Method for extracting adipic acid from fermentation liquor through aqueous two-phase extraction

Abstract

The invention relates to the technical field of biochemical engineering, and discloses a method for extracting adipic acid from an aqueous two-phase extraction fermentation broth, which comprises the following steps: S1, using an adipic acid fermentation broth produced by a microbial fermentation method as a raw material, carrying out solid-liquid separation on the adipic acid fermentation broth, taking a liquid phase, adding acid for acidification, and adjusting the pH value to 0.5-3; s2, adding inorganic salt into the acidified fermentation liquor, mixing, adding an organic solvent 1 and an organic solvent 2, uniformly mixing, standing for phase splitting, distilling the upper phase to recover the organic solvent, and concentrating the lower phase to recover the inorganic salt; s3, adding water into residues after upper-phase distillation, heating and dissolving, then cooling and crystallizing, filtering and drying to obtain adipic acid. According to the method provided by the invention, adipic acid can be rapidly and efficiently extracted and separated from the fermentation liquor, and the obtained product is high in purity.

Description

Technical Field

[0001] The present invention relates to the technical field of biochemistry, and particularly relates to a method for extracting adipic acid from fermentation broth by aqueous two-phase extraction. Background Art

[0002] Adipic acid is prepared by biological fermentation using microorganisms to metabolize adipic acid. Its raw material sources are extensive and can be biomass resources such as grapes and xylose, which have the advantages of environmental protection and sustainability. However, the biological fermentation broth contains impurities such as proteins and polysaccharides, and the separation and purification of products are difficult.

[0003] Aqueous two-phase extraction (ATPE, which can be a polymer / inorganic salt system or a polymer / polymer system) separates by using the difference in the partition coefficients of the extract in the two aqueous phases. It has mild conditions, simple operation, and easy scale-up, and has been well applied in the extraction of biological macromolecules (such as proteins). However, due to the high cost of polymers, its application in the extraction of bulk chemicals is limited. In recent years, researchers found during organic solvent salting-out crystallization that some hydrophilic solvents and inorganic salts can also form an aqueous two-phase system under suitable conditions. Compared with the traditional aqueous two-phase system, this new type of aqueous two-phase system has low viscosity, fast phase separation, and low cost.

[0004] CN 117430500 A discloses a method for extracting adipic acid from fermentation broth through a ceramic membrane, including inactivating the adipic acid fermentation broth, rough filtration by a plate and frame, and fine filtration by a ceramic membrane, followed by decolorization, acidification, and concentration, and finally preparing the product by low-temperature crystallization, but the purity of adipic acid is less than 93%. Summary of the Invention

[0005] The present invention provides a method for extracting adipic acid from fermentation broth by aqueous two-phase extraction, which can quickly and efficiently extract and separate adipic acid from fermentation broth, and the obtained product has high purity.

[0006] The technical solution of the present invention is to provide a method for extracting adipic acid from fermentation broth by aqueous two-phase extraction, including the following steps: S1. Using the adipic acid fermentation broth produced by microbial fermentation as the raw material, performing solid-liquid separation on it, taking the liquid phase and adding acid for acidification, and adjusting the pH to 0.5 - 3; S2. Adding an inorganic salt to the acidified fermentation broth for mixing, then adding organic solvent 1 and organic solvent 2, mixing evenly and then standing for phase separation, distilling the upper phase to recover the organic solvent, and concentrating the lower phase to recover the inorganic salt; S3. Adding water to the residue after distillation of the upper phase and heating to dissolve, then cooling for crystallization, and filtering and drying to obtain adipic acid.

[0007] Optionally, in S1, the concentration of the fermentation broth is 80 - 180 g / L, and the solid-liquid separation is centrifugation or ceramic membrane filtration.

[0008] Optionally, during centrifugation, the rotational speed is 4000RPM - 10000RPM; during ceramic membrane filtration, the filtration accuracy is 30 - 50nm.

[0009] Optionally, the acid in S1 is sulfuric acid, hydrochloric acid or nitric acid; adding acid to adjust the pH to 0.8 - 1.5.

[0010] Optionally, the inorganic salt in S1 is an acid salt or a neutral inorganic salt, and the final concentration mass fraction of the inorganic salt in the system after addition is 5 - 30wt%.

[0011] Optionally, the organic solvent 1 in S2 is a hydrophilic organic solvent, the organic solvent 2 is a hydrophobic organic solvent, and the mass ratio of the organic solvent 2 to the organic solvent 1 is 0.1 - 1.

[0012] Optionally, the organic solvent 1 is one or more of acetone, isopropyl alcohol, n - propanol, cyclohexanol, glycerol and ether.

