Screening High-yield Strains Producing Surfactin Through Mutation Breeding

Surfactin is a cyclic lipopeptide,can serve as a lipopeptide antibiotic,antimicrobial lipopeptide may also be referred to.Because of their special chemical structure it is given favorable surfactivity,as well as anti-pathogenic fungi and anti-bacteria and so on.In the field of food,cosmetics,medicine,agriculture and other fields it has broad application prospects.The Bacillus subtilis named 5#was first treated as the initial strain in the experiment.Atmospheric and room temperature plasma(ARTP for short)mutagenesis method was put to use.Basing on the lethality curve,180s was regarded as the optimum mutagenesis time.The instrument power was set to 100W,the gas flow rate set to 10SLM.After ARTP mutagenesis,a large number of suspected mutants gained were firstly sreened by blood-plate method and then secongly sreened by acid sinking turbidimetry.In secondary screening model,the culture mode of porous plate should be adopted,thereby improving throughput screening.By comparing the fermentation unit of 24-well,48-well microplate with that of shake flask,24-well microplate whose fermentation level is closer to that of shake flask was selected,reducing the screening error.And the orthogonal optimization of cultural condition of 24-well microplate was carried out,obtaining the optimal combination:inoculation amount was 1%,speed of 180rpm,liquid volume of 2.5mL.In this study,acid sinking turbidimetry combined with microplate reader was used to determine surfactin content,and the surfactin(separation and purification in the lab)standard curve measured by acid sinking turbidimetry combined with microplate reader and spectrophotometer were respectively drawn.The correlation coefficient,R value,was 0.99891 and 0.99561 respectively,showing a good linear correlation.Therefore,instead of spectrophotometer,the microplate reader can determine surfactin concentrations.By studying the correlation of fermentation yields of 26 mutant in the 24-well microplate using acid sinking turbidimetry and the relevant diameter of oil drain ring,obtaining the correlation coefficient,R value,which was 0.80068,indicating that the diameter of oil drain ring can be considered to be an indicator that can evaluate the results of secondary screening in subsequent verification of shake flask.A mutant Y1-19 obtained finally,whose crude product rate of surfactin was up to 10.15g/L,increasing 23.78 percent comparing with the initial strain 5#before mutagenesis.The oil discharge activity increased 26.7 percent.After several passages,it had good genetic stability.Finally,the fermentation conditions of the high-yield strain Y1-19 in shake flask need to be optimized.To determine that the optimal initial pH was 8.5 and culture temperature was 33?.Optimal carbon source was glucose and optimum nitrogen source was peptone.Afetr the orthogonal optimization of Zn2+,Fe2+ and Mn2+,the optimal combination was:Zn2+concentration was 1.0mg/L,Fe2+ concentration was 0.2mg/L,Mn concentration was 1.5mg/L.Under these optimal conditions,the oil discharge activity Y1-19 strain increased 20.69 percent.