WO2011001696A1 - 有機酸類の製造法 - Google Patents
有機酸類の製造法 Download PDFInfo
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- WO2011001696A1 WO2011001696A1 PCT/JP2010/004362 JP2010004362W WO2011001696A1 WO 2011001696 A1 WO2011001696 A1 WO 2011001696A1 JP 2010004362 W JP2010004362 W JP 2010004362W WO 2011001696 A1 WO2011001696 A1 WO 2011001696A1
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- C—CHEMISTRY; METALLURGY
- 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
- 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
- C12P7/44—Polycarboxylic acids
- C12P7/46—Dicarboxylic acids having four or less carbon atoms, e.g. fumaric acid, maleic acid
Definitions
- the present invention relates to a method for producing L-tartaric acid or a salt thereof and / or glycolic acid or a salt thereof from glucose.
- L-tartaric acid and its salts are useful in a wide range of industries such as acidification in the food field, pH adjusters, cosmetics, dyeing, detergents and plating in the industrial field, and pharmaceutical raw materials in the pharmaceutical field. It is a substance. Further, glycolic acid produced together with L-tartaric acid when producing L-tartaric acid from glucose is useful as a metal detergent, plating additive, cosmetic additive, raw material for hard biodegradable polymer, etc. It is a substance.
- acetic acid bacteria reduce not only enzymes that produce 5-ketogluconic acid from glucose via gluconic acid, but also 5-keto-D-gluconic acid reduction that converts 5-keto-D-gluconic acid to gluconic acid. It has an enzyme in the cytoplasm. Reduced gluconic acid is consumed by metabolic pathways such as the pentose / phosphoric acid pathway and the Entner / Doudoroff pathway, resulting in decreased production of 5-keto-D-gluconic acid as a raw material for L-tartaric acid and glycolic acid. It is also known that this occurs (see Non-Patent Document 1).
- the removed calcium carbonate needs to be treated as waste, it becomes a factor of high cost of the present production method.
- this production method there are problems such as that the yield of tartaric acid is as low as about 10%, and that unnecessary organic acids other than tartaric acid are produced as a by-product, so that they need to be separated. It cannot be used for industrial tartaric acid production.
- the production method when a method for producing tartaric acid from glucose is considered based on the conventional knowledge, the production method generates a calcium salt of 5-keto-D-gluconic acid from glucose by microbial fermentation, A step of separating 5-keto-D-gluconic acid calcium salt, a step of converting the obtained poorly water-soluble 5-keto-D-gluconic acid calcium salt into a water-soluble salt, and further water-soluble 5-keto-D -Consisting of a step of chemically converting gluconate to tartaric acid.
- this manufacturing method has a great disadvantage that it involves many steps and takes time and effort, and therefore requires a large manufacturing cost. Therefore, it is thought that the manufacturing method of tartaric acid which consists of the said process was not industrially realizable until now.
- the first reaction step (converting 5-keto-D-gluconate to tartaric acid) in the medium for converting glucose to 5-keto-D-gluconic acid by acetic acid bacteria in the first step
- a method for producing tartaric acid to which a vanadic acid catalyst necessary for the step is added has been reported (see Patent Document 4).
- Patent Document 4 in this production method, only 43 g (acquisition yield: 5.2% by weight) of tartaric acid is collected from 1000 g of glucose. Thus, since the yield of this production method is very low, this production method has not been utilized for the production of industrial tartaric acid.
- Patent Document 5 only one report has been known so far regarding a method for producing glycolic acid from 5-keto-D-gluconic acid by a chemical reaction (see Patent Document 5).
- Patent Document 5 since a large number of low molecular organic acids other than glycolic acid are by-produced in the reaction solution, only 11.6 g / L glycol from 796 g / L 5-keto-D-gluconic acid is produced. Only acid is produced. Thus, since the yield of this production method is very low, this production method has not been used for industrial production of glycolic acid.
- An object of the present invention is a method capable of producing L-tartaric acid or a salt thereof and / or glycolic acid or a salt thereof from glucose efficiently and at low cost, and can be used for industrial production. The purpose is to provide a highly reliable method.
- the present inventors are a method capable of producing L-tartaric acid or a salt thereof and / or glycolic acid or a salt thereof from glucose efficiently and at low cost, and can be used for industrial production. As a result of intensive research on the realization of a highly efficient method, it was found that the problem could be solved by adopting the following steps A to C, and the present invention was completed. .
- a microorganism producing 5-keto-D-gluconic acid from glucose is cultured in a glucose-containing culture medium in the presence of an alkali capable of converting 5-keto-D-gluconic acid to a water-soluble salt, Step A for obtaining a culture solution containing a water-soluble salt of keto-D-gluconic acid,
- B By adjusting and maintaining the pH of the culture solution containing the water-soluble salt of 5-keto-D-gluconic acid obtained in step A within the range of 7 to 12, 5-keto-D-gluconic acid
- C Step C for collecting L-tartaric acid or a salt thereof and / or glycolic acid or a salt thereof from the reaction solution obtained in Step B.
- the present invention comprises (1) a process for producing L-tartaric acid or a salt thereof and / or glycolic acid or a salt thereof from glucose, comprising the following steps A to C; (A) 5-keto-from glucose By culturing a microorganism producing D-gluconic acid in a glucose-containing culture solution in the presence of an alkali capable of converting 5-keto-D-gluconic acid into a water-soluble salt, the water-solubility of 5-keto-D-gluconic acid is obtained.
- Step A for obtaining a culture solution containing a salt (B) adjusting and maintaining the pH of the culture solution containing a water-soluble salt of 5-keto-D-gluconic acid obtained in Step A within a range of 7-12.
- a reaction solution in which a water-soluble salt of 5-keto-D-gluconic acid is converted to L-tartaric acid or a salt thereof and / or glycolic acid or a salt thereof;
- C obtained in Step B From the reaction solution, L-tartar Or the process C which collect
- cultivation liquid which further contained the transition metal catalyst for the culture liquid in the said process B is used (The above characterized by 1) The method described in 1) or (3) one or more selected from the group consisting of palladium, rhodium, ruthenium, platinum, manganese, copper, cobalt, nickel, zinc, vanadium and iron Or (4) the concentration of the transition metal catalyst is in the range of 0.00002 to
- the concentration of the transition metal catalyst is in the range of 0.0001 to 1%.