[0013] Optionally, the organic solvent 2 is benzene and / or n - butanol. [[ID=…]]

[0014] Optionally, the total addition amount of the organic solvent 1 and the organic solvent 2 in S2 is 0.5 - 5 times the volume of the fermentation broth. After adding the organic solvents, mix for 10 - 60min and then let it stand for phase separation.

[0015] Optionally, after adding water in S3, dissolve at 60℃ - 80℃, and then cool down to 4 - 15℃ to precipitate crystals.

[0016] The present invention has the following beneficial effects: The present invention performs acidification treatment on the fermentation broth after impurity removal, then adds inorganic salts and hydrophilic organic solvents to form a two - aqueous - phase system, and also adds a certain amount of benzene and / or n - butanol, making the phase separation effect better, and the hydrophilic organic solvents originally present in the lower phase are more distributed in the upper phase, thereby improving the recovery rate of the organic solvent. More denatured proteins remain in the aqueous phase, resulting in a higher product purity. Specific Embodiments

[0017] The experimental methods in the following examples are all conventional methods unless otherwise specified. The materials used in the following examples are all commercially available products unless otherwise specified. The embodiments of the present invention will be described in detail below in conjunction with the examples, but those skilled in the art will understand that the following examples are only used to illustrate the present invention and should not be regarded as limiting the scope of the present invention.

[0018] The adipic acid fermentation broth used in the following examples and comparative examples was prepared by fermenting genetically modified Escherichia coli, with the adipic acid content being 80 - 180 g / L. The sum of other substances such as proteins, secondary metabolites, and cytoplasm after deducting adipic acid was calculated as the solid content, and the solid content was 4% - 9%. The pH of the fermentation broth was 7.1 - 7.7.

[0019] During the acidification process, the hydrochloric acid solution added was a mixture of concentrated hydrochloric acid and water with a volume ratio of 1:1, and the nitric acid solution was a mixture of concentrated nitric acid and water with a volume ratio of 1:1.

[0020] Example 1 The method for extracting adipic acid from the fermentation broth by aqueous two - phase extraction mainly comprises the following steps: At 25°C, 100 g of adipic acid fermentation broth with the adipic acid content of 120 g / L, which had been centrifuged at 4000 RPM to remove the bacteria, was taken. While stirring, 30 g of hydrochloric acid solution was added for acidification. After acidification, the pH was 1.12, and flocs (denatured proteins and a small amount of bacteria) and adipic acid crystals slowly precipitated. It was poured into a separatory funnel, 10 g of ammonium sulfate was added, then 80 g of isopropanol and 20 g of benzene were added. The separatory funnel was shaken several times and left to stand for 10 hours for stratification. The upper layer was taken, and 95 g of organic solvents were recovered by rotary evaporation, with the organic solvent recovery rate being 95%. The remaining solid was added with water to 70 g, then heated to 75°C. After complete dissolution, it was cooled to 12°C in an ice - water bath, and crystals slowly precipitated. The crystals were separated and dried at 105°C to obtain 8.93 g of the product. After being detected by titration with 0.1 mol / L sodium hydroxide, the purity of adipic acid was 98.51% and the yield was 73.31%. After detection, it contained impurities such as hexadecanamide, sphingosine, diisooctylamine, and avocatin acetate. Recrystallization in a mixture of water and isopropanol could further improve the product purity.

[0021] Comparative Example 1 The method for extracting adipic acid from the fermentation broth by aqueous two - phase extraction mainly comprises the following steps: At 25°C, 100 g of adipic acid fermentation broth with the adipic acid content of 120 g / L, which had been centrifuged at 4000 RPM to remove the bacteria, was taken. While stirring, 30 g of hydrochloric acid solution was added for acidification. After acidification, the pH was 1.14, and flocs (denatured proteins and a small amount of bacteria) and adipic acid crystals slowly precipitated. It was poured into a separatory funnel, 10 g of ammonium sulfate was added, then 80 g of isopropanol was added. The separatory funnel was shaken several times and left to stand for 10 hours for stratification. The upper layer was taken, and 70 g of organic solvents were recovered by rotary evaporation, with the organic solvent recovery rate being 87.5%. The remaining solid was added with water to 70 g, then heated to 75°C. After complete dissolution, it was cooled to 12°C in an ice - water bath, and crystals slowly precipitated. The crystals were separated and dried at 105°C to obtain 8.11 g of the product. After being detected by titration with 0.1 mol / L sodium hydroxide, the purity of adipic acid was 96.02% and the yield was 64.89%.

