Long-chain dicarboxylic acid product and method for producing long-chain dicarboxylic acid through fermentation

Abstract

The invention belongs to the technical field of fermentation, and particularly relates to a long-chain dibasic acid product and a method for producing long-chain dibasic acid through fermentation, and the method comprises the following steps: when the fermentation time is t0, starting to supplement a carbon source into a fermentation liquid: when the fermentation time is greater than t0 and less than or equal to t1, controlling the numerical relationship between the material supplementing rate V1 of the carbon source and the fermentation time t to meet the following conditions: when the fermentation time is greater than t1 and less than or equal to t2, starting to supplement the carbon source; when the fermentation time is greater than t2 and less than or equal to t3, the numerical relationship between the feeding rate V3 of the carbon source and the fermentation time t is controlled to meet the condition that a fermentation substrate is selected from any one or a combination of more of fatty acid, fatty acid ester, fatty acid salt or alkane with the carbon atom number of 8-18. By supplementing the carbon source in the fermentation process, the substrate can be converted into the long-chain dicarboxylic acid as much as possible, the acid yield of the long-chain dicarboxylic acid with the target chain length is remarkably improved, and the method has the potential of industrial large-scale production.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of fermentation, and particularly relates to a long-chain dibasic acid product and a method for fermentatively producing long-chain dibasic acid. BACKGROUND

[0002] Long-chain dibasic acid (HOOC-(CH2) n -COOH, n = 7-16, abbreviated as DC9-DC18) has a wide range of application fields. Special nylon, high-grade perfume, high-grade hot melt adhesive, cold-resistant plasticizer, high-grade lubricating oil, high-grade antirust agent, high-grade paint and coating, etc. can be synthesized by using long-chain dibasic acid as raw material. Long-chain dibasic acid is usually synthesized by chemical or biological method. However, the route of chemical synthesis is relatively long, the reaction process requires high temperature and high pressure, and the catalyst has strict requirements. Therefore, the biological synthesis of long-chain dibasic acid is considered as a feasible way, which has the advantages of environmental friendliness and sustainable development. By synthesizing long-chain dibasic acid by biological method, not only the dependence on fossil resources can be reduced, but also the energy consumption and environmental pollution can be effectively reduced.

[0003] At present, the biological synthesis of long-chain dibasic acid is mainly based on petroleum-based alkanes as substrate, and long-chain dibasic acid is obtained by microbial transformation. However, petroleum-based alkanes are non-renewable. Therefore, it is urgent to replace or partially replace petroleum-based alkanes and other raw materials with new plant-based resources such as fatty acids, fatty acid salts and fatty acid esters. However, at present, the direct use of fatty acids and their derivatives as raw materials to convert long-chain dibasic acid mostly has the problems of low dibasic acid yield and low scale-up production efficiency, which is rarely applied in industry. SUMMARY

[0004] In order to overcome the defects existing in the production process of long-chain dibasic acid, the present application provides a long-chain dibasic acid product and a method for fermentatively producing long-chain dibasic acid. By supplementing carbon source during fermentation and controlling the feeding rate of carbon source and the fermentation time to meet a specific measurement relationship, not only the stable cell activity can be ensured, but also the induction time can be prolonged. In addition, the substrate can be as efficiently converted as possible into long-chain dibasic acid, and the acid yield of long-chain dibasic acid with target chain length can be significantly improved, which has the potential for industrial scale-up production.

[0005] The first aspect of the present application provides a method for fermentatively producing long-chain dibasic acid, which comprises: fermentatively culturing Candida yeast, adding fermentation substrate for fermentation, and supplementing carbon source into the fermentation broth when the fermentation time is t0:

[0006] When t0 0.5≤A1≤0.8, 0.5≤B1≤2.0;

[0007] When t1 < fermentation time ≤ t2, the numerical relationship between the carbon source feeding rate V2 and the fermentation time t satisfies: 0.1≤A2≤0.3, 0.5≤B2≤3.0;

[0008] When t2 < fermentation time ≤ t3, the numerical relationship between the carbon source feeding rate V3 and the fermentation time t satisfies: 0.0001≤A3≤0.003, 0.05≤B3≤0.08, 4.0≤C3≤8.0;

[0009] Wherein, 8≤t0≤14, 45≤t1≤55, 75≤t2≤85, 130≤t3≤145, the units of the carbon source feeding rate V1, V2, V3 are g / h, and the unit of the fermentation time t is h, V 初始 represents the volume of the fermentation medium, and the unit is L;

[0010] The fermentation substrate is selected from any one or combination of fatty acids, fatty acid esters, fatty acid salts or alkanes with carbon atom number of 8-18. In the present application, the carbon source is continuously added to the fermentation broth.

[0011] In some embodiments, V 初始 represents the volume of the fermentation medium, and is a constant value.

[0012] In the present application, the carbon source feeding rate V1, V2, V3 represents the mass of the continuously added carbon source per hour or the feeding amount of the carbon source per hour, for example, when the fermentation time is t, V1, V2, V3 respectively represent the mass of the carbon source added from (t-1) to t. The fermentation time t in the present application is the minimum unit of hours, and t is an integer.

[0013] In the present application, the carbon source feeding rate and the fermentation time t satisfy different relationships in different fermentation time periods, in which t is an integer and the unit is hour, and with the change of the fermentation time t, the carbon source feeding rate also changes constantly, for example, when the fermentation time is from 10h to 11h, the numerical relationship between the carbon source feeding rate V1 and the fermentation time t is: Wherein V 初始 , A1, B1 are constants; when the fermentation time is from 11h to 12h, the numerical relationship between the carbon source feeding rate V1 and the fermentation time t is: Wherein V 初始 , A1, B1 are constants; that is, the carbon source feeding rate is changed every 1 hour, so that it is in a dynamic change process. The other relationships are the same, and are not described here.

[0014] In some embodiments, 0.6≤A1≤0.7.

[0015] In some implementations, 0.5 ≤ B1 ≤ 1.5.

[0016] In some implementations, 0.15 ≤ A2 ≤ 0.2.

