Optimization And Control On The Fermentation Process Of Nosiheptide Produced By Streptomyces Actuosus

Nosiheptide is sulfur polypeptide antibiotic by S. actuosus production, mainly inhibits the growth of gram positive bacteria, that can regulate the harmful bacteria of the livestock and fish gut and promote the absorption of nutrients, promote the growth of mice, improve the utilization rate of feed. Compare nosiheptide with other antibiotics in feed additives, nosiheptide has the following characteristics:less dosage, difficult to generate resistance, no cross resistance, no residue, low toxicity, and small influence on environment. With the market demand for the expansion of nosiheptide, The existence of the traditional biological fermentation characteristics which are level low, long production cycle, high cost, contradiction between supply and demand increased with time, therefore, conducts the research to enhance nosiheptide fermentation titer is very important.This study is based on the above background, the strategy combined with the pH control method and under the guidance of surfactants and chemical additives, in order to improve the fermentation unit, optimized control has be used on the S. actuosus producing nosiheptide fermentation process. Design method was be used single factor experiment and multi factors response surface experimental, nosiheptide titer, mycelial biomass, pH, total sugar, reducing sugar and amino nitrogen metabolism as the index to study exogenous additives on the fermentation process of S. actuosus that to obtain the optimal surfactants and additives added conditions. Moreover, this thesis also based on 3L fermentation tank, pH two stage control strategies are explored. These have the positive scientific significance and realistic economic, social benefits in nosiheptide fermentation process control field. The main results are as follows:(1) Effects of surfactant on the Nosiheptide fermentated by S. actuosusComparison the effect of surfactants which are Triton X-100, CaCl2, DMSO, sorbitol, SDS, Tween-80, CTAB on the nosiheptide fermentated by S. actuosus, Using the single factor experiment to study the surfactants add concentration and the add time, and the add concentration are optimize by response surface. Study concluded that kinds of surfactants tested both significantly affected nosiheptide syntjesis and result in low concentration promoting and high concentration inhibiting, The effect of surfctants on nosiheptide production appeared intragroup and intergroup differences. DMSO CaCl2 and Triton X-100 significantly promoted the nosiheptide yield in low concentration range. When adding 0.808mg/mL of DMSO, 0.799mg/mL of CaCl2 and 0.574mg/mL of Triton X-100 at 24h,24h and 48h respectively, the nosiheptide value was up to 931.493 μg/mL, which was increased 32.63% than initial. The response surface optimization conditions was test at shake flask, the average the nosiheptide titer was 1147.502μg/mL of three parallel experiments that increased by 23.19% than before well microplates optimization. This indicates that surfactants to 500mL shake flask amplification is feasible.(2) Base on the regulatin of additives to improve the titer of the nosiheptide fermentationUsing the single factor experiment investigated the effect of chemicaladditives on nosiheptide fermentation, that to obtain their better add level and adding time. Studies show that the tested additives are different degree promotion nosiheptide fermentation level that enhance the effect of the order of EDTA> MnSO4> Ce(NO3)3> olive oil> La(NO3)3> chitosan> nystatin chitosan. The comprehensive results of single factor experiments, in order to nosiheptide titer as index, were optimized by using response surface method for adding concentration of MnSO4, La(NO3)3 and Ce(NO3)3. Research shows that the concentrations of MnSO4,La(NO3)3 and Ce(NO3)3 are respectively at 9.27μg/L,75.15μmol/L and 0.90mmol/L and add time are respectively in the fermentation of 24h,96h,96h, nosiheptide fermentation titer reached 948.08μg/mL that increased by 35.00% than before optimization. The experiments of additives added conditions on shake flask for verification, the average of nosiheptide titer was 1157.836μg/mL of three parallel experiments that increased by 22.12% than before optimization.(3) Researchs of surfactant on the nosiheptide fermentated by pH segmented controlBased on the dynamic analysis of biomass, nosiheptide titer, sugar, amino nitrogen and pH value during the synthesis of nosiheptide by S. actuosus, the fermentation process was constantly regulated by different single pH value to investigate the dynamic variation principles of biomass and specific growth rate of S. actuosus and the nosiheptide synthesis. Ultimately a two-stage pH control strategy was introduced. The results indicate that in nosiheptide synthetic metabolism of S. actuosus, pH fluctuated in the S-shaped, cell growth and nosiheptide synthetic corresponded to different pH values. Under the constant control pattern, S. actuosus has maximum specific growth rate with pH6.5 while nosiheptide synthesis titer was highest with pH7.5. Under C6H12O6/NH3H2O feeding mode to sustain the two-stage pH control, the capacity for nosiheptide synthesis was higher than that of H3PO4/NaOH and (NH4)2SO4/NaNO3 pH value was controlled constantly at 6.5 during 0-72h, thereafter at 7.5, under which the nosiheptide titer was improved 25.58% than that of the uncontrolled mode. Experiments with pH subsection control and optimal conditions of additives added that the nosiheptide titer was 1294.703μg/mL on 3L fermentation tank which increased by 32.21% than before optimization.