Decanedioic acid produced by microbial fermentation process and preparation method thereof

Abstract

The present invention provides a decanedioic acid produced by microbial fermentation process, in which the content of C10 aliphatic acid and C10 hydroxy aliphatic acid is maintained at a very low level. The present invention also provides a preparation method of the decanedioic acid and a polymer prepared by using the decanedioic acid as monomer. The decanedioic acid provided by the present invention is prepared by microbial fermentation process. The decanedioic acid product which is produced through the processes of microbial fermentation and separation has a higher purity, a higher thermal stability, and a lower impurity content. The decanedioic acid provided by the present invention could satisfy the requirements of high grade product of polyamide or polyester to produce polymer with excellent qualities. The preparation method of the decanedioic acid provided by the present invention which has many advantages of mild reaction conditions, environmental friendliness, high yield, and good product quality, may replace the chemical method to be used in industrial scale production of the decanedioic acid.

Claims

1. A method of preparing decanedioic acid, comprising: culturing Candida tropicalis to obtain a seed culture; inoculating the obtained seed culture with a fermentation medium; starting a fermentation process to produce fermentation broth by adding decane; and purifying the obtained fermentation broth to give the decanedioic acid; wherein the fermentation medium comprises the following ingredients: 1-10 g/L potassium nitrate, 1-10 g/L potassium phosphate monobasic, and 1-4 g/L ammonium sulfate, and wherein the content of aliphatic acid comprising 10 carbons is less than 200 ppm, and the content of hydroxy aliphatic acid comprising 10 carbons is less than 300 ppm.

2. The method according to claim 1, wherein the fermentation medium also comprises the following ingredients: 0-50 g/L glucose, 2-10 g/L corn steep liquor and 1-8 g/L yeast extract.

3. The method according to claim 1, wherein the fermentation medium comprises the following ingredients: 1.2-2.5 g/L potassium nitrate, 1.8-3.5 g/L potassium phosphate monobasic, and 1.2-2 g/L ammonium sulfate.

4. The method according to claim 1, wherein the decane is added when OD 620 of the fermentation medium with a dilution of 1:30 reaches more than 0.5.

5. The method according to claim 4, wherein the OD 620 of the fermentation medium with a dilution of 1:30 is maintained at 0.3-0.8 in the fermentation process.

6. The method according to claim 1, wherein relative dissolved oxygen is maintained at more than 10% (v/v).

7. The method according to claim 6, wherein the relative dissolved oxygen is maintained at 10% to 70% in the fermentation process.

8. The method according to claim 1, wherein pH value is maintained at 5.0-8.0 in the fermentation process.

9. The method according to claim 1, wherein the content of decane in fermentation system is maintained at more than or equal to 1% (v/v) by feeding the decane in a continuous way or in a batch way in the fermentation process, and wherein the content of decane in the obtained fermentation broth is maintained at less than or equal to 0.5% (v/v) when the fermentation process is terminated.

10. The method according to claim 1, wherein the Candida tropicalis is Candida tropicalis CAT N145.

11. The method according to claim 1, wherein purifying the obtained fermentation broth comprises the following steps: S1: removing impurities by one or more separation processes consisting of membrane filtration, plate and frame filtration, and centrifugation to obtain decanedioic acid crude product; S2: purifying the obtained decanedioic acid crude product by one or more crystallization processes to obtain decanedioic acid crystals; and S3: washing and drying the decanedioic acid crystals to yield the decanedioic acid.

12. The method according to claim 11, wherein a crystallization solvent is used in the one or more crystallization processes in the step S2; wherein the crystallization solvent is selected from the group consisting of water, ethanol, acetic acid, ethyl acetate, and butyl acetate.

13. The method according to claim 12, the crystallization process using water as solvent comprising: mixing the decanedioic acid crude product with a certain amount of water to obtain a mixture with a 5%-10% (w/v) decanedioic acid content; heating the mixture to a temperature of 95° C.-110° C., followed by cooling down to 25° C.-30° C. to crystallize; and obtaining the crystals through centrifugation or plate and frame filtration.