- (6) from glucose The method according to any one of (1) to (5) above, wherein the microorganism producing keto-D-gluconic acid is a microorganism belonging to the genus Gluconobacter or Acetobacter, )
- the microorganisms belonging to the genus Gluconobacter or Acetobacter are Gluconobacter oxydans NBRC 3172 and NBRC 3255, Gluconobacter pastorianus NBRC 3225, Gluconobacter albidas NBRC 3250, Gluconobacter suboxydans NBRC 3254, Gluconobacter industrious NBRC 3260, Gluconobacter cereus NBRC 3267, Gluconobacter dioxyacetonicus NBRC 3273, Gluconobacter monooxygluconicus NBRC 3276 Gluconobacter
- any one of (1) to (9) above wherein the pH of the culture solution in step B is adjusted and maintained within the range of 9 to 10.
- the temperature of the culture solution is further adjusted and maintained within a range of 0 ° C. to 70 ° C.
- the temperature of the culture solution is further set to 20 ° C to 60 ° C.
- the pH of the culture solution is adjusted and maintained in the step B.
- L-tartaric acid or a salt thereof and / or glycolic acid or a salt thereof can be produced from glucose very efficiently and at low cost. Therefore, the production method of the present invention can be used for industrial production, and is extremely practical.
- the “method for producing L-tartaric acid or a salt thereof and / or glycolic acid or a salt thereof from glucose” of the present invention refers to 5- By culturing a microorganism producing keto-D-gluconic acid in a glucose-containing culture solution in the presence of an alkali capable of converting 5-keto-D-gluconic acid into a water-soluble salt, Step A for obtaining a culture solution containing a water-soluble salt; adjusting and maintaining the pH of the culture solution containing the water-soluble salt of 5-keto-D-gluconic acid obtained in Step A within a range of 7 to 12 To obtain a reaction solution in which a water-soluble salt of 5-keto-D-gluconic acid is converted to L-tartaric acid or a salt thereof and / or glycolic acid or a salt thereof; and the reaction obtained in step B From liquid L- tartaric acid or a salt thereof, and / or, not particularly
- L-tartaric acid or a salt thereof and / or glycolic acid or a salt thereof (hereinafter referred to as “L-tartaric acid or a salt thereof and / or glycolic acid”) Alternatively, the salt thereof is simply indicated as “L-tartaric acid or the like”.)
- L-tartaric acid or a salt thereof and / or glycolic acid Alternatively, the salt thereof is simply indicated as “L-tartaric acid or the like”.
- microorganism producing 5-keto-D-gluconic acid from glucose (hereinafter also referred to as “microorganism in the present invention”) in the above step A is suitable for the growth of the microorganism. Under the conditions, it has the ability to produce 5-keto-D-gluconic acid from glucose by culturing in a glucose-containing culture solution (hereinafter also simply referred to as “5-keto-D-gluconic acid producing ability”). Means microorganism.
- acetic acid bacteria belonging to the genus Gluconobacter or Acetobacter can be preferably exemplified, and among them, public strain preservation such as the National Institute of Technology and Evaluation (NBRC) Gluconobacter oxydans NBRC 3172 and NBRC 3255, Gluconobacter pastorianus NBRC 3225, Gluconobacter albidas NBRC 3250, Gluconobacter Suboxydans NBRC 3254, Gluconobacternapss NBRC 3260, Gluconobacter cereus NBRC 3267, Gluconobacter dioxyacetonicus NBRC 3273, Gluconobacter -Monooxygluconicus NBRC 3276, Gluconobacter gluconicus NBRC 3285, Gluconobacter roseus NBRC 3990, Gluconobacter flavii NBRC 3265, Acetobacter aceti NBRC 3259, etc.
- NBRC National Institute of Technology and Evaluation
- -Gluconobacter oxydans NBRC 3172 strain can be more preferably exemplified because of its excellent ability to produce keto-D-gluconic acid.
- a strain belonging to the genus Gluconobacter or Acetobacter newly isolated from nature can be used as long as it has the ability to produce 5-keto-D-gluconic acid.
- 2-keto-D-gluconic acid low productivity or 5-keto-D-gluconic acid low examples of the microorganism further having consumability (preferably, a microorganism belonging to the genus Gluconobacter or Acetobacter) can be given, and more preferable microorganisms include 5-keto-D-gluconic acid production ability.
- microorganisms having low 2-keto-D-gluconic acid low productivity and 5-keto-D-gluconic acid low consumption preferably microorganisms belonging to the genus Gluconobacter or Acetobacter
- the microorganism of the present invention such as acetic acid bacteria produces not only 5-keto-D-gluconic acid but also 2-keto-D-gluconic acid, an unnecessary by-product, from glucose via gluconic acid. This is because having a low 2-keto-D-gluconic acid productivity is preferable in that the yield of 5-keto-D-gluconic acid is improved.
- the microorganism of the present invention such as acetic acid bacteria consumes a part of the produced 5-keto-D-gluconic acid for growth, it further has low consumption of 5-keto-D-gluconic acid. This is because the yield of 5-keto-D-gluconic acid is improved.
- microorganism having low productivity of 2-keto-D-gluconic acid means low productivity of 2-keto-D-gluconic acid (2-keto-from a specific amount of glucose). D-gluconic acid is produced in a small amount, or the ratio of 2-keto-D-gluconic acid is low relative to the amount of 5-keto-D-gluconic acid produced from a specific amount of glucose.
- the microorganism is an acetic acid bacterium, it means an acetic acid bacterium having a low productivity of 2-keto-D-gluconic acid as compared with Gluconobacter oxydans NBRC 3172 strain. From the viewpoint of further improving the yield of 5-keto-D-gluconic acid, 2-keto-D-gluconic acid non-productivity is preferable among 2-keto-D-gluconic acid low productivity.
- microbe having low consumption of 5-keto-D-gluconic acid means a microorganism that consumes less 5-keto-D-gluconic acid.
