A method for separating and purifying 3-hydroxybutyric acid from a fermentation broth

Abstract

This invention discloses a method for separating and purifying 3-hydroxybutyric acid (3-Hydroxybutyric acid) from fermentation broth, relating to the field of 3-Hydroxybutyric acid preparation technology. The method includes the following steps: S1, adjusting the pH of the supernatant of the 3-Hydroxybutyric acid fermentation broth to no higher than 7, adding a decolorizing agent to obtain a decolorized supernatant; S2, passing the decolorized supernatant through an alkaline anion exchange resin for adsorption, rinsing off impurities with water, and then eluting with an inorganic alkaline aqueous solution to collect the eluent; S3, passing the eluent through an acidic resin and collecting the effluent; S4, desalting the effluent. This method is simple, easily scalable, produces low levels of waste, conforms to green production trends, and yields high-purity 3-Hydroxybutyric acid.

Description

Technical Field

[0001] This invention relates to the field of 3-hydroxybutyric acid preparation technology, and in particular to a method for separating and purifying 3-hydroxybutyric acid from fermentation broth. Background Technology

[0002] 3-Hydroxybutyrate (3HB), an important ketone body molecule, is mainly found in the liver, muscles, and brain of the human body and has therapeutic effects on various diseases. For example, in patients with hypertension, 3HB inhibits nephritoprotein NIRp3. Supplementing with exogenous 1,3-butanediol, which is metabolized into 3HB by the liver, can have a positive effect on lowering arterial hypertension. In diabetic patients, 3HB can serve as an energy source for organs such as the heart, partially replacing glucose. Therefore, 3HB shows significant application value in diseases such as hypertension and diabetes.

[0003] In related technologies, the conversion of 3-hydroxybutyraldehyde to 3HB is mainly achieved by using cobalt acetate catalysis in organic acid ester solvents. However, 3-hydroxybutyraldehyde is highly reactive, expensive, and has low purity. Furthermore, the homogeneous catalyst used in liquid-phase oxidation suffers from difficulties in recovery, high solvent consumption, and excessively long reaction times. Another method involves the hydrolysis of poly(3-hydroxybutyrate) ester (PHB), but the extraction process for high-purity PHB is cumbersome and costly. Using acid or alkali to hydrolyze PHB results in a large amount of ionic impurities in the final product, increasing the difficulty of subsequent purification and complicating the process. A third method synthesizes 3HB through microbial fermentation. This method eliminates the need for the expensive intermediate 3-hydroxybutyraldehyde, avoids the difficult-to-recover catalyst and large amounts of solvent, and avoids the ionic impurities and strong acid / alkali waste liquid problems associated with PHB hydrolysis. It uses a low-cost mixed carbon source as raw material, but issues such as the difficulty in removing pigment / salt impurities remain.

[0004] Therefore, there is a need to provide a method for preparing 3HB. Summary of the Invention

[0005] The present invention aims to at least solve one of the technical problems existing in the prior art. To this end, the present invention proposes a method for separating and purifying 3-hydroxybutyric acid from fermentation broth.

[0006] A method for separating and purifying 3-hydroxybutyric acid from fermentation broth according to a first aspect of the present invention includes the following steps: S1. Adjust the pH of the supernatant of the fermentation broth of 3-hydroxybutyric acid to no higher than 7, add a decolorizing agent to treat it, and obtain a decolorized supernatant; S2. After the decolorized supernatant is adsorbed by alkaline anion exchange resin, impurities are washed away with water, and then eluted with sodium hydroxide aqueous solution to obtain the eluent. S3. The eluent is passed through an acidic resin, and the effluent is collected. S4. The effluent is subjected to desalination treatment.

[0007] The method according to embodiments of the present invention has at least the following beneficial effects: The method described in this embodiment is simple, easily scalable, and produces minimal waste, aligning with green production trends. By adjusting the pH of the fermentation supernatant to neutral or acidic, and combining this with a decolorization process, the non-specific adsorption of 3HB by the decolorizing agent (resulting in a 5%-10% loss) is reduced, thus minimizing the loss of 3HB (<3%). Subsequently, the solution is treated with anion exchange resin to exchange the 3-hydroxybutyrate anions in the 3HB with the anions in the resin, yielding 3-hydroxybutyrate. Then, acidic resin treatment converts the 3-hydroxybutyrate into an acid (3-hydroxybutyric acid), increasing the product concentration. Simultaneously, a large amount of alkali (such as NaOH) in the solution is converted into a salt (NaCl). Finally, concentration and desalination are performed, resulting in a high yield (up to 87%) and high purity (up to 99.5%).

