CN118405973A - Method for separating and extracting glycollic acid from fermentation liquor - Google Patents
Method for separating and extracting glycollic acid from fermentation liquor Download PDFInfo
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- CN118405973A CN118405973A CN202310067053.4A CN202310067053A CN118405973A CN 118405973 A CN118405973 A CN 118405973A CN 202310067053 A CN202310067053 A CN 202310067053A CN 118405973 A CN118405973 A CN 118405973A
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- AEMRFAOFKBGASW-UHFFFAOYSA-N Glycolic acid Chemical compound OCC(O)=O AEMRFAOFKBGASW-UHFFFAOYSA-N 0.000 title claims abstract description 322
- 238000000855 fermentation Methods 0.000 title claims abstract description 83
- 230000004151 fermentation Effects 0.000 title claims abstract description 83
- 238000000034 method Methods 0.000 title claims abstract description 53
- KFZMGEQAYNKOFK-UHFFFAOYSA-N Isopropanol Chemical compound CC(C)O KFZMGEQAYNKOFK-UHFFFAOYSA-N 0.000 claims abstract description 32
- QAOWNCQODCNURD-UHFFFAOYSA-N Sulfuric acid Chemical compound OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 claims abstract description 32
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 claims abstract description 31
- 239000000047 product Substances 0.000 claims abstract description 20
- 238000001816 cooling Methods 0.000 claims abstract description 16
- 235000011114 ammonium hydroxide Nutrition 0.000 claims abstract description 15
- AXCZMVOFGPJBDE-UHFFFAOYSA-L calcium dihydroxide Chemical compound [OH-].[OH-].[Ca+2] AXCZMVOFGPJBDE-UHFFFAOYSA-L 0.000 claims abstract description 15
- 239000000920 calcium hydroxide Substances 0.000 claims abstract description 15
- 229910001861 calcium hydroxide Inorganic materials 0.000 claims abstract description 15
- 230000001105 regulatory effect Effects 0.000 claims abstract description 13
- 239000002244 precipitate Substances 0.000 claims abstract description 12
- CHRHZFQUDFAQEQ-UHFFFAOYSA-L calcium;2-hydroxyacetate Chemical compound [Ca+2].OCC([O-])=O.OCC([O-])=O CHRHZFQUDFAQEQ-UHFFFAOYSA-L 0.000 claims abstract description 10
- 239000013049 sediment Substances 0.000 claims abstract description 4
- 238000002390 rotary evaporation Methods 0.000 claims abstract description 3
- 150000003839 salts Chemical class 0.000 claims abstract description 3
- 239000013078 crystal Substances 0.000 claims description 75
- 238000000967 suction filtration Methods 0.000 claims description 15
- VHUUQVKOLVNVRT-UHFFFAOYSA-N Ammonium hydroxide Chemical compound [NH4+].[OH-] VHUUQVKOLVNVRT-UHFFFAOYSA-N 0.000 claims description 14
- 238000004108 freeze drying Methods 0.000 claims description 14
- 238000003756 stirring Methods 0.000 claims description 14
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 claims description 6
- 239000008103 glucose Substances 0.000 claims description 6
- 101150046493 glcC gene Proteins 0.000 claims description 4
- 229910052799 carbon Inorganic materials 0.000 claims description 3
- 241000660147 Escherichia coli str. K-12 substr. MG1655 Species 0.000 claims description 2
- 238000004821 distillation Methods 0.000 claims description 2
- 238000005292 vacuum distillation Methods 0.000 claims description 2
- 238000011084 recovery Methods 0.000 abstract description 12
- 238000000926 separation method Methods 0.000 abstract description 8
- 238000000746 purification Methods 0.000 abstract description 5
- QGZKDVFQNNGYKY-UHFFFAOYSA-N Ammonia Chemical compound N QGZKDVFQNNGYKY-UHFFFAOYSA-N 0.000 abstract description 3
- 238000005265 energy consumption Methods 0.000 abstract description 2
- 238000000605 extraction Methods 0.000 abstract 1
- 239000002994 raw material Substances 0.000 description 22
- 239000006228 supernatant Substances 0.000 description 20
- 238000002425 crystallisation Methods 0.000 description 12
- 230000008025 crystallization Effects 0.000 description 12
- 238000002360 preparation method Methods 0.000 description 12
- 239000007788 liquid Substances 0.000 description 11
- 239000002002 slurry Substances 0.000 description 10
- 239000000126 substance Substances 0.000 description 6
- 239000000843 powder Substances 0.000 description 5
- 239000001888 Peptone Substances 0.000 description 4
- 108010080698 Peptones Proteins 0.000 description 4
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 4
- 238000001514 detection method Methods 0.000 description 4
- 235000019319 peptone Nutrition 0.000 description 4
- 238000011218 seed culture Methods 0.000 description 4
- 239000011780 sodium chloride Substances 0.000 description 4
- FAPWRFPIFSIZLT-UHFFFAOYSA-M sodium chloride Inorganic materials [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 4
- 240000008042 Zea mays Species 0.000 description 3
- 235000005824 Zea mays ssp. parviglumis Nutrition 0.000 description 3
- 235000002017 Zea mays subsp mays Nutrition 0.000 description 3
- 235000005822 corn Nutrition 0.000 description 3
- 229910052943 magnesium sulfate Inorganic materials 0.000 description 3
- CSNNHWWHGAXBCP-UHFFFAOYSA-L magnesium sulphate Substances [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 description 3
- 239000013028 medium composition Substances 0.000 description 3
- 229920001817 Agar Polymers 0.000 description 2
- NLXLAEXVIDQMFP-UHFFFAOYSA-N Ammonium chloride Substances [NH4+].[Cl-] NLXLAEXVIDQMFP-UHFFFAOYSA-N 0.000 description 2
- VTYYLEPIZMXCLO-UHFFFAOYSA-L Calcium carbonate Chemical compound [Ca+2].[O-]C([O-])=O VTYYLEPIZMXCLO-UHFFFAOYSA-L 0.000 description 2