14. The method according to claim 12, the crystallization process using acetic acid as solvent comprising: heating the solution of the decanedioic acid crude product in acetic acid to a temperature of 90° C.-95° C., then cooling down to 20° C.-30° C. to crystallize; wherein the mass ratio of the decanedioic acid crude product to acetic acid is 1/2 to 1/4.

15. The method according to claim 12, wherein the crystallization solvent is acetic acid, and the content of acetic acid in the obtained decanedioic acid crystals is maintained at less than 500 ppm.

16. A decanedioic acid product produced by the method according to claim 1.

17. The decanedioic acid product according to claim 16, wherein the thermal stability of the decanedioic acid is more than 90%.

18. A polymer prepared by using the decanedioic acid produced by the method according to claim 1 as monomer, wherein the polymer is selected from polyamide or polyester.

19. The polymer according to claim 18, wherein the polymer is polyamide polymerized by the decanedioic acid and aliphatic amine comprising 5 to 12 carbons, and wherein the aliphatic amine is selected from pentamethylene diamine, hexamethylene diamine, or decamethylene diamine.

Description

DETAILED DESCRIPTION

(1) In order to make the purpose, technical solutions and advantages of the present invention more clear, the technical solutions of the exemplary examples of the present invention will be further described below.

Example 1

(2) Candida tropicalis CAT N145 with a conservation number CCTCC M2011192 was inoculated with seed medium to conduct the process of seed culture at 30° C. As the growth rate of the seed proceeded to the logarithmic phase, the seed liquid obtained was inoculated with fermentation medium to carry out the fermentation process, with the inoculation amount of 10% (v/v). In the process of fermentation, the temperature was 30° C., the pH value was maintained at 5.0-8.0, the air ventilation rate was controlled at 0.5 VVM, the fermentor pressure was controlled at 0.05 MPa, and the relative dissolved oxygen was maintained at 30%.

(3) After the seed was inoculated into the fermentor, bacterium began to grow and multiply. When OD 620 of the fermentation medium reached more than 0.5 (with a dilution of 1:30), decane was added to conduct the microbial transformation process. The content of decane in the fermentation system was controlled at more than or equal to 1% (v/v) during the process of transformation. 24 hours before the end of the fermentation, the addition of decane was ended. Until the residual decane in the system all converted, the fermentation was terminated. The content of C10 aliphatic acid and C10 hydroxy aliphatic acid in the fermentation broth obtained was measured using gas chromatography.

(4) The seed medium comprised:

(5) 20 g/L sucrose; 10 g/L corn steep liquor; 3 g/L yeast extract; 6 g/L potassium phosphate monobasic; and 3 g/L urea.

(6) The fermentation medium comprised:

(7) ammonium sulfate, potassium nitrate, and potassium phosphate monobasic with various concentrations shown in Table 1, and other ingredients: 50 g/L glucose; 3 g/L corn steep liquor; and 1 g/L yeast extract.

(8) TABLE-US-00001 TABLE 1 1# 2# 3# 4# 3.2 g/L Ammonium 1.6 g/L Ammonium 0.96 g/LAmmonium 1.28 g/LAmmonium Sulfate; 3.4 g/L Sulfate; 1.7 g/L Sulfate; 1.02 g/L Sulfate; 1.36 g/L Potassium Nitrate; Potassium Nitrate; Potassium Nitrate; Potassium Nitrate; Initial 5 g/L Potassium 2.5 g/L Potassium 1.5 g/L Potassium 2 g/L Potassium Concentration Phosphate Monobasic. Phosphate Monobasic. Phosphate Monobasic. Phosphate Monobasic. Content of C10 Hydroxy 1.52% 0.76% 1.43% 0.87% Aliphatic Acid in Fermentation Broth (mass ratio, compared to decanedioic acid) Content of C10 0.70% 0.49% 1.18% 0.55% Aliphatic Acid in Fermentation Broth (mass ratio, compared to decanedioic acid) Average OD 620 0.773 0.466 0.339 0.426 during the process of transformation (with a dilution of 1:30)