[0022] Example 2 At 25°C, 75 g of adipic acid fermentation broth that had been centrifuged and flocculated to remove bacteria, with an adipic acid content of 110 g / L, was taken. While stirring, concentrated sulfuric acid was added for acidification until the pH reached 0.97. Flocs and crystals slowly precipitated out. It was poured into a separatory funnel, 7.29 g of ammonium sulfate was added, and then 32 g of isopropanol and 6 g of benzene were added. The separatory funnel was shaken multiple times, then centrifuged and decanted. After stratification, 43.49 g of the upper layer was taken. The organic solvents were recovered by rotary evaporation, with 34 g recovered and the recovery rate of organic solvents being 89.5%. The remaining solid was added with water to 20 g and heated to 75°C. After complete dissolution, it was cooled to 12°C in an ice-water bath, and crystals slowly precipitated out. The crystals were separated by filtration and dried at 105°C to obtain 4.42 g of the product. After titration with a 0.1 mol / L sodium hydroxide solution, the purity of adipic acid was 98.76% and the yield was 57.70%.

[0023] Comparative Example 2 At 25°C, 75 g of adipic acid fermentation broth that had been centrifuged and flocculated to remove bacteria, with an adipic acid content of 110 g / L, was taken. While stirring, concentrated sulfuric acid was added for acidification until the pH reached 0.95. Flocs and crystals slowly precipitated out. It was poured into a separatory funnel, 7.29 g of ammonium sulfate was added, and then 38 g of isopropanol was added. The separatory funnel was shaken multiple times, then centrifuged and decanted. After stratification, 45.47 g of the upper layer was taken. The organic solvents were recovered by rotary evaporation, with 29 g recovered and the recovery rate of organic solvents being 76.3%. The remaining solid was added with water to 20 g and heated to 75°C. After complete dissolution, it was cooled to 12°C in an ice-water bath, and crystals slowly precipitated out. The crystals were separated by filtration and dried at 105°C to obtain 4.42 g of the product. After titration with a 0.1 mol / L sodium hydroxide solution, the purity of adipic acid was 96.50% and the yield was 51.70%.

[0024] Example 3 At 25°C, 100 g of adipic acid fermentation broth that had been centrifuged at 4000 RPM to remove bacteria, with an adipic acid content of 120 g / L, was taken. While stirring, 30 g of hydrochloric acid solution was added for acidification, and the pH after acidification was 1.12. Flocs (denatured proteins and a small amount of bacteria) and adipic acid crystals slowly precipitated out. It was poured into a separatory funnel, 10 g of ammonium sulfate was added, and then 60 g of isopropanol and 20 g of benzene were added. The separatory funnel was shaken multiple times and allowed to stand for 3 hours for stratification. The upper layer was taken, and the organic solvents were recovered by rotary evaporation, with 78 g recovered and the recovery rate of organic solvents being 97.5%. The remaining solid was added with water to 70 g, then heated to 75°C. After complete dissolution, it was cooled to 12°C in an ice-water bath, and crystals slowly precipitated out. The crystals were separated, and dried at 105°C to obtain 8.54 g of the product. After titration with a 0.1 mol / L sodium hydroxide solution, the purity of adipic acid was 98.37% and the yield was 70.01%.

[0025] Comparative Example 3 At 25°C, 100 g of adipic acid fermentation broth with the bacteria removed by centrifugation at 4000 RPM was taken, and the adipic acid content was 120 g / L. While stirring, 30 g of hydrochloric acid solution was added for acidification. After acidification, the pH was 1.14, and flocculants (denatured proteins and a small amount of bacteria) and adipic acid crystals slowly precipitated. It was poured into a separatory funnel, 10 g of ammonium sulfate was added, and then 80 g of isopropanol was added. The separatory funnel was shaken multiple times and left to stand for 3 hours. After stratification, the upper layer was taken, and 70 g of the organic solvent was recovered by rotary evaporation. The recovery rate of the organic solvent was 87.5%. The remaining solid was added with water to 70 g, then heated to 75°C. After complete dissolution, it was cooled to 12°C in an ice-water bath, and crystals slowly precipitated. The crystals were separated and dried at 105°C to obtain 8.11 g of the product. After titration detection with 0.1 mol / L sodium hydroxide, the purity of adipic acid was 96.02%, and the yield was 64.89%.

[0026] Example 4 Same as Example 1, except that the pH was controlled at 2.8 during acidification.

[0027] Example 5 Same as Example 1, except that the pH was controlled at 1.5 during acidification.

[0028] Example 6 Same as Example 1, acidified with nitric acid solution, and the acidification pH was controlled at 0.8.

[0029] Example 7 Same as Example 1, acidified with nitric acid solution, and the acidification pH was controlled at 0.5.

[0030] Example 8 Same as Example 1, when adding organic solvents, it was 30 g of acetone, 30 g of isopropanol, and 20 g of n-butanol.