[0017] In some implementations, 1.0 ≤ B2 ≤ 2.0.

[0018] In some implementations, 0.00015 ≤ A3 ≤ 0.0003.

[0019] In some implementations, 0.06 ≤ B3 ≤ 0.08.

[0020] In some implementations, 5.0 ≤ C3 ≤ 7.0.

[0021] The inventors of this invention discovered in their research that, in order to solve the problem of low dicarboxylic acid yield in the existing fermentation and conversion of target long-chain dicarboxylic acids using alkanes, fatty acids and their derivatives as raw materials, a dynamic carbon source supplementation method is adopted. This not only maintains the cell activity at a high level, but also enables the substrate to be converted into the target long-chain dicarboxylic acid as much as possible, significantly increasing the acid production of the target long-chain dicarboxylic acid.

[0022] In some implementations, when t0 < fermentation time ≤ t1, control Further for In this invention, the feeding rate of the carbon source is different from the conventional feeding rate in the prior art. It is not a constant quantity, but rather it is constantly changing dynamically with the fermentation time.

[0023] In some implementations, when t1 < fermentation time ≤ t2, control Further for In this invention, the feeding rate of the carbon source each time is different from the conventional feeding rate in the prior art. It is not a constant quantity, but is constantly changing dynamically with the fermentation time.

[0024] In some implementations, when t2 < fermentation time ≤ t3, control Further for In this invention, the feeding rate of the carbon source is different from the conventional feeding rate in the prior art. It is not a constant quantity, but rather it is constantly changing dynamically with the fermentation time.

[0025] In some embodiments, the fatty acid is selected from straight-chain monocarboxylic acids having 8-18 carbon atoms, and more particularly from straight-chain monocarboxylic acids having 9-18 carbon atoms.

[0026] In some preferred embodiments, the fatty acid is selected from any one or a combination of several of the following: nonanoic acid, decaanoic acid, undecanoic acid, lauric acid (also known as dodecanoic acid), tridecanoic acid, myristic acid (also known as tetradecanoic acid), pentadecanoic acid, palmitic acid (also known as hexadecanoic acid), heptadecanoic acid, and octadecanoic acid.

[0027] In some embodiments, the fatty acid ester is selected from at least one of nonanoic acid ester, decaanoic acid ester, undecanoic acid ester, laurate, tridecanoic acid ester, myristate ester, pentadecanoic acid ester, palmitate ester, heptadecanoate, and octadecanoic acid ester.

[0028] In some embodiments, the fatty acid ester is selected from fatty acid esters with 8 or more atoms, and is further selected from at least one of fatty acid methyl ester, fatty acid ethyl ester, fatty acid propyl ester or fatty acid butyl ester with 9 to 18 carbon atoms.

[0029] In some specific embodiments, the fatty acid ester is selected from at least one of methyl laurate, ethyl laurate, butyl laurate, methyl myristate, ethyl myristate, butyl myristate, methyl palmitate, ethyl palmitate, and butyl palmitate. Since most fatty acid esters are liquid at room temperature, they disperse well and have characteristics similar to alkanes, making them a viable alternative to currently used alkanes as substrates. Fatty acid esters are mainly derived from the seeds of oil-rich tropical crops such as palm and coconut trees. This novel renewable resource can replace fossil fuels such as petroleum alkanes, which is of great significance for the sustainable development of industrial processes.

[0030] In some embodiments, the fatty acid salt is selected from fatty acid salts with 8 or more carbon atoms, and more specifically from at least one of sodium, potassium, ammonium, and calcium salts of fatty acids with 9 to 18 carbon atoms. The fatty acid has the same limitations as described above. Fatty acids are mainly derived from the seeds of oil-rich tropical crops such as palm and coconut trees. This novel renewable resource can replace fossil fuels such as petroleum and alkanes, and is of great significance to the sustainable development of industrial processes.

[0031] In some embodiments, the alkane is selected from one or more combinations of nonane, decane, undecane, dodecane, tridecane, tetradecane, pentadecane, hexadecane, heptadecane, and octadecane, preferably n-alkanes. The alkane may be petroleum-based alkanes or coal-derived alkanes derived from petroleum or coal, or bio-based alkanes obtained through the processing of vegetable oils.

[0032] The long-chain dicarboxylic acid of the present invention has the general formula HOOC(CH2)nCOOH, where n is 6 to 18. Specifically, the long-chain dicarboxylic acid is selected from any one of octanoic acid, azelaic acid, sebacic acid, undecanoic acid, dodecanoic acid, tridecanoic acid, tetradecanoic acid, pentadecanoic acid, hexadecanoic acid, heptadecanoic acid, and octadecanoic acid.

[0033] In some embodiments, the fermentation substrate is selected from one or more of the following combinations: a combination of fatty acids and fatty acid esters; a combination of fatty acids and alkanes; a combination of fatty acid esters and alkanes; a combination of fatty acid salts and alkanes; or a combination of fatty acids, fatty acid esters, and alkanes. In the above combinations consisting of two or three different types of substrates, the mass ratio of the different types of substrates is not particularly limited. For example, when the substrate is a combination of fatty acids and fatty acid esters, the mass ratio of fatty acids to fatty acid esters is not particularly limited, and can be, for example, 1:99 to 99:1.

[0034] In some embodiments, the fermentation substrate is a combination of fatty acids and fatty acid esters in a mass ratio of 1:99 to 99:1; a combination of fatty acids and alkanes in a mass ratio of 1:99 to 99:1; a combination of fatty acid esters and alkanes in a mass ratio of 1:99 to 99:1; a combination of fatty acid salts and alkanes in a mass ratio of 1:99 to 99:1; or, a combination of fatty acids, fatty acid esters, and alkanes in any mass ratio.

[0035] In some specific embodiments, when fermenting to produce dodecanoic acid, the fermentation substrate is selected from any one of dodecane, lauric acid, laurate, a combination of lauric acid and dodecane, a combination of laurate and dodecane, or a combination of lauric acid and laurate, wherein the laurate can be methyl laurate or ethyl laurate.

