CRYSTALS OF 3-HYDROXYPROPIONATE AND PROCESS OF RECOVERING 3-HYDROXYPROPIONIC ACID

Abstract

Provided is a process of recovering 3-hydroxypropionic acid and a crystal of 3-hydroxypropionate, wherein the process includes the steps of: forming crystals of 3-hydroxypropionate in a concentrate containing 3-hydroxypropionic acid in an amount of 300 g/L or more in the presence of an alkali metal salt; and separating the crystals of 3-hydroxypropionate from the concentrate and converting into 3-hydroxypropionic acid.

Claims

1. A process of recovering 3-hydroxypropionic acid, comprising the steps of: forming a crystal of 3-hydroxypropionate in a concentrate containing 3-hydroxypropionic acid in an amount of 300 g/L or more in the presence of an alkali metal salt; and separating the crystal of 3-hydroxypropionate from the concentrate; and converting it into 3-hydroxypropionic acid.

2. The process of claim 1, wherein the alkali metal salt comprises at least one cation selected from the group consisting of Na.sup.+, Mg.sup.2+ and Ca.sup.2+.

3. The process of claim 1, wherein the alkali metal salt is Ca(OH).sub.2, Mg(OH).sub.2 or a mixture thereof.

4. The process of claim 1, wherein the concentrate comprises the 3-hydroxypropionic acid in an amount of 350 g/L or more and 900 g/L or less.

5. (canceled)

6. The process of claim 1, wherein the crystal of 3-hydroxypropionate has a particle size distribution D.sub.50 of 20 μm or more and 90 μm or less, and (D.sub.90−D.sub.10)/D.sub.50 of 1.00 or more and 3.00 or less, wherein the crystal of 3-hydroxypropionate has a mean diameter of volume distribution of 30 μm or more and 100 μm or less, a mean diameter of number distribution of 1 μm or more and 30 μm or less, and a mean diameter of area distribution of 10 μm or more and 70 μm or less, and wherein the crystal of 3-hydroxypropionate has an average aspect ratio (LW ratio; length to width ratio) of 0.50 or more and 3.00 or less.

7.-8. (canceled)

9. The process of claim 1, wherein the crystal of 3-hydroxypropionate has a moisture content of 200 ppm or more and 5000 ppm or less as measured by a Karl Fischer method.

10. The process of claim 1, wherein the crystal of 3-hydroxypropionate has a radioactive isotope content of 20 pMC (percent modern carbon) or more and a biocarbon content of 20 wt % or more as measured by ASTM D6866-21 standard.

11. The process of claim 1, wherein the crystal of 3-hydroxypropionate has an interatomic spacing (d value) of 1.00 Å or more and 15.00 Å or less in a crystal derived from X-ray diffraction (XRD) analysis, and wherein the crystal of 3-hydroxypropionate shows a peak between crystal lattices in a 2θrange of 8 to 22° in an X-ray diffraction (XRD) analysis.

12. (canceled)

13. The process of claim 1, wherein the crystal of 3-hydroxypropionate has a glass transition temperature of −55° C. or more and −30° C. or less in a differential scanning calorimetry (DSC) analysis, wherein the crystal of 3-hydroxypropionate has a melting point of 30° C. or more and 170° C. or less in a differential scanning calorimetry (DSC) analysis, and wherein the crystal of 3-hydroxypropionate has a crystallization temperature of 25° C. or more and 170° C. or less in a differential scanning calorimetry (DSC) analysis.

14.-16. (canceled)

17. The process of claim 1, further comprising the steps of: producing a fermented solution of 3-hydroxypropionic acid by fermenting a strain having a 3-hydroxypropionic acid-producing capacity; and forming a concentrate containing the 3-hydroxypropionic acid in an amount of 300 g/L or more by concentrating the fermented solution.

18. The process of claim 17, further comprising the step of: after the production of the fermented solution of 3-hydroxypropionic acid, removing cells from the fermented solution; or filtering or purifying the fermented solution.

19. The process of claim 17, wherein in the forming of the concentrate, the concentration is performed by evaporating the fermented solution.

