch fermentation condition.4. Five macroporous resins including X-5, XDA-4, HPD300, HPD700 and D101 have been studied for their adsorption and recovery of Iturin A under room temperature. HPD300 shows the strongest adsorption capacity (87.3 mg/g) and recovery rate (91.0%) among the give resins, which is about 38.7%-64.3% and 34.2%-75.9% higher than that of other four resins, respectively. The static adsorption properties of HPD300 to different concentrations of Iturin A fit best to the Langmuir and Freundlich isotherms under various temperatures. The highest adsorption capacity appeared at the concentration of Iturin A 370 mg/L and 25℃. Based on the result of static adsorption, we have determined the dynamic adsorption properties of HPD300 resin to Iturin A in a chromatography column (diameter,2.0 cm). Result showed that the optimal purification parameter of Iturin A was:flow rate of sample loading 2 BV/h, the sample volume 25 BV, resin height to the diameter of the column 1:6 and eluted ethanol 90%. Furthermore, we have studied the optimal elution system based on recovery rate and purity of Iturin A in the final products. Under the following elution process, distilled water (5 BV)â†'45% ethanol (7.5 BV)â†'90% ethanol (7.5BV), we have obtained the highest recovery yield up to 91.2% and purity up to 56.2% of Iturin A. Compared with the concentration of Iturin A (3.6%) in the original extract, the content of Iturin A in the final product was increased about 15.6 fold after purification through HPD300 resin column.Fermentation conditions of B.amyloliquefaciens CC09 producting Iturin A and the extraction process of Iturin A have been explored, ultimately, we abtained the low-cost fermentation medium and efficient chromatographic separation method, which could provid reference to the industrial production of B.amyloliquefaciens CC09 and Iturin A.