Study On Anaerobic Basic Physiological And Metabolic Characteristics Of Bacillus Licheniformis

Dissolved oxygen is a metabolic substrate that is essential for cell growth,maintenance,metabolism,and production.Of course,oxygen is not necessary for microorganisms,obligate anaerobic bacteria,aerometric bacteria,and facultative anaerobic bacteria can grow,proliferate,and metabolize in an oxygen-free environment.Bacillus licheniformis is a facultative anaerobic gram-positive bacterium.At present,Bacillus licheniformis is mainly based on aerobic fermentation in industrial production,and its anaerobic basic physiological and metabolic characteristics are not well understood,which limits the further expansion of its application.In this project,the anaerobic basal physiological and metabolic characteristics of Bacillus licheniformis were systematically and comprehensively analyzed,and the changes of relevant parameters in the process of anaerobic fermentation were studied by detecting OD₆₀₀,residual sugar,p H,nitrate concentration and overflow metabolite amount in the fermentation process,taking Bacillus licheniformis wild-type strain Bacillus licheniformis as the research object.The main research contents and results of this project are as follows:（1）Establishment of anaerobic culture method and growth metabolic characteristics of B.licheniformis serum bottle.Firstly,the anaerobic culture method of serum bottle of Bacillus licheniformis was established,and then the anaerobic basal physiological and metabolic characteristics of the strain were explored on this basis.When nitrite was found as an electron acceptor,the volume of bacteria in the stable phase was about twice that of nitrate.The maximum value of the glucose specific consumption rate curve when KNO₃ was used as the electron acceptor was 0.34,which was about 1.5 times that of other conditions,and under the condition of adding 1.0 g·L^-1 KNO₃,the glucose consumption rate was the fastest,and its value was 0.94 g·L^-1·h^-1,and under this condition,the maximum value of the lactic acid specific synthesis rate curve was more than 2.5 times that of other conditions,that is,it was most suitable for lactic acid synthesis under this condition.（2）Effect of global anaerobic regulator Fnr on anaerobic growth and metabolism of B.licheniformis.Based on the understanding of the anaerobic physiological and metabolic characteristics of Bacillus licheniformis and the establishment of culture conditions that maintain relatively stable anaerobic growth and metabolism of cells,the effect of the global anaerobic regulator Fnr on the anaerobic growth and metabolism of Bacillus licheniformis was further explored.It was found that Fnr overexpression could increase the anaerobic activity of Bacillus licheniformis,and the lactic acid concentration was also increased by 3.5 times compared with the control strain（3）Effect of Fnr overexpression on transcription intensity of nitrate reductase nar GHJI gene cluster.After understanding that Fnr overexpression can increase the anaerobic activity of Bacillus licheniformis,the deep molecular mechanism behind it was further explored by RT-q PCR.It was found that the transcriptional intensity of nar G,nar H,nar J and nar I genes increased by 1.18,0.19,0.32 and 0.65 times compared with the control strains,respectively.It was shown that Fnr overexpression could increase the expression level of nitrate metabolism genes and further increase the supply of ATP,thereby improving the anaerobic activity of the strain.（4）Analysis of anaerobic growth and metabolic differences of B.licheniformis under different oxygen supply conditions in 5 L fermenter.The physiological and metabolic differences of this strain at different oxygen supply levels were further explored at the 5 L fermenter level.It was found that nitrate was used as nitrate respiration under anaerobic conditions more efficiently than nitrate assimilation under aerobic conditions,and it was also found that acetic acid accumulated rapidly during the exponential growth phase of cells under different oxygen supply conditions,and then the concentration of acetic acid decreased with the autolysis of cells.Acetic acid can be used as a secondary carbon source to maintain cell growth and metabolism.