CN117210343A - Candida tropicalis and application thereof, and production method of long-chain dibasic acid - Google Patents
Candida tropicalis and application thereof, and production method of long-chain dibasic acid Download PDFInfo
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- CN117210343A CN117210343A CN202311329545.2A CN202311329545A CN117210343A CN 117210343 A CN117210343 A CN 117210343A CN 202311329545 A CN202311329545 A CN 202311329545A CN 117210343 A CN117210343 A CN 117210343A
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- fermentation
- long
- percent
- dibasic acid
- chain dibasic
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- 239000002253 acid Substances 0.000 title claims abstract description 148
- 241000222178 Candida tropicalis Species 0.000 title claims abstract description 55
- 238000004519 manufacturing process Methods 0.000 title claims abstract description 43
- 238000000855 fermentation Methods 0.000 claims abstract description 216
- 230000004151 fermentation Effects 0.000 claims abstract description 216
- 238000000034 method Methods 0.000 claims abstract description 65
- 238000002360 preparation method Methods 0.000 claims abstract description 12
- 238000006243 chemical reaction Methods 0.000 claims description 49
- 239000007788 liquid Substances 0.000 claims description 36
- 150000001335 aliphatic alkanes Chemical class 0.000 claims description 28
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 claims description 24
- 239000008103 glucose Substances 0.000 claims description 24
- FGIUAXJPYTZDNR-UHFFFAOYSA-N potassium nitrate Chemical compound [K+].[O-][N+]([O-])=O FGIUAXJPYTZDNR-UHFFFAOYSA-N 0.000 claims description 24
- 150000007513 acids Chemical class 0.000 claims description 23
- 239000000243 solution Substances 0.000 claims description 20
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 claims description 19
- XSQUKJJJFZCRTK-UHFFFAOYSA-N Urea Chemical compound NC(N)=O XSQUKJJJFZCRTK-UHFFFAOYSA-N 0.000 claims description 17
- 230000012010 growth Effects 0.000 claims description 17
- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 claims description 16
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 14
- 229910000402 monopotassium phosphate Inorganic materials 0.000 claims description 14
- 235000019796 monopotassium phosphate Nutrition 0.000 claims description 14
- 238000001816 cooling Methods 0.000 claims description 13
- 239000007787 solid Substances 0.000 claims description 13
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 claims description 12
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 12
- 229940041514 candida albicans extract Drugs 0.000 claims description 12
- 239000004323 potassium nitrate Substances 0.000 claims description 12
- 235000010333 potassium nitrate Nutrition 0.000 claims description 12
- 239000012138 yeast extract Substances 0.000 claims description 12
- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical compound N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 claims description 11
- 229910052921 ammonium sulfate Inorganic materials 0.000 claims description 11
- 235000011130 ammonium sulphate Nutrition 0.000 claims description 11
- 230000003287 optical effect Effects 0.000 claims description 11
- 240000008042 Zea mays Species 0.000 claims description 10
- 235000005824 Zea mays ssp. parviglumis Nutrition 0.000 claims description 10
- 235000002017 Zea mays subsp mays Nutrition 0.000 claims description 10
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- 238000002425 crystallisation Methods 0.000 claims description 10
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- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 claims description 9
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 9
- 239000004202 carbamide Substances 0.000 claims description 9
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- 238000000926 separation method Methods 0.000 claims description 9
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 claims description 8
- 229910052943 magnesium sulfate Inorganic materials 0.000 claims description 8
- 235000019341 magnesium sulphate Nutrition 0.000 claims description 8
- GNSKLFRGEWLPPA-UHFFFAOYSA-M potassium dihydrogen phosphate Chemical compound [K+].OP(O)([O-])=O GNSKLFRGEWLPPA-UHFFFAOYSA-M 0.000 claims description 8
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- 230000000694 effects Effects 0.000 abstract description 6
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- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 21
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- 239000007853 buffer solution Substances 0.000 description 6
- KRKNYBCHXYNGOX-UHFFFAOYSA-N citric acid Chemical compound OC(=O)CC(O)(C(O)=O)CC(O)=O KRKNYBCHXYNGOX-UHFFFAOYSA-N 0.000 description 6
- 235000014113 dietary fatty acids Nutrition 0.000 description 6
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- DXNCZXXFRKPEPY-UHFFFAOYSA-N tridecanedioic acid Chemical compound OC(=O)CCCCCCCCCCCC(O)=O DXNCZXXFRKPEPY-UHFFFAOYSA-N 0.000 description 5
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- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/14—Fungi; Culture media therefor
- C12N1/16—Yeasts; Culture media therefor
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- C12N1/165—Yeast isolates
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- C12P7/00—Preparation of oxygen-containing organic compounds
- C12P7/64—Fats; Fatty oils; Ester-type waxes; Higher fatty acids, i.e. having at least seven carbon atoms in an unbroken chain bound to a carboxyl group; Oxidised oils or fats
- C12P7/6409—Fatty acids
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- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/645—Fungi ; Processes using fungi
- C12R2001/72—Candida
- C12R2001/74—Candida tropicalis
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Abstract
The application discloses candida tropicalis (Candida tropicalis) strain CAT H1614, a microbial inoculum comprising the candida tropicalis strain CAT H1614, application of the candida tropicalis strain CAT H1614, a method for producing long-chain dibasic acid by fermentation and a preparation method of the long-chain dibasic acid. The candida tropicalis strain CAT H1614 can keep high activity for a long time in an environment with the pH value lower than 7.0, so that the candida tropicalis strain CAT H1614 is particularly suitable for producing long-chain dibasic acid by a biological method, has fast acid production and high yield in the fermentation process, and can also reduce the fermentation period. The long-chain binary acid fermentation method and the preparation method provided by the application have obvious cost advantages, and can effectively relieve the pressure on resources and environment, so that the method has obvious industrial value advantages.
Description
The application relates to a Chinese patent application of application number 2018101720915, application date 2018, 3 months and 1 day, and the application name of candida tropicalis, application of candida tropicalis and a production method of long-chain dibasic acid.
Technical Field
The application relates to the field of fermentation, in particular to candida tropicalis and application thereof in the field of fermentation, and a production method of long-chain dibasic acid.
Background
The Long Chain Dibasic Acid (LCDA) has the general formula (HOOC (CH) 2 ) nCOOH, n is more than or equal to 7), has very wide application, and can synthesize special nylon, high-grade spice, high-grade hot melt adhesive, cold-resistant plasticizer, high-grade lubricating oil, high-grade antirust agent, high-grade paint, coating and the like by taking long-chain dibasic acid as a raw material. Long chain dibasic acids can be synthesized generally by chemical or biological methods. The chemical method has long synthetic route, high temperature and high pressure are needed for the reaction, and the requirements on the catalyst are more severe, so that the variety of long-chain dicarboxylic acid on the industrial scale is less, and only few varieties such as twelve-carbon long-chain dicarboxylic acid are needed. The biological method is to take long-chain alkane as a substrate and obtain the long-chain alkane through microbial transformation, and the production process is at normal temperature and normal pressure, so that various long-chain dibasic acids such as C9 to C18 can be produced in large scale.