20. (canceled)

21. A crystal of 3-hydroxypropionate of Structural formula 1 or Structural formula 2:
Cation(3HP).sub.n  [Structural formula 1]
Cation(3HP).sub.n.Math.mH.sub.2O  [Structural formula 2] wherein the Cation is a cation, the 3HP is 3-hydroxypropionic acid bonding to the cation; the n is a number of 3HPs bonding to the cation, which is an integer of 1 or more; and the m is a number of water molecules in a hydrate, which is an integer of 1 or more.

22. The crystal of 3-hydroxypropionate of claim 21, wherein the crystal of 3-hydroxypropionate has a particle size distribution D.sub.50 of 20 μm or more and 90 μm or less, and (D.sub.90−D.sub.10)/D.sub.50 of 1.00 or more and 3.00 or less, and wherein the crystal of 3-hydroxypropionate has an average aspect ratio (LW ratio; length to width ratio) of 0.50 or more and 3.00 or less.

23. (canceled)

24. The crystal of 3-hydroxypropionate of claim 21, wherein the crystal of 3-hydroxypropionate has a radioactive isotope content of 20 pMC (percent modern carbon) or more and a biocarbon content of 20 wt % or more as measured by ASTM D6866-21 standard.

25. The crystal of 3-hydroxypropionate of claim 21, wherein the crystal of 3-hydroxypropionate has an interatomic spacing (d value) of 1.00 Å or more and 15.00 Å or less in a crystal derived from X-ray diffraction (XRD) analysis, and wherein the crystal of 3-hydroxypropionate shows a peak between crystal lattices in a 2θrange of 8 to 22° in an X-ray diffraction (XRD) analysis.

26. (canceled)

27. The crystal of 3-hydroxypropionate of claim 21, wherein the crystal of 3-hydroxypropionate has a glass transition temperature of −55° C. or more and −30° C. or less in a differential scanning calorimetry (DSC) analysis, and wherein the crystal of 3-hydroxypropionate has a melting point of 30° C. or more and 170° C. or less in a differential scanning calorimetry (DSC) analysis.

28.-29. (canceled)

30. A crystal of 3-hydroxypropionate, comprising an alkali metal salt of 3-hydroxypropionic acid or a hydrate thereof, wherein a content of biocarbon is 20 wt % or more as measured by ASTM D6866-21 standard.

31. The crystal of 3-hydroxypropionate of claim 30, wherein the alkali metal salt of 3-hydroxypropionic acid is a calcium salt or magnesium salt of 3-hydroxypropionic acid.

32. The crystal of 3-hydroxypropionate of claim 30, wherein the crystal of 3-hydroxypropionate has a mean diameter of volume distribution of 30 μm or more and 100 μm or less, a mean diameter of number distribution of 1 μm or more and 30 μm or less, and a mean diameter of area distribution of 10 μm or more and 70 μm or less, and wherein the crystal of 3-hydroxypropionate has an average aspect ratio (LW ratio: length to width ratio) of 0.50 or more and 3.00 or less.

33. (canceled)

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0112] FIGS. 1 and 2 are graphs of showing results of an X-ray diffraction (XRD) analysis on the crystal of 3-hydroxypropionate (Ca(3HP).sub.2) of Example 1.

[0113] FIGS. 3 and 4 are graphs of showing results of an X-ray diffraction (XRD) analysis on the crystal of 3-hydroxypropionate (Mg(3HP).sub.2) of Example 2.

[0114] FIG. 5 is a graph of showing results of a differential scanning calorimetry (DSC) analysis on the crystal of 3-hydroxypropionate (Ca(3HP).sub.2) of Example 1.

[0115] FIG. 6 is a graph of showing results of a differential scanning calorimetry (DSC) analysis on the crystal of 3-hydroxypropionate (Mg(3HP).sub.2) of Example 2.

DETAILED DESCRIPTION

[0116] Hereinafter, the present disclosure will be described in more detail with reference to examples. However, the following examples are provided only for the purpose of illustrating the present disclosure, and thus the scope of the present disclosure is not limited thereto.

[0117] In this disclosure, all temperatures are described in degrees Celsius unless otherwise specified.

