Microbial Diversity Analysis During Mellowing Process Of Tobacco Waste Extract And Its Functional Strains Isolation, Identification, And Characteristics Research

Tobacco waste extract(TWE)is the main ingredient and the water discharge form of nicotine in the process of reconstituted tobacco production.So TWE is the direct source of nicotine pollution in modern tobacco industrial production.The mellowing process of TWE is an important step in the production of reconstituted tobacco.The mellowing process can improve the quality of reconstituted tobacco significantly,during which microorganisms may paly important roles.However,less report could be found about the study of microbial diversity analysis in the mellowing process of TWE.In this study,MiSeq high throughput sequencing was used to analyze the microbial diversity in the mellowing process of TWE concentrated solution.In each sample,Lactobacillus is the dominant bacteria,and Candida which can tolerate high density sugar is the dominant fungus.Moreover,the abundance of Bacillus and Pseudomonas,which can tolerate extreme environment and can degrade nicotine respectively,increased significantly after 5 days mellowing.So the process of mellowing has roles on the microbial community structure of TWE concentrated solution.Then,functional strains that can improve the flavor of tobacco or degrade the nicotine were isolated from TWE concentrated solution samples.A strain which has the best flavoring function was identified as one strain of Bacillus subtilis and denominated as Bacillus subtilis SM.The effect of strain SM on improving the flavor of TWE concentrated solution was evaluated.Finally,Bacillus subtilis SM was used to improve the flavor of reconstituted tobacco in 2 m~3 industrial biochemical reaction tank.The result showed that SM significantly improved the flavor of reconstituted tobacco.In addition,Pseudomonas sp.JY-Q,which was also isolated from TWE samples and exihibited the strongest ability of nicotine degradation,was used to degrade the nicotine of TWE in the process of reconstituted tobacco production.After degradation conditions optimization,the strain JY-Q can completely degrade the 1.5 g/L nicotine of 10%TWE within 9hours in a 30 L fermentation tank.In 2 m~3 industrial biochemical reaction tank,Pseudomonas sp.JY-Q was used to degrade the nicotine of 100 L10%TWE at 30?,45 rpm/min.After 24 hours,17%nicotine was degraded and the content of degraded nicotine reached 25 g.At last,in order to reduce the influence of glycometabolism on degradation of nicotine,Red thomologous recombination technology was used to knock down the pathway of glycometabolism.Glucose kinase gene which is the key enzyme gene in glycometabolism was chose to be knockout.The gene of kana was successfully integrated into the genome of Pseudomonas sp.JY-Q while the gene of glucokinase was not knockouted.The designed homologous arms might too short to cause the site of recombine offsets,suggesting that the appropriate length of homologous arms required to design to increase the specificity of homologous recombination to complete the knockout of glucokinase gene in subsequent study.