(9) As can be seen from Table 1, the concentration of the bacterium during the process of conversion from decane to decanedioic acid could be adjusted by varying the concentrations of ammonium sulfate, potassium nitrate, and potassium phosphate monobasic in the fermentation medium, thereby the produce rate of decanedioic acid and other metabolites obtained by microbial transformation of substrate, especially the produce rate of C10 aliphatic acid and C10 hydroxy aliphatic acid could be controlled.

(10) The fermentation process was conducted repeatedly in the condition of varying the initial concentration of glucose and using the same concentrations as solution 1# for other ingredients of the fermentation medium. The results are shown in Table 2.

(11) TABLE-US-00002 TABLE 2 1# 102# 202# 204# Initial Concentration of Glucose 50 g/L 20 g/L 30 g/L 40 g/L Concentration of C10 Hydroxy Aliphatic Acid in Fermentation 1.52% 0.60% 0.46% 0.49% Broth (mass ratio, compared to decanedioic acid)

(12) As can be seen from Table 2, the content of glucose in the fermentation medium also has an effect on the produce rate of metabolites, especially on the produce rate of C10 hydroxy aliphatic acid. When the concentration of glucose in the fermentation medium is from 25 g/L to 40 g/L, the content of C10 hydroxy aliphatic acid in the fermentation broth could be maintained at a lower level.

Example 2

(13) Used Fermentation Medium Comprised:

(14) 50 g/L glucose; 3 g/L corn steep liquor; 1 g/L yeast extract; 3.2 g/L ammonium sulfate; 3.4 g/L potassium nitrate; and 5 g/L potassium phosphate monobasic.

(15) The stirring rate and air ventilation rate was adjusted to control the relative dissolved oxygen in the process of microbial transformation, which had an effect on the content of C10 hydroxy aliphatic acid and C10 aliphatic acid in fermentation broth, and other steps and conditions were the same as Example 1. The results are shown in Table 3.

(16) TABLE-US-00003 TABLE 3 102# 103# 104# 203# Relative Dissolved Oxygen   10%   70%   50%   90% Fermentation Time (h) 200 h 182 h 167 h 171 h Content of C10 Hydroxy 1.82% 0.93% 1.25% 2.03% Aliphatic Acid (mass ratio, compared to decanedioic acid) Content of C10 Aliphatic Acid 0.90% 0.50% 0.65% 1.27% (mass ratio, compared to decanedioic acid)

(17) As can be seen from Table 3, the content of C10 hydroxy aliphatic acid and C10 aliphatic acid could be maintained at a lower level by controlling the relative dissolved oxygen below 90% in the process of microbial transformation, preferably from 10% to 70%, more preferably from 40% to 70%.

Example 3

(18) The decanedioic acid fermentation broths 2# and 4# listed in Table 1 in Example 1 were processed by membrane filtration respectively. The pH value of fermentation broth was adjusted to 9.0-11.0, then the fermentation broth was heated to 70° C.-80° C., followed by filtering through microfiltration membrane with 0.05 μm pore size. Feed pump was used to maintain the feed pressure into membrane at about 0.15 to 0.2 MPa and the discharge pressure out of membrane at about 0.1 MPa. The C10 aliphatic acid and most of the hydroxyl acid were efficiently intercepted through the membrane filtration. The results are shown in Table 4.

