Screening And Indentification Of A Strain Producing Glyceric Acid And Studies On Fermentation Process

Glyceric acid?GA?is a kind of organic acid,which widely exists in all kinds of plants in nature.Its derivatives also exist in the body as a metabolite.GA has many applications in chemical,medical,food,and so on.A method using a refractive index detector to detect glyceric acid and other metabolites of the fermentation broth was described.Glyceric acid,glycerol,and dihydroxyacetone were successfully separated by a sulfonated polystyrene divinyl benzene column?Aminex HPX-87H?with a refractive index detector.It is found that the optimal conditions of column temperature was 60?,and the mobile phase was5 mmol sulfuric acid containing 20%acetonitrile,which were applied to the quantification.The linear range for glyceric acid,glycerol and dihydroxyacetone were 0.2¹ g/L and the correlation coefficient of them were 0.999,0.992,and 0.994,respectively.Recoveries of glyceric acid glycerol,and dihydroxyacetone added to culture were?99.52±1.30?%,?97.61±1.33?%,and?98.52±1.58?%,respectively.The method is accurate rapid and suitable for routine analysis.A strain,Gluconobacter japonicus CGMCC 12425,producing GA was isolated from rotted fruit using the method of the color change of the medium.A series of morphological and biochemical characteristics and sequence analysis of 16s rDNA revealed that it belongs to Gluconobacter japonicus,which named Gluconobacter japonicus HD1025 and was preserved by China General Microbiological Culture Collection Center.The preservation number is CGMCC12425.A preliminary study of fermentation showed that the strain produced 49.47 g/L of glyceric acid when cultured in medium containing 150 g/L glycerol as carbon source.The orthogonal methodology was used to optimize the seed medium.It is found that the optimal seed medium contain glycerol 20 g/L,yeast extract powder 15 g/L and Ca CO₃ 1 g/L.The optimal inoculation time of seed was determined by drawing the growth curve of seed.The single factor experiments containing temperature,initial pH,rotational speed,the concentration of the glycerol,nitrogen source and inorganic salts and so on showed that the CaCO₃,soybean cake powder and glycerol significantly improved the production of glyceric acid.The Response Surface Methodology?RSM?based on a three-factor Box-Behnken design of experiments was used to build an analytical model and finally employed as a constraint in an obvious optimization process.The optimized parameters were glycerol 144.67 g/L,soybean cake powder 25.12 g/L,and CaCO₃ 22.08 g/L.Under those optimization conditions,it was predicted that the highest concentration of glyceric acid reached 67.14 g/L.After optimization,the glyceric acid production increased by 79.32%,and the experimental data basically in line with the predicted values.The rotate speed,pH value and the p H regulated way of the 5 L fermentor was studied by the result of optimized flask fermentation conditions.The result showed that the highest concentration of glyceric acid reached 73.04 g/L when cultured in medium containing 150 g/L glycerol as carbon source and when the rotate speed was 400 rpm and pH value was 5 and the p H was regulated by NaOH.