- fungi it means acetic acid bacteria that consume less 5-keto-D-gluconic acid compared to Gluconobacter oxydans NBRC 3172 strain. From the viewpoint of further improving the yield of 5-keto-D-gluconic acid, among the low consumption of 5-keto-D-gluconic acid, non-consumption of 5-keto-D-gluconic acid is preferable.
- microorganisms having low productivity of 2-keto-D-gluconic acid and microorganisms having low consumption of 5-keto-D-gluconic acid are referred to as microorganisms in the present invention (from glucose to 5-keto).
- the method for producing from -D-gluconic acid producing microorganism) is not particularly limited, but N-methyl-N-nitro-N'-nitrosoguanidine (hereinafter abbreviated as “NTG”) is not used for the microorganism in the present invention.
- NTG N-methyl-N-nitro-N'-nitrosoguanidine
- Example 1 The screening method described in Example 1 to be described later can be more preferably illustrated as a more specific method. Whether a certain microorganism has low productivity of 2-keto-D-gluconic acid is determined by, for example, spotting the supernatant of the culture solution of the microorganism on silica gel TLC (manufactured by Merck) and adding n-butanol-acetic acid.
- keto-D-gluconic acid can be confirmed by a method such as visual judgment, and whether or not it has a low consumption of 5-keto-D-gluconic acid is determined by the supernatant of the culture solution of the microorganism.
- Reaction carried out can be confirmed by a method such as examining the consumption of 5-keto -D- gluconate visually.
- the Gluconobacter oxydans TABT-200 strain can be preferably exemplified.
- This TABT-200 strain is a strain prepared by the present inventors by treating the NBRC 3172 strain with NTG and screening a strain with low productivity of 2-keto-D-gluconic acid (Example 1 described later). reference).
- This TABT-200 strain is a 2-keto-D-gluconic acid non-producing strain that hardly produces 2-keto-D-gluconic acid.
- the Gluconobacter oxydans TADK-267 strain is particularly preferably exemplified. Can do.
- This TADK-267 strain is a strain prepared by the present inventors by subjecting the TABT-200 strain to NTG treatment and screening a 5-keto-D-gluconic acid low-consumption strain (Examples described later). 1).
- the TADK-267 strain is a 2-keto-D-gluconic acid non-producing strain and a 5-keto-D-gluconic acid non-consuming strain that does not consume 5-keto-D-gluconic acid. is there.
- alkali capable of converting 5-keto-D-gluconic acid into a water-soluble salt means that the 5-keto-D-gluconic acid is converted into 5-keto-D-gluconic acid when contacted with 5-keto-D-gluconic acid in solution.
- the alkali is not particularly limited as long as it is an alkali that forms a water-soluble salt, and examples thereof include sodium hydroxide, potassium hydroxide, and ammonia.
- the amount of alkali used in the above step A is not particularly limited as long as the microorganism in the present invention can grow, but the pH of the culture solution during the culture is preferably in the range of 3 to 9, more preferably. An amount necessary to maintain within the range of 4 to 7, more preferably within the range of 5 to 6 can be used.
- the “glucose-containing culture solution” in the above step A is not particularly limited as long as it contains glucose and is a culture solution in which the microorganism in the above step A can grow, but a carbon source that can be assimilated by the microorganism, A culture solution containing a digestible nitrogen source, inorganic metal salts, and vitamins or antifoaming agents necessary for growth can be exemplified.
- calcium is not essential for the growth of the microorganism, it is an essential component for the 5-ketogluconic acid production reaction from glucose. Therefore, a trace amount of calcium is added to the extent that 5-ketogluconic acid calcium salt is not generated as much as possible. It is necessary to add it to the culture medium containing glucose.
- the upper limit of the concentration of calcium to be added is preferably a concentration (w / w (%)) of 1/20 or less of the glucose concentration (w / w (%)) in the culture solution, and 1/40
- concentration (w / w (%)) is more preferable, and as the lower limit, the concentration (w / w) of 1/500 or more of the glucose concentration (w / w (%)) in the culture solution w (%)) is preferable, and a concentration (w / w (%)) of 1/20 or more is more preferable.
- glucose, fructose, mannitol, sorbitol, sorbose, lactose, galactose, sucrose, maltose or glycerin may be used.
- Glucose is used as the carbon source of the culture solution, and its concentration is preferably in the range of 1% to 20%, preferably in the range of 5% to 16%.
- the culture solution in step A supplemented with fructose, mannitol, sorbitol, sorbose, lactose, galactose, sucrose, maltose or glycerin, specifically 0.1% to 2.0.
- % Is preferably added to the medium from the viewpoint of further improving the yield of 5-keto-D-gluconic acid.
- the nitrogen source for example, peptone, soybean powder, corn steep powder, corn steep liquor, meat extract, yeast extract, amino acids and other organic nitrogen sources or ammonium sulfate, ammonium nitrate
- Inorganic nitrogen sources such as urea can be used alone or as a mixture, but industrially preferred are corn steep powder and corn steep liquor because they are inexpensive.
- the inorganic metal salt for example, sulfates such as calcium, magnesium, zinc, manganese, cobalt, and iron, hydrochlorides, carbonates, or phosphates are preferably used.
- Step A when the microorganism is acetic acid bacteria, more specifically, 6% glucose, 0.09% ammonium chloride, 0.06% potassium dihydrogen phosphate, 0.18% corn steep Powder (manufactured by Sigma), 0.1% yeast extract (manufactured by Difco Laboratories), 0.015% magnesium sulfate heptahydrate, 0.0029% manganese sulfate pentahydrate, 0.12% calcium chloride dihydrate And MB medium composed of 0.1% Actol (manufactured by Takeda Chemical Industries) can be particularly preferably exemplified.
- the culture conditions in step A are not particularly limited as long as the microorganism can grow in the step and the microorganism can produce 5-keto-D-gluconic acid.
- strong acidic organic acids such as gluconic acid and 5-keto-D-gluconic acid are produced in large quantities, and the pH of the culture solution changes significantly. Therefore, while continuously adding an acid solution and an alkaline solution to the culture solution, It is preferable to maintain a pH suitable for the production of 5-keto-D-gluconic acid.