- 239000002253 acid Substances 0.000 description 2
- 238000005273 aeration Methods 0.000 description 2
- 239000008272 agar Substances 0.000 description 2
- 239000003242 anti bacterial agent Substances 0.000 description 2
- 229940088710 antibiotic agent Drugs 0.000 description 2
- 230000001276 controlling effect Effects 0.000 description 2
- 238000012258 culturing Methods 0.000 description 2
- 238000011033 desalting Methods 0.000 description 2
- 238000000909 electrodialysis Methods 0.000 description 2
- 239000000413 hydrolysate Substances 0.000 description 2
- 238000009776 industrial production Methods 0.000 description 2
- 239000000203 mixture Substances 0.000 description 2
- 108090000790 Enzymes Proteins 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 241000588724 Escherichia coli Species 0.000 description 1
- 241000589232 Gluconobacter oxydans Species 0.000 description 1
- 230000000844 anti-bacterial effect Effects 0.000 description 1
- 239000003899 bactericide agent Substances 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 239000011942 biocatalyst Substances 0.000 description 1
- 238000010170 biological method Methods 0.000 description 1
- 239000006227 byproduct Substances 0.000 description 1
- 229910000019 calcium carbonate Inorganic materials 0.000 description 1
- 159000000007 calcium salts Chemical class 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 238000004140 cleaning Methods 0.000 description 1
- 239000012043 crude product Substances 0.000 description 1
- 238000001085 differential centrifugation Methods 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 238000009713 electroplating Methods 0.000 description 1
- 238000003912 environmental pollution Methods 0.000 description 1
- 239000012535 impurity Substances 0.000 description 1
- 238000011065 in-situ storage Methods 0.000 description 1
- 238000011081 inoculation Methods 0.000 description 1
- 239000002054 inoculum Substances 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 239000002086 nanomaterial Substances 0.000 description 1
- 238000007670 refining Methods 0.000 description 1
- 238000007789 sealing Methods 0.000 description 1
- 239000007858 starting material Substances 0.000 description 1
- 239000000758 substrate Substances 0.000 description 1
- 238000004381 surface treatment Methods 0.000 description 1
- 238000003466 welding Methods 0.000 description 1
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C51/00—Preparation of carboxylic acids or their salts, halides or anhydrides
- C07C51/42—Separation; Purification; Stabilisation; Use of additives
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C51/00—Preparation of carboxylic acids or their salts, halides or anhydrides
- C07C51/42—Separation; Purification; Stabilisation; Use of additives
- C07C51/43—Separation; Purification; Stabilisation; Use of additives by change of the physical state, e.g. crystallisation
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C51/00—Preparation of carboxylic acids or their salts, halides or anhydrides
- C07C51/42—Separation; Purification; Stabilisation; Use of additives
- C07C51/47—Separation; Purification; Stabilisation; Use of additives by solid-liquid treatment; by chemisorption
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C51/00—Preparation of carboxylic acids or their salts, halides or anhydrides
- C07C51/42—Separation; Purification; Stabilisation; Use of additives
- C07C51/487—Separation; Purification; Stabilisation; Use of additives by treatment giving rise to chemical modification
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C59/00—Compounds having carboxyl groups bound to acyclic carbon atoms and containing any of the groups OH, O—metal, —CHO, keto, ether, groups, groups, or groups
- C07C59/01—Saturated compounds having only one carboxyl group and containing hydroxy or O-metal groups
- C07C59/06—Glycolic acid
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P7/00—Preparation of oxygen-containing organic compounds
- C12P7/40—Preparation of oxygen-containing organic compounds containing a carboxyl group including Peroxycarboxylic acids
- C12P7/42—Hydroxy-carboxylic acids
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
- C12R2001/185—Escherichia
- C12R2001/19—Escherichia coli
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Abstract
The invention discloses a method for separating and extracting glycolic acid from glycolic acid fermentation broth, and belongs to the technical field of glycolic acid separation and purification. The invention comprises the following steps: (1) Centrifuging and concentrating the glycollic acid fermentation liquor to obtain clarified fermentation liquor; (2) Regulating the pH of the clarified fermentation liquid ammonia water to be alkaline to obtain alkaline fermentation liquor; (3) Reacting the alkaline fermentation liquor with calcium hydroxide and centrifuging to obtain calcium glycolate sediment; (4) Reacting calcium glycolate precipitate with sulfuric acid, and centrifuging to obtain an acidified solution; (5) Adding activated carbon into the acidified solution for decoloring to obtain a decolored solution; (6) Adding absolute isopropanol into the decolorized solution to remove salt, centrifuging, and concentrating by rotary evaporation to obtain concentrated solution; and (7) cooling and crystallizing the concentrated solution to obtain a glycolic acid product. The glycolic acid extraction method is simple, easy to operate, low in energy consumption, high in recovery rate and high in product quality.
