Salting-out And Sugaring-out Extraction Of 2,3-butanediol From Fermentation Broths

2,3-Butanediol (2,3-BD) is a green platform chemical with wide applications in chemicals, pharmaceuticals, foods, fuels and aerospace. Recently, the microbial production of 2,3-butanediol with cheap non-food carbon sources (such as sugarcane molasses) has attracted much attention. Down-stream processing becomes the bottle-neck in the industrialization of 2,3-BD production due to the high hydrophilicity of 2,3-BD, comlexed components of fermentation broth and impurities from biomass. The salting-out extraction method, developed by our group, showed high efficiency in the separation of 2,3-BD from fermentation broth, while little work was done on the salting-out extraction of 2,3-butanediol from biomass-based fermentation broths, such as molasses fermentation broths. Sugaring-out is a novel phase separation method which was found in the separation of acetonitrile and was tried in the recovery of metal ions, biomolecules, drugs and proteins, but the application in separation of hydrophilic primary metabolites from fermentation broths was unknown.Therefore, separation of 2,3-BD from fermentation broths was investigated by salting-out and sugaring-out extraction, and the reuse of the bottom phase for 2,3-BD fermentation was explored.Firstly, salting-out extraction (SOE) of 2,3-butanediol from molasses-based fermentation broths was studied. Different systems formed by different solvent and inorganic salted were screened based on the recovery of 2,3-BD and removal of coloring matters, and the K2HPO4/ethyl acetate system was selected for the following study. The recovery of 2,3-BD reached 98.81%, and coloring matters were not detected in the top phase when SOE was carried out in the volume ratio of 1:1 for fermentation broth (4 mL) to ethyl acetate, ratio of 1:1 (w/v) for K2HPO4 to fermentation broth. When the volume of fermentation broths was 400 mL, the recovery rate of 2,3-BD was 91.32% in the K2HPO4/ethyl acetate system.Secondly, sugaring-out extraction of 2,3-BD was explored. The effects of sugars and solvents on phase separation and 2,3-butanediol distribution were investigated to obtain an appropriate sugaring-out extraction system. The t-butanol/glucose system was selected for the following study. The operation conditions on 2,3-butanediol distribution were studied, including glucose addition,2,3-butanediol concentration and (NH4)2HPO4 addition. When the fermentation broth, containing 60.3 g/L 2,3-butanediol and 18.0 g/L ammonium phosphate, was mixed with glucose (30%w/v) and equal volume of t-butanol, about 76.0% 2,3-butanediol was distributed into the top phase and 80.5% glucose into the bottom phase, and the removel of soluble proteins and lactic acid was 77.98% and 86.78%, respectively.Finally, the reuse of bottom phase and cells for 2,3-BD fermentation were tried. The effect of t-butanol on the bacterial growth was investigated by adding t-butanol into the fermention broth, and a concentration limit of 5 g/L was determined. The bottom phase could be diluted to prepare fermentation medium after removing part of the residual t-butanol by vacuum distillation, and wet cells could be used as seed for fermentation. At appropriate conditions, glucose consumption and 2,3-BD production were similar to those using normal fermentation medium and seed.In conclusion, the salting-out and sugaring-out extraction would be two potential methods for 2,3-BD separation from fermentation broths due to the simple operation and easy scale-up. It was feasible by coupling sugaring-out extraction with the 2,3-BD fermentation.