Biological production of long chain dibasic acids has been studied for many years. The scholars first screen strains from oil fields which can produce long-chain dicarboxylic acid, and mutagenesis is carried out on the strains to improve the yield of the long-chain dicarboxylic acid. Some scholars have also studied key enzymes in the synthesis of long chain dibasic acids. The literature for foreign research on long chain dibasic acids is mostly to genetically modify strains, and to enhance the yield of products by blocking or weakening the relevant enzyme system for beta oxidation of fatty acids and strengthening the enzyme system for alpha-omega oxidation of fatty acids. The published patents such as CN 1071951A, CN 1067725C, CN 1259424C, 200410018255.7, 200610029784.6 etc. provide methods for microbial fermentation production of long chain dibasic acids, which all require that the pH of the fermentation broth be adjusted to above 7.0 during the fermentation process, especially during the acidogenesis, mainly because: firstly, the fermentation strain candida tropicalis has higher enzyme activity in a slightly alkaline environment, so that the catalytic efficiency of the alkaline condition is high; secondly, long-chain dibasic acid can exist in the form of salt of dibasic acid dissolved in water under alkaline environment, so that good mass transfer is maintained in the fermentation process. Therefore, in the conventional method for producing long-chain dibasic acid by fermentation, the pH value of a fermentation system is required to be more than 7.0 during fermentation, particularly during the acid production period (conversion period). In addition, in the fermentation process, as the accumulation amount of long-chain dibasic acid increases, a large amount of alkali is required to neutralize the long-chain dibasic acid produced. In the fermented fermentation liquor, long-chain dibasic acid exists in the fermentation liquor in the form of salt, and a large amount of acid is needed to convert the salt of the long-chain dibasic acid in the system into long-chain dibasic acid products in the process of extracting and purifying the dibasic acid, so that the concentration of the salt in the fermentation treatment liquor system is up to 50000-70000ug/ml, the high-salt wastewater is difficult to treat, the environmental challenge is very important, and the development of the biological long-chain dibasic acid industry is seriously influenced.
Patent US 6569670B2 reports a method for producing long chain dibasic acid from fatty acid as a raw material in an environment of pH 5.8, which is mainly because above pH 7.0, fatty acid exists in the form of fatty acid salt, which is particularly easy to foam, and fermentation cannot be performed, so that fermentation must be performed at a lower pH, but strain metabolism is slow at a low pH, and production efficiency is low.
Disclosure of Invention
In order to overcome the defects in the existing fermentation production process of long-chain dibasic acid, the first object of the invention is to provide a novel candida tropicalis which has high activity in a fermentation system with the pH value below 7.0 and is very suitable for fermentation production of long-chain dibasic acid.
The second object of the invention is to provide a microbial inoculum comprising the candida tropicalis.
A third object of the invention is to provide the use of said candida tropicalis.
A fourth object of the present invention is to provide a method for producing long chain dibasic acid by fermentation.
The fifth object of the invention is to provide a method for preparing long chain dibasic acid.
It is a sixth object of the present invention to provide a long chain dibasic acid.
The candida tropicalis (Candida tropicalis) strain CAT H1614 provided by the invention has a preservation number of CCTCC NO: m2013143, which was preserved in China Center for Type Culture Collection (CCTCC) at 11 days of 4.2013 in Jiuqiu 299 Wuhan university of Wuhan district of Wuhan, china.
The invention also provides a microbial inoculum comprising the candida tropicalis strain CAT H1614.
The invention also provides application of the candida tropicalis strain CAT H1614 in preparing long-chain dibasic acid by fermentation.
The method for producing long-chain dibasic acid by fermentation adopts the candida tropicalis strain CAT H1614 to produce long-chain dibasic acid by fermentation.
In the method for producing long-chain dibasic acid by fermentation, the fermentation process comprises a strain growth period and a conversion period, wherein the pH value of a fermentation system is controlled to be below 7.0 in the conversion period; preferably 4.0 to 6.8; more preferably 5.0 to 6.5.
In the method for producing long-chain dibasic acid by fermentation provided by the invention, the strain grows to the optical density OD of the thalli after dilution by 30 times 620 When the pH value of the fermentation system is more than 0.5, controlling the pH value of the fermentation system to be below 7.0; preferably 4.0 to 6.8; more preferably 5.0 to 6.5.
In the method for producing long-chain dibasic acid by fermentation provided by the invention, the substrate used in the fermentation process comprises alkane, preferably comprises C9-C22 n-alkane; more preferably, the n-alkane comprises C9 to C18; most preferably, the n-alkane comprises C10, C11, C12, C13, C14, C15 or C16;
And/or, the medium used in the fermentation process comprises the following components (w/v): 1 to 5 percent of glucose, 0.1 to 0.9 percent of corn steep liquor, 0.1 to 0.5 percent of yeast extract, 0.05 to 1.2 percent of potassium nitrate, 0.05 to 1.0 percent of monopotassium phosphate, 0.05 to 0.3 percent of urea, 0.05 to 0.3 percent of ammonium sulfate and 0.05 to 0.2 percent of sodium chloride; or comprises the following components (w/v): 1 to 5 percent of glucose, 0.05 to 0.6 percent of potassium nitrate, 0.02 to 0.6 percent of monopotassium phosphate, 0.05 to 0.3 percent of ammonium sulfate and 0.05 to 0.3 percent of magnesium sulfate.
In the method for producing long-chain dibasic acid by fermentation, the inoculation amount of the strain is 10-30% in the fermentation process;
and/or, the temperature is 28-32 ℃;
and/or the air quantity is 0.3-0.7 vvm;
and/or the pressure is 0.05-0.14 MPa;
and/or the pH value of the fermentation system is not lower than 3.0, preferably 3.5-6.5 in the growth period of the strain.
The preparation method of the long-chain dibasic acid provided by the invention comprises the following steps:
s1: fermenting to obtain long-chain binary acid fermentation liquor; and
s2: extracting and purifying the obtained long-chain dibasic acid fermentation liquor to obtain the long-chain dibasic acid;
wherein, the step S1 adopts the method for producing long-chain dibasic acid by fermentation according to any one of the technical schemes to prepare the long-chain dibasic acid fermentation liquor.
In the preparation method of the long-chain dibasic acid provided by the invention, the extraction and purification treatment of the step S2 comprises the following steps: acidifying the obtained long-chain dibasic acid fermentation liquor, separating to obtain a solid, dissolving the solid in an organic solvent, separating to obtain clear liquid, and crystallizing to obtain the long-chain dibasic acid.
In the preparation method of the long-chain dibasic acid provided by the invention, the pH value of acidification is 2.5-5, preferably 3-4;
and/or, the separation method is centrifugation or filtration;
and/or, the organic solvent comprises: one or more of alcohols, acids, ketones, and esters; wherein the alcohol comprises one or more of methanol, ethanol, isopropanol and n-butanol; the acid comprises acetic acid; the ketone comprises acetone; the ester comprises ethyl acetate and/or butyl acetate;
and/or, after the solid is dissolved in an organic solvent, decoloring, and separating to obtain clear liquid, wherein the decoloring method is preferably activated carbon decoloring; the addition amount of the activated carbon is not more than 5% of the volume of the solution; the decoloring temperature is 85-100 ℃; the decoloring time is 15-165 min;
and/or, the crystallization is cooling crystallization; the cooling crystallization comprises the following steps: cooling to 65-80 ℃, preserving heat for 1-2 hours, cooling to 25-35 ℃ and crystallizing;
And/or, after said crystallization, separating the crystals, thereby obtaining the long chain dibasic acid; the separation method is centrifugal separation.
