Effect Analysis Of Nitrate Reduction On Poly-Gamma-Glutamate Acid Biosynthesis In Bacillus Licheniformis

Nitrate is an important inorganic nitrogen sources for microorganism. Nitrate is first reduced to nitrite and then transformed to ammonium. This process has some kinds of physiological functions for bacterium. It can help cell to stabilize the membrane proton gradient as electron acceptor, or to maintain balance of reducing power, or to supple diet for growth as nitrogen source. Nitrate reduction has been exploited in many bacteria, however the function of nitrate reduction on poly-gramma-glutamate acid (y-PGA) in Bacillus licheniformis was not research. In this, the effect of nitrate on y-PGA fermentation was first study. At the same time, the metabolomics sample handling process of y-PGA fermentation broth was set up. On this basic, the relationship of nitrate reduction and y-PGA synthesis was exploit in metabolism level by using GC-MS. At last, the specific physiological functions of two nitrate reductases was study through analysis the divergence of nitrate reductase deletion mutant with original strain. The conclusions are as follows:1, Direct centrifugation and membrane filtration as conventional method in metabolomics were not suit for cell collect from y-PGA fermentation broth. Acid treat would cause the death of cell and the percent of mortality had a negative correlation with pH. Six solvent was choose, including pure methanol, pure ethanol, methanol-ethanol mixture,10M HC1, saturated trichloroacetic acid and perchloric acid, and the quenching effect was compared. Pure ehanol was the most suitable quenching solven. Otherwise, five extract methods were compared included hot pure methanol, hot pure ethanol, cold pure methanol, cold pure ethanol and freeze-thaw. Hot ethanol methanol was the best method. This metabolomics sample handling methods based on ethanol could provide more separation effect, more metabolites and better stability for GC-MS detect.2, Nitrate could great improve the y-PGA synthesis capability of B. licheniformis WX-02and the maximum production increased from22.28mg/mL to35.23mg/mL. Nitrate reduction improved the utilization of extracellular L-glutamate, but also strengtheneds the synthesis of intracellular L-glutamate. Nitrate reduction was close link with glycolysis and nitrogen cycle in y-PGA synthesis. Nitrate reduction was significant effected by dissolved oxygen level. Meanwhile, the tolerance of B. licheniformis WX-02to nitrate and nitrite (the lowest inhibition concentration) was lower in nutrient-pool medium than in nutrient-rich medium. 3, Glycolysis, tricarboxylic acid cycle, organic acids and amino acids were both response to the y-PGA synthesis and nitrate reduction. The metabolism activity of glycolysis and tricarboxylic acid cycle were improved during y-PGA synthesis and nitrate reduction would further strengthen this function. So, the extent of metabolism change caused by y-PGA synthesis was deeper than that caused by nitrate reduction. The change of intracellular concentration of6-phophate glucose, glutamate and glutamine indicated that the three metabolites maybe were common response metabolite note for y-PGA synthesis and nitrate reduction. In addition, the accumulation of some metabolites was dependence on special condition. L-cysteine was only found in nitrate reduction. Glyceraldehyde3-phosphate, glutarate and L-alanine were only accumulated in y-PGA synthesis. Acetoacetic acid, malonic acid and L-valine were detected in non-y-PGA synthesis. Propionic acid was the only intracellular metabolites which required the exit of y-PGA and nitrate reduction at the same time.4, The result of nitrate and nitrite reduction revealled that respiratory nitrate reductase carried out the nitrate reduction and assimilatory nitrate reductase effected the transform from nitrite to ammonia during y-PGA fermentation. The nitrate reduction of B. Hcheniformis WX-02was a respiration-assimilatory process during y-PGA fermentation. Integral nitrate reduction pathway helped cell to use more exogenous glutamate. Furthermore, the physiological functions of these two reductase had a relevant with the component of inorganic nitrogen source in medium. In addition, the integrity of the nitrate reduction pathway had a significant impact on uptake of extracellular citric acid, acetic acid and acetoin metabolism in B. Hcheniformis WX-02.