| Acetoin (AC) is a valuable flavor naturally existing in corn, grapes, cocoa, apples, bananas, cheese, meat and many other foods. Acetoin can be widely used in food industry, pharmaceutical industry, tobacco industry, chemical industry, paint industry, IT industry, plastics industry and other industries.In this study, the optimal fermentation medium with corn steep liquor as substrate for acetoin production from B. licheniformis MEL-09, a high acetoin-producing strain screened by our laboratory, was investigated. The optimal fermentation medium contains (g/L):glucose 100, corn steep liquor 33.0, (NH4)2SO4 9.00, K2HPO41.00, MgSO4 1.50, NaCl 0.50, ZnCl2 0.12, FeCl3 0.01, MnSO4 0.01, pH 7.0. Using the optimal fermentation parateres, the acetoin yield and unit cell yield (YP/X) reached 37.85 g/L and 1.36 g/(L-OD6oo) respectively in flasks when B. licheniformis MEL-09 was grown at 37℃and 200 r/min, with inoculum age 10 h and the inoculum amount 1%. The acetoin yield and YP/X in the optimal condition had increased by 23.91% and 21.85% respectively compared with that in the unoptimized medium. On the basis of flaskes trials, the effect of the agitation rate on acetoin production in a 3 L bioreactor was investigated. The acetoin yield and YP/X were respectively 38.75 g/L and 1.69 g/(L-OD6oo) at agitation rate(600 r/min).In order to investigate the mechanism of corn steep liquor during the acetoin production by B. licheniformis MEL-09, the single-addition technique and the single-omission technique were applied to the five main nutrients (ammonium salt, water-soluble vitamin, lactic acid, amino acid and phosphorus) of corn steep liquor in order to investigate the effects of these components on cell growth and acetoin fermentation. The results showed that lactic acid, amino acid and phosphorus were considered to have significantly improved the cell growth and acetoin yield. Ammonium salt played smaller role in improving the cell growth and acetoin yield, while the water-soluble vitamin basically had no effect. The amino acids may be used as organic nitrogen source or growth factor to enhance the cell growth. Phosphorus provided organic phosphorus source which can be easily used. Lactic acid improved the cell growth and acetoin yield because lactic acid may be a carbon source to provide precursors for acetoin production on one hand, and lactic acid may activate the TCA cycle to provide energy for the cell growth on the other hand. Meanwhile, the buffering capacity of corn steep liquor may be another important factor for high-yield acetoin production. Under the action of these factors, the biomass of B. licheniformis MEL-09 and acetoin yield were respectively 77.06% and 73.42% of those in the optimum medium. Because of the rich nutrients in corn steep liquor, the experiments were only simple analysis of the main nutrients in corn steep liquor and exploration of the effect of these nutrients on acetoin production. There may be many other unknown factors, such as nucleotides, improving the growth of MEL-09 and acetoin production.Based on the batch fermentation in shake flask, different concentrations of glucose were added at stationary phase (24 h) to investigate the effect of glucose supplementation on acetoin production. It was shown that glucose supplementation can improve the acetoin synthesis. Especially when 60g/L of glucose was added and simultaneously pH was intermittently maintained at 6.0, the acetoin yield and YP/X were 55.82 g/L and 1.93 g/(L-OD6oo) respectively, increased by 45.18% and 41.91% respectively. However, the cell appeared secondary growth when 60g/L of glucose was added one time in 3 L fermenter and simultaneously pH was maintained at 6.0. The biomass (30.45(OD600)) and acetoin yield (43.66 g/L) increased by 32.97% and 12.67% respectively compared with those in batch fermentation. However, YP/X and YP/S both decreased because of the organic acid systhesis. |
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