The invention also provides the long-chain dibasic acid prepared by the preparation method of the long-chain dibasic acid according to any one of the technical schemes.
The novel candida tropicalis strain CAT H1614 is obtained through screening, and can keep high activity for a long time in an environment with the pH value lower than 7.0, so that the candida tropicalis strain CAT H1614 is particularly suitable for producing long-chain dibasic acid by a biological method, is quick in acid production and high in yield, and can also reduce the fermentation period.
The long-chain diacid fermentation method and the preparation method provided by the invention can be used for fermentation in an acidic environment, on one hand, a large amount of precipitated particles (comprising a large amount of diacid crystals) can be directly obtained, on the other hand, the dosage of alkali liquor and the dosage of acid in the subsequent long-chain diacid extraction process can be effectively reduced, the extraction process of diacid products is simplified, and the amount of salt generated in the long-chain diacid production process is greatly reduced (reduced to one tenth or lower of the original salt production content). In addition, the candida tropicalis CAT H1614 has high catalytic activity, so that the process method provided by the invention has the advantages of shortening the fermentation time, improving the acid yield, reducing the using amount of a culture medium, being suitable for producing various long-chain dibasic acids, and the like. Compared with the existing production process, the method has the obvious cost advantage, and can effectively relieve the pressure on resources and environment, so that the method has very obvious industrial value advantage.
The invention relates to candida tropicalis (Candida tropicalis) strain CAT H1614, which has a preservation number of CCTCC NO: m2013143, which was preserved in China Center for Type Culture Collection (CCTCC) at 11 days of 4.2013 in Jiuqiu 299 Wuhan university of Wuhan district of Wuhan, china.
Drawings
FIG. 1 is a flow chart showing the preparation of the strain CAT H1614 of Candida tropicalis.
Detailed Description
The first aspect of the invention provides a candida tropicalis (Candida tropicalis) strain CAT H1614 which is preserved in China Center for Type Culture Collection (CCTCC), and is provided by the university of Wuhan, china, and has a preservation number of CCTCC NO: m2013143.
The candida tropicalis CAT H1614 provided by the invention can be prepared from candida tropicalis CCTCC NO: m203052 is obtained by mutagenesis and screening, and the mutagenesis and screening process is shown in figure 1.
The specific process can be as follows: candida tropicalis cctccc NO: m203052 (starting strain) glycerol tube is connected into a shake flask filled with YPD culture solution and is added with 0.001-0.02% of 5-fluorouracil, and shake-cultured for 16-24 h at 28-32 ℃ and 200-250 rpm. The grown culture was collected by centrifugation and washed several times with physiological saline, and finally cells were suspended with an equal amount of aqueous LiCl solution. Placing a proper amount of cell suspension in a sterile plate, irradiating and mutagenizing by adopting an ultraviolet lamp with the power of 15W and the irradiation distance of 20-30 cm for 60-120 s. Diluting the mutagenized cell suspension, coating a flat plate containing YPD culture medium, culturing for 48-72 h at 28-30 ℃, selecting a monoclonal with good growth to a pore plate containing a primary screening culture medium (including substrate long chain alkane in the culture medium), wherein the initial pH value is about 6.0-6.8, the pH value is reduced along with the accumulation of long chain dibasic acid in the process, alkali is not added in the process, the natural pH value is generally 4.5-6.8, the fermentation is carried out at 28-32 ℃, the rotation speed of a shaking table is 200-250 rpm, the fermentation period is 60-72 h, the strain with relatively high yield of long chain dibasic acid is selected from the strain, the strain is subjected to re-screening through the pore plate, the re-screened strain is subjected to 500ml shaking bottle fermentation re-screening, the 500ml shaking bottle fermentation is carried out at 28-32 ℃, the rotation speed is 200-250 rpm, the pH in the fermentation process is natural, the fermentation period is 96-120 h, and the tropical candida oil pipe strain with high yield of long chain dibasic acid is selected and is stored in the shaking table.
YPD medium was composed of the following components: 20g/L glucose, 10g/L yeast extract, 20g/L peptone and 2% agar.
The culture medium for the primary screening and the secondary screening comprises the following components: 1 to 5 percent of glucose, 0.1 to 0.9 percent of corn steep liquor, 0.1 to 0.5 percent of yeast extract, 0.05 to 1.2 percent of potassium nitrate, 0.05 to 1.0 percent of monopotassium phosphate, 0.05 to 0.3 percent of urea, 0.05 to 0.3 percent of ammonium sulfate and 0.05 to 0.2 percent of sodium chloride (w/v). The primary screening and the secondary screening can be performed by using common operation methods in the field, and the specific components of the culture medium can be adjusted or selected by a person skilled in the art according to the actual conditions of the process.
The candida tropicalis strain CAT H1614 can still keep high enzyme activity for a long time under the environment that the pH value is lower than 7.0, so that the candida tropicalis strain CAT H1614 is particularly suitable for producing long-chain dibasic acid by a biological method, has fast acid production and high yield, and can also reduce the fermentation period.
In a second aspect the invention provides a microbial agent comprising said candida tropicalis strain CAT H1614. The microbial inoculum can also comprise various nutrient substances which are conventional in the art and ensure the activity of candida tropicalis strains.
The third aspect of the invention provides the application of the candida tropicalis strain CAT H1614. The application is the application of candida tropicalis strain CAT H1614 in preparing long-chain dibasic acid by fermentation.
In a fourth aspect, the invention provides a method for producing long-chain dibasic acid by fermentation, which comprises the step of fermenting with the candida tropicalis strain CAT H1614 to produce long-chain dibasic acid.
In one embodiment of the method for producing long chain dibasic acid by fermentation according to the present invention, the process of fermentation may include a strain growth phase and a conversion phase, wherein the pH of the fermentation system is controlled to 7 or less, preferably 4.0 to 6.8, more preferably 5.0 to 6.5 in the conversion phase of fermentation.
Alternatively, when the strain grows to a cell optical density (OD 620 ) Above 0.5 (30 times dilution), the pH of the fermentation system is controlled to 7.0 or less, preferably 4.0 to 6.8, more preferably 5.0 to 6.5; specific examples of pH values may be: 3,3.1,3.2,3.3,3.4,3.5,3.6,3.7,3.8,3.9,4.0,4.1,4.2,4.3,4.4,4.5,4.6,4.7,4.8,4.9,5.0,5.1,5.2,5.3,5.4,5.5,5.6,5.7,5.8,5.9,6.0,6.1,6.2,6.3,6.4,6.5,6.6,6.7,6.8,6.9,7.0. The manner of adjusting or controlling the pH is not particularly limited, and may be one or a combination of several of a constant control of a certain pH value, an uncontrolled pH value, an no lower pH value, an no higher pH value, an up-regulation of a pH value, a down-regulation of a pH value, a control from outside the range into or naturally into the range of the pH value. The method for regulating and controlling the pH value is not limited, and common means in the field of fermentation, such as adding alkali liquor with proper concentration, can be adopted.