Description
Technical Field
The invention belongs to the technical field of glycolic acid separation and purification, and particularly relates to a method for separating and extracting glycolic acid from glycolic acid fermentation liquor.
Background
Glycolic acid is also called glycolic acid, and has wide application due to excellent physicochemical properties, and is mainly applied to the fields of bactericides, welding agents, chemical assistants, daily chemicals, electroplating surface treatment, chemical cleaning, nano material surface sealing and the like. At present, the industrial production of the glycollic acid is commonly performed by a chemical method and biological fermentation, wherein crude products obtained by the chemical method often contain unreacted complete substrates, reacted byproducts and other impurities, the separation and refining of final products are difficult, the environmental pollution is serious, and economic burden is brought to enterprises and society. Compared with the chemical method, the glycolic acid product obtained by the biological method has high purity, but the separation and industrialization of the high-purity bio-based glycolic acid are not realized at present due to the complex components of the fermentation broth.
Disclosure of Invention
In order to solve the technical problems, the invention provides the following technical scheme: a method for separating and extracting glycolic acid from a glycolic acid fermentation broth, comprising the steps of:
(1) Centrifuging and concentrating the glycollic acid fermentation liquor to obtain clarified fermentation liquor;
(2) Regulating the clarified fermentation broth to be alkaline to obtain alkaline fermentation broth;
(3) Reacting alkaline fermentation liquor with calcium hydroxide and centrifuging to obtain calcium glycolate sediment;
(4) Reacting calcium glycolate precipitate with sulfuric acid, and centrifuging to obtain an acidified solution;
(5) Adding activated carbon into the acidified solution for decoloring to obtain a decolored solution;
(6) Adding anhydrous isopropanol into the decolorized solution to remove salt, carrying out suction filtration, and concentrating by rotary evaporation to obtain concentrated solution;
(7) Cooling, crystallizing and freeze-drying the concentrated solution to obtain a glycolic acid product.
As a preferred embodiment of the method for separating and extracting glycolic acid from a glycolic acid fermentation broth according to the present invention, there is provided a method comprising: the centrifugation uses a refrigerated high-speed centrifuge.
As a preferred embodiment of the method for separating and extracting glycolic acid from a glycolic acid fermentation broth according to the present invention, there is provided a method comprising: (1) The medium centrifugal rotating speed is 9000-11000 rpm, and the time is 10-20 min; concentrating by vacuum distillation at 50-70deg.C and vacuum degree of 50-200 mbar until glycolic acid concentration is 50g/L.
As a preferred embodiment of the method for separating and extracting glycolic acid from a glycolic acid fermentation broth according to the present invention, there is provided a method comprising: and (2) regulating the pH value of the clarified fermentation broth to 8-10 by using ammonia water.
As a preferred embodiment of the method for separating and extracting glycolic acid from a glycolic acid fermentation broth according to the present invention, there is provided a method comprising: (2) adjusting the pH value of the clarified fermentation broth to 8.5-9.5.
As a preferred embodiment of the method for separating and extracting glycolic acid from a glycolic acid fermentation broth according to the present invention, there is provided a method comprising: (3) The adding amount of the medium calcium hydroxide is 40-50 g/L, the reaction temperature is 45-60 ℃ and the time is 4-7 h; the centrifugal speed is 9000-11000 rpm, and the time is 10-20 min.
As a preferred embodiment of the method for separating and extracting glycolic acid from a glycolic acid fermentation broth according to the present invention, there is provided a method comprising: (4) The concentration of the medium sulfuric acid is 1.84mol/L, the addition amount is 75-125 mL/L, the reaction temperature is 40-70 ℃ and the time is 0.5-2 h; the centrifugal speed is 9000-11000 rpm, and the time is 10-20 min.
As a preferred embodiment of the method for separating and extracting glycolic acid from a glycolic acid fermentation broth according to the present invention, there is provided a method comprising: (5) The addition amount of the medium active carbon is 15-25 g/L, the temperature is 45-60 ℃, and the stirring time is 0.5-2 h.