[0008] According to some embodiments of the present invention, the method for preparing the fermentation broth supernatant includes the following steps: Microorganisms capable of synthesizing 3-hydroxybutyric acid are cultured to obtain a fermentation broth. The bacteria are then isolated to obtain the supernatant of the fermentation broth.

[0009] According to some embodiments of the present invention, the microorganism includes at least one of bacteria and fungi.

[0010] According to some embodiments of the present invention, the bacteria include at least one of Escherichia coli and Bacillus subtilis.

[0011] According to some embodiments of the present invention, the fungus includes yeast.

[0012] According to some embodiments of the present invention, the culture medium for culturing the microorganisms includes a carbon source, a nitrogen source, and inorganic salts.

[0013] According to some embodiments of the present invention, the carbon source includes at least one of glucose and acetate.

[0014] According to some embodiments of the present invention, the acetate includes sodium acetate.

[0015] According to some embodiments of the present invention, the nitrogen source includes at least one of yeast powder and ammonium chloride.

[0016] According to some embodiments of the present invention, the inorganic salt includes at least one of a magnesium source, a potassium source, a sodium source, and a calcium source.

[0017] According to some embodiments of the present invention, the magnesium source includes magnesium sulfate.

[0018] According to some embodiments of the present invention, the potassium source includes potassium dihydrogen phosphate.

[0019] According to some embodiments of the present invention, the sodium source includes sodium chloride.

[0020] According to some embodiments of the present invention, the calcium source includes calcium chloride.

[0021] According to some embodiments of the present invention, the culture medium for culturing the microorganisms comprises 40-50 g / L carbon source, 5-7 g / L nitrogen source, 0.1-0.15 g / L magnesium source, 8-10 g / L potassium source, 0.4-0.6 g / L sodium source, and 0.005-0.015 g / L calcium source. The pH of the culture medium is 7.0.

[0022] According to some embodiments of the present invention, the mass ratio of glucose to acetate is 30:(0-30). For example, it can be 30:0, 30:2, 30:4, 30:6, 30:8, 30:10, 30:12, 30:14, 30:16, 30:18, 30:20, 30:22, 30:24, 30:26, 30:28 or 30:30.

[0023] According to some embodiments of the present invention, the temperature for culturing the microorganisms is preferably suitable for their growth. The temperature for culturing the microorganisms is 30℃-40℃. For example, it can be 30℃, 31℃, 32℃, 33℃, 34℃, 35℃, 36℃, 37℃, 38℃, 39℃, or 40℃.

[0024] According to some embodiments of the present invention, the aeration rate for culturing the microorganisms is 2 L / min to 5 L / min. For example, it can be 2 L / min, 2.1 L / min, 2.2 L / min, 2.3 L / min, 2.4 L / min, 2.5 L / min, 2.6 L / min, 2.7 L / min, 2.8 L / min, 2.9 L / min, 3 L / min, 3.1 L / min, 3.2 L / min, 3.3 L / min, 3.4 L / min, 3.5 L / min, 3.6 L / min, 3.7 L / min, 3.8 L / min, 3.9 L / min, 4 L / min, 4.1 L / min, 4.2 L / min, 4.3 L / min, 4.4 L / min, 4.5 L / min, 4.6 L / min, 4.7 L / min, 4.8 L / min, 4.9 L / min, or 5 L / min.

[0025] According to some embodiments of the present invention, the rotation speed for culturing the microorganisms is 300 rpm to 800 rpm. For example, it can be 300 rpm, 320 rpm, 340 rpm, 360 rpm, 380 rpm, 400 rpm, 420 rpm, 440 rpm, 460 rpm, 480 rpm, 500 rpm, 520 rpm, 540 rpm, 560 rpm, 580 rpm, 600 rpm, 620 rpm, 640 rpm, 660 rpm, 680 rpm, 700 rpm, 720 rpm, 740 rpm, 760 rpm, 780 rpm, or 800 rpm.