- Such pH is preferably exemplified in the range of 3 to 9, more preferably exemplified in the range of 4 to 7, and further preferably exemplified in the range of 5 to 6. it can.
- the acid solution examples include acid aqueous solutions such as hydrochloric acid, sulfuric acid, and nitric acid.
- the alkali aqueous solution preferably include aqueous alkali solutions such as sodium hydroxide, potassium hydroxide, and ammonia. be able to.
- a temperature within a range of 10 ° C. to 40 ° C. can be preferably exemplified, and a temperature within a range of 25 ° C. to 35 ° C. can be more suitably exemplified.
- the oxygen conditions for the culture aerobic conditions such as aeration and agitation culture are preferable, and more specifically, the ratio of the air supply amount to the culture solution amount per minute is within the range of 0.2 to 2.0. It can be preferably exemplified that it is within the range of 0.5 to 1.5.
- a continuously controllable stirred fermenter can be suitably used. If the microorganism in the present invention is cultured in the step A under suitable culture conditions, the culture in the step A is usually completed in 1 to 7 days, preferably 1 to 4 days, and a molar yield of 70% to 95%. Makes it possible to produce 5-keto-D-gluconic acid.
- step A When the culture in step A is carried out, a water-soluble salt of 5-keto-D-gluconic acid is produced in the culture solution.
- Step B the method for adjusting and maintaining the pH of the culture solution containing the water-soluble salt of 5-keto-D-gluconic acid obtained in Step A within the range of 7 to 12 is not particularly limited.
- an acid aqueous solution such as hydrochloric acid, sulfuric acid or nitric acid, or an alkali such as sodium hydroxide, potassium hydroxide or ammonia
- a method of adding the aqueous solution to the culture medium obtained in step A can be suitably exemplified.
- the pH adjusted and maintained in Step B is preferably within the range of 8 to 11, and preferably within the range of 9 to 10, from the viewpoint of obtaining excellent conversion efficiency to L-tartaric acid and the like. Is more preferable.
- the air supply rate is such that the ratio of the air supply amount to the culture solution amount per minute is in the range of 0.2 to 5. This can be preferably exemplified, and can be more preferably exemplified as being within the range of 0.5 to 3. If the reaction in Step B is carried out under suitable conditions, the reaction is usually completed in 1 to 14 days, preferably 1 to 10 days, and a mole of 10 to 30% (preferably 20 to 30%) with respect to glucose. L-tartaric acid and glycolic acid can be obtained in a yield.
- the culture solution is selected from the group consisting of palladium, rhodium, ruthenium, platinum, manganese, copper, cobalt, nickel, zinc, vanadium and iron.
- the addition of one or more (preferably two or more) transition metal catalysts is particularly preferred from the viewpoint of obtaining better conversion efficiency to L-tartaric acid or the like.
- a combination of palladium and vanadium can be exemplified, and among them, a combination of palladium carbon and vanadate is more preferably exemplified. be able to.
- the addition amount of the transition metal catalyst is preferably in the range of 0.00002% (w / v) to 2% (w / v) with respect to the culture solution (reaction solution), and is 0.0001% ( More preferably, it is within the range of w / v) to 1% (w / v).
- the reaction rate to L-tartaric acid and the like in step B is increased, so that the reaction (maintenance of pH) is usually completed in 1 to 7 days, preferably 1 to 4 days, and glucose is converted.
- L-tartaric acid and glycolic acid can be obtained in a molar yield of 40 to 70% (preferably 50 to 70%, more preferably 60 to 70%), respectively.
- the transition metal catalyst may be a water-soluble metal salt such as hydrochloride, sulfate, nitrate, acetate, etc., for example, palladium oxide, rhodium oxide, ruthenium oxide, platinum oxide, manganese oxide, Transition metal oxides such as copper oxide, cobalt oxide, nickel oxide, zinc oxide, vanadium oxide, iron oxide; and palladium carbon, rhodium carbon, ruthenium carbon, platinum carbon, nickel carbon, palladium alumina, When a water-insoluble metal compound such as rhodium alumina, ruthenium alumina, platinum alumina or the like, a compound in which one or more transition metal catalysts are adsorbed on a water-insoluble carrier (carbon, alumina, etc.); Can be easily recovered by centrifuging the reaction solution after completion of the reaction, and the recovered solution can be used repeatedly.
- a water-insoluble metal compound such as hydrochloride, sulfate, nitrate, a
- the amount of the transition metal catalyst adsorbed on the water-insoluble support is preferably such that the transition metal catalyst is adsorbed on the water-insoluble support so that the content is 0.1 wt% to 30 wt%. It is more preferable to adsorb so that it may become 15 weight% of content rate.
- step B when the pH of the culture solution containing the water-soluble salt of 5-keto-D-gluconic acid obtained in step A is adjusted and maintained within the range of 7 to 12, A reaction solution in which the water-soluble salt is converted to L-tartaric acid or a salt thereof and / or glycolic acid or a salt thereof can be obtained. Note that a water-soluble salt of 5-keto-D-gluconic acid may remain in the reaction solution.
- the method for collecting L-tartaric acid or a salt thereof and / or glycolic acid or a salt thereof from the reaction solution obtained in the step B is not particularly limited.
- L-tartaric acid or a salt thereof In the case of collecting L-tartaric acid, it is possible to easily isolate and purify L-tartaric acid and the like by using a known purification method such as a method using the fact that monopotassium tartrate is sparingly soluble in water.
- a known purification method such as a method using the fact that monopotassium tartrate is sparingly soluble in water.
- an insoluble component a microbial cell residue used in Step A or a compound in which a transition metal catalyst is adsorbed on a water-insoluble carrier in Step B is used from the reaction solution obtained in Step B, the compound is used.
- a known purification method such as an ion exchange method using an anion exchange resin or an electrodialysis method can be used.
- the pH of the reaction solution obtained in Step B or the reaction solution after collecting L-tartaric acid from the reaction solution is adjusted to 7.0 or higher with an alkali solution such as sodium hydroxide,
- the solution is passed through a strongly basic anion exchange resin (eg, formic acid type) column to remove cations such as sodium ions, and the non-adsorbed fraction containing glycolic acid and formic acid is then added with aqueous ammonia (eg, 2M aqueous ammonia).