Preparation Example 1. Preparation of Strains for Production of 3-Hydroxypropionic Acid

[0118] Prepared was a recombinant vector incorporating a gene encoding glycerol dehydratase and aldehyde dehydrogenase, which are known to produce 3-hydroxypropionic acid (3HP) using glycerol as a substrate. The prepared recombinant vector was introduced into the E. coli W3110 strain to prepare a 3-hydroxypropionic acid-producing strain.

[0119] More specifically, pCDF_J23101_dhaB_gdrAB_J23100_aldH_btuR vector, obtained by cloning BtuR gene encoding adenosyltransferase into plasmid pCDF including a gene (dhaB) encoding glycerol dehydratase, a gene (aldH) encoding aldehyde dehydrogenase and a gene (gdrAB) encoding glycerol dehydratase reactivase, was introduced into W3110 strain (KCCM 40219) by electroporation using an electroporation device (Bio-Rad, Gene Pulser Xcell) to prepare a 3-hydroxypropionic acid-producing strain. The process of preparing the 3-hydroxypropionic acid-producing strain of this Preparation Example 1, and the vectors, primers and enzymes used were performed with reference to Example 1 of Korean Patent Publication No. 10-2020-0051375 (incorporated herein by reference).

Example 1. Preparation of Ca(3HP).SUB.2 .Crystals

[0120] The strain for producing 3-hydroxypropionic acid prepared in Preparation Example 1 was fermented and cultured at 35° C. in a 5 L fermenter by using unrefined glycerol as a carbon source, so as to produce 3-hydroxypropionic acid. In order to prevent a decrease in pH due to the production of 3-hydroxypropionic acid, calcium hydroxide (Ca(OH).sub.2), an alkali metal salt, was added to maintain a neutral pH during fermentation.

[0121] After fermentation culture, cells were removed by centrifugation (4000 rpm, 10 minutes, 4° C.), and primary fermented solution purification (primary purification) was performed by using activated carbon. Specifically, activated carbon was added to the fermented solution, from which the cells were removed by centrifugation, mixed well, and then centrifuged again to separate the activated carbon. After that, the fermented solution, from which the activated carbon was separated, was filtered through a 0.7 μm filter paper with a vacuum pump to purify the 3-hydroxypropionic acid fermented solution.

[0122] The concentration of 3-hydroxypropionic acid in the fermented solution obtained after the primary purification was at a level of 50 to 100 g/L, and the fermented solution was concentrated at a concentration of 600 g/L using a rotary evaporator (50° C., 50 mbar) to prepare a concentrate, which was then stirred at room temperature (300 rpm) to produce Ca(3HP).sub.2 crystals. At this time, the concentration of the alkali metal salt in the concentrate was 493.3 g/L (based on Ca(OH).sub.2).

[0123] The resulting crystals were washed three times with ethanol (EtOH) and dried in an oven at 50° C. to finally recover crystals. The weight of the crystals was measured, the crystals were dissolved in water, and the amount of 3-hydroxypropionic acid (3HP) was quantified with high performance liquid chromatography (HPLC) to measure the amount of 3-hydroxypropionic acid in the crystals, after which a recovery rate was measured (Equation 1) compared to the amount of 3-hydroxypropionic acid measured by HPLC in the fermented solution.

3HP recovery rate (%)={(3HP content in crystal)/(3HP content in fermented solution before crystallization)}*100  [Equation 1]

[0124] The recovery rate of 3-hydroxypropionic acid recovered as crystals was measured to be about 92%.

Example 2. Preparation of Mg(3HP).SUB.2 .Crystals

[0125] A fermentation process was performed in substantially the same manner as in above Example 1, except for using Mg(OH).sub.2 instead of Ca(OH).sub.2 as an alkali metal salt, followed by concentration to produce and recover Mg(3HP).sub.2 crystals.

[0126] Table 1 below shows the recovery rates of Mg(3HP).sub.2 crystals for each concentration of each concentrate.

TABLE-US-00001 TABLE 1 Concentration 3HP recovery rate (g/L) through crystals (%) 461.2 92.4 527.9 94.3 576.2 95.9 800.0 96.5

[0127] As can be seen in Table 1 above, at least 90% of 3-hydroxypropionic acid was recovered as a result of crystallization after concentration and thus it was confirmed that the recovery rate of 3-hydroxypropionic acid through crystallization was excellent.