[0026] According to some embodiments of the present invention, the pH of the fermentation broth supernatant is 3-7. For example, it can be 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, or 7.

[0027] According to some embodiments of the present invention, the decolorizing agent includes activated carbon. The porous structure of the activated carbon can adsorb pigments and impurities, thereby removing pigments and other impurities from the supernatant of the fermentation broth.

[0028] According to some embodiments of the present invention, the amount of the decolorizing agent added is 0.4 (w / v)%-2.4 (w / v)%. For example, it can be 0.4 (w / v)%, 0.6 (w / v)%, 0.8 (w / v)%, 1 (w / v)%, 1.2 (w / v)%, 1.4 (w / v)%, 1.6 (w / v)%, 1.8 (w / v)%, 2 (w / v)%, 2.2 (w / v)%, or 2.4 (w / v)%.

[0029] According to some embodiments of the present invention, the treatment time of the decolorizing agent is 4 h to 6 h. For example, it can be 4 h, 4.1 h, 4.2 h, 4.3 h, 4.4 h, 4.5 h, 4.6 h, 4.7 h, 4.8 h, 4.9 h, 5 h, 5.1 h, 5.2 h, 5.3 h, 5.4 h, 5.5 h, 5.6 h, 5.7 h, 5.8 h, 5.9 h, or 6 h.

[0030] According to some embodiments of the present invention, the basic anion exchange resin includes at least one of a strong basic anion exchange resin and a weak basic anion exchange resin.

[0031] According to some embodiments of the present invention, the basic anion exchange resin includes at least one of gel-type basic ion exchange resin and macroporous basic ion exchange resin.

[0032] According to some embodiments of the present invention, the strongly basic anion exchange resin uses a trimethyl quaternary ammonium group as a functional group. The strongly basic anion exchange resin includes at least one of gel-type anion exchange resin and macroporous anion exchange resin. The type of the strongly basic anion exchange resin includes at least one of LX2014 (purchased from Xi'an Lanxiao Technology New Materials Co., Ltd.), D201 (purchased from Ningbo Zhengguang Resin Co., Ltd.), and YKTA52 (purchased from Tianjin Yunkai Resin Technology Co., Ltd.). The trimethyl quaternary ammonium group remains completely dissociated over a wide pH range and carries a stable positive charge; 3HB dissociates into 3-hydroxybutyrate anion in aqueous solution. The trimethyl quaternary ammonium group and the 3-hydroxybutyrate anion undergo a highly efficient ion exchange reaction through electrostatic attraction (ionic bond), the 3-hydroxybutyrate anion is adsorbed by the resin, and the exchangeable anions on the resin are released into the solution.

[0033] According to some embodiments of the present invention, the weakly basic anion exchange resin uses tertiary ammonium groups as functional groups. The weakly basic anion exchange resin includes at least one of gel-type anion exchange resin and macroporous anion exchange resin. The type of the weakly basic anion exchange resin includes at least one of LX6700 (purchased from Xi'an Lanxiao Technology New Materials Co., Ltd.), YKTA1 (purchased from Tianjin Yunkai Resin Technology Co., Ltd.), and D354 (purchased from Ningbo Zhengguang Resin Co., Ltd.).

[0034] According to some embodiments of the present invention, the degree of crosslinking of the gel-type anion exchange resin is 4%-8%. For example, it can be 4%, 4.1%, 4.2%, 4.3%, 4.4%, 4.5%, 4.6%, 4.7%, 4.8%, 4.9%, 5%, 5.1%, 5.2%, 5.3%, 5.4%, 5.5%, 5.6%, 5.7%, 5.8%, 5.9%, 6%, 6.1%, 6.2%, 6.3%, 6.4%, 6.5%, 6.6%, 6.7%, 6.8%, 6.9%, 7%, 7.1%, 7.2%, 7.3%, 7.4%, 7.5%, 7.6%, 7.7%, 7.8%, 7.9%, or 8%. Thus, the crosslinking network of the resin is relatively loose, and after swelling in aqueous solution, it forms abundant macroporous or mesoporous structures. The 3HB molecular weight is small and has low steric hindrance, enabling it to quickly pass through the pores and reach the functional group sites inside the resin, thereby significantly improving the adsorption rate and adsorption capacity.