In one embodiment of the method for fermentatively producing long chain dibasic acids according to the present invention, the fermentation conversion process may be performed in a fermentation medium or a buffer solution. Specifically, when candida tropicalis (Candida tropicalis) strain CAT H1614 is cultured until the strain is diluted by thirty times, OD 620 Above 0.5, at this point, the addition of long chain alkane substrate may begin to initiate fermentation conversion. When the fermentation and conversion process is carried out in a fermentation medium, a substrate can be directly added into the medium, or a strain can be transferred into a fermentation tank, and then the fermentation medium and the substrate are added for fermentation and conversion; when the fermentation and conversion process is carried out in the buffer solution, the thalli obtained by culture can be firstly separated and transferred into the buffer solution, and the substrate is added for fermentation and conversion.
In one embodiment of the method for fermentatively producing long chain dibasic acids according to the present invention, the substrate for fermentation comprises an alkane, preferably an n-alkane comprising C9 to C22, more preferably an n-alkane comprising C9 to C18, most preferably an n-alkane comprising C10, C11, C12, C13, C14, C15 or C16.
In one embodiment of the method for producing long chain dibasic acid by fermentation according to the present invention, when fermentation conversion is performed in a fermentation medium, the components of the medium used for fermentation optionally include a carbon source, a nitrogen source, an inorganic salt, a nutritional factor, and the like. Wherein the carbon source may be candida fermentable sugars, comprising: glucose, sucrose, maltose, etc., the carbon source may be added in an amount of 1% to 10% (w/v); the nitrogen source may be organic nitrogen and/or inorganic nitrogen, the organic nitrogen including: one or more of yeast extract, peptone and corn steep liquor, wherein the inorganic nitrogen comprises: urea, ammonium sulfate and/or potassium nitrate, wherein the addition amount of the nitrogen source can be 0.1% -3% (w/v); the inorganic salts include: one or more of potassium dihydrogen phosphate, potassium chloride, magnesium sulfate, calcium chloride, ferric chloride and copper sulfate, wherein the addition amount of the inorganic salt can be 0.1-1.5% (w/v); the nutritional factors include: one or more of vitamin B1, vitamin B2, vitamin C and biotin, wherein the addition amount of the nutritional factors can be 0% -1% (w/v).
In a preferred embodiment of the method for producing a long-chain dibasic acid by fermentation according to the present invention, an aqueous medium (hereinafter referred to as "fermentation medium 1") comprising the following components: 1 to 5 percent of glucose, 0.1 to 0.9 percent of corn steep liquor, 0.1 to 0.5 percent of yeast extract, 0.05 to 1.2 percent of potassium nitrate, 0.05 to 1.0 percent of monopotassium phosphate, 0.05 to 0.3 percent of urea, 0.05 to 0.3 percent of ammonium sulfate and 0.05 to 0.2 percent of sodium chloride (w/v). The fermentation medium 1 can be suitable for fermentation production on a scale from a shake flask of several tens of milliliters to a fermenter of several hundred tons.
In another preferred embodiment of the method for producing long-chain dibasic acid by fermentation according to the present invention, an aqueous medium with a low nutrient concentration (hereinafter referred to as "fermentation medium 2") may also be selected depending on the pH of the fermentation system, which comprises the following components: 1 to 5 percent of glucose, 0.05 to 0.6 percent of potassium nitrate, 0.02 to 0.6 percent of monopotassium phosphate, 0.05 to 0.3 percent of ammonium sulfate and 0.05 to 0.3 percent of magnesium sulfate (w/v). The fermentation medium 2 reduces complex components such as corn steep liquor, yeast extract and the like, has more definite components, and can better control fermentation indexes and product quality. It can be prepared with water, sterilized at 121deg.C for 20min, and cooled to appropriate temperature for fermentation culture.
In one embodiment of the method for producing long-chain dibasic acid by fermentation according to the invention, fermentation conversion rate can be further improved by supplementing aqueous sugar solution as carbon source in fermentation conversion process. The sugar water solution can be fed in batch or continuous. The sugar can be common sucrose or glucose, and the concentration of the additional sugar water solution can be 10% -70% (w/v). The sugar concentration of the fermentation conversion system can be controlled to be 0.1% -1% (w/v) by supplementing the sugar water solution.
In one embodiment of the method for fermentatively producing a long chain dibasic acid according to the present invention, when the fermentative conversion process is performed in a buffer solution, the buffer solution may be a phosphate buffer solution. In a preferred embodiment, the buffer solution may be a potassium dihydrogen phosphate-disodium hydrogen phosphate buffer solution.
In one embodiment of the method for fermentatively producing long chain dibasic acids according to the present invention, the fermentation conditions may be controlled as follows: the inoculation amount of the candida tropicalis strain CAT H1614 can be 10-30 percent; in another embodiment, the temperature may be 28 to 32 ℃; in another embodiment, the air volume may be 0.3 to 0.7vvm; in another embodiment, the pressure may be 0.05 to 0.14MPa; in another embodiment, the dissolved oxygen during the fermentation conversion is not less than 10%.
In one embodiment of the method for producing a long-chain dibasic acid by fermentation according to the present invention, the pH of the system is not less than 3.0, preferably 3.5 to 6.5, during the growth phase of the strain culture.
In a preferred embodiment of the method for fermentatively producing long chain dibasic acids according to the present invention, the following steps may be included:
the seed bottle culture process comprises the following steps:
the glycerol tube strain of candida tropicalis is inoculated into a seed bottle filled with YPD culture solution, the pH is natural, and the culture is carried out for 1-2 days by shaking at 200-250 rpm at 28-32 ℃.
The seed tank culture process comprises the following steps:
taking shaking bottle seeds, inoculating the shaking bottle seeds into a seed tank filled with a seed culture medium, wherein the inoculum size is 10% -30%, the initial pH value of a fermentation system after inoculation is 6.0-6.8, the ventilation rate is 0.3-0.7 vvm, the tank pressure is 0.05-0.14 MPa, a certain stirring speed is kept, DO in the seed culture process is controlled to be not lower than 10%, the culture is carried out for 15-30 h, and the standard of the mature seeds is OD after dilution by 30 times 620 Greater than 0.5, more preferably OD 620 0.5 to 1.0.
Preferably, an aqueous seed medium is used, comprising the following components: 1 to 3 percent of sucrose, 0.15 to 1 percent of corn steep liquor, 0.2 to 1.5 percent of yeast extract and KH 2 PO 4 0.4 to 1.5 percent and 0.05 to 0.5 percent of urea.
The fermentation and conversion process comprises the following steps:
inoculating the seed liquid obtained by culturing in a seed tank into a fermentation tank containing a fermentation medium, wherein the initial volume after inoculation is 4-6L, the inoculum size is 10-30% (v/v, relative to the initial volume of fermentation), alkane is added in the initial of fermentation by 0-10% (v/v, relative to the initial volume of fermentation), the fermentation process is controlled at 28-32 ℃, the ventilation rate is about 0.3-0.7 vvm, the tank pressure (gauge pressure) is about 0.05-0.14 MPa, a certain stirring speed is kept, and the dissolved oxygen is controlled to be not lower than 10%. The pH value of the fermentation liquid is controlled to be about 5.0 to 6.8, the pH value of the fermentation liquid is gradually reduced along with the growth of microorganisms, the pH value is controlled to be not lower than 3.0, and the Optical Density (OD) of the thalli is controlled 620 ) Above 0.5 (30-fold dilution), the pH is controlled to 7.0 or less, preferably about 4.0 to 6.8, more preferably 5.0 to 6.5, until the fermentation is completed. Alkane is added in batches when the fermentation period is 10-20 hours, the alkane content in the fermentation liquid is controlled to be not more than 10%, and the total fermentation period is about 100-180 hours. Optionally, by supplementing during fermentationAdding sugar water solution to control the sugar concentration of the fermentation liquor to be 0.1% -1% (w/v).