Comparative Example 1. Titration with Sulfuric Acid after Fermentation

[0128] A fermentation process was performed in the same manner as in above Example 1, and a fermented solution was titrated to pH 2 with sulfuric acid (H.sub.2SO.sub.4). The gypsum (Ca(SO).sub.4) generated in the titration process was removed to proceed with protonation of 3-hydroxypropionic acid.

[0129] The fermented solution, from which the gypsum had been removed, was first purified and concentrated in substantially the same manner as in above Example 1, so as to confirm whether crystals were formed or not.

[0130] However, in the case of concentration at 600 g/L (based on 3HP) during the concentration process, no crystals were formed, and even when concentration was performed up to 1000 g/L (based on 3HP), formation of crystals was not confirmed.

Test Example

[0131] 1. Analysis of Purity of 3-Hydroxypropionate in Crystals

[0132] Ion chromatography (IC) analysis (Dionex Aquion; Eluent: 20 mM MethanesulfonicAcid; Column: Dionex IonPac CS12A; Flow Rate: 1.0 ml/min; Suppressor: Dionex CERS 500 4 mm; Current: 59 mA) was performed for a cation analysis of the 3-hydroxypropionate crystals obtained in above Examples 1 and 2, so as to confirm the purity of each 3-hydroxypropionate in the crystals.

[0133] Table 2 below shows the results of cation analysis of Ca(3HP).sub.2 crystals of Example 1, and table 3 below shows the results of measuring purity of 3-hydroxypropionate for each concentration of the Mg(3HP).sub.2 crystals of Example 2.

TABLE-US-00002 TABLE 2 Ca(3HP).sub.2 crystals Cation type ppm Composition (%) Na.sup.+ 3.06 0.81 Mg.sup.2+ 1.26 0.33 Ca.sup.2+ 374.42 98.53 K.sup.+ 1.26 0.33 Total Cation 380.00 100.00

TABLE-US-00003 TABLE 3 Concentration (g/L) of Purity of Example 2 Mg(3HP).sub.2 461.2 93.2 527.9 92.9 576.2 93.0 800.0 93.5

[0134] Referring to above Tables 2 and 3, it was confirmed that the purity of 3-hydroxypropionate in the crystals in Examples 1 and 2 was 92.9% or more. The purity of Ca(3HP).sub.2 was confirmed by a proportion of Ca.sup.2+ among Cation types in above table 2, and as a result, it was confirmed that the purity was 98% or more, which is very excellent.

[0135] 2. Analysis of 3-Hydroxypropionic Acid in Crystals

[0136] High performance liquid chromatography (HPLC) analysis was performed to analyze the content of 3-hydroxypropionic acid in the crystal of 3-hydroxypropionate obtained in Examples 1 and 2. Specific conditions for HPLC performance are shown in table 4 below.

TABLE-US-00004 TABLE 4 Category Quality Detector RI/UV detector Column Bio-Rad Aminex HPX-87H Ion Exclusion Column 300 mm × 7.8 mm Mobile Phase 0.5 mM H.sub.2SO.sub.4 Flow Rate 0.4 mL/min Run Time     35 min Column temperature 35° C. Detector temperature 35° C. Injection Volume 10 μl

[0137] Table 5 below shows the results of HPLC performance for the crystals of Examples 1 and 2. In Table 5 below, 3-hydroxypropionate in the crystals can refer to Ca(3HP).sub.2 in Example 1 and Mg(3HP).sub.2 in Example 2.

TABLE-US-00005 TABLE 5 3HP 3HP salt Crystal Crystal mass in mass in purity Type mass (g) crystal (g) crystal (g) (%) Example 0.2426 0.1880 0.2302 94.89 1 Example 0.2580 0.2118 0.2404 93.20 2

[0138] Referring to Table 5 above, it was confirmed that the purity of 3HP salt in the crystals shown as a result of performing HPLC on the crystals of Examples 1 and 2 was 93.20% or more.

[0139] 3. Analysis of Particle Size of 3-Hydroxypropionate Crystals

[0140] With regard to crystals of 3-hydroxypropionate (Ca(3HP).sub.2) of Example 1 and crystals of 3-hydroxypropionate (Mg(3HP).sub.2) of Example 2, D.sub.10, D.sub.50, D.sub.90, mean diameters of the volume distribution, number distribution and area distribution were measured respectively with a size and shape particle analyzer (Microtrac TurboSync), and the results are shown in Table 6 below.

