Screening And Breeding Of Bacillus Licheniformis Strains Highly Producing 2,3-butanediol

2,3-butanediol is an important chemical raw material.It is widely used in food,chemical industry,energy and other fields.Currently the main production strains are pathogenic bacterium,like Klebsiella sp.This article intends to separate the strains that produces 2,3-butanediol from the environmental samples and to increase the 2,3-butanediol production by ultraviolet mutagenesis.Acetoin is the precursor of 2,3-butanediol.The environmental samples were kept in 80℃ for 20 min and 12 red strains were separated on creatine naphthol screening plates.The strains can produce acetoin.The 2,3-butanediol production was analyzed with gas chromatography.The strain with the highest production of 2,3-butanediol was named as 127-7.The strain 127-7 was identified as bacillus licheniformis by 16s rRNA gene and physiological and biochemical methodThe high glucose-resistant strains were screened by UV mutagenesis.The mutant strains can lower substrate inhibition and shorten fermentation time.There were 2406 mutant strains in screening medium of 250 g/L glucose concentration.105 mutant strains with the colony diameter longer than 1.5 mm were selected to do the creatine colorimetric experiments,and measure the acetoin production.Then selected 10 strains of high acetoin production made the shake flask fermentation.The mutat strain BL41 had the highest production of 2,3-butanediol.Compared with the parent strain 127-7,the production was increased by 41.1%.The analysis of the relationship between lactic acid and pH showed that lactic acid yield can be significantly reduced if the pH of fermentation broth is not controlled.In the experiment,the lactic acid production reduced from 21.0 g/L to 0 g/L in shake flask fermentation.In 5 L fermentor,2,3-butanediol production of bacillus licheniformis BL41 was 81.4 g/L in 72 h through the fed-batch fermentation.Furthermore,adjust to carbohydrate supplement strategy and maintain the minimum residual sugar concentration of 30 g/L,the 2,3-butanediol of bacillus licheniformis BL41 was 83.4 g/L in 5 L fermentor.The fermentation time shortened to 46 h.In the fermentation process,fermentation broth viscosity of bacillus licheniformis BL41 increased gradually.However,extracellular polysaccharides production was only 1.0 g/L in fermentation broth.There was no poly glutamic acid production.Further study found that the later period of fermentation,cell number decreased,the cell autolyzed and intracellular DNA was outflowed,which caused the fermented broth to become sticky.Then strains BL41 was screened by UV mutagenesis.There were 61 mutant strains with low level of autolysis on autolysis screening plate.Furthermore,there were 7 mutant strains with high acetoin production were obtained by creatine colorimetric method.The mutant strain BL41-103 production of 2,3-butanediol was the highest in shake flask fermentation,9.0%higher than bacillus licheniformis BL41.The fermented broth pH of mutant strains were all higher than 7.0.The glucose utilization efficiency was different for 7 mutant strains in shaking flask fermentation of different initial glucose concentrations.The 2,3-butanediol production of BL41-196,BL41-103 and BL41-213 were highest.Then the 3 mutant strains were made the batch fermentation in multifors of 1L.The strain BL41-196 got the highest 2,3-butanediol production in multifors of 44 h,65.1g/L,36.0%higher than the parent strain BL41.