Strain Isolation And Optimization Of Flask Fermentation Parameters For Efficient Production Of Acetoin By Bacillus Licheniformis And Primary Investigation Of 2,3-Butanediol Dehydrogenase

Acetoin is one C4 compound, naturally existing in wine, cocoa, honey, etc, which is a food flavor allowed by national food regulations. Furthermore, acetoin can be widely used in pharmaceutical, tobacco industry and cosmetics fields. The high cost of general chemical synthesis, the low purity of acetoin and low yield by fermentation from microorganisms at present, the main purpose of this paper is to obtain a relatively high acetion productive strain by screening, to further improve the acetion production through optimization of fermentation process, and to preliminary research 2,3-butanediol deydrogenase(BDH)in the acetoin synthesis pathway.In this study, a high acetion productive strain, osmophilic bacterium MEL-09 was isolated from the Zhenjiang cuchang soil samples with acetoin yield of 22.32 g/L. By morphology, physiological and biochemical property and 16S rDNA sequence analysis, the strain was identified as Bacillus licheniformis. The osmophilic ability of MEL-09 was investigated, it demonstrated that the strain could grow in 100 g/L NaCl or 300 g/L glucose. This was the first report of isolation of a high acetoin productive strain, B. licheniformis.The flaskes fermentation medium and cultivation conditions were optimized by B. licheniformis MEL-09. The optimal fermentation medium contains(g/L):glucose 100.00, yeast extract 12.00, peptone 1.00, (NH4)2SO48.00, K2HPO42.50, MgSO41.50, NaCl 0.50, ZnCl2 0.12. The optimal cultivation conditions:the volum 30 mL/250 mL flask, initial pH 6.5, the inoculum amount 1.0%, inoculum age 10 h, stirring speed 200 r/min and temperature 37℃. Using the optimal fermentation parameteres, the acetoin production, glucose conversion efficiency and productivity were achieved at 41.26 g/L,41.26% and 1.15 g/(L·h), respectively, which was increased by 84.86%,47.89% and 85.48% as compared with those of the initial cultural conditions. Simultaneously, the cell growth(OD6oo)significantly increased from 15.90 to 25.26), and Ya/x(g/(L-OD6oo)) increased to 1.63 from 1.40. Moreover, it found that there was no diacetyl detected during acetoin fermentation from the strain MEL-09 with 2,3-butanediol as a major by-product, and it was known that 2,3-butanediol is catalyzed by the BDH from acetoin by investigation of the acetoin pathway in B. licheniformis, but there is no report of BDH in B. licheniformis up to the present.Refer to the amino acid sequences of B. subtilis 168 BDH, B. cereus ATCC 14579 BDH and B. cereus YUF-4 NADH-linked acetylacetoin reductase/2,3-butanediol dehydrogenase(AACRII/BDH), homology sequence search and analysis was performed by BLASTP in the global proteome of B. licheniformis ATCC14580 to obtain a series of highly homologus protein sequence. The highest similar sequence YJMD was selected as the candidate protein of BDH in B. licheniformis and the function of its gene yjmD was explored by using gene deletion method. The homologous deletion vector pYJMD was constructed using the upstream and downstream of homologous segment gene yjmD and was electrotransformation to B.licheniformis MEL-09, no transformant.