As a preferred embodiment of the method for separating and extracting glycolic acid from a glycolic acid fermentation broth according to the present invention, there is provided a method comprising: (6) The addition amount of the anhydrous isopropanol is 2-5 times of the mass of the glycolic acid in the decolorized solution, the concentration adopts a reduced pressure distillation mode, the temperature is 40-70 ℃, the vacuum degree is 50-200 mbar, and the concentration of the glycolic acid is 50-90%.
As a preferred embodiment of the method for separating and extracting glycolic acid from a glycolic acid fermentation broth according to the present invention, there is provided a method comprising: the suction filtration in (6) adopts a Buchner funnel.
As a preferred embodiment of the method for separating and extracting glycolic acid from a glycolic acid fermentation broth according to the present invention, there is provided a method comprising: (7) And (3) cooling to a temperature lower than the saturation temperature of the concentrated solution by 0.5-5 ℃, adding glycolic acid crystals with purity not lower than 99% as seed crystals, wherein the adding amount of the seed crystals is 0.5-5% of the mass of the glycolic acid in the concentrated solution, maintaining the constant temperature after adding the seed crystals, cooling to-5 to-18 ℃, crystallizing for 2-4 hours, and carrying out suction filtration and freeze drying to obtain the glycolic acid crystals.
As a preferred embodiment of the method for separating and extracting glycolic acid from a glycolic acid fermentation broth according to the present invention, there is provided a method comprising: the purity of the seed crystal is not less than 99.5%.
As a preferred embodiment of the method for separating and extracting glycolic acid from a glycolic acid fermentation broth according to the present invention, there is provided a method comprising: the adding amount of the seed crystal is 1-3% of the mass of the glycollic acid in the concentrated solution.
As a preferred embodiment of the method for separating and extracting glycolic acid from a glycolic acid fermentation broth according to the present invention, there is provided a method comprising: cooling to a temperature which is 2-4 ℃ lower than the saturation temperature of the concentrated solution.
As a preferred embodiment of the method for separating and extracting glycolic acid from a glycolic acid fermentation broth according to the present invention, there is provided a method comprising: the crystallization temperature is-15 to 5 ℃.
Another object of the present invention is to provide glycolic acid obtained by the above-mentioned preparation method by separation and purification, wherein the purity is 99 to 99.7%.
Another object of the present invention is to provide a process for producing a glycolic acid fermentation broth used in the above-mentioned production method, wherein fermentation is carried out using Escherichia coli MG1655 (DE 3) (ΔldhA ΔglcB ΔaceB ΔaldA Δ glcDEF ΔicdA Δ glcC) with glucose as a carbon source to obtain a glycolic acid fermentation broth.
As a preferable scheme of the preparation method of the glycollic acid fermentation liquid, the method specifically comprises the following steps:
s1, plate culture: culturing the strain preserved in advance on an agar plate by streaking, and culturing in a constant temperature incubator at 30 ℃ overnight;
s2, seed culture: the single colony is picked to 20mL of LB liquid medium, the seed liquid is cultivated at 37 ℃ and 250rpm for overnight, the cultivated seed liquid is inoculated to 50mL of modified M9 fermentation medium with 2% of inoculation amount, and corresponding antibiotics are added for cultivation at 37 ℃ and 250 rpm.
S3, fermenting to produce acid: 450mL of seed culture solution, which was cultured in advance, was inoculated into a 5L fermenter containing 3L of fermentation medium. The initial sugar content is 8g/L, and the sugar concentration is controlled to be 1-4g/L by controlling the feeding speed in the later stage. The pH in the fermenter was maintained at about 7.0 with an aqueous ammonia solution, the fermentation temperature was 37℃and the stirring speed was 400rpm, and the aeration rate was 2vvm.
As a preferable scheme of the preparation method of the glycollic acid fermentation liquid, provided by the invention, the preparation method comprises the following steps: the strain was E.coli MG1655 (DE 3) (ΔldhA ΔglcB ΔaceB ΔaldA Δ glcDEF ΔicdA. DELTA. glcC).
As a preferable scheme of the preparation method of the glycollic acid fermentation liquid, provided by the invention, the preparation method comprises the following steps: the composition of the modified M9 fermentation medium is 6.78g·L-1Na2HPO4,3g·L-1KH2PO4,1g·L-1NH4Cl,0.5g·L-1NaCl,0.24g·L- 1MgSO4(, and the modified M9 fermentation medium is independently sterilized, and is composed of 0.011 g.L -1CaCl2,8g·L-1 peptone, 2 g.L -1 yeast powder and 8 g.L -1 glucose.
As a preferable scheme of the preparation method of the glycollic acid fermentation liquid, provided by the invention, the preparation method comprises the following steps: antibiotics were 50uL 100 mg/mL -1 Amp and 50uL 50 mg/mL -1 Str.