[0141] In addition, with regard to crystals of 3-hydroxypropionate of Examples 1 and 2, an aspect ratio (LW Ratio; length to width ratio) and an average aspect ratio at each of D.sub.10, D.sub.50 and D.sub.90 were measured with the size and shape particle analyzer, and the results are shown in Table 7 below.

TABLE-US-00006 TABLE 6 Mean Mean Mean diameter of diameter of diameter of volume number area Concentration D.sub.10 D.sub.50 D.sub.90 distribution distribution distribution (g/L) (μm) (μm) (μm) (MV, μm) (MN, μm) (MA, μm) Example 1 600 27.41 71.40 170.40 88.71 18.99 53.45 Example 2 800 13.68 36.50  74.02 41.30  5.13 24.81

TABLE-US-00007 TABLE 7 D.sub.10 aspect D.sub.50 aspect D.sub.90 aspect Average ratio ratio ratio aspect ratio Example 27.41 71.40 170.40 88.71 1 Example 13.68 36.50 74.02 41.30 2 [0142] Aspect ratio (LW Ratio; Length to width ratio)

[0143] Referring to above Tables 6 and 7, in Examples 1 and 2, it was confirmed that the particle size distribution D.sub.10 was 13.68 to 27.41 μm; the particle size distribution D.sub.50 was 36.50 to 71.40 μm; the particle size distribution D.sub.90 was 74.02 to 170.40; (D.sub.90−D.sub.10)/D.sub.50 was 2.00 for Example 1 and was 1.65 for Example 2; the mean diameter of the volume distribution was 41.30 to 88.71 μm; the mean diameter of the number distribution was 5.13 to 18.99 μm; the mean diameter of the area distribution was 24.81 to 53.45 μm; the D.sub.10 aspect ratio (LW Ratio; length to width ratio) was 13.68 to 27.41; the D.sub.50 aspect ratio was 36.50 to 71.40; the D.sub.90 aspect ratio was 74.02 to 170.40; and the average aspect ratio was 41.30 to 88.71.

[0144] 4. Analysis of Moisture Content of 3-Hydroxypropionate Crystals

[0145] The moisture content in 3-hydroxypropionate crystals (Ca(3HP).sub.2) of Example 1 and in 3-hydroxypropionate crystals (Mg(3HP).sub.2) of Example 2 was analyzed with Karl-Fischer titrator from Metrohm and is shown in Table 8 below.

TABLE-US-00008 TABLE 8 Concentration Moisture (g/L) content (ppm) Example 1 600 351 Example 2 800 4370

[0146] Referring to Table 8 above, it was confirmed that the moisture content of Examples 1 and 2 was 351 to 4370 ppm.

[0147] 5. Analysis of Biocarbon Content in 3-Hydroxypropionate Crystals

[0148] A radioactive isotope ratio (pMC) and a biocarbon content in 3-hydroxypropionate crystals (Ca(3HP).sub.2) of Example 1 and in 3-hydroxypropionate crystals (Mg(3HP).sub.2) of Example 2 were analyzed with ASTM D 6866-21 (Method B) and the results are shown in Table 9 below.

[0149] The biocarbon content can refer to the content of biocarbon contained in the 3-hydroxypropionate crystals of Examples 1 and 2, respectively, and the radioactive isotope ratio (pMC) can refer to the ratio of the radioactive isotope (.sup.14C) contained in the 3-hydroxypropionate crystals and the radioactive isotope (.sup.14C) of the modern reference material.

TABLE-US-00009 TABLE 9 Radioactive Biocarbon Concentration isotope ratio content (g/L) (pMC) (%) Example 1 600 101.52 100 Example 2 800 102.23 100

[0150] pMC: Percent Modern Carbon

[0151] Referring to Table 9 above, it was confirmed that all crystals of 3HP salt of above Examples 1 and 2 had 101 pMC or more, and a biocarbon content of 100%.

[0152] 6. X-Ray Diffraction (XRD) Analysis of 3-Hydroxypropionate Crystals

[0153] The crystals prepared in Examples 1 and 2 were irradiated with Cu-Kα rays having a wavelength of 1.54 Å to measure an X-ray diffraction (XRD) pattern in a reflection mode.