Or centrifuging seed liquid obtained by culturing a seed tank, adding thalli into a conversion tank containing phosphate buffer solution, adding alkane in batches in the process, controlling the alkane in the conversion liquid to be not more than 10%, controlling the temperature to be 28-32 ℃, the ventilation quantity to be about 0.3-0.7 vvm, the tank pressure (gauge pressure) to be about 0.05-0.14 MPa, maintaining a certain stirring speed, and controlling the dissolved oxygen to be not less than 10%. The pH value of the liquid alkali conversion liquid is controlled to be below 7.0, preferably 4.0-6.8, more preferably 5.0-6.5 by adding 10-40 percent of liquid alkali until the conversion is finished, and the total conversion period is about 100-180 hours.
The fifth aspect of the present invention provides a method for preparing a long chain dibasic acid, comprising the steps of:
s1: fermenting to obtain long-chain binary acid fermentation liquor; and
s2: extracting and purifying the obtained long-chain dibasic acid fermentation liquor to obtain the long-chain dibasic acid;
wherein, step S1 adopts the method for producing long-chain dibasic acid by fermentation in the fourth aspect of the invention to prepare the long-chain dibasic acid fermentation broth.
In the preparation method of the long-chain dibasic acid, the obtained long-chain dibasic acid fermentation broth can be subjected to the existing extraction and purification process to prepare the final long-chain dibasic acid product.
In one embodiment of the method for producing a long-chain dibasic acid according to the present invention, the extraction and purification treatment of step S2 may include the following processes: acidifying the obtained long-chain dibasic acid fermentation liquor, separating to obtain a solid, dissolving the solid in an organic solvent, separating to obtain clear liquid, and crystallizing to obtain the long-chain dibasic acid.
In a preferred embodiment, the pH of the acidification may be between 2.5 and 5, preferably between 3 and 4; in another preferred embodiment, the separation mode of separating the solid matters after acidification can be centrifugation, filtration and the like; in another preferred embodiment, the organic solvent used to dissolve the solids may be one or more of alcohols including but not limited to methanol, ethanol, isopropanol, n-butanol, etc., acids including but not limited to acetic acid, etc., ketones including but not limited to acetone, etc., and esters including but not limited to ethyl acetate, butyl acetate, etc.; in another preferred embodiment, the solid is decolorized after being dissolved in an organic solvent, and clear liquid is obtained after separation, and the decolorization method preferably uses activated carbon for decolorization, wherein the addition amount of the activated carbon is not more than 5% of the volume of the solution, the decolorization temperature is 85-100 ℃, and the decolorization time is 15-165 min; in another preferred embodiment, after separation of the supernatant, the cooled crystallization may comprise the steps of: firstly, cooling to 65-80 ℃, preserving heat for 1-2 hours, then cooling to 25-35 ℃ and crystallizing; in another preferred embodiment, after crystallization, the resulting crystals are separated, whereby long chain dibasic acid is obtained, and the crystals may be separated by centrifugation.
In a sixth aspect, the present invention provides a long chain diacid product prepared by the method of preparing a long chain diacid as described above.
The long-chain dicarboxylic acids (LCDA; also referred to as long-chain dicarboxylic acids and long-chain diacids) according to the invention comprise the formula HOOC (CH) 2 ) A dibasic acid of nCOOH, wherein n is not less than 7; preferably, 20.gtoreq.n.gtoreq.7; more preferably, 16.gtoreq.n.gtoreq.7. Examples of LCDA according to the present invention include: : azelaic acid (HOOC (CH) 2 ) 7 COOH), sebacic acid (HOOC (CH) 2 ) 8 COOH), undecanedioic acid (HOOC (CH) 2 ) 9 COOH,1, 9-nonadicarboxylic acid or 1, 11-undecanedioic acid, labeled "DC11" in the present invention, dodecadioic acid (HOOC (CH) 2 ) 10 COOH,1, 10-decadicarboxylic acid or 1, 12-dodecadicarboxylic acid, labeled "DC12" in the present invention), tridecanedioic acid (HOOC (CH) 2 ) 11 COOH,1, 11-undecanedicarboxylic acid or 1, 13-tridecanedioic acid, labeled "DC13" in the present invention), tetradecanedioic acid (HOOC (CH) 2 ) 12 COOH,1, 12-dodecanedioic acid or 1, 14-tetradecanedioic acid, labeled "DC14" in the present invention), pentadecanedioic acid (HOOC (CH) 2 ) 13 COOH,1, 13-tridecanediolCarboxylic acid or 1, 15-pentadecanoic acid, labeled "DC15" in the present invention, hexadecanoic acid (HOOC (CH) 2 ) 14 COOH,1, 14-tetradecanedicarboxylic acid or 1, 16-hexadecanedicarboxylic acid, labeled "DC16" in the present invention), heptadecanedicarboxylic acid (HOOC (CH) 2 ) 15 COOH,1, 15-pentadecanedicarboxylic acid or 1, 17-heptadecanedicarboxylic acid, labeled "DC17" in the present invention), octadecanedioic acid (HOOC (CH) 2 ) 16 COOH,1, 16-hexadecanedicarboxylic acid or 1, 18-octadecanedioic acid, labeled "DC18" in the present invention), and the like.
In some embodiments of methods of fermentatively producing long chain dibasic acids according to the present invention, in a 10L fermenter fermentation process, undecanedioic acid may be produced in an acid yield of at least 110mg/g, dodecadioic acid of at least 150mg/g, tridecanedioic acid of at least 130mg/g, tetradecanedioic acid of at least 150mg/g, pentadecanodioic acid of at least 140mg/g, or hexadecanedioic acid of at least 130 mg/g.
In some embodiments of the method of fermentatively producing long chain dibasic acids according to the present invention, at least 60% of the base usage may be saved in a 10L fermenter fermentation process as compared to conventional processes; in other embodiments, about 90% of the alkali usage can be saved.
In some embodiments of the methods of fermentatively producing long chain dibasic acids according to the present invention, in a 10L fermenter conversion process, twelve carbon dibasic acids may be produced in an acid yield of at least 150mg/g and a weight conversion (w/w, weight percent conversion of alkane to dibasic acid) of 92% or greater is achieved.
In some embodiments of the methods of fermentatively producing long chain dibasic acids according to the present invention, the fermentation is performed at 200M 3 In the fermentation process of the fermentation tank, the dodecadiacid with the acid yield of at least 150mg/g can be produced, and the weight conversion rate (w/w, weight percent of alkane converted into diacid) of more than 92 percent is achieved.
In some embodiments of the methods of fermentatively producing long chain dibasic acids according to the present invention, the fermentation conversion may be increased by supplementing the secondary carbon source, and a weight conversion (w/w, weight percent conversion of alkane to dibasic acid) of greater than 95% may be achieved.
In some embodiments of the methods of fermentatively producing long chain dibasic acids according to the present invention, the fermentation is performed at 200M 3 In the fermentation process of the fermentation tank, tridecanedioic acid with the acid yield of at least 140mg/g can be produced, and the weight conversion rate (w/w, weight percent of alkane converted into the dicarboxylic acid) of more than 85 percent is achieved.
