Sample Preparation For Metabolomics And Effect Of Environmental Factors On γ-PGA Biosynthesis By Bacillus Licheniformis

In this paper, a high-yield poly-y-glutamic acid and salt-tolerant Bacillus licheniformis strain, WX-02, was used, to clarify the effects of ultrasound on the B. licheniformis WX-02 fermentation process of y-PGA, to establish a optimizing preliminary treatment of B. licheniformis WX-02 metabolism samples, to study the effect of environmental factors on y-PGA production. The main findings are as follows:(1) HPLC was used to set up the condition for analysis of intracellular nucleotides of B. licheniformis WX-02. According to the physical and chemical properties of energy substances (ATP, ADP and AMP), and different types and content of metabolite components, HPLC parameters were optimized:mobile phase,0.1 mol/L pH 6.25 phosphate buffer,5% methanol,15 mmol/L formic acid; isocratic elution flow rate,1 mL/min; detection wavelength:254 nm; injection volume:15μL; column temperature:35℃. The results showed that high precision and repeatability were obtained for ATP, ADP and AMP detection, relative standard deviation (RSD) was 0.70%,0.90% and 0.28%, respectively; the average recovery of 110%,102%, and 100%, respectively.(2) Preliminary treatment of Bacillus licheniformis WX-02 fermentation sample was established. Parameters such as quenching agents, quenching time, pressure conditions for cells broken were optimized for better detection:quenching agent 60%MeOH/70 mM HEPES, quenching time 30 min, crushing pressure 2000 bar. The total nucleotide 0.6317μmol/g, energy charge was 0.93.(3) The effect of Environmental factors on y-PGA biosynthesis production by B. licheniformis WX-02. Different environmental factors conditions like cold, ethanol, H2O2, sodium acetate and ultrasonic were applied to the y-PGA fermentation. It was found that the ultrasonic treatment and sodium acetate increase the yield of y-PGA. Under the optimal confitions of sodium acetate treatment (supplement 0.6% of sodium acetate at the beginning of the fermentation) and ultrasonic treatment(120 W, 40 kHz),γ-PGA yield increased by 112.7% and 96.2% respectively, as compared with the control. In this study, sodium acetate and ultrasonic treatment had been used in B. licheniformis WX-02 to improveγ-PGA biosynthesis production. Because of its low cost and simple operation, this method could be used inγ-PGA fermentation industrial production.