[0154] The measurement equipment used was a Bruker AXS D4 Endeavor XRD. The voltage and current used are 40 kV and 40 mA, respectively, and the optics and detector used are as follows: [0155] Primary (incident beam) optics: motorized divergence slit, soller slit 2.3° [0156] Secondary (diffracted beam) optics: soller slit 2.3° [0157] LynxEye detector (1 D detector)

[0158] FIGS. 1 and 2 are graphs of showing results of an X-ray diffraction (XRD) analysis on the crystal of 3-hydroxypropionate of Example 1, and FIGS. 2 and 3 are graphs of showing results of an X-ray diffraction (XRD) analysis on the crystal of 3-hydroxypropionate of Example 2.

[0159] The 2θ value and lattice plane spacing (d values) of the numbered peaks in the XRD graphs of FIGS. 1 and 2 are shown in Table 10, and the 2θ value and lattice plane spacing of the numbered peaks in the XRD graphs of FIGS. 3 and 4 are shown in Table 11 below.

TABLE-US-00010 TABLE 10 Lattice Lattice plane plane Peak 2θ spacing (d Peak 2θ spacing (d No. value (º) value, Å) No. value (º) value, Å) 1 10.0-11.0 8.37 8 20.6-21.2 4.24 2 11.1-11.6 7.75 9 21.5-22.0 4.10 3 11.6-12.4 7.38 10 22.0-22.4 4.01 4 12.9-13.3 6.75 11 23.2-23.7 3.78 5 14.1-14.5 6.17 12 23.9-24.3 3.69 6 17.2-17.6 5.08 13 26.9-27.2 3.29 7 19.2-19.8 4.55 14 29.2-29.6 3.03

TABLE-US-00011 TABLE 11 Lattice Lattice plane plane Peak 2θ spacing (d Peak 2θ spacing (d No. value (º) value, Å) No. value (º) value, Å) 1 8.5-9.2 9.84 6 16.8-17.3 5.29 2  9.5-10.3 8.82 7 20.7-21.0 4.24 3 12.0-12.7 7.10 8 23.3-23.7 3.85 4 12.9-13.2 6.77 9 24.0-24.4 3.66 5 13.5-14.0 5.95 — — —

[0160] Referring to Tables 10 and 11, it was confirmed that the crystal of 3-hydroxypropionate prepared in Example 1 was Ca(3HP).sub.2 and the crystal of 3-hydroxypropionate prepared in Example 2 was Mg(3HP).sub.2.

[0161] 7. Differential Scanning Calorimetry (DSC) Analysis of 3-Hydroxypropionate Crystals

[0162] With regard to the crystals of Examples 1 and 2, a melting point (Tm) of each crystal was confirmed with a differential scanning calorimeter (DSC (Q2000) manufactured by TA Instruments Co., Ltd.). In the case of Example 1, a glass transition temperature, a crystallization temperature, etc. were measured in the range of −60° C. to 150° C. at a temperature increasing rate of 10° C./min. In the case of Example 2, a glass transition temperature, a crystallization temperature, etc. were measured in the range of −70° C. to 30° C. at a temperature increasing rate of 10° C./min, and the results are shown in Table 12 below.

[0163] FIG. 5 is a graph of showing results of a differential scanning calorimeter (DSC) analysis on the crystal of 3-hydroxypropionate (Ca(3HP).sub.2) of Example 1, and FIG. 6 is a graph of showing results of a differential scanning calorimeter (DSC) analysis on the crystal of 3-hydroxypropionate (Mg(3HP).sub.2) of Example 2.

TABLE-US-00012 TABLE 12 Glass Crystal- transition lization Crystal- temper- Melting temper- lization ature Point ature stable (Tg, ° C.) (Tm, ° C.) (Tc, ° C.) region Metal Example 1 −45 32 30 −40~30 Ca Example 2 −40 155 150  −20~150 Mg

[0164] Referring to Table 12, it was confirmed that the glass transition temperature of Examples 1 and 2 was −45 to −40° C., the melting point was 32 to 155° C., and the crystallization temperature was 30 to 150° C.