[0036] In some specific embodiments, when fermenting to produce tetradecanoic acid, the fermentation substrate is selected from any one of tetradecane, myristic acid, myristic acid ester, a combination of myristic acid and tetradecane, a combination of myristic acid ester and tetradecane, or a combination of myristic acid and myristic acid ester, wherein the myristic acid ester can be methyl myristicate or ethyl myristicate.

[0037] In some specific embodiments, when fermenting to produce hexadecanoic acid, the fermentation substrate is selected from any one of hexadecane, palmitic acid, palmitate, a combination of palmitic acid and hexadecane, a combination of palmitate and hexadecane, or a combination of palmitic acid and palmitate, wherein the palmitate can be methyl palmitate or ethyl palmitate.

[0038] In some embodiments, the added carbon source is selected from at least one of glucose, sucrose, lactose, maltose, fructose, molasses, glycerol, sorbitol, arabinose, rhamnose, cellobiose, sophorose, and gentiobiose, more preferably from at least one of glucose, lactose, and sucrose. In this invention, the carbon source is added in the form of an aqueous sugar solution with a concentration of 10 wt% to 70 wt%.

[0039] In some embodiments, the sugar concentration in the fermentation broth is maintained in the range of 0.1-1 g / L, preferably in the range of 0.1-0.5 g / L, by supplementing a carbon source.

[0040] In some embodiments, when the optical density value OD of the strain 620 When diluted 30 times to reach 0.5-1.0, add the fermentation substrate to start fermentation.

[0041] In some embodiments, the substrate is added in a single addition, batch addition, or continuous feeding manner. Preferably, during fermentation culture, the concentration of the long-chain substrate is controlled below 8% (v / v), more preferably 0.01 to 5% (v / v), for example below 0.5%, below 1.5%, or below 2%.

[0042] In some embodiments, during fermentation culture, the inoculum size of Candida virescens is 5 to 50% (v / v) of the fermentation medium, that is, the volume ratio between the seed culture and the fermentation medium is (5 to 50): 100.

[0043] In some embodiments, the Candida species includes at least one of Candida viswanathii, Candida albicans, Candida tropicalis, Candida sake, or Yarrowia lipolytica.

[0044] In some implementations, the fermentation temperature is controlled at 27-35°C, for example, 27°C, 28°C, 29°C, 30°C, 31°C, 32°C, 33°C, or 35°C.

[0045] In some implementations, the fermentation airflow is controlled to be 0.2-0.8 vvm, for example, 0.2 vvm, 0.3 vvm, 0.4 vvm, 0.5 vvm, 0.6 vvm, 0.7 vvm or 0.8 vvm.

[0046] In some implementations, the fermentation pressure is controlled at 0.05-0.20 MPa during the fermentation process, for example, 0.05 MPa, 0.08 MPa, 0.11 MPa, 0.12 MPa, 0.15 MPa, or 0.2 MPa.

[0047] In one embodiment, during fermentation, the pH value of the fermentation is controlled to be 4.5-7.5, for example, 5, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0.

[0048] In one embodiment, during fermentation, the dissolved oxygen level is controlled to be 5%-90%, more specifically 25%-70%, for example 10%, 20%, 30%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, or 80%.

[0049] In some embodiments, when the fermentation substrate is solid, it is first heated to dissolve it before being added to the fermentation tank; wherein, the temperature for heating the fermentation substrate can be 45 to 65°C.

[0050] In some implementations, the fermentation time is 130 to 180 hours.

[0051] In some implementations, the culture medium for fermentation contains at least a carbon source, a nitrogen source, and / or inorganic salts.

[0052] In some preferred embodiments, the carbon source is selected from one or more combinations of glucose, sucrose, lactose, maltose, fructose, molasses, glycerol, sorbitol, arabinose, rhamnose, cellobiose, sophorose, and gentiobiose, more preferably from one or more combinations of glucose, lactose, and sucrose.

[0053] In some preferred embodiments, the nitrogen source is selected from one or more combinations of yeast extract, peptone, corn steep liquor, urea, ammonium salts, and nitrates.

[0054] In some preferred embodiments, the inorganic salt is selected from one or more combinations of sulfates, hydrochlorides, nitrates and phosphates, more preferably from one or more combinations of potassium dihydrogen phosphate, dipotassium hydrogen phosphate, ammonium dihydrogen phosphate, sodium dihydrogen phosphate, disodium hydrogen phosphate, magnesium sulfate, calcium chloride and potassium nitrate.

[0055] In some preferred embodiments, the culture medium comprises a seed culture medium and a fermentation culture medium, both containing the carbon source, the nitrogen source, and the inorganic salts. *Candida virescens* is first cultured in the seed culture medium, and then inoculated into the fermentation culture medium for further culture and fermentation transformation.

[0056] In some preferred embodiments, the culture medium further contains an antifoaming agent, which includes polyether antifoaming agents, silicone antifoaming agents, silicone-ether composite antifoaming agents, mineral oil antifoaming agents, or nonionic surfactants. Examples include commercially available polyether antifoaming agents, Dow DF-103 antifoaming agent, Sigma Aldrich antifoaming agent 204, polyethylene glycol monostearate, or dimethicone.

[0057] In some specific embodiments, the seed culture medium comprises 1% to 3.5% sucrose, 0.1% to 1.5% corn steep liquor, 0.1% to 1.0% yeast extract, 0.2% to 2.0% potassium dihydrogen phosphate, 0.1% to 0.8% urea, and 0.01% to 0.10% (w / v) defoamer.

[0058] In some specific embodiments, the fermentation medium comprises 1%–5% glucose, 0.1%–1.0% corn steep liquor, 0.1%–0.6% yeast extract, 0.1%–0.8% potassium nitrate, 0.1%–0.8% potassium dihydrogen phosphate, 0.05%–0.35% ammonium sulfate, 0.02%–0.30% urea, 0.05%–0.40% sodium chloride, and 0.01%–0.10% (w / v) defoamer.

