Strain Mutation Via Satellite, Process Optimization And Scale-up For Industrial Natamycin Production

In this work, the research and development of the whole process to industrialize the natamycin fermatation was performed in depth. With Streptomyces gilvosporeus ATCC13326 as a starting strain, the conventional and space-flight carried mutations were carried out to obtain mutated strains with higher natamycin yield. The medium composition and fermentation process were optimized to further enhance the natamycin productivity. By controlling the dissolved oxygen above the critical DO level, the natamycin fermentation was successively scaled up from laboratory scale to 18 m~3 fermenter. Based on the stability and solubility of natamycin in fermentation broth, an environment-friendly separation technology was adopted without toxic organic solvent. The main results of this work are as followings.(1) A dual-wavelength ultraviolet spectrophotography method was developed to analyze the concentration of natamycin in fermentation broth. The method is simple, fast and accurate with low cost and is suitable for the quick analysis for large amount of samples. The method was reliable and had been used both in the high throughput screening of high productive strain and fermentation process optimization.(2) With Streptomyces gilvosporeus ATCC13326 as a starting strain, its mutations were carried out by UV, UV plus LiCl, space flight. After mutation, a rational screening procedure was designated to screen a 2-deoxy-glucose resistant strain, named as LK04-119. The natamycin yield of this strain reached 1920mgA in shaking flask when glucose was used as the unique carbon source, which was 5-fold higher than that of the starting strain ATCC 13326(318mg/l).(3) We studied the optimal cultivation temperature and the cultivation time of the slant, the flask seed amd flask fermentation; we studied the initial pH of the fermentation medium and the quantity of innoculum also. The temperature for slant cultivation, the temperature, cultivation time and inoculum amount in seed preparation, as well as the temperature, initial pH and fermentation period in natamycin fermentation were determined experimentally. Both orthogonal and rotatable central composite design stretagy were adopted to optimize the medium compositions for saint, seed and fermentation, respectively. The results showed that the highest natamycin yield in shake flask was 5054 mg/1, which is 1.56 times higher than 1975mg/l without medium optimization.(4) The effects of short-chain precursors for natamycin synthesis, such as acids and their salts, alcohols and esters, with 2 to 4 carbon atoms, on the natamycin biosynthesis were evaluated. The results indicated that the biosynthesis of natamycin was inhibited by the addition of the studied acids and their salts, whereas, promoted by the alcohols and esters. The strongest inhibitor was sodium proponiate and the most positive precursor was propanol. There were certain effects of the addition of amino acids on the natamycin biosynthesis, in which, the addition of 0.5 g/1 L-valine can increase natamycin yield up to 27.0%, while the addition of 2 g/l L-asparigin may get 51.5% of natamycin yield.(5) The batch fermentation was performed in a 30 liter fermenter with high-yield strain LK04-196. The fermentation conditions were optimized and the average natamycin yield reached 3784 mg/1, while the highest was 3902 mg/1. The metabolic features of the batch fermentation were analyzed and a fed-batch fermentation stretagy was adopted. The glucose concentration should be maintained above 35 g/1 and the continuous feed time was 24 hours. After 120 hours fed-batch fermentation, the natamycin yield (5094 mg/l) was 34.6% higher than that in batch fermentation.(6) From the results of batch and fed-batch fermentation in 30 1 fermenter, the natamycin fermentation was successively scaled up based on the controlling dissolved oxygen higher than the critical DO value by regulating air supply and stirring rate. In batch fermentation in 1 m~3 ,4m~3 and 18 m~3 fermenter, the natamycin productivity were 4.26 g/1, 4.05 g/l and 3.83 g/l, respectively. A support vector machine model was applied to simulate and predict the fermentation processes in different fermenter scales. The fed-batch fermentation was also carried out in 18 m~3 fermenter and the natamycin yield was 5.07g/l, which was almost the same as that in 30 l fermenter.(7) The effects of pH value, temperature, solvent ratio and buffer solution on the stability of natamycin were evaluated and the hydrolization kinetics was studied. It was found that the natamycin in fermentation broth was much more stable than that in pure water. Also the effect of pH value on the solubility of natamycin was examined. Based on above results, a evironment-friendly separation process with simple operation and low cost was proposed without the application of organic solvent. The high-purity sample of natamycin was prepared for structure identification. The results showed that the product is the same as the commercial natamycin.The creative research works involved in this paper are: the establishment of a new and rapid analysis method of natamycin in the fermentation broth; a high production ability strain without glucose effects by using space flight mutation and rational screening; the fermentation process scale up to 18m~3 fermenter by controlling the level of dissolved oxygen; the development of a new and green natamycin separation process without organic solvent based on the study of stability and solubility of natamycin in fermentation broth.The natamycin production has been successfully industrialized in Lu Kang.