[0031] Example 9 Same as Example 1, when adding organic solvents, it was 70 g of cyclohexanol and 10 g of n-butanol.

[0032] Example 10 Same as Example 1, when adding organic solvents, it was 40 g of isopropanol and 40 g of benzene.

[0033] Comparative Example 4 Same as Example 1, when adding organic solvents, it was 30 g of isopropanol and 50 g of benzene.

[0034] Comparative Example 5 Same as Example 1, when adding organic solvents, it was 60 g of isopropanol and 20 g of ethyl acetate.

[0035] Comparative Example 6 Same as Example 1, when adding organic solvents, it was 60 g of isopropanol and 20 g of n-heptane.

[0036] Comparative Example 7 Same as Example 1. When adding organic solvents, it was 60 g of isopropanol and 20 g of cyclohexane.

[0037] The adipic acid yields, purities and other data obtained from the above Examples 4 to 10 and Comparative Examples 4 to 7 are shown in Table 1.

[0038] Table 1

[0039] Examples 1 and Comparative Example 1 show that, compared with the aqueous two-phase system for extracting adipic acid fermentation broth simply constructed with isopropanol and ammonium sulfate, the product yield, purity and organic solvent recovery rate are all improved in the system after adding benzene. The same conclusion can be obtained from Examples 2 and Comparative Example 2. Examples 1, 4 and 5 show that a lower pH is more beneficial to the improvement of adipic acid yield.

[0040] Example 10 and Comparative Example 4 show that a higher ratio of benzene / isopropanol is not conducive to the improvement of adipic acid yield; Examples 3 and Comparative Examples 5, 6, 7 show that adding ethyl acetate, n-heptane or cyclohexane to the aqueous two-phase system constructed with isopropanol - ammonium sulfate - fermentation broth will instead reduce the adipic acid yield and purity.

[0041] The above examples describe the preferred embodiments of the present invention, but the present invention is not limited thereto. Within the technical concept scope of the present invention, various simple modifications can be made to the technical solutions of the present invention, including any other combinations of each technical feature. These simple modifications and combinations should also be regarded as the content disclosed by the present invention and fall within the protection scope of the present invention. Therefore, the protection scope of this invention patent shall be subject to the appended claims.

Claims

1. A method for extracting adipic acid from fermentation broth by aqueous two-phase extraction, characterized in that, It includes the following steps: S1: Using the adipic acid fermentation broth produced by microbial fermentation method as raw material, performing solid-liquid separation on it, adding acid to the liquid phase for acidification, and adjusting the pH to 0.5 - 3; S2: Adding inorganic salts to the acidified fermentation broth for mixing, then adding organic solvent 1 and organic solvent 2, mixing evenly and then standing for phase separation. The upper phase is distilled to recover organic solvent 1 and organic solvent 2, and the lower phase is concentrated to recover inorganic salts; S3: Adding water to the residue after distillation of the upper phase and heating to dissolve it, then cooling for crystallization, and filtering and drying to obtain adipic acid.

2. The method according to claim 1, characterized in that: In S1, the concentration of the fermentation broth is 80 - 180 g / L, and the solid-liquid separation is centrifugation or ceramic membrane filtration.

3. The method according to claim 2, wherein: During centrifugation, the rotation speed is 4000 RPM - 10000 RPM; during ceramic membrane filtration, the filtration accuracy is 30 nm - 50 nm.

4. The method according to claim 1, characterized in that: The acid in S1 is sulfuric acid, hydrochloric acid or nitric acid; adding acid to adjust the pH to 0.8 - 1.

5.

5. The method according to claim 1, characterized in that: The inorganic salt in S1 is acidic inorganic salt or neutral inorganic salt, and the final concentration mass fraction of the inorganic salt in the system after addition is 5 - 30 wt%.

6. The method according to any one of claims 1 to 5, characterized in that: In S2, organic solvent 1 is a hydrophilic organic solvent, organic solvent 2 is a hydrophobic organic solvent, and the mass ratio of the two is 0.1 - 1.

7. The method according to claim 6, characterized in that: Organic solvent 1 is one or more of acetone, isopropanol, n-propanol, cyclohexanol, glycerol and ether.

8. The method according to claim 6, characterized in that: Organic solvent 2 is benzene and / or n-butanol.

9. The method according to claim 6, wherein: In S2, the total addition amount of organic solvent 1 and organic solvent 2 is 0.5 - 5 times the volume of the fermentation broth, and after adding the organic solvents, mix for 10 - 60 min and then stand for phase separation.

10. The method according to claim 1, wherein: In S3, add water and dissolve at 60°C - 80°C, then cool to 4 - 15°C to precipitate crystals.

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