Salting-out And Sugaring-out Extraction Of Acetoin From Fermentation Broths

Acetoin is a natural flavor widely existed in nature it is an important intermediate in the production of related products in pharmaceutical, chemical, liquid crystal, paint industry, and so on. In recent years, the acetoin research focuses on strain screening and improvement, and optimization of fermentation conditions, while the separation is rarely studied. One of the key problems for large-scale production of acetoin is the downstream processing, because it is difficult. In our previous study, a high-production acetoin strain of marine Bacillus subtilis has been screened, and the fermentation characteristics were studied. Based on the situation of acetoin fermentation study and our previous work, in this thesis acetoin production process was studied from three aspects in fermentation medium, separation, coupling of fermentation and separation.First, effects of metal ions on acetoin formation was studied using Bacillus subtilis DL01. A Plackett-Burman design was used to evaluate the influence of seven metal ions. The result showed that Mo6+, Zn2+, Mg2+, Fe2+ and Ni2+ had significant influence on acetoin formation, and Mo6+ and Mg2+ were positive factors. Then, the path of steepest aseent and central composite design were used for further optimization on these two factors, and the optimal concentration of the variables were determined as 1.85 mmol/L Mg2+ and 3 mmol/L Mo6+ according to the analysis of Design Expert. Finally, acetoin production was verified by shaking-flask cultivation and 47.79 g/L was obtained, which was 16% and 13% higher than the control (without metal ions) and the test that just added Mn2+ (0.05 g/L), respetively.Secondly, salting-out extraction (SOE) of acetoin from fermentation broth by mixed solvent was explored. The distribution of acetoin in different SOE systems was investigated, and the system composed by B-ethanol (8:2, v/v)/K.2HPO4 was selected. In a SOE system formed by fermentation broth (4 ml), B-ethanol (4 mL) and K2HPO4 (4 g), the recovery of acetoin reached 95.59%. When the system was magnified 25 times, the recovery of acetoin both reached 95% if the amount of K2HPO4was 50% and 75% (m/v) of fermentation broth, respectively, and stirring time was 3 min. The recovery coefficients of acetoin was 85% when the organic solvent reuse once. The recovery coefficients of acetoin decreased as recovery times of phosphate increased, indicating that the impurities in the recovered salt had significant impact on the distribution of acetoin in two phases.Finally, sugaring-out extraction coupled with acetoin fermentation was studied. First, the distribution of acetoin and glucose in different sugaring-out extraction system was investigated, and growth inhibition of organic solvent on B. subtilis was examined by shaking-flask cultivation. Considiering the distribution of acetoin and glucose and the IC50 value of organic solvents, the ethyl acetate system was selected which showed low toxicity and moderate recovery. Secondly, the effects of glucose concentration, standing time and the amount of ethyl acetate on acetoin distribution were checked. When the mass to volume ratio of glucose and fermentation liquid was 100%, the volume ratio of fermentation broth and ethyl acetate was 1:2 and 1:3, the recovery of acetoin was 58.31% and 65.35%, respectively, sugar recovery in bottom phase was nearly 100%, and the coloring matters and organic acids in top phase were not detected. The condition of volume ratio of 1:2 for fermentation broth and ethyl acetate was selected for amplification, and the recovery was similar to the above results. Finally, acetoin fermentation using bottom phase as carbon source was tried. Acetoin production was similar to that of the control, cell density was a little higher, while sugar conversion rate was slightly lower, confirmed that sugaring-out exaction coupled with acetoin fermentation was feasible.In conclusion, metal ions are very important for acetoin production. Acetoin could be recovered from fermentation broth efficiently by salting-out extraction based mixed solvent, while lower yield was obtained by sugaring-out extraction. However, the residual nutrients were reused and the amount of wastewater for treating and discharging was greatly reduced by the method of sugaring-out extraction coupled with fermentation. The obtained results in this work will provide an experimental basis for the development of acetoin production by fermentation.