- aqueous ammonia eg, 2M aqueous ammonia
- Step A Whether or not it is an intermediate product (gluconic acid) in Step A is determined by the high performance liquid chromatography (hereinafter referred to as “HPLC”) method reported by Herrmann et al. (Herrmann, U., Merfort, M., Jeude, M ., Bringer-Meyer, S., and Sahm, H., Appl. Microbiol. Biotechnol. Specific examples of the preferable method include the following methods.
- a DE-613 column manufactured by Shodex Co., Ltd.
- the absolute coordination and purity assay of tartaric acid is an enzymatic assay for L-tartaric acid using D-malate dehydrogenase which is not active on D-tartaric acid and meso-tartaric acid but only on L-tartaric acid. (T. Tsukatani, K. Matsumoto, Biosci. Biotechnol. Biochem., 63, 1730-1735, 1999).
- the type of salt of L-tartaric acid in Step C is not particularly limited because it depends on the type of alkali used in Step A or Step B.
- potassium hydroxide is used as the alkali in Step A or Step B.
- the monopotassium salt of L-tartaric acid is obtained in Step C.
- examples of the salt of L-tartaric acid in Step C include L-tartaric acid dipotassium salt, L-tartaric acid sodium salt, L-tartaric acid sodium potassium salt, and L-tartaric acid ammonium salt.
- the type of glycolic acid salt in Step C is not particularly limited because it depends on the type of alkali used in Step A or Step B, the type of acid used in Step C, etc., but hydrochloric acid is used in Step C. When used, a glycolic acid free acid is obtained.
- Other examples of the salt of glycolic acid in Step C include potassium glycolate, sodium glycolate, and ammonium glycolate.
- Gluconobacter oxydans NBRC 3172 strain was inoculated in a 300 mL Erlenmeyer flask containing 30 mL of MA medium that had been autoclaved at 121 ° C. for 20 min. After shaking culture, cells were aseptically obtained from the culture by centrifugation. The obtained cells were washed once with 0.85% sterilized physiological saline and then suspended in sterilized physiological saline.
- the NTG solution at a concentration of 2,000 ⁇ g / mL was added to make 5 mL.
- These test tubes were subjected to mutation treatment by reciprocally shaking at 30 ° C. and 250 rpm for 30 minutes. The reaction solution in the test tube was washed twice with sterile physiological saline and then resuspended.
- the suspension was diluted stepwise up to 107 times, and each diluted solution was applied to an MA agar medium and cultured in a thermostat at 30 ° C. for 3 to 5 days. Screening was performed using 2,600 colonies randomly picked from the grown colonies on the same agar medium and cultured at 30 ° C. for 1-2 days.
- the parent strain Gluconobacter oxydans NBRC 3172, produces 2-keto-D-gluconic acid and 5-keto-D-gluconic acid at a ratio of approximately 1: 1, while Seven strains that produced only keto-D-gluconic acid and hardly produced 2-keto-D-gluconic acid (non-producing 2-keto-D-gluconic acid strain) were obtained.
- Gluconobacter oxydans TADK-267 obtained in Example 1 above was sterilized by heating at 121 ° C. for 20 minutes, 5 mL of MB medium [2.5% mannitol, 0.5% yeast extract (Difco Laboratories, Inc.) ), 0.3% peptone (manufactured by Difco Laboratories)] was inoculated into 3 test tubes and cultured with reciprocal shaking for 17 hours at 28 ° C. and 250 rpm. The culture media in these three test tubes were combined to make a seed culture solution.
- MB medium 2.5% mannitol, 0.5% yeast extract (Difco Laboratories, Inc.)
- peptone manufactured by Difco Laboratories
- 1% fermenter manufactured by Able Co., Ltd.
- 6% glucose 0.09% ammonium chloride, 0.06% potassium dihydrogen phosphate, 0.18% corn steep powder (manufactured by Sigma)
- 0.1% yeast extract 0.015% magnesium sulfate heptahydrate, 0.0029% manganese sulfate pentahydrate, 0.12% calcium chloride dihydrate, and 0.1% 500 mL of a medium composed of Actol (manufactured by Takeda Chemical Industry Co., Ltd.) was added, and the mixture was sterilized by heating in the same manner.
- the potassium hydroxide solution After adding a 6M potassium hydroxide solution to the culture solution to raise the pH of the culture solution to 9.6, the potassium hydroxide solution is added as it is to maintain the pH of the culture solution at 9.6 or higher.
- 12.1 g / L tartaric acid and 6.0 g / L glycolic acid were produced.
- the molar yield of tartaric acid from glucose is 23.0%
- the glycolic acid from glucose is The molar yield was 22.5%.
- Example 3 The whole reaction solution obtained in Example 3 above was centrifuged at 5,000 rpm together with the washing solution of the fermenter to separate insoluble fractions such as cell residue and palladium carbon, and then washed once with about 100 mL of water. The centrifugal supernatants were combined and purified from a 635 mL solution. It was confirmed by HPLC analysis that the obtained solution contained 13.1 g of tartaric acid and 5.9 g of glycolic acid. Concentrated hydrochloric acid was gradually added to the obtained solution and the pH was lowered to 3.7. As a result, a precipitate was formed, which was allowed to stand overnight at 5 ° C. and then centrifuged to obtain a precipitate and a supernatant. separated. The precipitate contained tartaric acid and the supernatant contained glycolic acid.
- the precipitate contained fine palladium carbon and was slightly gray, but it was confirmed by HPLC analysis that the peak area value of tartaric acid accounted for 94% of the total area value.
- This precipitate was suspended in water, dissolved by gradually adding potassium hydroxide solution, and then recrystallized by slowly lowering the pH to 3.7 with 1M hydrochloric acid to obtain 14.1 g of monopotassium tartrate as a white powder. It was. The collected tartaric acid was assayed by the enzymological method described below.