[0035] According to some embodiments of the present invention, the anion exchange resin uses at least one of a styrene polymer and an acrylic polymer as a matrix.

[0036] According to some embodiments of the present invention, the column adsorption rate of the decolorized supernatant is 1.5 BV / h-2 BV / h. For example, it can be 1.5 BV / h, 1.55 BV / h, 1.6 BV / h, 1.65 BV / h, 1.7 BV / h, 1.75 BV / h, 1.8 BV / h, 1.85 BV / h, 1.9 BV / h, 1.95 BV / h, or 2 BV / h.

[0037] According to some embodiments of the present invention, the amount of water used is 3 BV-5 BV. For example, it can be 3 BV, 3.5 BV, 4 BV, 4.5 BV, or 5 BV.

[0038] According to some embodiments of the present invention, the water flow rate is 1 BV / h to 4 BV / h. For example, it can be 1 BV / h, 1.5 BV / h, 2 BV / h, 2.5 BV / h, 3 BV / h, 3.5 BV / h, or 4 BV / h.

[0039] According to some embodiments of the present invention, the concentration of sodium hydroxide in the sodium hydroxide aqueous solution is 0.1 M-0.5 M. For example, it can be 0.1 M, 0.15 M, 0.2 M, 0.25 M, 0.3 M, 0.35 M, 0.4 M, 0.45 M or 0.5 M.

[0040] According to some embodiments of the present invention, the inorganic alkaline aqueous solution is used at an elution flow rate of 1.5 BV / h to 2 BV / h. For example, the flow rate can be 1.5 BV / h, 1.55 BV / h, 1.6 BV / h, 1.65 BV / h, 1.7 BV / h, 1.75 BV / h, 1.8 BV / h, 1.85 BV / h, 1.9 BV / h, 1.95 BV / h, or 2 BV / h.

[0041] According to some embodiments of the present invention, the acidic resin includes at least one of a strong acidic cation exchange resin, a weak acidic cation exchange resin, and a weak acidic adsorption resin.

[0042] According to some embodiments of the present invention, the strongly acidic cation exchange resin uses sulfonic acid groups as functional groups. The strongly acidic cation exchange resin includes at least one of macroporous cation exchange resin and gel-type cation exchange resin. The type of the strongly acidic cation exchange resin includes at least one of LX0017 (purchased from Xi'an Lanxiao Technology New Materials Co., Ltd.), D001 (purchased from Ningbo Zhengguang Resin Co., Ltd.), and YKC100 (purchased from Tianjin Yunkai Resin Technology Co., Ltd.). The dissociation of sulfonic acid groups is not affected by the solution pH, and it maintains high exchange activity over a wide pH range (typically pH 1~14). The macroporous structure can significantly reduce the molecular diffusion resistance of small molecule compounds such as 3HB-Na and 3HB, allowing sodium ions in the feed solution to rapidly diffuse to the active sites inside the resin and exchange with hydrogen ions; the generated 3HB can also rapidly desorb into the solution, avoiding the "retention effect" caused by slow mass transfer and significantly improving the conversion reaction rate.

[0043] According to some embodiments of the present invention, the strongly acidic cation exchange resin uses at least one of a styrene-divinylbenzene crosslinked backbone and a styrene polymer crosslinked backbone as the matrix.

[0044] According to some embodiments of the present invention, the weakly acidic cation exchange resin has at least one functional group selected from acetic acid group and phenolic hydroxyl group. The weakly acidic cation exchange resin includes at least one selected from macroporous cation exchange resin and gel-type cation exchange resin. The type of the weakly acidic cation exchange resin includes at least one selected from LXB40 (purchased from Xi'an Lanxiao Technology New Material Co., Ltd.), ZGC151 (purchased from Ningbo Zhengguang Resin Co., Ltd.), and YKTA100 (purchased from Tianjin Yunkai Resin Technology Co., Ltd.).

[0045] According to some embodiments of the present invention, the weakly acidic cation exchange resin is based on a styrene polymer.

[0046] According to some embodiments of the present invention, the weakly acidic adsorption resin adsorbs through hydrogen bonds and van der Waals forces.

[0047] According to some embodiments of the present invention, the weakly acidic adsorption resin includes a macroporous adsorption resin.

