Development Of Fed-batch Culturing Process Of Bacillus Subtilis For Producing Amylase

Amylase is widely used in various industrial fields,and its market occupies most of the industrial enzyme catalysis market.This work focused on the culture of genetic engineered Bacillus subtilis strain for amylase fermentation production,from several issues like,growth properties of strain,fermentation optimization on both flask and fermenter scale,and optimized fed-batch process,a series of results were gotten:Firstly,the genetic stability of the recombinant plasmid of the used strain was first examined,together with the checking of amylase productivity following several generations of the studied strain.It was found that there were 100%stable plasmid contained strain after 84h of fermentation under the screening pressure of kanamycin,while it dropped to 65%without addition of kanamycin.It was also found that after 72 h of seed culture the productivity of the strain decreased significantly.Secondly,on the level of flask fermentation,optimizing culture conditions were carried out:single parameter experiment was designed to get optimized broth ingredient,filling volume ratio,seeding time etc.Based on these,the medium composition was optimized through the single factor experiment and the orthogonal experiment.The optimized condition was as followed:inoculation ratio to be 10%,filling volume to be 50 mL in 500 mL flask,seed transfer time to be 24h.The best carbon source was corn starch,and best nitrogen source was cotton meal with their optimized concentration to be 112g/L(corn starch),and 51.75 g/L(cotton meal),the corresponding final enzyme titer reached 880 U/mL,which was 41.9%higher than before.Lastly,we investigated the rotation speed effect on the cell growth in the 5 L bioreactor scale.It found that higher or lower rotating speed were harmful to the cell growth at the early stage of the fermentation.However,the cell grew well with DO not lower than 40%saturation.The batch culture was developed.,and the amylase activity at the end of fermentation was 850 U/mL.Based on these findings,two fed-batch processes both for broth medium before optimization and after optimization were developed,and their effects were compared.Fed with only glucose or full medium composition were investigated,for which the final enzyme titer reached 1039 U/mL(18%higher than batch)or 1193 U/mL(35.5%higher than batch)for the broth medium without optimization.The same two strategies were carried out for optimized medium composition,and it found that only fed glucose showed no effect,however,feed full medium could reach to 1200 U/mL of final enzyme titer,which was 41%higher than the corresponding batch process.This work pave the road for industrialization of the fed-batch process using the Bacillus subtilis to produce amylase,based on the whole research work from culture condition optimization,medium composition screening,to the fed-batch culture process that optimize the amylase fermentation.