[0059] In some implementations, after a certain fermentation time, the addition of fermentation substrate is stopped, and fermentation continues using the remaining substrate in the fermentation system until the fermentation substrate is completely consumed or no longer long-chain dicarboxylic acids are produced. The total fermentation time is calculated.

[0060] The fermentation broth produced by fermentation can be used directly as a product, or long-chain dicarboxylic acids can be extracted from the fermentation broth as a final product.

[0061] A second aspect of the present invention provides a method for preparing a long-chain dicarboxylic acid, the method comprising:

[0062] S1: Obtain a long-chain dicarboxylic acid fermentation broth by fermentation production method according to any of the above-mentioned methods;

[0063] S2: Extract and purify the long-chain dicarboxylic acid fermentation broth to obtain the long-chain dicarboxylic acid product.

[0064] In some specific embodiments, the extraction and purification includes: acidifying the fermentation broth, separating the solids, dissolving the solids in an organic solvent, crystallizing and separating the solids, washing and drying the separated solids to obtain a long-chain dicarboxylic acid product.

[0065] In some other specific embodiments, the extraction and purification includes: acidifying the fermentation broth, separating the solid, dissolving the solid in an organic solvent, crystallizing and separating the solid, and then treating the separated solid with hot water, washing and drying to obtain a long-chain dicarboxylic acid product.

[0066] In some embodiments, the pH of the acidification is 2-5, preferably 3.5-4.5. Acidification causes the long-chain dicarboxylic acids in the fermentation broth to crystallize.

[0067] In some embodiments, the above-described solid-liquid separation method includes at least one of filtration or centrifugation.

[0068] In some embodiments, the mass ratio of solids to organic solvent is 1:(3-6).

[0069] In some embodiments, the organic solvent includes one or more of acids, alcohols, esters, and ketones; wherein the alcohol includes one or more of methanol, ethanol, n-butanol, and isopropanol, the acid includes acetic acid, the ketone includes acetone, and the ester includes at least one of ethyl acetate and butyl acetate. In some embodiments, the solid is decolorized after being dissolved in the organic solvent and before crystallization.

[0070] In some embodiments, the amount of decolorizing agent added during the decolorization process is 0.5%-10% of the mass of the long-chain dicarboxylic acid contained in the decolorizing solution, more specifically 1%-5%, and more specifically 1.2%-4.8%.

[0071] In some embodiments, the decolorizing agent is activated carbon.

[0072] In some embodiments, the decolorization temperature is 80-110°C, and the decolorization time is 30-190 min.

[0073] In some embodiments, the temperature of the hot water treatment is 70-150°C, and more specifically 70-135°C.

[0074] In some embodiments, the hot water treatment includes the following steps: contacting the separated solids with hot water to reduce the impurity content, wherein the temperature of the hot water is 70-150°C, or more specifically 70-135°C.

[0075] In some embodiments, the hot water treatment includes the following steps: mixing solid and water at a mass ratio of 1:(1-10), keeping the mixture at 70-150°C for 30-180 minutes, cooling it to 25-60°C, separating the solid and liquid to obtain the solid.

[0076] In some embodiments, the hot water treatment includes the following steps: mixing solid and water at a mass ratio of 1:(1-10), keeping the mixture at 70-135°C for 30-180 minutes, cooling it to 25-60°C, separating the solid and liquid to obtain the solid.

[0077] In some embodiments, the solid obtained after hot water treatment is washed and dried.

[0078] In some embodiments, the crystallization is cooling crystallization, and the endpoint temperature of the cooling crystallization is 20-40°C.

[0079] A third aspect of the present invention also provides a long-chain dicarboxylic acid product, which is prepared by the above-described preparation method, wherein the purity of the long-chain dicarboxylic acid product is 98% or more, and further 99% or more.

[0080] The bio-based content of long-chain dicarboxylic acid products can be adjusted according to the fermentation substrate, ranging from 1% to 100%, further to 5% to 100%, further to 5% to 99%, further to 5% to 80%, and further to 5% to 50%, for example, 100%, 98%, 95%, 80%, 70%, 50%, 45%, 35%, 30%, 25%, 15%, 10%, etc. When the fermentation substrate is derived from vegetable oils, such as coconut oil or palm oil, the corresponding long-chain dicarboxylic acid obtained through fermentation will have the corresponding bio-based content. For example, lauric acid can be obtained through the hydrolysis or high-temperature and high-pressure decomposition of coconut oil, and methyl laurate can be prepared from lauric acid and methanol through an esterification reaction. Dodecanoic acid produced by fermentation using this type of lauric acid or methyl laurate as the fermentation substrate will have the corresponding bio-based content.

[0081] The technical solution of this invention has the following technical effects:

[0082] This invention uses green raw materials such as fatty acids, fatty acid salts, fatty acid esters, and bio-based alkanes as substrates, partially or completely replacing fossil energy sources such as petroleum-based alkanes. It can obtain long-chain dicarboxylic acid products with different bio-based contents, which is in line with the concept of sustainable development of material sources. In addition, by adding carbon sources during the fermentation process and ensuring that the carbon source feeding rate meets the specific stoichiometric relationship with the fermentation time, it is beneficial to the efficient conversion of fermentation substrates by Candida albicans, thereby significantly increasing the yield of the target long-chain dicarboxylic acid. Through further extraction and purification of the fermentation broth, high-purity long-chain dicarboxylic acid products can be obtained to meet the requirements of polymerization reactions. Detailed Implementation

[0083] The preferred embodiments of the present invention will now be described in detail with reference to specific examples. It should be understood that the following examples are given for illustrative purposes only, to make the features and advantages of the present invention clearer, and are not intended to limit the scope of the present invention. Those skilled in the art can make various modifications and substitutions to the present invention without departing from its spirit and essence; the scope of the present invention is not limited to the embodiments listed herein.

[0084] Unless otherwise specified, the experimental methods used in the following examples are all known methods and conventional conditions in the art. Unless otherwise specified, the materials and reagents used in the following examples are commercially available. Raw materials requiring sterilization were sterilized at 121°C for 20 minutes. Unless otherwise specified, the percentages used to characterize the component content in the culture media of this invention follow general conventions in the fermentation field; percentages represent weight-volume ratios (w / v), i.e., % represents g / 100mL.