In some embodiments of the methods of fermentatively producing long chain dibasic acids according to the present invention, at 450M 3 In the fermentation process of the fermentation tank, the dodecadiacid with the acid yield of at least 150mg/g can be produced, and the weight conversion rate (w/w, weight percent of alkane converted into diacid) of more than 90 percent is achieved.
The present invention will be described in detail with reference to the following examples to make the features and advantages of the present invention more apparent. It should be noted that the examples are intended to understand the concept of the present invention and the scope of the present invention is not limited only to the examples listed herein.
According to common knowledge in the field of fermentation, the percentages in the invention are mass-to-volume ratios, unless otherwise specified, namely: w/v; % represents g/100mL.
The examples and comparative examples of the present invention employ techniques well known to those skilled in the art, such as the measurement method disclosed in chinese patent ZL 95117436.3 to measure the concentration of dibasic acid in fermentation broth, and the specific measurement process is as follows: adjusting the pH of the fermentation liquor to 3.0 by using a hydrochloric acid solution, adding 100mL of diethyl ether for extracting the dibasic acid in the fermentation liquor, evaporating to remove the diethyl ether, obtaining dibasic acid powder, dissolving the obtained dibasic acid powder in ethanol, and titrating by using a 0.1mol/L NaOH solution to finally obtain the dibasic acid titration amount in the fermentation liquor.
The specific determination process of the salt content of the fermentation treatment liquid in the embodiment and the comparative example comprises the following steps: and (3) placing the fermentation treatment liquid in a glass evaporation dish after drying and constant weight, and evaporating in a water bath. If the residue has color, dropwise adding hydrogen peroxide until bubbles disappear, evaporating in water bath, repeatedly treating for several times until the color becomes white or the color is stable, drying the evaporating dish until the weight is constant, weighing, and calculating the salt content.
EXAMPLE 1 obtaining of candida tropicalis CAT H1614 Strain
Starting strain cctccc NO: m203052 glycerol was added to a shake flask containing YPD medium and 0.01% 5-fluorouracil was added thereto, followed by shaking culture at 30℃and 200rpm for 16 to 24 hours. The grown culture was collected by centrifugation and washed several times with physiological saline, and finally cells were suspended with an equal amount of aqueous LiCl solution. Placing proper amount of cell suspension in a sterile plate, irradiating with ultraviolet lamp with power of 15W for 90s at a distance of 20 cm. The strain after mutagenesis is cultivated on a flat plate containing YPD culture medium, the cultivation temperature is 29 ℃, the cultivation time is 50 hours, a monoclonal with good growth is selected to a pore plate containing a primary screening culture medium (the culture medium comprises substrate long-chain alkane), fermentation is carried out, alkali is not added in the process, the natural pH value is kept, the fermentation is carried out at 29 ℃, the rotation speed of a shaking table is 220rpm, the fermentation period is 70 hours, and the primary screening is carried out after the fermentation is finished. The strains with relatively high yield of long chain dibasic acid are selected from the strain, and then the strains are re-screened through an orifice plate, and the re-screened strains are re-screened through 500ml shaking flask fermentation, wherein 500ml shaking flask fermentation is carried out at 29 ℃, the rotation speed of a shaking table is 220rpm, the pH of the fermentation process is natural, and the fermentation period is 110 hours. Finally, the stable high-yield strain is obtained and named CAT H1614 and is stored in an glycerol pipe. The flow is referred to in fig. 1.
EXAMPLE 8 production of Long chain dibasic acid by fermentation of candida tropicalis CAT H1614 in 10L fermentors
The glycerol tube strain of candida tropicalis CAT H1614 was inoculated into a seed bottle containing 25ml of liquid medium (glucose 2%, yeast extract 1%, peptone 2%), and cultured on a shaker at 30 ℃ for 2 days at 230rpm at natural pH. Inoculating shake flask seed into 6L seed culture medium (sucrose 2%, corn steep liquor 0.3%, yeast extract 0.5%, KH) 2 PO 4 0.8 percent and 0.3 percent of urea) in a 10L seed tank, the inoculation amount is 20 percent, the initial pH value of a system after inoculation is 6.2, the ventilation amount is 0.5vvm at 30 ℃, the tank pressure is 0.1MPa, the culture is carried out for 20 hours, and the pH value naturally drops to 3 in the culture process. OD (optical density) 620 Inoculating to fermentation medium 1 (glucose 2%, corn steep liquor 0.2%, yeast extract 0.2%, potassium nitrate 0.08%, potassium dihydrogen phosphate 0.3%, urea 0.2%, and sulfuric acid) when the fermentation time reaches 0.6In a fermentation tank with 0.1% of ammonium and 0.1% of sodium chloride, the initial volume after inoculation is 6L, the inoculation amount is 15%, 6% (v/v) of dodecane is added at the beginning of fermentation, the fermentation process is controlled at 28 ℃, the ventilation rate is about 0.4vvm, the tank pressure (gauge pressure) is about 0.11MPa, the dissolved oxygen is controlled to be not lower than 20%, and the pH value of the fermentation liquid is controlled by adding 32% of liquid alkali. The early stage of fermentation mainly comprises thallus growth, the initial pH of fermentation is about 6.6, the pH of fermentation liquid gradually decreases along with the growth of microorganism, the pH is controlled to be not lower than 3.0, and the Optical Density (OD) of thallus is controlled 620 ) When the concentration of the alkane is more than 0.5 (30 times of dilution), controlling the pH value to be 5.0 until the fermentation is finished, starting to add the alkane in batches when the fermentation period is 10-20 hours, and controlling the alkane content in the fermentation liquid to be not more than 10%.
Comparative example 1 candida tropicalis cctccc NO: production of long chain dibasic acid by fermentation of M203052 (starting Strain) in 10L fermenter
Candida tropicalis cctccc NO: m203052 (starting strain) substitution strain CAT H1614 was also fermented using the same procedure as in example 8.
Comparative example 2 candida tropicalis cctccc NO: production of long chain dibasic acid by fermentation of M203052 (starting strain) in 10L fermenter by conventional process
Candida tropicalis cctccc NO: m203052 (starting strain) was fermented using the existing conventional process: mature seeds cultivated in a seed tank are inoculated into a fermentation tank containing a fermentation medium (3% of glucose, 0.5% of monopotassium phosphate, 0.2% of yeast extract, 0.15% of corn steep liquor, 0.25% of urea, 0.2% of sodium chloride and 0.7% of potassium nitrate), and C12 alkane and feed sugar are eliminated. Culturing at 29℃under a aeration rate of 0.5vvm and a pot pressure of 0.1 MPa. pH is natural 20 hours before fermentation, mainly the bacterial growth, when the bacterial growth optical density (OD 620 ) The addition of C12 alkane is started to be carried out in batch and is carried out once every 8 hours, the alkane concentration in the fermentation liquor is controlled to be kept at about 5 percent (V: V), the pH is adjusted to be 6.5 after 48 hours, the pH is adjusted to be 7.0 by NaOH solution every 4 hours, 48-72 hours, the pH is adjusted to be 7.5 by NaOH solution every 4 hours, 72-120 hours, the pH is adjusted to be 7.8 by NaOH solution every 4 hours, and the pH is adjusted to be 8.0 by NaOH solution every 4 hours. Fermenting to 24, 48, 72 hr Glucose was fed in 1% (W: V) in time-series.
