Studies On Fermentation Performance Of A Marine Bacillus Subtilis Strain For Acetoin Production

Bacillus subtilis is generally recogonized as safe by the Food and Drug Administration, and has been allowed to be used as feed additives by Agriculture Ministry, with characteristics of easy cultivation, fast growth, easy preservation, high adaptability, non-toxic and non-pollution. In recent years, it has been widely used in the field of industrial biology, biological control of plant pathogens, animal production and purification micro-polluted water. Acetoin is a platform chemical which can be used in cosmetic products, foods, pharmaceutical industry, chemical synthesis, tobacco, coating products and agricultur. Sugarcane molasses is the by-product of sugar produced, and is a cheap raw materials for fermentation with abundant nutrients. The purpose of this work was to explore the fermentation charcheristic of a newly isolated marine Bacillus subtilis strain DL01, and the mutation breeding and sepatation of acetoin.Firstly, the effects of nutrition components and initial culture conditions (such as divalent metal ions, nitrogen sources, initial sugar concentration and pH, etc.) on the growth and metabolism of strain DL01 was investigated, The experiments showed that (NH4)2HPO4 and MnS04 were the optimum nitrogen source and metal ion. The strain grew well in a simple fermentation medium only containing molasses, (NH4)2HPO4, MnS04. D-Acetoin concentration of 52 g/L was obtained after 72 h shaking-flask culture with an initial sugar concentration of 180 g/L and initial pH of 6.5.Secondly, effects of oxygen supply to the growth and metabolism of Bacillus subtilis was determined by batch fermentation. The oxygen was supplied by constant agitation and aeration, constant agitation under stage-enhanced aeration, and constant aeration under stage-enhanced agitation. The optimum aeration rate was 0.4 vvm with an agitation speed of 200 rpm in 0 h-6 h,300 rpm in 6 h-24 h and 400 rpm after 24 h. Under such a oxygen supply mode,58.2 g/L D-acetoin,61.15 g/L acetoin was achieved from molasses-based medium with initial sugar concentration of 200 g/L, and the yield was 0.35 g/g, productivity 0.80 g/(1-h); 60.91 g/L D-acetoin,76.04 g/L acetoin was obtained with initial glucose concentration at 210 g/L, and the yield was 0.42 g/g, productivity 1.00 g/(1h).Thirdly, acetoin seperation from molasses-based fermentation medium was explored by salting-out extraction. Three kinds of salting-out extraction systems have been compared, and ethanol/K2HPO4 system was the best. When the salting-out extraction was carried out under a volume ratio of 2:1 for fermentation broth to ethanol, ratio of 4:3 for fermentation broth to K2HPO4, the acetoin recovery was 96.7% with 94.5% coloring matters removed.Finally, a two-stage induced mutation of UV-LiCl and low temperature plasma-LiCl was tried, and a mutated strain was screened by which acetoin concentration was 21% higher.