- L-tartaric acid manufactured by Sigma-Aldrich
- D-tartaric acid manufactured by Sigma-Aldrich
- meso-tartaric acid manufactured by Sigma-Aldrich
- the micro cuvette set on the sample side includes 20 mM manganese chloride, 2 mM dithiothreitol (manufactured by Wako Pure Chemical Industries), 15 mM oxidized nicotinamide adenine dinucleotide (NAD +, manufactured by Oriental Yeast), 5 units of D-apple Acid dehydrogenase (EC 1.1.1.183, manufactured by Megazyme), 60 mM glycylglycine buffer (adjusted to pH 9.0 with potassium hydroxide), and L-tartaric acid and D-tartaric acid at each concentration prepared above.
- a mixture containing meso-tartaric acid (total amount 150 ⁇ L), and a micro cuvette set on the reference side is filled with 150 ⁇ L of water, and a UV2200 spectrophotometer (manufactured by Shimadzu Corporation) is used at a wavelength of 340 nm.
- the increase in absorbance per minute was measured.
- L-tartaric acid showed an increase in absorbance of 3.75 at a concentration of 7.5 mM.
- the increase in absorbance of each of the 0, 2.5, 5, and 7.5 mM solutions was plotted on the vertical axis. When the concentration was plotted on the horizontal axis, all L-tartaric acid standard solutions were plotted on a straight line passing through the origin.
- Transition metal catalysts described in Table 4 described below were added to 500 mM potassium carbonate buffer (pH 10.0) containing 10% 5-keto-D-gluconic acid to examine the production of tartaric acid and glycolic acid.
- a large test tube (diameter 21 mm ′ length 200 mm) containing 2 mL of the reaction solution with a urethane stopper was shaken back and forth at 28 ° C. and 250 rpm.
- 0.5 mL of 3M potassium carbonate solution was added, and the pH of the reaction solution was maintained at around 9.5.
- a reaction solution using 0.2 g / L of 10% palladium carbon and 0.2, 0.4, or 0.8 g / L ammonium vanadate as a mixed catalyst was 0.2 g / L of 10% palladium carbon alone or Compared with the reaction solution of 0.2, 0.4 or 0.8 g / L ammonium vanadate alone, the production amounts of L-tartaric acid and glycolic acid were remarkably increased, and their productivity was remarkably improved.
- L-tartaric acid or a salt thereof and / or glycolic acid or a salt thereof can be produced from glucose very efficiently and at low cost. Therefore, the production method of the present invention can be used for industrial production, and is extremely practical. Therefore, it is particularly useful in industrial fields using L-tartaric acid, glycolic acid and the like, for example, in the food field, industrial field, and pharmaceutical field.
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Abstract
Description
(A)グルコースから5-ケト-D-グルコン酸を生産する微生物を、5-ケト-D-グルコン酸を水溶性塩とし得るアルカリの存在下、グルコース含有培養液で培養することにより、5-ケト-D-グルコン酸の水溶性塩を含む培養液を得る工程A、
(B)工程Aで得られた5-ケト-D-グルコン酸の水溶性塩を含む培養液のpHを7~12の範囲内に調整及び維持することにより、5-ケト-D-グルコン酸の水溶性塩がL-酒石酸若しくはその塩、及び/又は、グリコール酸若しくはその塩に変換した反応液を得る工程B、
(C)工程Bで得られた反応液から、L-酒石酸若しくはその塩、及び/又は、グリコール酸若しくはその塩を採取する工程C。
グルコノバクター・オキシダンスNBRC 3172株由来の2-ケト-D-グルコン酸非生産菌株を作製するために、NBRC 3172株についてNTG変異処理を行い、2-ケト-D-グルコン酸非生産菌のスクリーニングを試みた。具体的には、以下のような方法で行なった。