As a preferable scheme of the preparation method of the glycollic acid fermentation liquid, provided by the invention, the preparation method comprises the following steps: the fermentation medium composition was 6.78g·L-1Na2HPO4,3g·L-1KH2PO4,1g·L-1NH4Cl,0.5g·L-1NaCl,0.24g·L-1MgSO4( sterilized alone), 0.011 g.L -1CaCl2,8g·L-1 peptone, 2 g.L -1 yeast powder, concentrated corn cob hydrolysate (115 ℃ C., 15 min), concentrated corn steep liquor (115 ℃ C., 7 min).
As a preferable scheme of the preparation method of the glycollic acid fermentation liquid, provided by the invention, the preparation method comprises the following steps: the initial sugar was 8 g.L -1 glucose.
Compared with the prior art, for example CNll4854796A uses gluconobacter oxydans as a biocatalyst for fermentation, excessive calcium carbonate powder is added in the fermentation process, differential centrifugation is adopted for in-situ separation to respectively obtain calcium glycolate and cells, calcium glycolate is recrystallized to obtain calcium glycolate crystals, sulfuric acid is used for acidifying calcium glycolate solution for electrodialysis, and the high-purity glycolic acid solution is obtained, and the specific purity is improved to 98%. The invention has the following beneficial effects:
According to the invention, firstly, the glycollic acid in the fermentation broth is replaced by a calcium salt method, and the purity of glycollic acid crystal products is further improved by a mode of desalting isopropanol, concentrating and crystallizing. Compared with the product with the purity of 98 percent obtained by electrodialysis, the glycolic acid crystal product obtained by desalting and concentrating has the purity of more than or equal to 99.5 percent, thus not only improving the yield, but also guaranteeing the purity of the product, and further improving the efficiency of the glycolic acid crystallization process. In addition, the separation and purification method provided by the invention has the advantages of simple and convenient operation process, easiness in operation, high separation and purification effect, low energy consumption, low cost and high recovery rate, and is suitable for industrial production.
Detailed Description
In order that the above-recited objects, features and advantages of the present invention will become more apparent, a more particular description of the invention will be rendered by reference to specific embodiments thereof which are illustrated in the appended drawings.
In the following description, numerous specific details are set forth in order to provide a thorough understanding of the present invention, but the present invention may be practiced in other ways other than those described herein, and persons skilled in the art will readily appreciate that the present invention is not limited to the specific embodiments disclosed below.
Further, reference herein to "one embodiment" or "an embodiment" means that a particular feature, structure, or characteristic can be included in at least one implementation of the invention. The appearances of the phrase "in one embodiment" in various places in the specification are not necessarily all referring to the same embodiment, nor are separate or alternative embodiments mutually exclusive of other embodiments.
The starting materials used in the examples were commercially available unless otherwise specified and the purity was either chemically pure or analyzed.
The raw material glycolic acid fermentation broth 1 used in the following examples was prepared as follows:
S1, plate culture: MG1655 (DE 3) (ΔldhA ΔglcB ΔaceB ΔaldA Δ glcDEF ΔicdA glcC) strain preserved in advance was streaked on an agar plate and cultured overnight in a constant temperature incubator at 30 ℃;
S2, seed culture: single colonies were picked up to 20mL of LB liquid medium, cultured overnight at 37℃and 250rpm, the cultured seed liquid was inoculated into 50mL of modified M9 fermentation medium (modified M9 medium composition 6.78g·L-1Na2HPO4,3g·L-1KH2PO4,1g·L-1NH4C1,0.5g·L-1NaCl,0.24g·L-1MgSI4( was sterilized alone), 0.011 g.L -1CaCl2,8g·L-1 peptone, 2 g.L -1 yeast powder, 8 g.L -1 glucose) at an inoculum size of 2%, and 50uL of 100 mg.mL -1 Amp and 50uL of 50 mg.mL -1 Str were added and cultured at 37℃and 250 rpm.
S3, fermenting to produce acid: 450mL of seed culture solution cultured in advance was inoculated into a 5L fermenter containing 3L of fermentation medium (fermentation medium composition 6.78g·L-1Na2HPO4,3g·L-1KH2PO4,1g·L-1NH4Cl,0.5g·L-1NaCl,0.24g·L- 1MgSO4( was sterilized alone), 0.011 g.L -1CaCl2,8g·L-1 peptone, 2 g.L -1 yeast powder, concentrated corncob hydrolysate (115 ℃ C., 15 min), concentrated corn steep liquor (115 ℃ C., 7 min)). The initial glucose is 8g/L, and the sugar concentration is controlled to be 1-4g/L by controlling the feeding speed in the later stage. The pH in the fermenter was maintained at about 7.0 with an aqueous ammonia solution, the fermentation temperature was 37℃and the stirring speed was 400rpm, and the aeration rate was 2vvm.