[0085] The strain used in the following examples and comparative examples is Candida viswanathii CAES2113, which has been disclosed in patent application CN111748480A and deposited on February 24, 2020, at the China Center for Type Culture Collection (address: Wuhan University, Wuhan, China), with accession number CCTCC M 2020048, and classified as Candida viswanathii.

[0086] YPD medium (w / v): 2.0% peptone, 2.0% glucose and 1.0% yeast extract (OXOID, LP0021). Solid medium should also contain 1.5% agar powder.

[0087] Seed culture medium (w / v): sucrose 3.3%, yeast extract 0.6%, corn steep liquor 0.6%, potassium dihydrogen phosphate 0.8%, urea 0.5%, defoamer (polyether) 0.05%.

[0088] Fermentation medium (w / v): glucose 3.5%, corn steep liquor 0.6%, yeast extract 0.5%, potassium nitrate 0.5%, potassium dihydrogen phosphate 0.4%, urea 0.3%, ammonium sulfate 0.15%, defoamer (polyether) 0.04%, and sodium chloride 0.2%.

[0089] In the following examples and comparative examples, gas chromatography was used to determine the content of dicarboxylic acids in the fermentation broth and the purity of the dicarboxylic acid products.

[0090] The following example describes the method for determining the bio-based content of dicarboxylic acid products: the method is performed by determining the content of radioactive C14, such as the American Society for Testing and Materials standard ASTM-D6866 method.

[0091] In the following examples and comparative examples, when some of the fermentation substrate was solid, it was first heated and dissolved at 45–65°C before being added to the fermenter.

[0092] Example 1

[0093] (1) Activation culture: The pH of 100 mL of malt extract at 10 Baume degrees was adjusted to about 5.5, and then glycerol seed tubes of Candida virescens (strain CAES2113) were inoculated. The cells were activated and cultured on a rotary shaker at 31℃ and 210 rpm for 40 h. The cell OD 620 When the value reaches 0.75 (30-fold dilution), the activation culture ends and the shake flask seeds are obtained.

[0094] (2) Seed culture: The shake flask seeds obtained in step (1) were inoculated into a seed tank containing seed culture medium at an inoculation rate of 1.8% (v / v). The seed culture was carried out at a temperature of 28°C, an air flow rate of 0.3 vvm, a pressure of 0.12 MPa, and a dissolved oxygen content of 15% until the OD of the seed culture was 30 times diluted. 620 The culture was terminated when the value reached 0.8.

[0095] (3) Fermentation culture: The seed culture obtained in step (2) was inoculated into a fermenter containing 5L of fermentation medium at an inoculation rate of 15% (v / v). Fermentation culture was carried out under the conditions of 30℃, air flow rate of 0.4vvm, pressure of 0.15MPa, and dissolved oxygen of 50%. When the OD of the strain... 620 When the pH reaches 0.75 (30-fold dilution), fermentation substrate is added to the fermenter. The fermentation substrate is lauric acid and petroleum-based n-dodecane in a mass ratio of 1:6. The fermentation pH is controlled at 6.3, and the substrate concentration in the fermentation broth is controlled below 1.5% (v / v). After 150 hours of fermentation, the addition of substrate is stopped, and the remaining substrate in the fermentation system is used for fermentation until the substrate is completely consumed or no more dodecanoic acid is produced.

[0096] V 初始 The volume of the fermentation medium is 5L. When fermentation has been going on for 14 hours, a 50wt% glucose solution is continuously added to the fermentation broth.

[0097] When the fermentation time is between 14h and 50h, the dynamic carbon source feeding system adjusts the glucose feeding rate V1 in real time according to the detected fermentation time, so that its numerical relationship with the fermentation time t satisfies the following:

[0098] A1 = 0.65, B1 = 1.0;

[0099] When the fermentation time is between 50h and 80h, the dynamic carbon source feeding system adjusts the glucose feeding rate V2 in real time according to the detected fermentation time, so that its numerical relationship with the fermentation time t satisfies the following:

[0100] A² = 0.19, B² = 1.2;

[0101] When the fermentation time is between 80h and 145h, the dynamic carbon source feeding system adjusts the glucose feeding rate V3 in real time according to the detected fermentation time, so that its numerical relationship with the fermentation time t satisfies the following: A3 = 0.0002, B3 = 0.063, C3 = 5.1, the sugar concentration in the fermentation broth was 0.5 g / L, and glucose was stopped after 145 h of fermentation.

[0102] Example 2

[0103] The fermentation process was basically the same as in Example 1, except that: in step (3), the fermentation substrate was lauric acid and bio-based n-dodecane, with a mass ratio of 1:8; after 10 hours of fermentation, a 50wt% glucose solution was continuously added to the fermentation broth.

[0104] When the fermentation time is between 10h and 50h, the dynamic carbon source feeding system adjusts the glucose feeding rate V1 in real time according to the detected fermentation time, so that its numerical relationship with the fermentation time t satisfies the following:

[0105] A1 = 0.6, B1 = 0.8;

[0106] When the fermentation time is between 50h and 80h, the dynamic carbon source feeding system adjusts the glucose feeding rate V2 in real time according to the detected fermentation time, so that its numerical relationship with the fermentation time t satisfies the following:

[0107] A² = 0.16, B² = 1.5;

[0108] When the fermentation time is between 80h and 145h, the dynamic carbon source feeding system adjusts the glucose feeding rate V3 in real time according to the detected fermentation time, so that its numerical relationship with the fermentation time t satisfies the following: A3 = 0.00025, B3 = 0.07, C3 = 5.6, the sugar concentration in the fermentation broth was 0.5 g / L, and glucose was stopped after 145 h of fermentation.

[0109] Example 3

[0110] The fermentation process for producing dodecanoic acid was essentially the same as in Example 1, except that in step (3), the fermentation substrate was methyl laurate and petroleum-based n-dodecane in a mass ratio of 1:8; and after 14 hours of fermentation, a 50 wt% glucose solution was continuously added to the fermentation broth.