(19) TABLE-US-00004 TABLE 4 C10 Hydroxy C10 Hydroxy Aliphatic Acid C10 Aliphatic Acid Aliphatic Acid C10 Aliphatic (compared to (compared to (compared to Acid (compared to decanedioic acid) decanedioic acid) decanedioic acid) decanedioic acid) (ppm) (ppm) (ppm) (ppm) Number Content Before Membrane Filtration Content After Membrane Filtration Fermentation 8724 5524 3925 828 Broth 4# Fermentation 7640 4987 3438 798 Broth 2#

Example 4

(20) The decanedioic acid fermentation broths 2# and 4# listed in Table 1 in Example 1 were heated to 70° C.-80° C. respectively, then processed by centrifugation to remove the C10 aliphatic acid and C10 hydroxyl aliphatic acid. The rotational speed of the centrifugation was 3000 rpm. The C10 aliphatic acid and some of the hydroxyl acid were efficiently removed through the centrifugation. The results are shown in Table 5.

(21) TABLE-US-00005 TABLE 5 C10 Hydroxy C10 Hydroxy Aliphatic Acid C10 Aliphatic Aliphatic Acid C10 Aliphatic (compared to Acid (compared to (compared to Acid (compared to decanedioic acid) decanedioic acid) decanedioic acid) decanedioic acid) (ppm) (ppm) (ppm) (ppm) Number Content Before Centrifugation Content After Centrifugation Fermentation 8724 5524 4362 1657 Broth 4# Fermentation 7640 4987 3972 1445 Broth 2#

Example 5

(22) Centrifuged fermentation broths 2# and 4# in Example 4 were diluted with 10 to 15 times deionized water respectively, followed by heating gradually to 100° C. to 105° C., then cooling down gradually to maintain the process of crystal growth to give decanedioic acid crystal crystallized by using water as solvent. The decanedioic acid crystal was processed by plate and frame filtration and dried to obtain decanedioic acid product. The contents of the C10 aliphatic acid and hydroxyl acids in the product are shown in Table 6.

(23) TABLE-US-00006 TABLE 6 C10 Hydroxy C10 Hydroxy Aliphatic Acid C10 Aliphatic Acid Aliphatic Acid C10 Aliphatic Acid (compared to (compared to (compared to (compared to decanedioic acid) decanedioic acid) decanedioic acid) decanedioic acid) (ppm) (ppm) (ppm) (ppm) Number Content Before Crystallization Content After Crystallization Sample 4# 4362 1657 2399 1242 Sample 2# 3972 1445 2264 1127

Example 6

(24) The decanedioic acid products obtained through crystallization using water as solvent in Example 5 were marked as 4#-water and 2#-water respectively. Acetic acid was separately added to the samples 4#-water and 2#-water to conduct solvent crystallization, the mass ratio of decanedioic acid to acetic acid was maintained at 1/2-1/4. The obtained solutions were heated to 80° C.-100° C., and then cooled down to 20° C.-35° C. gradually to control the time of crystal growth. After precipitation of the crystal, liquid solid separation was carried out. The resulting solid was washed by solvent and water, and dried to give the product. The contents of impurities are shown in Table 7.

(25) TABLE-US-00007 TABLE 7 C10 Hydroxy C10 Hydroxy Aliphatic Acid C10 Aliphatic Aliphatic Acid C10 Aliphatic (compared to Acid (compared (compared to Acid (compared Purity of decanedioic to decanedioic decanedioic to decanedioic Decanedioic acid) (ppm) acid) (ppm) acid) (ppm) acid) (ppm) Acid (%) Number Content Before Crystallization Content After Crystallization Product 4#-water 2399 1242 285 50 99.72% 2#-water 2264 1127 272 46 99.85%

(26) Ethyl acetate was used instead of acetic acid to carry out the above-mentioned crystallization procedure. The contents of impurities in the resulting decanedioic acid product are shown in Table 8.