[0048] According to some embodiments of the present invention, the weakly acidic adsorption resin is based on a styrene polymer.

[0049] The sulfonic acid group is a typical strong acid group that can completely dissociate in aqueous solution, stably releasing hydrogen ions. The resin skeleton is negatively charged. Sodium 3-hydroxybutyrate is a strong base-weak acid salt, which dissociates in solution into 3-hydroxybutyrate and sodium ions. When the feed solution flows through the resin column, the hydrogen ions on the resin undergo an equivalent ion exchange reaction with the sodium ions in the feed solution. Compared with weakly acidic cation exchange resins (such as carboxylic acid resins), the dissociation of the sulfonic acid group is not affected by the solution pH and maintains high exchange activity over a wide pH range (usually pH 1~14). Its exchange capacity for sodium ions is stable, and it can almost completely convert sodium 3-hydroxybutyrate in the feed solution into 3-hydroxybutyric acid, with a conversion rate approaching 100%.

[0050] According to some embodiments of the present invention, the column flow rate of the eluent is 1 BV / h-2 BV / h. For example, it can be 1 BV / h, 1.1 BV / h, 1.2 BV / h, 1.3 BV / h, 1.4 BV / h, 1.5 BV / h, 1.6 BV / h, 1.7 BV / h, 1.8 BV / h, 1.9 BV / h, or 2 BV / h.

[0051] According to some embodiments of the present invention, the effluent needs to be concentrated before the desalination process is performed.

[0052] According to some embodiments of the present invention, the concentration process includes vacuum concentration.

[0053] According to some embodiments of the present invention, the temperature for vacuum concentration is 40℃-55℃. For example, it can be 40℃, 41℃, 42℃, 43℃, 44℃, 45℃, 46℃, 47℃, 48℃, 49℃, 50℃, 51℃, 52℃, 53℃, 54℃, or 55℃. Although 3-hydroxybutyric acid has good thermal stability, prolonged high temperatures (such as exceeding 60℃) may cause some molecules to undergo dehydration polymerization (generating 3-hydroxybutyric acid dimers), or produce trace amounts of degradation products due to localized overheating, thereby affecting the purity and bioactivity of the product. Therefore, efficient separation of solvent and solute can be achieved while maintaining 3HB activity.

[0054] According to some embodiments of the present invention, the vacuum pressure of the reduced pressure concentration is less than 0.01 MPa.

[0055] According to some embodiments of the present invention, the desalination process includes mixing the effluent with alcohol.

[0056] According to some embodiments of the present invention, the alcohol includes ethanol.

[0057] According to some embodiments of the present invention, the volume ratio of the effluent to the alcohol is 1:(8-10). For example, it can be 1:8, 1:8.5, 1:9, 1:9.5 or 1:10.

[0058] According to some embodiments of the present invention, the desalination treatment temperature is 20℃-30℃. For example, it can be 20℃, 21℃, 22℃, 23℃, 24℃, 25℃, 26℃, 27℃, 28℃, 29℃ or 30℃.

[0059] According to some embodiments of the present invention, the desalination treatment time is 0.5 h to 2 h. For example, it can be 0.5 h, 0.6 h, 0.7 h, 0.8 h, 0.9 h, 1 h, 1.1 h, 1.2 h, 1.3 h, 1.4 h, 1.5 h, 1.6 h, 1.7 h, 1.8 h, 1.9 h, or 2 h.

[0060] According to some embodiments of the present invention, the desalting product needs to be subjected to a solvent removal treatment after the desalting process. The solvent removal treatment includes, but is not limited to, rotary evaporation.

[0061] Other features and advantages of the invention will be set forth in the description which follows, and will be apparent in part from the description, or may be learned by practicing the invention. Detailed Implementation

[0062] The following will describe the concept and technical effects of the present invention clearly and completely with reference to embodiments, so as to fully understand the purpose, features and effects of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are all within the scope of protection of the present invention.

[0063] Unless otherwise specified in the examples, the procedures should be performed under standard conditions or conditions recommended by the manufacturer. Reagents or instruments whose manufacturers are not specified are all commercially available products.

