| α-Ketoglutaric acid（α-KG）and pyruvic acid（PYR）are important organic acids widely used in the food,chemical and pharmaceutical industries.These two kinds of keto acids have high commercial value(12-15$·kg-1),which are much higher than that of other organic acids.At present,the production ofα-KG is a competitive method by Yarrowia lipolytica WSH-Z06.However,the accumulation of PYR and the long period of fermentation was unavoidable that increased the cost of production directly during the production ofα-KG.In order to maximize productivity,co-production ofα-KG and PYR was investigated by Yarrowia lipolytica WSH-Z06.Then,effects of three different methods on the separation of keto acids from fermentation broth were compared.Then a simple and effective method was established for the separation ofα-KG and PYR.The main results were presented as below:（1）Co-production ofα-KG and PYR were conducted with Yarrowia lipolytica WSH-Z06C3 screened in our previous work.Effects of nitrogen source concentration and inoculation volume on the co-production of keto acids were investigated in shake flasks.That the optimum concentration of seed culture nitrogen source was 4 g·L-1 and inoculation quantity was 12%were determined.Effects of dissolved oxygen control parameters and the thiamine concentration on the co-production of keto acids were investigated in 15 L fermentor.Results showed that the total mass of keto acids was 53.4 g·L-1 when dissolved oxygen level was controlled in 50%for 96 h.With the strategy of two-stage oxygen controlling,the total of keto acids reached 64.7 g·L-11 and the yield of keto acids improved 21.2%compared with that of the control.The titer ofα-KG and PYR were 24.5 g·L-1 and 53.7 g·L-1 when the optimum concentration of thiamine concentration was 0.2?g·L-1.The total production was 57.9%higher than that of the control.（2）The ion exchange method was conducted to separate theα-KG and PYR from the fermentation broth.Effects of eight different types of exchange resins on the separation were investigated.The D318 resin was selected due to a highly adsorption capacity ofα-KG.Static adsorption（contact time,temperature and pH）and dynamic desorption（eluent selection,eluent concentration,p H and flow rate）process were optimized.The results showed thatα-KG and PYR could not be separated effectively by D318 resin.（3）The calcium salt precipitation method and organic solvent extraction method were used to separate theα-KG and PYR from the fermentation broth.Effects of three different calcium salts on the separation of keto acids were compared.CaCO3 was the optimum precipitant ofα-KG.The recovery and purity rate ofα-KG were 84.5%and 99.2%,respectively.However,the result showed that precipitation method could not be used for the separation of PYR,because PYR was unstable and easy to polymerize under the partial neutrality and high temperature.Effects of different organic solvents on the separation of keto acids were compared as well.Ethyl acetate was found to be the optimum solvent for separatingα-KG.The recovery ofα-KG and PYR were 92.5%and 89.2%.Then PYR was distilled under high vacuum condition.Finally the purity ofα-KG and PYR reached 99.2%and 98.5%,respectively.（4）The adsorption chromatography method was applied to separate theα-KG and PYR from the fermentation broth.Effects of two adsorbents and three solvent systems on the separation of keto acids were compared.Silica gel was identified as the optimum adsorbent,mixture of ethyl acetate and petroleum ether was the optimum elution system.Effects of the ratio of height to diameter,flow rate,silica gel mesh and loading method on the separation of keto acids were investigated.Adsorption chromatography process parameters after optimization:the ratio of height to diameter was 1/4,the flow rate was 7 m L·min-1,silica gel mesh was 200-300,the loading method was wet.Finally,the purity and yield of KGA/PYR reached 98.7%/99.1%and 86.7%/70.9%when the size of chromatography column was expanded from?30?300 mm to?50?500 mm. |
PDF Full Text Request