Study On Optimization Of High-yield Spore Process And Regulation Of Sporulation By Bacillus Subtilis Q409 Strain

Bacillus subtilis is a plant rhizosphere beneficial strain with significant control over various plant diseases,and has widely been used in agricultural production as a biological agent.Spore content of this agent is a crucial factor affecting its agricultural application.This study aims to optimize the fermentation process,increase the spore content of this agent,thereby reducing the production costs.With the cell dry weight as an indicator,through the single factor experiment,Plackett-Burman test,the steepest grade test and Box-Behnken experimental,the optimal culture condition was confirmed:culture temperature,37?;shaking speed,200 r/min;inoculum rate,10%;incubation time,30h;culture pH,7.0?7.2;the optimized medium for B.subtilis Q409:glucose,75.88 g/L;corn syrup,12.23 g/L;calcium carbonate,13.19 g/L;ammonium sulfate,5 g/L;MgSO4,0.40g/L;FeSO4,0.20g/L;pH 7.0?7.2.The biomass of B.subtilis Q409 obtained in optimized medium is 13.37 g/L,23.91%higher than that cultured in initial medium.Control cultivation conditions and medium composition for promoting sporulation of B.subtilis Q409.After 30-hour fermentation,incubation temperature was changed from 37? to 20?,rotation speed dropped from 200 r/min to 140 r/min,and medium pH increased from 6.46 to 7.5.At the end of the fermentation,the highest sporulation rate were obtained as 89.23%,85.86%,93.00%accordingly;Similarly,varying concentrations of MnC12,MgC12and 6-nitrogen uracil were added to the fermentation broth.When their final concentrations are 3.00 mM,8.00 mM,1.50 mM respectively,The highest sporulation rate and spore number(cfu/ml)can be reached with 85.31%,83.14%,89.66%and 5.46×1011,7.45×1011,5.81×1011 respectively,at the end of the fermentation.In 5 L fermentor,the effect of optimized medium compositions,obtained in shake flask cultivation,on the spore formation of B.subtilis Q409 was identified.Bacterial cell number,spore number and sporulation rates in optimized medium increased by 13.25%,22.89%and 7.35%,compared with that in initial medium.In fed-batch fermentation of B.subtilis Q409,the cell number,spore number,sporulation rate were 23.85%,27.95%,4.63%than that in batch fermentation respectively;the increase of 29.49%,33.24%,4.01%in cell number,spore number,sporulation rate were observed in a continuous feeding cultivation of B.subtilis Q409 in comparison with batch fermentation.After 30h fermentation,certain amounts of MgCl2 and MnCl2 were added into the fermentation broth.At 72h,the final sporulation rates and spore number(cfu/ml)were 72.73%,78.23%and 6.4×101,1.2×1012;After 30h fermentation of B.subtilis Q409,the culture pH were changed from 7.0 to 7.5,and the temperature decreased from 37? to 30?,the spore formation rate and the spore number(cfu/ml)were 75.00%,81.71%and 6.3×1011,6.7×1011 respectively,at the final of the 72-hour fermentation.