上記実施例1で得られたグルコノバクター・オキシダンスTADK-267を、121℃、20分間加熱殺菌した5mLのMB培地[2.5%マンニトール、0.5%酵母エキス(ディフコ・ラボラトリー社製)、0.3%ペプトン(ディフコ・ラボラトリー社製)]入り試験管3本に植菌し、28℃、250回転で17時間往復振とう培養した。これら3本の試験管中の培養液を合わせて種培養液とした。本培養は、1Lの醗酵槽(エイブル株式会社製)に、6%グルコース、0.09%塩化アンモニウム、0.06%リン酸二水素カリウム、0.18%コーンスティープパウダー(シグマ社製)、0.1%酵母エキス(ディフコ・ラボラトリー社製)、0.015%硫酸マグネシウム7水塩、0.0029%硫酸マンガン5水塩、0.12%塩化カルシウム2水塩、及び、0.1%アクトコール(武田化学工業社製)からなる培地500mLを入れ、同様に加熱殺菌したところへ、前述の種培養液10mLを接種して開始した。6M水酸化カリウム溶液でpH5.5以上に調整しながら、30℃、1vvm、800回転で通気攪拌培養を行ない、培養液を1M塩酸溶液で51倍希釈後、遠心上清のHPLC分析を行って生成物を経時的に定量した。培養開始後50時間目に培養液中のグルコースは完全に消費され、培養液中には、2.2g/Lのグルコン酸が残るものの、70.5g/Lの5-ケト-D-グルコン酸が生成した。この培養液に6M水酸化カリウム溶液を添加して培養液のpHを9.6に上げた後、そのままその水酸化カリウム溶液を添加して培養液のpHを9.6以上に維持しつつ、144時間目まで通気攪拌を継続したところ、12.1g/Lの酒石酸及び6.0g/Lのグリコール酸が生成した。蒸発及び水酸化カリウム溶液添加による液量変化、並びに、途中サンプリングによる液量減少分を考慮して計算すると、グルコースからの酒石酸のモル収率は23.0%であり、グルコースからのグリコール酸のモル収率は22.5%であった。
上記実施例2と同様にグルコノバクター・オキシダンスTADK-267を、醗酵槽を用いて50時間培養したところ、グルコースは完全に消費され、2.8g/Lのグルコン酸が残るものの、67.4g/Lの5-ケト-D-グルコン酸が溶液中に生成した。この培養液に6M水酸化カリウム溶液を添加して培養液のpHを9.6に上げた後、10gのパラジウム活性炭素(10%、和光純薬工業社製)を添加し、以降は水酸化カリウム溶液を添加してpH9.6以上に維持しつつ、144時間目まで通気攪拌を継続したところ、31.9g/Lの酒石酸及び15.9g/Lのグリコール酸を含む反応液を得た。蒸発及び水酸化カリウム溶液添加による液量変化、並びに、途中サンプリングによる液量減少分を考慮して計算すると、グルコースからの酒石酸のモル収率は61%であり、グルコースからのグリコール酸のモル収率は60%であった。
上記実施例3で得られた反応液全量を醗酵槽の洗液と共に5,000回転で遠心処理し、菌体残渣、パラジウム炭素などの不溶性画分を分離した後、約100mLの水で一度洗浄し、遠心上澄を合わせて635mLの溶液から精製を行った。得られた溶液中には13.1gの酒石酸と5.9gのグリコール酸が含まれていることをHPLC分析で確認した。前述の得られたこの溶液に、濃塩酸を徐々に加えてpHを3.7に下げたところ、沈澱が生成したので、5℃で一晩放置してから遠心処理し沈殿と上澄とに分離した。沈殿には酒石酸が、上澄にはグリコール酸が含まれていた。
1%の5-ケト-D-グルコン酸カリウム塩を含む5mLの0.45M炭酸ナトリウム緩衝液(pH9.55)に40mgのパラジウム炭素を添加し、9日間振とうした。その結果4g/LのL-酒石酸と1g/Lのグリコール酸が生成し、0.7g/Lの5-ケト-D-グルコン酸が残存した。この3.5mLの反応液を遠心してパラジウム炭素を回収し、0.5Mの同緩衝液3.15mLと0.10%の5-ケト-D-グルコン酸カリウム35mLを加えて7日間振とうしたところ、5g/LのL-酒石酸と1.2g/Lのグリコール酸が生成し、2.1g/Lの5-ケト-D-グルコン酸が残存した。
1%の5-ケト-D-グルコン酸を含むpH6、7、8若しくは9の450mMリン酸カリウム緩衝液、又は、pH 9、10、11若しくは11.5の炭酸ナトリウム緩衝液に、パラジウム炭素(10%)添加又は無添加の条件で、酒石酸及びグリコール酸の生成を検討した。反応はシリコセンをした10mL容試験管に1.5mLの液量で行い、2日間28℃、250rpmで往復振とうした後にpHを測定し、生成した酒石酸とグリコール酸を定量した。結果を以下の表1に示すが反応中のpHが維持されにくかったことから、酒石酸及びグリコール酸の生成にはpH8から11が適していると考えられた。特にpH9から10で顕著な生産が認められた。また、いずれのpHでも、パラジウム炭素を添加すると、酒石酸及びグリコール酸の生産性が著しく向上することが分かる。
1%の5-ケト-D-グルコン酸を含む450mM炭酸ナトリウム緩衝液(pH9.55)に、パラジウム炭素(10%)添加又は無添加の条件で、30℃、40℃、50℃又は60℃での酒石酸、及び、グリコール酸の生成を検討した。反応はシリコセンをした30mLフラスコに3mLの液量にて、各温度で2日間、80回転の往復振とうして行った。得られた反応液を分析した結果を以下の表2に示すが、30℃から60℃、特に30℃から40℃で酒石酸およびグリコール酸の良好な生産が認められた。また、いずれの温度でも、パラジウム炭素を添加すると、酒石酸及びグリコール酸の生産性が著しく向上することが分かる。
1%の5-ケト-D-グルコン酸を含む450mM炭酸ナトリウム緩衝液(pH9.55)に後述の表3記載の触媒を加えて酒石酸及びグリコール酸の生成を検討した。反応はシリコセンをした30mLフラスコに5mLの液量にて、24℃で2日間、往復振とうして行なった。得られた反応液を分析した結果を以下の表3に示す。検討したいずれの触媒も、酒石酸及びグリコール酸の生産性の向上に有効であったが、特に、パラジウム、ロジウム及びマンガンが優れていた。
10%の5-ケト-D-グルコン酸を含む500mM炭酸カリウム緩衝液(pH10.0)に、後述の表4記載の遷移金属触媒をそれぞれ加えて酒石酸及びグリコール酸の生成を検討した。反応はウレタン栓をした2mLの反応液を含む大試験管(直径21mm ´ 長さ200mm)を28℃、250回転/分で往復振とうを行なった。19時間目に3M炭酸カリウム溶液を0.5mL添加し、反応液のpHを9.5付近に維持した。反応開始から3日目の反応液を分析した結果を以下の表4に示す。検討したいずれの触媒も、酒石酸及びグリコール酸の生産性の向上に有効であった。用いた触媒の中でも、硫酸銅(II)5水和物や、バナジン酸アンモニウムが特に優れていた。
10%の5-ケト-D-グルコン酸を含む500mM炭酸カリウム緩衝液(pH10.0)に、後述の表5記載の濃度の硫酸銅(II)6水和物あるいはバナジン酸アンモニウムを加えて酒石酸及びグリコール酸の生成を検討した。遷移金属塩なしを対照区とした。反応はウレタン栓をした5mLの反応液を含む100mLフラスコを28℃で1分間あたり250回転にて回転振とうを行なった。反応開始から19時間目に3M炭酸カリウム溶液を0.5mL加え、反応液のpHを9.5付近に維持した。得られた反応液を分析した結果を以下の表5に示す。0.075g/Lの濃度の硫酸銅(II)6水和物で、また0.2g/Lの濃度のバナジン酸アンモニウムで、酒石酸、グリコール酸の生成量は最大に達し、いずれの触媒も、それ以上の濃度では酒石やグリコール酸のほぼ同じ生成量であった。