The fermentation results of the above fermentation process are shown in Table 2.
TABLE 2 fermentation results of different strains under different technologies
As shown in Table 2, the fermentation result of the candida tropicalis CAT H1614 is obviously superior to that of the conventional process and the existing strain at a low pH value, and the high catalytic activity can be maintained for a long time.
EXAMPLE 10 Candida tropicalis CAT H1614 at 200M 3 Fermentation DC12 in fermenter
The glycerol tube strain of candida tropicalis CAT H1614 was taken and seed cultured as described in example 8, with the pH naturally dropping to 3 during the culture. OD (optical density) 620 Inoculating to a fermentation tank containing fermentation medium 2 (glucose 4%, potassium nitrate 0.1%, potassium dihydrogen phosphate 0.1%, ammonium sulfate 0.1% and magnesium sulfate 0.1%) at a time of 0.8%, starting volume after inoculation being 6L, inoculum size being 22%, adding 4% (v/v) of dodecane relative to the fermentation starting volume) at fermentation start, controlling fermentation process at 28deg.C, ventilation volume at about 0.6vvm, tank pressure (gauge pressure) at about 0.10MPa, and controlling dissolved oxygen at not less than 20%. Adding 33% pH value of liquid alkali to control pH value of fermentation liquid, wherein the fermentation is mainly thallus growth, the fermentation initial pH is about 6.7, the pH of fermentation liquid gradually decreases along with the growth of microorganism, the pH is controlled to be not lower than 3.0, and the Optical Density (OD) of thallus is reduced 620 ) When the concentration of the sugar solution is more than 0.5 (30 times of dilution), the pH is controlled to be about 6.0, and the sugar solution with the concentration of 25% is fed in, so that the sugar concentration of the fermentation liquid is controlled to be 0.5%. And (3) starting to add alkane in batches when the fermentation period is 10-20 hours, and controlling the alkane content in the fermentation liquid to be not more than 10%. The total fermentation period is 122 hours, the acid yield is 182.3mg/g, the weight conversion rate of dibasic acid to alkane is 100.4 percent, and the alkali adding amount is 2.5 tons.
EXAMPLE 11 Candida tropicalis CAT H1614 at 200M 3 Fermentation DC13 in fermenter
Taking glycerol tube strain of candida tropicalis CAT H1614,seed culture was performed as described in example 8, with the pH naturally dropping to 3 during the culture. OD (optical density) 620 Inoculating to a fermentation tank containing fermentation medium 2 (glucose 3.8%, potassium nitrate 0.12%, potassium dihydrogen phosphate 0.12%, ammonium sulfate 0.12% and magnesium sulfate 0.12%) at a time of 0.8%, inoculating 25%, adding tridecane alkane at a fermentation start time of 4% (v/v, relative to the fermentation start volume), controlling the fermentation process at 28deg.C, ventilating at about 0.6vvm, and controlling dissolved oxygen at not lower than 20% under a tank pressure (gauge pressure) of about 0.10 MPa. Adding 30% liquid alkali to control pH value of fermentation liquid, wherein the fermentation is mainly thallus growth, the fermentation initial pH is about 6.6, the pH of fermentation liquid gradually decreases with the growth of microorganism, the pH is controlled to be not lower than 3.0, and the Optical Density (OD) of thallus is reduced 620 ) When the pH is more than 0.5 (diluted by 30 times), controlling the pH to be about 6.5 until the fermentation is finished, starting to add alkane in batches when the fermentation period is 10-20 hours, and controlling the alkane content in the fermentation liquid to be not more than 10%. The total fermentation period is 132 hours, the acid yield is 147.3mg/g, the weight conversion rate of dibasic acid to alkane is 85.4 percent, and the alkali addition amount is 3.7 tons.
EXAMPLE 15 preparation of Long chain dibasic acid product
Regulating pH value of the fermentation broth prepared in example 11 to 3.2 with sulfuric acid, acidifying, filtering to obtain solid, dissolving the solid in acetic acid, adding active carbon with volume not exceeding 5% of the clear liquid, decolorizing at 85deg.C for 70min, filtering to obtain clear liquid, cooling the clear liquid to 65deg.C, maintaining the temperature for 1 hr, cooling to 35deg.C, crystallizing, and centrifuging to obtain diacid product.
Comparative example 3 fermentation production of DC13 by candida tropicalis H5343ALK2-1
The culture medium (including 27.0g/L of glucose, 7.0g/L of ammonium sulfate, 5.1g/L of potassium dihydrogen phosphate, 0.5g/L of magnesium sulfate, 0.1g/L of calcium chloride, 0.06g/L of citric acid, 0.023g/L of ferric trichloride, 0.0002g/L of biotin, 0.0009g/L of trace metals such as boric acid, 0.00007g/L of copper sulfate, 0.00018g/L of potassium iodide, 0.00036g/L of ferric trichloride, 0.00072g/L of manganese sulfate, 0.00036g/L of sodium molybdate, 0.00072g/L of zinc sulfate, 0.8ml of SAG471 antifoaming agent and the balance of water) was sterilized by heating in an appropriate manner so as to avoid precipitation reaction, and was added to a sterilized fermenter after cooling. The uninoculated complete medium was found to be very clear and light straw-colored with no strong odor. Slight cloudiness resulted after addition of the antifoam. Candida tropicalis H5343ALK2-1 was cultured under aseptic conditions using the above medium by stirring and aeration of a fermenter having an initial liquid volume of 12L. The sterile medium was inoculated with 5% of an inoculum of candida tropicalis H5343ALK2-1 and cultured with stirring at 35 ℃ at a ph of 5.8 and aeration rate sufficient to maintain dissolved oxygen above 20% for about 10 hours. When the culture terminates the exponential growth, dissolved oxygen begins to rise, and the conversion phase is initiated by: the feed stream of Exxon Developmental Fluid137 (a hydrocarbon containing about 94.4% tridecane, the remainder being predominantly dodecane) was continuously fed at a rate of 0.7 g/L/hr and mixed with the fermentation aid ingredients 1.25% emersol 267 (a technical grade oleic acid) and 1.25% emery 2203 (a technical grade methyl oil (talowate)). At the same time, the temperature in the fermenter was reduced from 35℃to 30℃and the aeration rate was reduced to 0.4vvm, and a back pressure of 0.4 bar was applied to the tank. The pH was maintained between 5.8 and 5.9 during the growth and conversion phases using 6N KOH. When the biomass concentration reached about 10g/L, glucose was continuously fed to the fermenter at a rate of 1.58g glucose/L/hr. The glucose supplementation rate during the conversion phase was reduced to 0-15% based on daily microscopic observations and evaluation of storage vesicles accumulated in yeast cells. 7ml of PPG (polypropylene glycol) antifoam was added to the fermenter during the conversion period to control slight foam. After 50 hours of the conversion period, the entire fermentation broth in the fermenter contained 41.5g/Kg (i.e., 41.5 mg/g) of 1,13-tridecanedioic acid (1, 13-tridecanedioic acid).