Example 1
The glycolic acid fermentation broth 1 is centrifugated and concentrated to 50g/L of glycolic acid content, 80mL of solution is taken, the PH is regulated to 8 by using ammonia water, 2.1g of calcium hydroxide is added for reaction, then centrifugated, the sediment reacts with 20mL of 1.84mol/L of sulfuric acid, centrifugated, and the recovery rate of the glycolic acid is 76 percent after detection. And (3) decoloring the supernatant by using activated carbon, adding isopropanol with the mass of 2 times of that of the glycolic acid, stirring for 30min, centrifuging, and concentrating the supernatant to 70% of the glycolic acid content by using a rotary evaporator. Cooling and crystallizing, and adding glycolic acid crystals as seed crystals when the solution temperature is lower than the saturation temperature of the raw materials by 2 ℃, wherein the purity of the seed crystals is 99%, and the adding amount of the seed crystals is 1.5% of the mass of the glycolic acid in the raw materials; the total crystallization time is 3 hours, and the glycolic acid crystal is obtained after the crystal slurry is obtained through suction filtration and freeze drying, and the purity of the product is 99.5 percent.
Example 2
The glycolic acid fermentation broth 1 is centrifugated and concentrated to 50g/L of glycolic acid content, 80mL of solution is taken, the PH is regulated to 9.5 by using ammonia water, 1.97g of calcium hydroxide is added for reaction, then centrifugated, the precipitate is reacted with 20mL of 1.84mol/L of sulfuric acid, centrifugated, and the recovery rate of the detected glycolic acid is 74%. And (3) decoloring the supernatant by using activated carbon, adding isopropanol with the mass of 2 times of that of the glycolic acid, stirring for 30min, centrifuging, and concentrating the supernatant to the content of 60% of the glycolic acid by using a rotary evaporator. Cooling and crystallizing, and adding glycolic acid crystals as seed crystals when the solution temperature is lower than the saturation temperature of the raw materials by 2 ℃, wherein the purity of the seed crystals is 99%, and the adding amount of the seed crystals is 1.5% of the mass of the glycolic acid in the raw materials; the total crystallization time is 3 hours, and the glycolic acid crystal is obtained after the crystal slurry is obtained through suction filtration and freeze drying, and the purity of the product is 99.5 percent.
Example 3
The glycolic acid fermentation broth 1 is centrifugated and concentrated to 50g/L of glycolic acid content, 80mL of solution is taken, the PH is regulated to 9 by using ammonia water, 2.1g of calcium hydroxide is added for reaction, then centrifugated, the precipitate is reacted with 25mL of 1.84mol/L of sulfuric acid, centrifugated, and the recovery rate of the glycolic acid is detected to be 81%. And (3) decoloring the supernatant by using activated carbon, adding isopropanol with the mass 3 times of that of the glycolic acid, stirring for 30min, centrifuging, and concentrating the supernatant to 80% of the glycolic acid content by using a rotary evaporator. Cooling and crystallizing, and adding glycolic acid crystals as seed crystals when the solution temperature is lower than the saturation temperature of the raw materials by 2 ℃, wherein the purity of the seed crystals is 99%, and the adding amount of the seed crystals is 2% of the mass of the glycolic acid in the raw materials; the total crystallization time is 3 hours, and the glycolic acid crystal is obtained after the crystal slurry is obtained through suction filtration and freeze drying, and the purity of the product is 99.5 percent.
Example 4
The glycolic acid fermentation broth 1 is centrifugated and concentrated to 50g/L of glycolic acid content, 80mL of solution is taken, the PH is regulated to 8.5 by using ammonia water, 2.2g of calcium hydroxide is added for reaction, then centrifugated, the precipitate is reacted with 20mL of 1.84mol/L of sulfuric acid, centrifugated, and the recovery rate of the glycolic acid is 76 percent after detection. And (3) decoloring the supernatant by using activated carbon, adding isopropanol with the mass 3 times of that of the glycolic acid, stirring for 30min, centrifuging, and concentrating the supernatant to 80% of the glycolic acid content by using a rotary evaporator. Cooling and crystallizing, and adding glycolic acid crystals as seed crystals when the solution temperature is lower than the saturation temperature of the raw materials by 2 ℃, wherein the purity of the seed crystals is 99.5%, and the adding amount of the seed crystals is 2% of the mass of the glycolic acid in the raw materials; the total crystallization time is 4 hours, and the glycolic acid crystal is obtained after the crystal slurry is obtained through suction filtration and freeze drying, and the purity of the product is 99.6 percent.
Example 5
The glycolic acid fermentation broth 1 is centrifugated and concentrated to 50g/L of glycolic acid content, 80mL of solution is taken, the PH is regulated to 9 by using ammonia water, 2.3g of calcium hydroxide is added for reaction, then centrifugated, the precipitate is reacted with 25mL of 1.84mol/L sulfuric acid, centrifugated, and the recovery rate of the glycolic acid is detected to be 84 percent. And (3) decoloring the supernatant by using activated carbon, adding isopropanol with the mass 3 times of that of the glycolic acid, stirring for 30min, centrifuging, and concentrating the supernatant to 80% of the glycolic acid content by using a rotary evaporator. Cooling and crystallizing, and adding glycolic acid crystals as seed crystals when the solution temperature is lower than the saturation temperature of the raw materials by 0 ℃, wherein the purity of the seed crystals is 99.5%, and the adding amount of the seed crystals is 2% of the mass of the glycolic acid in the raw materials; the total crystallization time is 4 hours, and the glycolic acid crystal is obtained after the crystal slurry is obtained through suction filtration and freeze drying, and the purity of the product is 99.7 percent.