[0064] When a numerical range is disclosed herein, the range is considered continuous and includes the minimum and maximum values ​​of the range, as well as every value between the minimum and maximum values. Furthermore, when the range refers to integers, it includes every integer between the minimum and maximum values ​​of the range. Additionally, when multiple ranges are provided to describe a feature or characteristic, the ranges may be combined. In other words, unless otherwise specified, all ranges disclosed herein should be understood to include any and all subranges to which they are incorporated.

[0065] Unless otherwise specified, in this invention, "left and right" means that the allowable error is within ±2%.

[0066] Unless otherwise specified, "room temperature" in this invention means (25±5)℃.

[0067] Unless otherwise specified, the term "not higher than" means less than or equal to, and should be understood to include the number itself.

[0068] "And / or" is used to indicate that one or both of the described situations may occur, for example, A and / or B includes (A and B) and (A or B).

[0069] Unless otherwise specified, the formulation of "LB medium" in this invention is as follows: 0.5 g / L yeast extract, 1 g / L tryptone, 1 g / L sodium chloride, pH adjusted to 7.0; if it is a solid medium, 1.8 g / L agar powder is added.

[0070] "Engineering" Escherichia coli Nissle 1917" for "Hyperproduction of 3-hydroxybutyrate using engineered probiotic E. coli EY15 in Nissle 1917 from glucose andCO2-derived acetate.

[0071] Example 1 (1) Engineering Escherichia coli Nissle 1917 was streaked on LB plates and incubated at 37°C for 24 hours. Single clones were then selected and inoculated into 12 mL shake tubes (containing 5 mL of LB liquid medium) and incubated at 37°C for 12 hours to obtain the primary seed culture. The primary seed culture was then inoculated into LB liquid medium at an inoculation rate of 5 (v / v)% and incubated at 37°C for 12 hours to obtain the secondary seed culture.

[0072] (2) 200 mL of secondary seed culture was inoculated into a 5 L fermenter containing 2.8 L of fermentation medium. During fermentation, the pH was adjusted to 7.0 using phosphoric acid and sodium hydroxide. A 50 (w / v)% glucose aqueous solution and a 40 (w / v)% sodium acetate aqueous solution were used as feed carbon sources. OD was measured every 2 hours. 600 The carbon source concentration and 3HB concentration were adjusted accordingly, and the pH was adjusted (keeping the pH at 7.0). The aeration rate was kept at 3 L / min, the rotation speed at 600 rpm, and the total fermentation time was 48 hours.

[0073] OD 600 The methods for determining carbon source concentration and 3HB concentration are as follows: OD 600 Determination: Take 1 mL of fermentation broth, dilute it appropriately, and measure the OD value in a spectrophotometer; Determination of glucose, sodium acetate, and 3HB during fermentation: The fermentation broth was diluted appropriately and centrifuged at 12,000 rpm for 5 min at 4℃. The supernatant was filtered through a 0.22 μm aqueous filter and injected into a sample bottle. The contents of glucose, sodium acetate, and 3HB were then measured in the liquid chromatography system. Bio-rad HPX was used for liquid chromatography analysis. An 87-hour column was used with 0.5 mM sulfuric acid as the mobile phase, and the column oven temperature was 60°C. The injection volume was 10 μL. Glucose, sodium acetate, and 3HB were quantitatively analyzed using the external standard method, and their residues and yields were calculated based on peak areas.

[0074] The basic formulation of the fermentation medium consisted of 30 g / L glucose, 15 g / L sodium acetate, 5 g / L yeast extract, 0.12 g / L anhydrous magnesium sulfate, 9 g / L potassium dihydrogen phosphate, 0.5 g / L sodium chloride, 1 g / L ammonium chloride, and 0.011 g / L calcium chloride, with the pH adjusted to 7.0. The effects of different carbon source ratios and potassium salt contents on the synthesis of 3HB were investigated by adjusting the sodium acetate and potassium dihydrogen phosphate contents.

[0075] Table 1. Effect of different carbon source ratios on 3HB yield

[0076] Table 2 Effects of different potassium salt concentrations on 3HB yield

[0077] The results are shown in Tables 1 and 2 (all values ​​are mean plus variance). With a glucose to sodium acetate ratio of 2:1, the yield of 3HB reached 148.9 g / L after 48 hours of fermentation. With a potassium dihydrogen phosphate content of 9 g / L, the yield of 3HB reached 150.0 g / L after 48 hours of fermentation. After approximately 10 replicate batches, the content remained between 145-155 g / L, therefore the process under these conditions can be applied to subsequent industrial-scale production.