10%の5-ケト-D-グルコン酸を含む500mM炭酸カリウム緩衝液(pH10.0)に、後述の表6記載の濃度の10%パラジウム炭素とバナジン酸アンモニウムを加えて酒石酸及びグリコール酸の生成を検討した。パラジウム炭素単独あるいは各濃度のバナジン酸アンモニウム単独を対照区とした。反応はウレタン栓をした5mLの反応液を含む100mLフラスコを28℃で1分間あたり250回転で回転振とうを行なった。反応開始から19時間目に3M炭酸カリウム溶液を0.5mL加え、反応液のpHを9.5付近に維持した。3日目の反応液を分析した結果を以下の表6に示す。0.2g/Lの10%パラジウム炭素と0.2、0.4あるいは0.8g/Lのバナジン酸アンモニウムを混合触媒として用いた反応液は、0.2g/Lの10%パラジウム炭素単独あるいは0.2、0.4あるいは0.8g/Lのバナジン酸アンモニウム単独の反応液に比べてL-酒石酸、グリコール酸の生成量が著しく増加し、それらの生産性が著しく向上した。
[グルコースから酒石酸およびグリコール酸の製造方法(5-ケト-D-グルコン酸から1M 水酸化カリウム存在下で反応]
実施例2と同様にグルコノバクター・オキシダンスTADK-267を用いて得られた67.3g/Lの5-ケト-D-グルコン酸を含む培養液約430mLに、24gの水酸化カリウムを約30mLの水溶液として加え、溶液の水酸化カリウム終濃度を1Mとした。この時のpHは13.7であった。その後通気攪拌を継続したところ55時間目に3.1g/Lの酒石酸及び1.5g/Lのグリコール酸が生成し、微量の5-ケト-D-グルコン酸が残存していた。また、144時間目まで反応させたが酒石酸は3.6g/L、グリコール酸は1.7g/Lで、蒸発及び途中サンプリングによる減少分を考慮して計算すると、グルコースからの酒石酸のモル収率は6.6%であり、グルコースからのグリコール酸のモル収率は6.2%であった。すなわち、pHが12以上の場合は、酒石酸及びグリコール酸の収率が著しく低かった。
Claims (16)
- 以下の工程A~Cを備え、グルコースからL-酒石酸若しくはその塩、及び/又は、グリコール酸若しくはその塩を製造する方法。
(A)グルコースから5-ケト-D-グルコン酸を生産する微生物を、5-ケト-D-グルコン酸を水溶性塩とし得るアルカリの存在下、グルコース含有培養液で培養することにより、5-ケト-D-グルコン酸の水溶性塩を含む培養液を得る工程A、
(B)工程Aで得られた5-ケト-D-グルコン酸の水溶性塩を含む培養液のpHを7~12の範囲内に調整及び維持することにより、5-ケト-D-グルコン酸の水溶性塩がL-酒石酸若しくはその塩、及び/又は、グリコール酸若しくはその塩に変換した反応液を得る工程B、
(C)工程Bで得られた反応液から、L-酒石酸若しくはその塩、及び/又は、グリコール酸若しくはその塩を採取する工程C。 - 上記工程Bにおける培養液に、遷移金属触媒をさらに含有させた培養液を用いることを特徴とする請求項1に記載の方法。
- 遷移金属触媒が、パラジウム、ロジウム、ルテニウム、白金、マンガン、銅、コバルト、ニッケル、亜鉛、バナジウム及び鉄からなる群から選択される1種又は2種以上の遷移金属触媒であることを特徴とする請求項2に記載の方法。
- 遷移金属触媒の濃度が0.00002~2%の範囲内であることを特徴とする請求項2又は3に記載の方法。
- 遷移金属触媒の濃度が0.0001~1%の範囲内であることを特徴とする請求項2~4のいずれかに記載の方法。
- グルコースから5-ケト-D-グルコン酸を生産する微生物が、グルコノバクター属又はアセトバクター属に属する微生物であることを特徴とする請求項1~5のいずれかに記載の方法。
- グルコノバクター属又はアセトバクター属に属する微生物が、グルコノバクター・オキシダンスNBRC 3172およびNBRC 3255、グルコノバクター・パストリアヌスNBRC 3225、グルコノバクター・アルビダスNBRC 3250、グルコノバクター・サブオキシダンスNBRC 3254、グルコノバクター・インダストリアスNBRC 3260、グルコノバクター・セレウスNBRC 3267、グルコノバクター・ジオキシアセトニカスNBRC 3273、グルコノバクター・モノオキシグルコニカスNBRC 3276、グルコノバクター・グルコニカスNBRC 3285、グルコノバクター・ロゼウスNBRC 3990、グルコノバクター・フラトイリNBRC 3265、アセトバクター・アセチNBRC 3259、及び、それらの変異株であってグルコースから5-ケト-D-グルコン酸を生産する株からなる群から選択されるいずれか1種又は2種以上の微生物であることを特徴とする請求項6に記載の方法。
- グルコノバクター属又はアセトバクター属に属する微生物が、さらに2-ケト-D-グルコン酸低生産性及び/又は5-ケト-D-グルコン酸低消費性の株であることを特徴とする請求項7に記載の方法。
- 上記工程Bにおける培養液のpHを8~11の範囲内に調整及び維持することを特徴とする請求項1~8のいずれかに記載の方法。
- 上記工程Bにおける培養液のpHを9~10の範囲内に調整及び維持することを特徴とする請求項1~9のいずれかに記載の方法。
- 上記工程Bにおいて、培養液のpHを調整及び維持する際に、さらに、培養液の温度を0℃~70℃の範囲内に調整及び維持することを特徴とする請求項1~10のいずれかに記載の方法。
- 上記工程Bにおいて、培養液のpHを調整及び維持する際に、さらに、培養液の温度を20℃~60℃の範囲内に調整及び維持することを特徴とする請求項1~11のいずれかに記載の方法。
- 上記工程Bにおいて、培養液のpHを調整及び維持する際に、さらに、培養液に対する空気供給量の割合を0.2~5の範囲内に調整及び維持することを特徴とする請求項1~12のいずれかに記載の方法。
- 上記工程Bにおいて、培養液のpHを調整及び維持する際に、さらに、培養液に対する空気供給量の割合を0.5~3の範囲内に調整及び維持することを特徴とする請求項1~13のいずれかに記載の方法。
- 工程Cにおいて採取されたL-酒石酸の塩が、L-酒石酸の1カリウム塩であることを特徴とする請求項1~14のいずれかに記載の方法。
- 工程Cにおいて採取されたグリコール酸若しくはその塩が、グリコール酸のフリーな酸若しくはその塩であることを特徴とする請求項1~15のいずれかに記載の方法。
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CN110408802B (zh) * | 2019-09-04 | 2022-01-25 | 贵州理工学院 | 一种浸取含稀土磷石膏回收稀土的方法 |
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