Studies On Technological Conditions For Production Of Glycolic Acid From Ethylene Glycol By Gluconobacter Frateurii

Glycolic acid（GA）is the acid with the simplest molecular structure in the aliphatic group.It is less abundant in nature and exists in plants such as lemons,sugar beets and sugarcane.etc.have a wide range of uses.In the experiment,a high-performance liquid chromatograph was used to establish a method for the simultaneous detection of the substrate ethylene glycol and the product glycolic acid.The Bio Rad Aminex HPX-87H ion chromatographic column using sulfonated styrene-divinyl copolymer as the stationary phase was used as the analytical column,combined with the refractive index detector and UV detector of the high performance liquid chromatograph,to achieve the simultaneous detection of ethylene glycol and Purpose of Glycolic Acid.After comparing different chromatographic conditions,2 mmol/L H₂SO₄ was selected as the mobile phase,the column temperature was 30°C,the detection wavelength was 210 nm,the flow rate was0.6 m L/min,and the injection volume was 20μL.The linear ranges of ethylene glycol and glycolic acid in the samples were 0.01～2 g/L,and the correlation coefficients R² in the linear ranges were 0.9993 and 0.9996,respectively,and the linear results were good.Under laboratory conditions,the method has a large detection range,good repeatability and stability.In this paper,the technological conditions of Gluconobacter frateurii HD924 for converting ethylene glycol to glycolic acid were studied.In the experiment,the composition of the seed medium was optimized by the method of orthogonal experimental design,and the optimal composition of the seed medium（g/L）was determined:sorbitol 10,yeast powder 15,Ca CO₃ 1.5,and then drawn strains The logarithmic growth phase was determined by the growth curve of 1.Then,single factor test and Plackett-Burman test design were used to screen out the three main factors affecting the production of glycolic acid:Sorbitol concentration,yeast powder concentration,and ethylene glycol concentration.On this basis,the steepest climbing path method was used to approach the maximum response value range,and the response surface methodology was used to determine the interaction and optimal conditions between these main factors.The results showed that the optimization concentration were:Sorbitol 40.30 g/L,yeast powder 36.90 g/L,Ca CO₃ 2.50 g/L,ethylene glycol 28.14 g/L,culture temperature 30°C,p H 7,inoculum size 10%（v/v）,rotating speed 200r/min,fermentation period 48 h.Under the optimized condition,the titer of glycolic acid reached 21.04 g/L.After three parallel experiments,the actual average titer was close to the predicted titer.The conversion rate of glycolic acid was increased by 28.25%,reaching 74.77%,and the productivity was 10.52 g/（L·d）.On the basis of shake flask transformation,a 20 L fermenter experiment was performed.The concentration of the substrate was controlled by feeding batches,the oxygen supply was improved with compressed air,and the p H was controlled online by adding acid and alkali.The fermentation conditions were optimized.The initial ethylene glycol concentration was 28 g/L,and the stirring speed was 400 r/min,the ventilation volume was 1.2 vvm,the tank pressure was 0.05 Mpa,and the p H was controlled at 6 with sodium hydroxide.After 72 h of reaction,the output of glycolic acid reached 45.83 g/L,and the production intensity reached 15.28 g/（L·d）,a 45%improvement over shake flask fermentation results.The extraction process of glycolic acid was preliminarily explored.The bacteria and other impurities in the fermentation broth were initially screened by centrifugation,and then the residual bacterial protein and other small molecular particles in the fermentation broth were removed by membrane equipment,and activated carbon was used to adsorb the fermentation broth.The pigments in the product are finally vacuum filtered and recrystallized,and dried to obtain fine glycolic acid.