CN113943760B - Method for producing long-chain dibasic acid by fermentation of candida viscidosa, product and strain - Google Patents
Method for producing long-chain dibasic acid by fermentation of candida viscidosa, product and strain Download PDFInfo
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- 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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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- 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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- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
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- Health & Medical Sciences (AREA)
- Biotechnology (AREA)
- Genetics & Genomics (AREA)
- Bioinformatics & Cheminformatics (AREA)
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- Tropical Medicine & Parasitology (AREA)
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Abstract
The product relates to a method for synthesizing and producing long-chain dibasic acid by utilizing candida viscidosa biological method, which comprises a fermentation culture stage and a product separation and recovery stage after candida viscidosa is connected into a fermentation culture medium, wherein the preservation number of candida viscidosa is CCTCC M2021097. The candida viscidosa strain and the fermentation method can realize mass acid production within 40 hours when the DC12 is produced from nC12 by fermentation in an industrial fermentation tank. In addition, the content of monoacids is particularly low, and the monoacids are particularly suitable for downstream applications such as high-performance nylon, plasticizer and lubricating oil.
Description
Technical Field
The invention belongs to the field of biochemical engineering, and relates to a method for producing long-chain dibasic acid by fermentation of candida viscidosa, a product and a strain.
Background
Long chain dicarboxylic acids refer to dicarboxylic acids having a carbon number of from 6 to 24 carbon atoms, and generally employ a relatively large number of linear dicarboxylic acids having from 10 to 18 carbon atoms. Wherein, the dodecadiacid (1, 10-Dodecanedioic, DDA) is also called DC12 and is mainly applied to products such as synthetic engineering plastics, long-chain nylon and the like. The main processes for producing long chain dicarboxylic acids include vegetable oil catalysis, chemical synthesis and biological fermentation. Catalytic vegetable oils, chemical synthesis have been eliminated from the market for reasons of cost raw materials, etc. For example, the use of a chemical synthesis method for DC12 requires at least 9 complex reaction steps and requires fire, explosion and poison protection equipment for production. The biological fermentation method can be used for production at normal temperature and normal pressure, and has the advantage of cost.
Although various microorganisms, in particular candida, can be fermented to produce the dodecadiacid by a biological fermentation method, the method has low strain density and phase change to prolong the whole fermentation period due to the problem of absorbing raw alkane, so that the production efficiency is low and the problem of the diacid industry is solved.
Disclosure of Invention
In view of the above, the present invention aims to provide a strain and a method capable of obtaining a plurality of long chain dibasic acid products in a short time. Although many microorganisms such as bacteria, mold, and yeasts have a certain ability to oxidize alkanes to produce dicarboxylic acids, yeasts are the most powerful yeasts of all microorganisms to oxidize alkanes to produce dicarboxylic acids, and yeasts capable of converting alkanes to long chain dicarboxylic acids include: candida (Candida), rhodotorula (Rhodotorula), torulopsis (torulopsis), cryptococcus (Cryptococcus), endospora (Endomyces), candida (Trichosporon), pichia (Pichia), geotrichum (Geotrichum candidum), saccharomyces (Brettanomyces), saccharomyces (Saccharomyces), endomyzides (endomyzips). Among them, many species of candida genus, such as candida tropicalis, candida lipolytica, candida vista, etc., are excellent species for converting alkanes to dicarboxylic acids, and many of them are industrially used at present, including candida tropicalis and candida vista, which are the strongest species of all candida genus in terms of alkane utilization ability, and also are species for industrially producing long-chain dicarboxylic acids.