Example 6
The glycolic acid fermentation broth 1 is centrifugated and concentrated to 50g/L of glycolic acid content, 80mL of solution is taken, the PH is regulated to 8 by using ammonia water, 2.1g of calcium hydroxide is added for reaction, then centrifugated, the precipitate is reacted with 15mL of 1.84mol/L of sulfuric acid, centrifugated, and the recovery rate of the glycolic acid is 72 percent after detection. And (3) decoloring the supernatant by using activated carbon, adding isopropanol with the mass of 2 times of that of the glycolic acid, stirring for 30min, centrifuging, and concentrating the supernatant to 70% of the glycolic acid content by using a rotary evaporator. Cooling and crystallizing, and adding glycolic acid crystals as seed crystals when the solution temperature is lower than the saturation temperature of the raw materials by 1 ℃, wherein the purity of the seed crystals is 99.5%, and the adding amount of the seed crystals is 2% of the mass of the glycolic acid in the raw materials; the total crystallization time is 2 hours, and the glycolic acid crystal is obtained after the crystal slurry is obtained through suction filtration and freeze drying, and the purity of the product is 99.3 percent.
Example 7
The glycolic acid fermentation broth 1 is centrifugated and concentrated to 50g/L of glycolic acid content, 80mL of solution is taken, the PH is regulated to 8.5 by using ammonia water, 2.2g of calcium hydroxide is added for reaction, then centrifugated, the precipitate is reacted with 20mL of 1.84mol/L of sulfuric acid, centrifugated, and the recovery rate of the detected glycolic acid is 82%. And (3) decoloring the supernatant by using activated carbon, adding isopropanol with the mass 4 times of that of the glycolic acid, stirring for 60min, centrifuging, and concentrating the supernatant to 80% of the glycolic acid content by using a rotary evaporator. Cooling and crystallizing, and adding glycolic acid crystals as seed crystals when the solution temperature is lower than the saturation temperature of the raw materials by 0 ℃, wherein the purity of the seed crystals is 99.5%, and the adding amount of the seed crystals is 1.5% of the mass of the glycolic acid in the raw materials; the total crystallization time is 2 hours, and the glycolic acid crystal is obtained after the crystal slurry is obtained through suction filtration and freeze drying, and the purity of the product is 99.5 percent.
Example 8
The glycolic acid fermentation broth 1 is centrifugated and concentrated to 50g/L of glycolic acid content, 80mL of solution is taken, ammonia water is used for adjusting the PH to 9.5, 2.1g of calcium hydroxide is added for reaction, then centrifugated, the precipitate reacts with 20mL of 1.84mol/L of sulfuric acid, centrifugated, and the recovery rate of the detected glycolic acid is 85.6 percent. And (3) decoloring the supernatant by using activated carbon, adding isopropanol with the mass 5 times of that of the glycolic acid, stirring for 40min, centrifuging, and concentrating the supernatant to 75% of the glycolic acid content by using a rotary evaporator. Cooling and crystallizing, and adding glycolic acid crystals as seed crystals when the solution temperature is lower than the saturation temperature of the raw materials by-2 ℃, wherein the purity of the seed crystals is 99.5%, and the adding amount of the seed crystals is 2% of the mass of the glycolic acid in the raw materials; the total crystallization time is 4 hours, and the glycolic acid crystal is obtained after the crystal slurry is obtained through suction filtration and freeze drying, and the purity of the product is 99.7 percent.
Example 9
The glycolic acid fermentation broth 1 is centrifugated and concentrated to 50g/L of glycolic acid content, 80mL of solution is taken, the PH is regulated to 8.5 by using ammonia water, 2.4g of calcium hydroxide is added for reaction, then centrifugated, the precipitate is reacted with 25mL of sulfuric acid with 1.84mol/L, centrifugated, and the recovery rate of the detected glycolic acid is 79 percent. And (3) decoloring the supernatant by using activated carbon, adding isopropanol with the mass 3 times of that of the glycolic acid, stirring for 60min, centrifuging, and concentrating the supernatant to the content of 85% of the glycolic acid by using a rotary evaporator. Cooling and crystallizing, and adding glycolic acid crystals as seed crystals when the solution temperature is lower than the saturation temperature of the raw materials by-1 ℃, wherein the purity of the seed crystals is 99%, and the adding amount of the seed crystals is 1.5% of the mass of the glycolic acid in the raw materials; the total crystallization time is 3 hours, and the glycolic acid crystal is obtained after the crystal slurry is obtained through suction filtration and freeze drying, and the purity of the product is 99.6 percent.