Comparative example 4 production of Mixed Long chain dibasic acid by fermentation of candida tropicalis H5343HDC23-3
Culture medium (including 27.0g/L glucose, 4.9g/L potassium dihydrogen phosphate, 0.6g/L magnesium sulfate, 0.1g/L calcium chloride, 0.06g/L citric acid, 0.023g/L ferric trichloride, 0.000012g/L biotin, trace metals including 0.00007g/L copper sulfate, 0.00432g/L manganese sulfate, 0.00072g/L zinc sulfate, 0.00708g/L citrate, 0.6ml SAG471 antifoaming agent, and water balance) for the preparation of a beverageSterilization is performed in a manner so as to avoid any precipitation reaction, and then added to the sterilized fermenter. The uninoculated complete medium was found to be very clear and light straw-colored with no strong odor. Candida tropicalis H5343HDC23-3 was cultured under aseptic conditions using the above medium by stirring and aeration of a fermenter having an initial liquid volume of 12L. The sterile medium was inoculated with 3% of an inoculum of candida tropicalis H5343HDC23-3 and incubated with stirring at 35 ℃ and aeration rate sufficient to maintain dissolved oxygen above 20% for about 12 hours. By addition of NH of 6N 4 OH adjusts and maintains the pH in the growth phase at 5.8-5.9, said NH 4 OH is also an inorganic nitrogen source in the medium. When the culture stopped the exponential growth, dissolved oxygen began to rise, the conversion phase was initiated by the addition of an inducer substance and simultaneously the feed stream of sunflower fatty acids with high oil content (the remainder of 84.4% oleic acid, 5.2% linoleic acid, 4.7% stearic acid, 3.9% palmitic acid, and small amounts of eicosanoic acid (20:0), eicosanoicacid (20:1), pentadecanoic acid, lunoic acid, tetradecanoic acid) was continuously fed at a rate of 2.0 g/L/hr. At the same time, the temperature in the fermenter was reduced from 35℃to 30℃and the aeration rate was reduced to 0.4vvm, and the pH control reagent NH was added 4 OH was exchanged for NaOH. The pH of the conversion stage was adjusted and maintained at 5.8-5.9 using 6N NaOH. When the biomass concentration reached about 10g/L, glucose was continuously fed to the fermenter at a rate of 1.22g glucose/L/hr. The glucose supplementation rate during the conversion phase was reduced to 0-45% based on daily microscopic observations and evaluation of storage vesicles accumulated in yeast cells. No antifoam is added during the conversion period. After 50 hours of conversion, the total fermentation broth in the fermenter contained 71g/Kg (i.e., 71 mg/g) of total dicarboxylic acid.
Based on the foregoing examples and comparative examples, compared with the existing fermentation process, when the candida provided by the invention is used for long-chain diacid fermentation, the fermentation time is short, the consumption of alkali is low, the yield of diacid is high, the acid consumption in the subsequent extraction process is greatly reduced, the salt content in the wastewater is greatly reduced, the production cost of long-chain diacid can be greatly reduced as a whole, and the environment is friendly.
It will be apparent to those skilled in the art that various modifications and variations can be made in the present invention without departing from the scope or spirit of the invention, and combinations of the features described above, and other technical modifications made in accordance with the foregoing, are within the scope of the invention.
Claims (10)
1. A candida tropicalis (Candida tropicalis) strain CAT H1614 with a preservation number of cctccc NO: m2013143.
2. A microbial agent comprising candida tropicalis strain CAT H1614 as claimed in claim 1.
3. Use of candida tropicalis strain CAT H1614 as claimed in claim 1 for the fermentative preparation of long chain dibasic acids.
4. A method for producing long-chain dibasic acid by fermentation, which is characterized in that the candida tropicalis strain CAT H1614 as defined in claim 1 is adopted for fermentation to produce long-chain dibasic acid.
5. The method of claim 4, wherein the fermentation process comprises a strain growth phase and a conversion phase, wherein the conversion phase controls the pH of the fermentation system to 7.0 or less; preferably 4.0 to 6.8; more preferably 5.0 to 6.5.
6. The method according to claim 5, wherein the strain is grown to a cell optical density OD after 30-fold dilution 620 When the pH value of the fermentation system is more than 0.5, controlling the pH value of the fermentation system to be below 7.0; preferably 4.0 to 6.8; more preferably 5.0 to 6.5.
7. The method according to any one of claims 4-6, wherein the substrate for the fermentation process comprises an alkane, preferably an n-alkane comprising C9-C22; more preferably, the n-alkane comprises C9 to C18; most preferably, the n-alkane comprises C10, C11, C12, C13, C14, C15 or C16;
And/or, the medium used in the fermentation process comprises the following components (w/v): 1 to 5 percent of glucose, 0.1 to 0.9 percent of corn steep liquor, 0.1 to 0.5 percent of yeast extract, 0.05 to 1.2 percent of potassium nitrate, 0.05 to 1.0 percent of monopotassium phosphate, 0.05 to 0.3 percent of urea, 0.05 to 0.3 percent of ammonium sulfate and 0.05 to 0.2 percent of sodium chloride; or comprises the following components (w/v): 1 to 5 percent of glucose, 0.05 to 0.6 percent of potassium nitrate, 0.02 to 0.6 percent of monopotassium phosphate, 0.05 to 0.3 percent of ammonium sulfate and 0.05 to 0.3 percent of magnesium sulfate;
and/or, in the fermentation process, the inoculation amount of the strain is 10% -30%;
and/or, the temperature is 28-32 ℃;
and/or the air quantity is 0.3-0.7 vvm;
and/or the pressure is 0.05-0.14 MPa;
and/or the pH value of the fermentation system is not lower than 3.0, preferably 3.5-6.5 in the growth period of the strain.
8. A method for preparing a long chain dibasic acid comprising:
s1: fermenting to obtain long-chain binary acid fermentation liquor; and
s2: extracting and purifying the obtained long-chain dibasic acid fermentation liquor to obtain the long-chain dibasic acid;
wherein, the step S1 adopts the method of any one of claims 4 to 7 to prepare the long-chain dibasic acid fermentation broth.
9. The method according to claim 8, wherein the extraction and purification process of step S2 comprises: acidifying the obtained long-chain dibasic acid fermentation liquor, separating to obtain a solid, dissolving the solid in an organic solvent, separating to obtain clear liquid, and crystallizing to obtain long-chain dibasic acid;
the pH value of the acidification is 2.5-5, preferably 3-4;
and/or, the separation method is centrifugation or filtration;
and/or, the organic solvent comprises: one or more of alcohols, acids, ketones, and esters; wherein the alcohol comprises one or more of methanol, ethanol, isopropanol and n-butanol; the acid comprises acetic acid; the ketone comprises acetone; the ester comprises ethyl acetate and/or butyl acetate;
and/or, after the solid is dissolved in an organic solvent, decoloring, and separating to obtain clear liquid, wherein the decoloring method is preferably activated carbon decoloring; the addition amount of the activated carbon is not more than 5% of the volume of the solution; the decoloring temperature is 85-100 ℃; the decoloring time is 15-165 min;
and/or, the crystallization is cooling crystallization; the cooling crystallization comprises the following steps: cooling to 65-80 ℃, preserving heat for 1-2 hours, cooling to 25-35 ℃ and crystallizing;
And/or, after said crystallization, separating the crystals, thereby obtaining the long chain dibasic acid; the separation method is centrifugal separation.
10. A long chain diacid product made by the process of claim 8 or 9.
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