[0111] When the fermentation time is between 14h and 50h, the dynamic carbon source feeding system adjusts the glucose feeding rate V1 in real time according to the detected fermentation time, so that its numerical relationship with the fermentation time t satisfies the following:

[0112] A1 = 0.68, B1 = 1.2;

[0113] When the fermentation time is between 50h and 80h, the dynamic carbon source feeding system monitors the fermentation time and adjusts the glucose feeding rate V2 in real time to ensure that its relationship with the fermentation time t satisfies the following: A2 = 0.2, B2 = 1.1;

[0114] When the fermentation time is between 80h and 145h, the dynamic carbon source feeding system adjusts the glucose feeding rate V3 in real time according to the detected fermentation time, so that its numerical relationship with the fermentation time t satisfies the following: A3 = 0.00028, B3 = 0.08, C3 = 6.0, the sugar concentration in the fermentation broth was 0.3 g / L, and glucose was stopped after 145 h of fermentation.

[0115] Example 4

[0116] The fermentation process for producing dodecanoic acid was basically the same as in Example 1, except that in step (3), the fermentation substrate was methyl laurate and lauric acid in a mass ratio of 5:1; and after 10 hours of fermentation, a 50wt% glucose solution was continuously added to the fermentation broth.

[0117] When the fermentation time is between 10h and 50h, the dynamic carbon source feeding system adjusts the glucose feeding rate V1 in real time according to the detected fermentation time, so that its numerical relationship with the fermentation time t satisfies the following:

[0118] A1 = 0.7, B1 = 1.3;

[0119] When the fermentation time is between 50h and 80h, the dynamic carbon source feeding system adjusts the glucose feeding rate V2 in real time according to the detected fermentation time, so that its numerical relationship with the fermentation time t satisfies the following:

[0120] A² = 0.17, B² = 1.2;

[0121] When the fermentation time is between 138 and 80h, the dynamic carbon source feeding system adjusts the glucose feeding rate V3 in real time according to the detected fermentation time, so that its numerical relationship with the fermentation time t satisfies the following: A3 = 0.00018, B3 = 0.065, C3 = 5.7, the sugar concentration in the fermentation broth was 0.4 g / L, and glucose was stopped after 138 h of fermentation.

[0122] Example 5

[0123] The fermentation process for producing dodecanoic acid was essentially the same as in Example 1, except that in step (3), the fermentation substrate was methyl laurate; and after 12 hours of fermentation, a 50 wt% glucose solution was continuously added to the fermentation broth.

[0124] When 12h < fermentation time ≤ 50h, the dynamic carbon source feeding system adjusts the glucose feeding rate V1 in real time according to the detected fermentation time, so that its numerical relationship with the fermentation time t satisfies:

[0125] A1 = 0.67, B1 = 1.1;

[0126] When the fermentation time is between 50h and 80h, the dynamic carbon source feeding system adjusts the glucose feeding rate V2 in real time according to the detected fermentation time, so that its numerical relationship with the fermentation time t satisfies the following:

[0127] A² = 0.18, B² = 1.2;

[0128] When the fermentation time is between 80h and 140h, the dynamic carbon source feeding system adjusts the glucose feeding rate V3 in real time according to the detected fermentation time, so that its numerical relationship with the fermentation time t satisfies the following: A3 = 0.0002, B3 = 0.072, C3 = 6.3, the sugar concentration in the fermentation broth was 0.3 g / L, and glucose was stopped after 140 h of fermentation.

[0129] Example 6

[0130] The fermentation process was basically the same as in Example 1, except that in step (3), the fermentation substrate was myristic acid and bio-based n-tetradecane in a mass ratio of 1:6; and after 12 hours of fermentation, a 50 wt% glucose solution was continuously added to the fermentation broth.

[0131] When 12h < fermentation time ≤ 50h, the dynamic carbon source feeding system adjusts the glucose feeding rate V1 in real time according to the detected fermentation time, so that its numerical relationship with the fermentation time t satisfies:

[0132] A1 = 0.63, B1 = 0.7;

[0133] When the fermentation time is between 50h and 80h, the dynamic carbon source feeding system adjusts the glucose feeding rate V2 in real time according to the detected fermentation time, so that its numerical relationship with the fermentation time t satisfies the following:

[0134] A² = 0.16, B² = 1.3;

[0135] When the fermentation time is between 80h and 140h, the dynamic carbon source feeding system adjusts the glucose feeding rate V3 in real time according to the detected fermentation time, so that its numerical relationship with the fermentation time t satisfies the following: A3 = 0.00022, B3 = 0.075, C3 = 6.2, the sugar concentration in the fermentation broth was 0.5 g / L, and glucose was stopped after 140 h of fermentation.

[0136] Example 7

[0137] The fermentation process was basically the same as in Example 1, except that in step (3), the fermentation substrate was palmitic acid and bio-based n-hexadecane in a mass ratio of 1:6; and after 12 hours of fermentation, a 50 wt% glucose solution was continuously added to the fermentation broth.

[0138] When 12h < fermentation time ≤ 50h, the dynamic carbon source feeding system adjusts the glucose feeding rate V1 in real time according to the detected fermentation time, so that its numerical relationship with the fermentation time t satisfies:

[0139] A1 = 0.65, B1 = 0.8;

[0140] When the fermentation time is between 50h and 80h, the dynamic carbon source feeding system adjusts the glucose feeding rate V2 in real time according to the detected fermentation time, so that its numerical relationship with the fermentation time t satisfies the following:

[0141] A² = 0.17, B² = 1.3;

[0142] When the fermentation time is between 80h and 140h, the dynamic carbon source feeding system adjusts the glucose feeding rate V3 in real time according to the detected fermentation time, so that its numerical relationship with the fermentation time t satisfies the following: A3 = 0.0002, B3 = 0.07, C3 = 6.0, the sugar concentration in the fermentation broth was 0.5 g / L, and glucose was stopped after 140 h of fermentation.

