Research On Fermentation Regulation Of Riboflavin And The Metabolic Mechanism Of Bacillus Subtilis

Riboflavin is the precursor of FAD and FMN which plays an important role in oxidation and electron transfer,and promotes the metabolism of protein,fat and carbohydrate.Therefore,riboflavin becomes an essential vitamin in vivo.In this work,we primarily optimized the medium of recombinant Bacillus subtilis and studied the metabolic mechanisms preliminarily.Plackett-Burman design and Response Surface Methodology design were employed to conform the optimal medium composed of glucose 100 g/L,corn steep 30 g/L,yeast extract 5 g/L,urea 3 g/L MgSO4 1 g/L,(NH4)2SO4 5 g/L,KH2PO4 1.5 g/L,K2HPO4 2.42 g/L.This optimum medium led the concentration of riboflavin from 529 IU/mL to 639 IU/mL in shake flasks,which was 20.8%higher than control.The fermentation was performed in a 7 L bioreactor with a working volume of 2 L.2.5%-5%inoculums(about 10 h)was inoculated into fermentor in the condition of 40℃,pH 6.8-7.0 and dissolved oxygen above 20%.Then intermittent feeding mode and continuous feeding mode were carried out to study the impact on biomass and production.Finally,the continuous feeding strategy achieved the maximum yield of 4825 IU/mL by maintaining a constant glucose concentration at 10 g/L,which caused the volume of production to increase by 65.5%and 23.0%compared to the batch mode and intermittent mode,respectively.In flasks,the effect of different nitrogen sources on the yield of vitamin B2 was studied and found that ammonia could promote the biomass but inhibite the productivity.On the contrary,urea and nitrate could improve the productivity of vitamin B2 but had no significant effects on biomass.Therefore,this work employed the nitrate and ammonium by a proper ratio,and reached a good result both in biomass and production.Ammonia was usually made use of to accommodate pH in 7 L bioreactor.The influence of ammonium ion concentration on metabolic flux was investigated.We adjusted pH with 2 mol/L NaOH and ammonia to keep the ammonium concentration under 10mmol/L.Consequently,the yield of riboflavin achieved 5326 IU/mL and was higher 11.0%than control.