Optimization of Effective Minerals on Riboflavin Production by Bacillus subtilis subsp. subtilis ATCC 6051 Using Statistical Designs

BACKGROUND: Riboflavin (vitamin B(2)) is an essential component of the basic metabolism, and an important nutritional and growth factor in humans, animals, plants and micro-organisms. It has been widely used in the fields of pharmaceuticals, feed and food additives. The industrial production of riboflavin mostly relies on the microbial fermentation. Designing an appropriate fermentation medium is of crucial importance to improve the riboflavin production. METHODS: In this study, sequential methodology combining a screening test of minerals by Plackett-Burman (PB) and an optimization test by Central Composite Design (CCD) was applied to enhance riboflavin production by Bacillus subtilis ATCC 6051 in shake flasks. RESULTS: Initially, one-factor-at-a-time approach was applied to evaluate the effect of different carbon sources. The results showed that fructose was significantly most effective on biomass and riboflavin production. After that, 13 minerals [CaCl(2), CuCl, FeCl(3), FeSO(4), AlCl(3), Na(3)MoO(4), Co(NO(3))(2), NaCl, KH(2)PO(4), K(2)HPO(4), MgSO(4), ZnSO(4), and MnSO(4)] were studied with the screening test. The results revealed that concentration of MgSO(4), K(2)HPO(4), and FeSO(4) had greater influence on riboflavin production (p< 0.05). A CCD with five factors (concentration of fructose, MgSO(4), K(2)HPO(4), FeSO(4), and yeast extract) at five levels was then used to determine the maximum riboflavin concentration. The optimal concentrations (g/l) of these variables determined by Response Surface Methodology (RSM) were fructose, 38.10; MgSO(4), 0.85; K(2)HPO(4), 2.27; FeSO(4), 0.02; and yeast extract, 4.37. CONCLUSION: Statistical experimental design offers a practicable approach to the implementation of medium optimization. From an industrial view point, our optimum medium, besides fructose and a small amount of yeast extract, is mainly composed of common and cheap inorganic salts, which are available to the industrial riboflavin production.