[0143] Comparative Example 1

[0144] The fermentation process for producing dodecadicarboxylic acid is essentially the same as in Example 1, with the difference being:

[0145] When the fermentation time is between 14h and 50h, the dynamic carbon source feeding system adjusts the glucose feeding rate V1 in real time according to the detected fermentation time, so that its numerical relationship with the fermentation time t satisfies the following:

[0146] A1 = 0.45, B1 = 1.0;

[0147] When the fermentation time is between 50h and 80h, the dynamic carbon source feeding system adjusts the glucose feeding rate V2 in real time according to the detected fermentation time, so that its numerical relationship with the fermentation time t satisfies the following:

[0148] A² = 0.08, B² = 1.2;

[0149] When the fermentation time is between 80h and 145h, the dynamic carbon source feeding system adjusts the glucose feeding rate V3 in real time according to the detected fermentation time, so that its numerical relationship with the fermentation time t satisfies the following: A3 = 0.0002, B3 = 0.063, C3 = 5.1, and the rest are the same as in Example 1.

[0150] Comparative Example 2

[0151] The fermentation process for producing tetradecanoic acid is essentially the same as in Example 6, with the difference being:

[0152] When 12h < fermentation time ≤ 50h, the dynamic carbon source feeding system adjusts the glucose feeding rate V1 in real time according to the detected fermentation time, so that its numerical relationship with the fermentation time t satisfies:

[0153] A1 = 0.48, B1 = 0.7;

[0154] When the fermentation time is between 50h and 80h, the dynamic carbon source feeding system adjusts the glucose feeding rate V2 in real time according to the detected fermentation time, so that its numerical relationship with the fermentation time t satisfies the following:

[0155] A² = 0.05, B² = 1.3;

[0156] When the fermentation time is between 80h and 140h, the dynamic carbon source feeding system adjusts the glucose feeding rate V3 in real time according to the detected fermentation time, so that its numerical relationship with the fermentation time t satisfies the following: A3 = 0.00022, B3 = 0.075, C3 = 6.2; other values ​​are the same as in Example 6.

[0157] Comparative Example 3

[0158] The fermentation process for producing hexadecanoic acid is essentially the same as in Example 7, except that:

[0159] When 12h < fermentation time ≤ 50h, the dynamic carbon source feeding system adjusts the glucose feeding rate V1 in real time according to the detected fermentation time, so that its numerical relationship with the fermentation time t satisfies:

[0160] A1 = 0.82, B1 = 0.8;

[0161] When the fermentation time is between 50h and 80h, the dynamic carbon source feeding system adjusts the glucose feeding rate V2 in real time according to the detected fermentation time, so that its numerical relationship with the fermentation time t satisfies the following:

[0162] A² = 0.33, B² = 1.3;

[0163] When the fermentation time is between 80h and 140h, the dynamic carbon source feeding system adjusts the glucose feeding rate V3 in real time according to the detected fermentation time, so that its numerical relationship with the fermentation time t satisfies the following: A3 = 0.0002, B3 = 0.07, C3 = 6.0; the rest is the same as in Example 7.

[0164] Comparative Example 4

[0165] The fermentation process for producing dodecadicarboxylic acid is essentially the same as in Example 1, except that when the fermentation time is between 12 hours and 50 hours, the fermentation time is controlled...

[0166] When 50h < fermentation time ≤ 80h, control

[0167] When fermentation time is between 80h and 140h, control Everything else is the same as in Example 1.

[0168] The fermentation substrates, total amount of substrate added, relationship between sugar addition rate and fermentation time at different time periods, acid production, and total fermentation time of Examples 1-7 and Comparative Examples 1-4 are shown in Table 1 below. Acid production represents the concentration of the target long-chain dicarboxylic acid detected in the fermentation broth.

[0169]

[0170]

[0171] Preparation of long-chain dicarboxylic acid products

[0172] Sulfuric acid was added to the fermentation broth of the long-chain dicarboxylic acid prepared in Examples 1-7 to adjust the pH to 4.0 for acidification and crystallization. The solid was separated to obtain crude long-chain dicarboxylic acid.

[0173] The crude long-chain dicarboxylic acid was placed in a decolorization tank, and acetic acid was added, with the mass ratio of crude long-chain dicarboxylic acid to acetic acid controlled at 1:3. Activated carbon was added at a concentration of 3.5% of the mass of the long-chain dicarboxylic acid in the decolorization solution. The mixture was heated to 92°C and decolorized for 40 minutes. The resulting liquid was filtered through a plate and frame filter press. The clarified liquid was cooled to 30°C, and crystals were precipitated. The crystals were then separated by centrifugation. The solid obtained was added to water at a mass ratio of 1:10, and the mixture was kept at 105°C for 90 minutes. After cooling to 32°C, the mixture was filtered to obtain a solid. The solid was washed and dried to obtain the long-chain dicarboxylic acid product. The purity and bio-based content of the dodecanoic acid product are shown in Table 2.

[0174] Table 2

[0175] No. Diacid type Purity wt% Biobased content (wt%) Example 1 DC12 99.91 12.41 Example 2 DC12 99.89 100 Example 3 DC12 98.86 9.04 Example 4 DC12 99.83 100 Example 5 DC12 99.75 100 Example 6 DC14 99.81 100 Example 7 DC16 98.88 100

[0176] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for fermentative production of long-chain diacids, characterized in that, It comprises: fermenting a culture of Candida utilis, adding a fermentation substrate to perform fermentation, when the fermentation time is t0, start to add carbon source to the fermentation broth: When t0 < fermentation time ≤ t1, the numerical relationship between the carbon source feeding rate V1 and the fermentation time t satisfies: 0.5≤A1≤0.8, 0.5≤B1≤2.0; When t1 < fermentation time ≤ t2, the numerical relationship between the control carbon source feeding rate V2 and the fermentation time t satisfies: 0.1≤A2≤0.3, 0.5≤B2≤3.0; When t2 < fermentation time ≤ t3, the numerical relationship between the control carbon source feeding rate V3 and the fermentation time t satisfies: 0.0001≤A3≤0.003, 0.05≤B3≤0.08, 4.0≤C3≤8.0; Wherein, 8≤t0≤14, 45≤t1≤55, 75≤t2≤85, 130≤t3≤145, the feeding rate V1, V2, V3 of the carbon source is g / h, the fermentation time t is h, V 初始 represents the volume of the fermentation medium, and the unit is L; The fermentation substrate is selected from any one or combination of fatty acids, fatty acid esters, fatty acid salts or alkanes with carbon atom number of 8-18.

