Production Of Acetoin By Microbial Fermentation Method

Acetoin is an important flavor compound which is widely used in food, chemical,pharmaceutical and tobacco industries. Currently, acetoin is produced by chemicalconversion from butanedione or2,3-butanediol, but the shortage of raw materials, thecomplexity and the pollution problems of the processes limited the large-scaleproduction of acetoin. By contrast, microbial fermentation for acetoin production hasadvantages of high efficiency, mild conditions and environment-friendly and the like,which can change the pressure from resource and environment that acetoin productionis faced with and is a competitive method for acetoin production. Therefore, acetoinproduction by microbial fermentation attracts more and more attention. This papermainly studied isolation of acetoin-producing strain, optimization of fermentationmedium and conditions and extraction of acetoin from fermentation broth, the maincontents and results were as follows:(1) An acetoin-producing bacterial strain SF4-3was isolated from Japanesetraditional food-natto. When incubated in the flask for96h on a rotary shaker at180r/min and37℃, acetoin concentration of strain SF4-3could reach33.9g/L usingglucose as the main carbon source. The fermentation broths of strain SF4-3wereanalyzed by gas chromatography (GC) and gas chromatography-mass spectrometry(GC-MS), the main product was acetoin and purity was more than91%, withoutbutanedione and2,3-butanediol produced which were commonly present asby-products. Based on morphology, physiological and biochemical characteristics aswell as the16S rDNA gene sequence, strain SF4-3was identified as Bacillus subtilis.(2) To improve the acetoin-producing ability of B. subtilis SF4-3, single factor test,orthogonal test, Plackett-Burman design and response surface methodology (RSM)were employed to optimize the medium compositions and fermentation conditions inshake flasks fermentation experiments. The optimal culture medium and fermentationconditions for the acetoin-producing B. subtilis SF4-3were obtained (glucose160g/L,yeast extract8.5g/L, corn steep liquor14.6g/L, urea3.8g/L, manganese sulfate0.05g/L, ferrous sulfate0.10g/L, temperature37°C, inoculum size5%, inoculum ages7h,media amount30mL/250mL, shaking speed180r/min, culture time96h). Under theoptimized cultural conditions, the time course of shaken flasks fermentation wasconstructed and the maximum acetoin concentration achieved was56.2g/L, theacetoin concentration was highly increased compared with the initial condition. (3) A novel aqueous two-phase system (ATPS) composed of hydrophilic solventsand inorganic salts was developed for the extraction of acetoin from fermentationbroths. Based on the partition coefficient and the recovery, the ethanol and dipotassiumhydrogen phosphate system could be used to extract acetoin from fermentation broths.According to the phase diagram of the aqueous two-phase system, the influences ofphase composition on partition of acetoin were investigated. The maximum partitioncoefficient（16.81）and recovery（98.2%）of acetoin were obtained, when25%(w/w)dipotassium hydrogen phosphate and26%(w/w) ethanol was used. The amplicationexperiment indicated the aqueous two-phase system had good stability and the changesof the scale of the system would not affect the partition coefficient and recovery foracetoin, which showed the ethanol/dipotassium hydrogen phosphate aqueoustwo-phase system could be applied to large scale industrial production.