Candida conversion of alkanes generally goes through two steps:
iα oxidation reaction process: that is, after the alkane is absorbed by the cell, the methyl at the tail end of the alkane is firstly oxidized into hydroxyl by the reductase consisting of cytochrome P450 enzyme and cytochrome in the microsome, and the hydroxyl is sequentially oxidized into aldehyde group and carboxyl by alcohol dehydrogenase and aldehyde dehydrogenase in the cytoplasm to generate monoacid; and
II omega oxidation reaction process: the monoacid is continuously subjected to omega oxidation to obtain omega hydroxy acid, and then is finally oxidized into dicarboxylic acid; secondly, monobasic acid can also continue to enter the micro body for beta oxidation and finally be metabolized into carbon dioxide and water. The two metabolic pathways depend on the strength of the ω and β oxidative capacity of the strain. The omega oxidizing ability is strong, the dicarboxylic acid yield obtained by fermentation is high, the alkane consumed by the strain with the beta oxidizing ability is high, and the accumulation amount of the dicarboxylic acid of the final product is small. In addition, dicarboxylic acids produced by ω -oxidation enter β -metabolic processes when they accumulate too much in the cell, and are eventually metabolized. Thus, it is effective in the actual production process to allow easy absorption of alkane by cells and efficient secretion of dicarboxylic acid product outside cells while reducing unnecessary β metabolic processes.
Based on the findings, the invention screens out candida vista (preservation number CCTCC M2021097, preservation date 2021, 1 month and 8 days, preservation place: china Center for Type Culture Collection (CCTCC), eight paths of Lojia mountain in Wuchang district of Wuhan, hubei province) by using a method of combining morphological observation with manual screening in waste residues containing alkane (waste paraffin oil is used for production), which can realize fermentation production of various diacid products on one hand, and on the other hand, has better cell permeability, can perform alkane fermentation at the initial stage of fermentation without seed tank fermentation, and can greatly reduce initial inoculation fermentation culture time.
The culture characteristics of candida viscidosa (cctccc M2021097) are as follows:
the feedstock comprises a mixture of carbon sources comprising one or more carbon sources selected from sugars, celluloses, alkanes, fatty acids, triacylglycerols, paraffins, and the like, or combinations thereof.
The nitrogen source may be derived from an inorganic (e.g., (NH) 4 ) 2 SO 4 ) Or an organic source (e.g., urea or glutamic acid). In addition to suitable carbon and nitrogen sources, the medium may also contain suitable minerals, salts, cofactors, buffers, vitamins, metal ions (e.g., mn +2 、Co +2 、Zn +2 、Mg +2 ) And other components. Or by culturing Candida vista in a yeast complex medium (e.g., yeast extract-peptone-dextrose broth (YPD)) or other commercially available general purpose yeast medium.
In one embodiment of the invention, a wort agar medium, such as YPD medium, may be selected: 10g/L yeast extract; 20g/L peptone; glucose at 20g/L, natural pH. The method is mainly used for activating and preserving strains.
The liquid fermentation medium comprises: 10-90g/L of carbon source, 4-15g/L of sodium phosphate, preferably 6-10 g/L, 3-8 g/L of yeast extract, 3-8 g/L of corn steep liquor, 0.5-1.5g/L of urea, 0.5-1g/L of NaCl, 1-10g/L of KCl and 0.5-1.5g/L of defoamer.
The method for producing long-chain dibasic acid by fermenting and converting alkane takes candida viscidosa (CCTCC M2021097) as a fermentation strain, and synchronously ferments in a fermentation medium added with n-alkane to produce alpha, omega-long-chain dibasic acid, and the method comprises the following steps:
directly inoculating the planar culture seeds of candida viscidosa (CCTCC M2021097) into a fermentation culture medium with the pH value of 5.5-9.0. Fermenting the mixed solution for 20-40 hours at 24-29 ℃, and then separating and purifying the produced long-chain dibasic acid. More preferably, the mixed solution is fermented at the pH of 6.0-7.5 and the temperature of 24-29 ℃ for 24-36 hours, and then the produced long-chain dibasic acid is separated and purified. More preferably, the n-alkane concentration in the fermentation broth is always > 40% (v/v) by supplementing n-alkane during the initial stage of the culture.
Preferably, the invention also comprises a step of separating and recovering long-chain dibasic acid in the fermentation liquor, preferably, the invention also comprises a step of removing thalli by membrane separation, placing clear liquid, cooling to 20 ℃, collecting crystals of DC12 sodium salt, directly acidifying mother liquor for crystallization, collecting DC12 sodium salt and DC12, and recrystallizing with water or an organic solvent to obtain DC12 white crystals.