(27) TABLE-US-00008 TABLE 8 C10 Hydroxy C10 Hydroxy Aliphatic Acid C10 Aliphatic Aliphatic Acid C10 Aliphatic (compared to Acid (compared (compared to Acid (compared Purity of decanedioic to decanedioic decanedioic to decanedioic Decanedioic acid) (ppm) acid) (ppm) acid) (ppm) acid) (ppm) Acid (%) Number Content Before Crystallization Content After Crystallization Product 4#-water 2399 1242 352 152 99.65% 2#-water 2264 1127 339 137 99.75%

Example 7

(28) Different batches of the decanedioic acid products obtained from the solvent crystallization were separately processed through same crystallization procedure using acetic acid as solvent in Example 6 to further reduce the content of impurities and enhance the product quality. The test results are shown in Table 9.

(29) TABLE-US-00009 TABLE 9 C10 Hydroxy Aliphatic Acid C10 Aliphatic Acid Batch (compared to decanedioic (compared to decanedioic Number acid) (ppm) acid) (ppm) 1 48 13 2 52 5 3 54 16 4 36 11 5 30 14 6 27 10 7 50 14 8 36 11 9 54 16 10 89 19 11 64 9

Application Example

(30) PA 610 was prepared according to below steps by using the decanedioic acid product of present invention and commercial decanedioic acid produced by conventional chemical method as material respectively. The performances of the decanedioic acid products and the PA 610 prepared are separately shown in the tables below.

(31) The preparation process of PA 610 was as follows:

(32) Step (1)

(33) A 100 L-polymerizer was subjected to nitrogen replacement, followed by charging with 20 kg of purified water. 11 kg of hexamethylene diamine was added with stirring, and then 19 kg of decanedioic acid was added. A small amount of hexamethylene diamine and decanedioic acid was added to adjust pH value of the resulting solution to 8.2 (a test result of the solution diluted to 10% w/w), and nylon salt aqueous solution was obtained.

(34) Step (2)

(35) In nitrogen atmosphere, the polymerizer was heated gradually to a oil bath temperature of 280° C., and started to exhaust air when the pressure in the polymerizer rose to 1.73 MPa. Sodium pyrophosphate aqueous solution containing 90 g of titanium dioxide was added when the temperature in the polymerizer was 245° C. When the temperature in the polymerizer was 270° C., the polymerizer was vacuumized to a pressure of −0.08 MPa, followed by maintaining for 20 minutes to give nylon 610.

(36) Step (3)

(37) Nitrogen was introduced to the polymerizer until the pressure was 0.6 MPa. The melted product began to be extruded and granulated by pelletizer, followed by drying in vacuo at 100° C. for 24 hours and packing.

(38) TABLE-US-00010 TABLE 10 Comparison of Decanedioic Acid (DC10) Products in Performance and Composition Water Melting Content Content of Ash C10 Hydroxy C10 Yellow Content Point of DC10 Ferric Salts Content Thermal Aliphatic Acid Aliphatic Acid Index Sample (%) (° C) (%) (ppm) (ppm) Stability (ppm) (ppm) (YI) Chemical 0.06 129.9-134.1 99.58 1.8 223 90.85 82 121 3.49 Method Biological 0.02 130.1-134.4 99.83 1.3 31 97.32 69 17 0.67 Method

(39) TABLE-US-00011 TABLE 11 Comparison of PA 610 in Performance Viscosity Yellow Number Index Appearance PA 610 Prepared by Biological 168 mL/g −2 White Method PA 610 Prepared by Chemical 156 mL/g 3 White Method

(40) As can be seen from the results of Tables 10 and 11, compared with the product prepared by chemical method, the decanedioic acid product of the present invention that is of higher qualities, especially in the respects of impurity content and thermal stability, is suitable for application of preparing polymer to improve the qualities and properties of the polymer. The decanedioic acid product and preparation method of the present invention make the decanedioic acid prepared by biological method more potential in application, which offers a possibility for replacing the decanedioic acid prepared by chemical method in industrial scale production.

(41) Unless otherwise specified, the terms used in the present invention have the meanings as commonly understood by those skilled in the art.

(42) While the present invention has been described with reference to the preferred embodiments thereof, it should be understood by those skilled in the art that various modifications, additions and substitutions may be made without departing from the true spirit and scope of the invention.