2. The method for fermentative production of long-chain diacids according to claim 1, characterized in that, 0.6≤A1≤0.7; and / or, 0.5≤B1≤1.5; and / or, 0.15≤A2≤0.2; and / or, 1.0≤B2≤2.0; and / or, 0.00015≤A3≤0.0003; and / or, 0.06≤B3≤0.08; and / or, 5.0≤C3≤7.

0.

3. The method for fermentative production of long-chain diacids according to claim 1, characterized in that, When t0 < fermentation time < t1, control Further to When t1 < fermentation time < t2, control Further to When t2 < fermentation time < t3, control Further to 4. The method for fermentative production of long-chain diacids according to claim 1, characterized in that, The fatty acid comprises a straight-chain monobasic acid with carbon atom number of 8-18; preferably, the fatty acid is selected from a combination of one or more of nonaenoic acid, decanoic acid, undecanoic acid, lauric acid, tridecanoic acid, myristic acid, pentadecanoic acid, palmitic acid, heptadecanoic acid and octadecanoic acid; and / or, The fatty acid ester is selected from a combination of one or more of fatty acid methyl ester, fatty acid ethyl ester, fatty acid propyl ester or fatty acid butyl ester; and / or, The fatty acid ester is selected from a combination of one or more of nonaenoic acid ester, decanoic acid ester, undecanoic acid ester, lauric acid ester, tridecanoic acid ester, myristic acid ester, pentadecanoic acid ester, palmitic acid ester, heptadecanoic acid ester or octadecanoic acid ester, more preferably from a combination of one or more of lauric acid methyl ester, lauric acid ethyl ester, lauric acid butyl ester, lauric acid glycerol ester, myristic acid methyl ester, myristic acid ethyl ester, myristic acid butyl ester, palmitic acid methyl ester, palmitic acid ethyl ester or palmitic acid butyl ester; and / or, The fatty acid salt is selected from a combination of one or more of sodium salt, potassium salt, ammonium salt and calcium salt of fatty acid; and / or, The alkane is selected from a combination of one or more of nonane, decane, undecane, dodecane, tridecane, tetradecane, pentadecane, hexadecane, heptadecane and octadecane, preferably normal alkane; and / or, HOOC(CH2) n COOH, wherein n is 6 to 18, including any of suberic acid, azelaic acid, sebacic acid, undecanedioic acid, dodecanedioic acid, tridecanedioic acid, tetradecanedioic acid, pentadecanedioic acid, hexadecanedioic acid, heptadecanedioic acid, and octadecanedioic acid.

5. The method for fermentative production of long-chain diacids according to claim 1, characterized in that, The fermentation substrate is selected from any one or more of the following combinations: (1) a combination of fatty acid and fatty acid ester; (2) a combination of fatty acid and alkane; (3) a combination of fatty acid ester and alkane; (4) a combination of fatty acid salt and alkane; or, (5) a combination of fatty acid, fatty acid ester and alkane; Preferably, the fermentation substrate is a combination of fatty acid and fatty acid ester with mass ratio of 1:99-99:1; a combination of fatty acid and alkane with mass ratio of 1:99-99:1; a combination of fatty acid ester and alkane with mass ratio of 1:99-99:1; a combination of fatty acid salt and alkane with mass ratio of 1:99-99:1; or, a combination of fatty acid, fatty acid ester and alkane with any mass ratio.

6. The method for fermentative production of long-chain diacids according to claim 1, characterized in that, The added carbon source is selected from at least one of glucose, sucrose, lactose, maltose, fructose, molasses, glycerol, sorbitol, arabinose, rhamnose, cellobiose, sophorose and gentiobiose, more preferably from at least one of glucose, lactose and sucrose; and / or, the sugar concentration in the fermentation broth is maintained in the range of 0.1-1 g / L by adding the carbon source.

7. The method of fermentative production of long-chain dibasic acids according to claim 1, characterized in that, When the optical density value OD of the strain 620 When diluted 30 times to reach a concentration of 0.5-1.0, add the fermentation substrate to begin fermentation; and / or, the Candida includes at least one of Candida viswanathii, Candida albicans, Candida tropicalis, Candida sake or Yarrowia lipolytica; and / or, during the fermentation, the air flow for the fermentation is controlled to be 0.2-0.8vvm; and / or, the pressure for the fermentation is controlled to be 0.05-0.20MPa; and / or, the temperature for the fermentation is controlled to be 27-35℃; and / or, the pH value for the fermentation is controlled to be 4.5-7.5; and / or, the dissolved oxygen for the fermentation is controlled to be 5%-90%, further 25%-70%.

8. A method for producing long-chain dibasic acid, characterized by, the method comprises: S1: obtaining long-chain dicarboxylic acid fermentation liquor by the method for producing long-chain dicarboxylic acid by fermentation according to any one of claims 1-7; S2: acidifying the long-chain dicarboxylic acid fermentation liquor, separating to obtain solid, dissolving the solid in an organic solvent, crystallizing, solid-liquid separating, washing and drying the separated solid to obtain long-chain dicarboxylic acid product.

9. The production method according to claim 8, wherein In step S2, before washing the separated solid, the separated solid is treated with hot water, and then washed and dried; Preferably, the temperature for the hot water treatment is 70-150℃; Preferably, the hot water treatment comprises the following steps: mixing the solid and water according to a mass ratio of 1:(1-10), keeping the mixture at 70-150℃ for 30-180min, cooling to 25-60℃, and then solid-liquid separating to obtain the solid.

10. A long-chain dibasic acid product characterized by, The purity of the long-chain dicarboxylic acid product is above 98%, further above 99%; and / or, the bio-based content is 1-100%, further 5-100%.

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