Influence Of Osmotic Pressure On Sodium Gluconate Fermentation By Aspergillus Niger And Its Own Physiological Metabolism

During sodium gluconate fermentation,the high initial concentration of glucose and a large amount of sodium hydroxide added to maintain the pH resulted in strong osmotic pressure at the end of the process,which might affect the cell growth and metabolism.Therefore,the influence of osmotic pressure on sodium gluconate production by Aspergillus niger and its own physiological metabolism were investigated in this study.Generally,microorganism could respond to negative stimuli by regulating intracellular metabolism.At first,a detection platform for the map of intracellular metabolites was successfully constructed.This was the first time taking different internal standard as the starting point to quantitatively quantify the carbon central metabolites by gas chromatography-mass spectrometry(GC/MS)with selected ion mode(SIM).Finally,norleucine(NLE),adipic acid(HEX)and monobutyl phosphate(MBP)were obtained as the optimal internal standard for the assay of.amino acids,organic acids and phosphosaccharides,respectively.The results showed that this method was in high accuracy,good reproducibility and stable performance with the standard curve R2 mostly to 0.99 or more.Sodium chloride was not involved in sodium gluconate biosynthesis but could enhance the osmotic pressure of broth.The effects of osmotic pressure on sodium gluconate fermentation were investigated by regulating the sodium chloride concentration in fermentation medium.The results showed that the group with 0.1 mol/L NaCl addition to the initial fermentation medium,corresponding to osmolality of 2400 mOsm/kg,was much more favorable to the sodium gluconate production.The hightest average glucose consumption rate and sodium gluconate production rate were obtained under 0.1 mol/L NaCl,as 15.83 and 18.67 g/L/h,respectively,and the latter was about 1.3 times as that of other groups.Particularly,compared to the control without NaCl addition,the concentration of main extracellular byproduct,citric acid at the initial stage of optimized fermentation reduced by 80%or so.Besides,we found that hyperosmolality,greater or equal to 2400 mOsm/kg,could stimulate the synthesis and accumulation of glutamate and proline used as osmoprotectants,while the TCA cycle was upregulated and EMP pathway was almost not influenced by osmotic pressure.High quality seeds were the prerequisite of an efficient bioprocess.While as the key substrate in the sodium gluconate fermentation,glucose could not only provide the carbon source,but also increase osmotic pressure of the fermentation broth.Based on oxygen consumption rate(OUR),the effects of different initial glucose concentrations on the seed culture by A.niger were studied.The optimum initial glucose concentration was 300 g/L,corresponding to 1900 mOsm/kg,with OUR level about 70%higher than that of other groups.Besides,the cells from optimized seed culture accumulated more osmoprotectants of alanine and glutamatela.It was interesting that osmolality could induce glucose oxidase(GOD)activity possibly by affecting the synthesis of histidine,one key component of active site of GOD.Prominently,the fermentation yield using the optimized seed culture was up to 1.198 g/g,99%of the theoretical value,which was the best in literature.