Screening Of Acetoin-Producing Strain From Distiller’s Yeast

Acetoin is a widely used food grade spices. Microbial fermentation production of acetoin has become the dominant direction of future research and production of acetoin. Combined with the actual production problem, in this study, an acetoin-producing bacterial strain was isolated from distiller’s yeast firstly. Then the original strain was mutagenized by using atmospheric and room temperature plasma(ARTP) and the strain mutant was preliminarily screened in 48-well microplates and further screened in shake flask. At last,this paper studied the fermentation mendium and condition of this high acetoin-producing strain in 48-well microplates.(1) Screening the strain producing acetoin and its preliminary identification. An acetoin-producing bacterial strain G7 was isolated from distiller’s yeast by preliminarily screening in 48-well microplates and further screening in shake flask. When it was incubated for 96h at 38℃ with shaking by 180r/min, acetoin yield of G7 could reach 24.30g/L.Based on physiological, morphology characteristics and 16S rDNA gene sequence, G7 strain was identified as Bacillus methylotrophicus strain.(2) Mutation breeding of acetoin producing strain of G7. As the original strain, G7 was mutagenized by ARTP. The mutant was preliminarily screened in 48-well microplates and further screened in shake flask, and evaluated for genetic stability. A high acetoin-producing strain named G7-6 with stable genetic characters was obtained. The acetoin production was 31.65 g/L, which was 30.25% higher than the original strain G7, and the conversion of acetoin could reach 27.31%. We also verified the significant relationship between culturing in 48-well microplates and shake flasks. The experimental results showed that 48-well microplates can simply and rapidly screen high acetoin-producing strains.It also laid the foundation for high-throughput selecting and breeding of acetoin-producing strains in future.(3) Optimizating of fermentation medium components of G7-6 in 48-well microplates.It studied the impact of fermentation medium components to the acetoin-producing of strain G7-6, including carbon sources, nitrogen sources and metal ions. Experiments reveled that the best carbon sources was glucose and the best nitrogen source was yeast powder and soybean meal mixture. Based on the study of single factor tests, yeast powder, soybean meal and (NH4)2SO4 were found to be the most important factors to the acetoin production by Plackett-Burman experiment. Then, the design of steepest ascent experimental was used to approach the best concentration range of the three key factors. Box-Benhnken Design was used to optimize the fermentation medium components, and confirm its experimental validity. The results showed that the optimal fermentation medium components were:yeast powder 11.64g/L, soybean meal 36.86g/L, (NH4)2SO4 4.87g/L.Under these conditions, the acetoin production reached 41.17g/L,30.08% higher than before.(4) Optimization of the fermentation conditions of G7-6 in 48-well microplates. In optimized fermentation medium components, the levels of fluid volume, initial pH, inoculum, culture time and temperature, rotating speed, etc were obtained via single factor tests, with fluid volume, culture time and rotating speed in orthogonal experiments to follow. The optimum culture conditions for G7-6 in 48-well microplates were:volume 1mL, inoculability quantity 5%, culture time 84h, initial pH 7.0, culture temperature 32℃, rotating speed 180r/min. Under the above conditions, the acetoin production could reach 45.46g/L,10.42% higher than before. Meanwhile, the conversion of acetoin had reached 35.08% from 33.12%. To further understand the fermentation of acetoin-producing G7-6, it also investigated the flask fermentation of G7-6 using the optimized parameters of 48-well plates, and the maximum acetoin yield reached 46.21 g/L.