Example 10
The glycolic acid fermentation broth 1 as a raw material is centrifuged and concentrated to have the glycolic acid content of 50g/L, 80mL of solution is taken, the pH is regulated to 9 by using ammonia water, 2.1g of calcium hydroxide is added for reaction, the mixture is centrifuged, the precipitate is reacted with 15mL of sulfuric acid of 1.84mol/L, and the recovery rate of the glycolic acid is 78% after the detection. And (3) decoloring the supernatant by using activated carbon, adding isopropanol with the mass 4 times of that of the glycolic acid, stirring for 30min, centrifuging, and concentrating the supernatant to 75% of the glycolic acid content by using a rotary evaporator. Cooling and crystallizing, and adding glycolic acid crystals as seed crystals when the solution temperature is lower than the saturation temperature of the raw materials by 0 ℃, wherein the purity of the seed crystals is 99.5%, and the adding amount of the seed crystals is 1.5% of the mass of the glycolic acid in the raw materials; the total crystallization time is 3 hours, and the glycolic acid crystal is obtained after the crystal slurry is obtained through suction filtration and freeze drying, and the purity of the product is 99.5 percent.
It should be noted that the above embodiments are only for illustrating the technical solution of the present invention and not for limiting the same, and although the present invention has been described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that the technical solution of the present invention may be modified or substituted without departing from the spirit and scope of the technical solution of the present invention, which is intended to be covered in the scope of the claims of the present invention.
Claims (10)
1. A method for separating and extracting glycollic acid from fermentation broth, which is characterized by comprising the following steps:
(1) Centrifuging and concentrating the glycollic acid fermentation liquor to obtain clarified fermentation liquor;
(2) Regulating the clarified fermentation broth to be alkaline to obtain alkaline fermentation broth;
(3) Reacting alkaline fermentation liquor with calcium hydroxide and centrifuging to obtain calcium glycolate sediment;
(4) Reacting calcium glycolate precipitate with sulfuric acid, and centrifuging to obtain an acidified solution;
(5) Adding activated carbon into the acidified solution for decoloring to obtain a decolored solution;
(6) Adding anhydrous isopropanol into the decolorized solution to remove salt, carrying out suction filtration, and concentrating by rotary evaporation to obtain concentrated solution;
(7) Cooling, crystallizing and freeze-drying the concentrated solution to obtain a glycolic acid product.
2. The method for separating and extracting glycolic acid from fermentation broth according to claim 1, wherein the centrifugal rotation speed in (1) is 9000-11000 rpm for 10-20 min; concentrating by vacuum distillation at 50-70deg.C and vacuum degree of 50-200 mbar until glycolic acid concentration is 50g/L.
3. The method for separating and extracting glycolic acid from fermentation broth according to claim 1, wherein (2) the pH of the clarified fermentation broth is adjusted to 8-10 using ammonia water.
4. The method for separating and extracting glycolic acid from fermentation broth according to claim 1, wherein the calcium hydroxide addition amount in (3) is 40-50 g/L, the reaction temperature is 45-60 ℃ and the time is 4-7 h; the centrifugal speed is 9000-11000 rpm, and the time is 10-20 min.
5. The method for separating and extracting glycolic acid from fermentation broth according to claim 1, wherein the concentration of sulfuric acid in (4) is 1.84mol/L, the addition amount is 75-125 mL/L, the reaction temperature is 40-70 ℃ and the time is 0.5-2 h; the centrifugal speed is 9000-11000 rpm, and the time is 10-20 min.
6. The method for separating and extracting glycolic acid from fermentation broth according to claim 1, wherein the addition amount of the activated carbon in (5) is 15-25 g/L, the temperature is 45-60 ℃, and the stirring time is 0.5-2 h.
7. The method for separating and extracting glycolic acid from fermentation broth according to claim 1, wherein the addition amount of anhydrous isopropanol in (6) is 2-5 times of the mass of the glycolic acid in the decolorized solution, the concentration is carried out by adopting a reduced pressure distillation mode, the temperature is 40-70 ℃, the vacuum degree is 50-200 mbar, and the concentration is 50-90% of the mass concentration of the glycolic acid.
8. The method for separating and extracting glycolic acid from fermentation broth according to claim 1, wherein in (7), the temperature is reduced to 0.5-5 ℃ below the saturation temperature of the concentrated solution, glycolic acid crystals with purity not lower than 99% are added as seed crystals, the adding amount of the seed crystals is 0.5-5% of the mass of the glycolic acid in the concentrated solution, the seed crystals are added, the temperature is maintained at constant temperature, then the temperature is reduced to-5 to-18 ℃, the temperature is reduced to 2-4 hours, and the glycolic acid crystals are obtained through suction filtration and freeze drying.
9. Glycolic acid isolated and purified by the method of any one of claims 1 to 8, having a purity of 99 to 99.7%.
10. A process for producing a glycolic acid fermentation broth used in the process according to any one of claims 1 to 8, wherein glucose is used as a carbon source, and fermentation is carried out using Escherichia coli MG1655 (DE 3) (ΔldhA ΔglcB ΔaceB ΔaldA Δ glcDEF ΔicdA Δ glcC) to obtain a glycolic acid fermentation broth.
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