[0078] Example 2 The fermentation broth obtained in Example 1 on the fermentation medium (basic formula) was collected and centrifuged at 4000 rpm for 30 minutes at room temperature to obtain a clear fermentation broth supernatant (pH 7.0, 3HB content 155 g / L). The fermentation broth supernatant was adjusted to different pH values, and different concentrations of activated carbon (Fujian Taining Herun 767 type activated carbon) were added. After treatment for different times, the color intensity of the fermentation broth after treatment was evaluated.

[0079] Color intensity = (X-X0) / (X1-X0)×100%; Where X represents the sample at OD 310 The absorbance value at point X1 represents the original fermentation broth at OD. 310 The absorbance value at the OD value, X0 represents the absorbance of water at the OD value. 310 Absorbance value at the location.

[0080] 3HB yield = Z1 / Z0 * 100%; Where Z1 represents the 3HB content in the treated sample, and Z0 represents the 3HB content (155 g / L) in the original fermentation broth.

[0081] Table 3

[0082] The results are shown in Table 3.

[0083] Adding an appropriate proportion of activated carbon and adjusting the pH can significantly remove pigments from the fermentation broth supernatant, preventing pigments from competing with 3HB for resin units, thus reducing the adsorption capacity of 3HB and affecting the 3HB yield. The decolorization effect improves with increasing activated carbon proportion, decreasing pH, and extending treatment time.

[0084] Example 3 (1) Collect the fermentation broth obtained in Example 1 on the fermentation medium (basic formula), centrifuge at room temperature and 4000 rpm for 30 minutes to obtain a transparent fermentation broth supernatant. Adjust the fermentation broth supernatant to 6, add activated carbon to a final concentration of 2.4%, treat for 6 h, separate the activated carbon, and obtain the decolorized supernatant (3HB content is 150 g / L).

[0085] (2) The activated resin was loaded into a 6 L purification column with a loading volume of 4 L (1 BV). 2.5 L of the decolorized supernatant from step (1) was passed through the column at a flow rate of 1.75 BV / h for adsorption. After adsorption, the column was rinsed with 4 BV of deionized water at a flow rate of 2-3 BV / h to remove unadsorbed cationic impurities, which may be in excess to obtain the target product and other impurities. Gradient elution was performed using 0.1 M, 0.2 M, 0.3 M, 0.4 M, and 0.5 M NaOH aqueous solutions at a flow rate of 1.75 BV / h. The eluents from each gradient were collected in segments, and the 3HB-Na content in the eluents was measured and converted into the amount of 3HB.

[0086] (3) Rinse the resin used in step (2) with 2 BV of 0.1 M NaOH aqueous solution at a flow rate of 2-3 BV / h, and then wash with 2 BV of deionized water until the pH is about 10.5, in preparation for the next use.

[0087] Table 4

[0088] The results are shown in Table 4 (all contents in the table are mean plus variance). 3HB-Na was mainly concentrated in 0.2 M NaOH aqueous solution and partially eluted in 0.3 M NaOH aqueous solution. Compared with weakly basic anion exchange resins, strongly basic anion exchange resins showed better adsorption of 3-hydroxybutyrate. Among them, the LX2014 strongly basic anion exchange resin from Xi'an Lanxiao Technology New Materials Co., Ltd. showed the highest adsorption effect for 3-hydroxybutyrate, with a 3HB content of 346.1 g after alkali elution and a total recovery rate of 92.3%.

[0089] Example 4 (1) Collect the eluent collected after treatment with LX2014 strong alkaline anion exchange resin from Xi'an Lanxiao Technology New Material Co., Ltd. in Example 3.

[0090] (2) The activated resin was loaded into a 6 L purification column with a loading volume of 4 L (1 BV). The eluent from step (1) was passed through the column at a rate of 1.5 BV / h, and the effluent was collected at the same time. The 3HB content of the collected effluent was measured.

[0091] (3) Rinse the resin used in step (2) with 2 BV of 0.1 M HCl at a flow rate of 2-3 BV / h, and then rinse with 2 BV of deionized water until the pH is about 3.0, in preparation for the next use.

[0092] Table 5

[0093] The results are shown in Table 4 (all contents in the table are mean plus variance). The LX0017 type strong acid cation exchange resin from Xi'an Lanxiao Technology New Materials Co., Ltd. showed the best conversion effect on sodium 3-hydroxybutyrate, with almost no loss, achieving a 3HB content of 342.0 g and a total recovery rate of 98.8%.

[0094] Example 5 (1) Collect the eluent collected in Example 4 after treatment with LX0017 type strong acid cation exchange resin from Xi'an Lanxiao Technology New Materials Co., Ltd. Place the eluent in a vacuum distillation apparatus, set the condensate temperature to 8°C, the rotary evaporation temperature to 50°C, and the vacuum pressure to less than 0.01 MPa. After vacuum distillation, the eluent concentration can reach more than 50 times. A concentrated solution is obtained.

[0095] (2) Mix the concentrate with 9 times the volume of anhydrous ethanol and stir at room temperature for 1 hour. A large amount of salt precipitates out. Remove the salt deposited at the bottom by centrifugation, and then pass the supernatant through a rotary evaporator once. Finally, a high-purity 3HB solution is obtained. The content is 326 g, the yield of 3HB is 84.1%, and the purity reaches 99.5%.

[0096] The embodiments of the present invention have been described in detail above with reference to the examples. However, the present invention is not limited to the above embodiments. Within the scope of knowledge possessed by those skilled in the art, various changes can be made without departing from the spirit of the present invention.

Claims

1. A method for separating and purifying 3-hydroxybutyric acid from a fermentation broth, characterized by, Includes the following steps: S1. Adjust the pH of the supernatant of the fermentation broth of 3-hydroxybutyric acid to no higher than 7, add a decolorizing agent to treat it, and obtain a decolorized supernatant; S2. After the decolorized supernatant is adsorbed by alkaline anion exchange resin, impurities are washed away with water, and then eluted with an inorganic alkaline aqueous solution to obtain the eluent. S3. The eluent is passed through an acidic resin, and the effluent is collected. S4. The effluent is subjected to desalination treatment.

2. The method according to claim 1, characterized in that, The decolorizing agent includes activated carbon; and / or, the amount of the decolorizing agent added is 0.4 (w / v)%-2.4 (w / v)%; and / or, the treatment time of the decolorizing agent is 4 h-6 h.

3. The method according to claim 1, characterized in that, The basic anion exchange resin includes at least one of a strongly basic anion exchange resin and a weakly basic anion exchange resin.

4. The method according to claim 1, characterized in that, The column adsorption rate of the decolorized supernatant is 1.5 BV / h-2 BV / h; and / or, the amount of water used is 3 BV-5 BV; and / or, the flow rate of the water is 1 BV / h-4 BV / h; and / or, the concentration of sodium hydroxide in the sodium hydroxide aqueous solution is 0.1 M-0.5 M; and / or, the inorganic alkali aqueous solution is used at an elution flow rate of 1.5 BV / h-2 BV / h.

5. The method according to claim 3, characterized in that, The strongly basic anion exchange resin has a trimethyl quaternary ammonium group as a functional group; and / or, the weakly basic anion exchange resin has a tertiary ammonium group as a functional group.

6. The method according to claim 1, characterized in that, The acidic resin includes at least one of a strong acidic cation exchange resin, a weak acidic cation exchange resin, and a weak acidic adsorption resin.

7. The method according to claim 6, characterized in that, The strongly acidic cation exchange resin has sulfonic acid groups as functional groups; and / or, the weakly acidic cation exchange resin has at least one of acetic acid groups and phenolic hydroxyl groups as functional groups; and / or, the weakly acidic adsorption resin adsorbs through hydrogen bonds and van der Waals forces.

8. The method according to claim 1, characterized in that, The column flow rate of the eluent is 1 BV / h-2 BV / h.

9. The method according to claim 1, characterized in that, The desalination process includes mixing the effluent with alcohol.

10. The method according to claim 9, characterized in that, The volume ratio of the effluent to the alcohol is 1:(8-10); and / or the desalination treatment time is 0.5 h-2 h.

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