Compared with the prior art, the invention has the following advantages:
by using the candida viscidosa (Candida viswanathii CCTCC M2021097) strain and the fermentation method, 162g/L of acid production can be realized only by culturing for less than 40 hours when DC12 is produced from nC12 in an industrial fermentation tank. And after fermentation is finished for 40 hours, the acid production rate exceeds 4.05 g/h.L. In addition, the content of monoacids is less than 0.0001%, and the monoacids are particularly suitable for downstream application such as high-performance nylon, plasticizer and lubricating oil.
Detailed Description
For the purposes of making the objects, technical solutions and advantages of the embodiments of the present disclosure more apparent, the technical solutions in the embodiments of the present disclosure will be clearly and completely described in the following embodiments of the present disclosure, and it is apparent that the described embodiments are some embodiments of the present disclosure, but not all embodiments. All other embodiments, which can be made by one of ordinary skill in the art without undue burden from the disclosure, are within the scope of the disclosure.
In one embodiment of the present invention, a method for the production of long chain dibasic acids by biological synthesis is provided, comprising the steps of:
(1) The candida viscidosa is inoculated into a fermentation culture medium;
(2) Fermentation culture stage of candida viscidosa;
(3) A stage of separating and recovering long-chain dibasic acid in the fermentation liquor, wherein,
the preservation number of the candida viscidosa is CCTCC M2021097.
Preferably, according to one embodiment of the invention, the candida viscidosa fermentation culture stage comprises a yeast liquid medium.
Preferably, according to one embodiment of the present invention, the yeast liquid medium comprises: 10-90g/L of carbon source, 4-15g/L of sodium phosphate, preferably 6-10 g/L, 3-8 g/L of yeast extract, 3-8 g/L of corn steep liquor, 0.5-1.5g/L of urea, 0.5-1g/L of NaCl, 1-10g/L of KCl and 0.5-1.5g/L of defoamer.
Preferably, according to one embodiment of the invention, the candida viscidosa fermentation culture stage is supplemented with n-alkanes at the initial stage of the culture.
Preferably, according to one embodiment of the invention, the addition of n-alkane concentration in the fermentation culture stage of candida viscidosis comprises making the n-alkane concentration in the fermentation broth always > 40% (v/v).
Preferably, according to one embodiment of the present invention, the fermentation culture stage of candida viscidosis is fermented at pH6.0 to 7.5 and 24 to 29 ℃ for 20 to 40 hours.
Preferably, according to one embodiment of the present invention, the fermentation and cultivation stage of candida viscidosis is fermentation and production for 24-36 hours at a pH of 6.0-7.5 and a temperature of 24-29 ℃.
Preferably, according to one embodiment of the invention, the seed culture stage is a fermentation culture in a fermenter comprising a bottom aeration device and/or a stirring device, and an alkane input device arranged in the middle of the fermenter.
Preferably, according to one embodiment of the invention, the product comprises one or several of the straight chain dicarboxylic acids of 10 to 18 carbon atoms.
Preferably, according to one embodiment of the invention, said bacterial species comprise candida viscidosa cctccc M2021097.
The technical solutions of the present invention will be clearly and completely described in the following in connection with the embodiments of the present invention. It will be apparent that the described embodiments are only some, but not all, embodiments of the invention. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
Example 1
The process of fermentatively synthesizing long chain dibasic acid (DC 12) is as follows:
1. preparation of culture medium
(1) Slant culture medium: wort agar medium;
(2) the fermentation medium comprises 60g/L glucose, 10g/L sodium hydrogen phosphate, 5g/L yeast extract, 6g/L corn steep liquor, 1.5g/L urea, 0.5g/L NaCl, 1g/L KCl and 0.5g/L Tween.
2. Stage of inoculating activated Strain
A Candida cyclovirosa (Candida viswanathii CCTCC M2021097) was spread on a large tube solid slant medium of 20X 180mm from a strain preservation freezer, and cultured at 27℃for 8 hours for activation.
3. Fermentation and culture stage:
inoculating the solid culture medium into a 10L fermentation tank filled with 5L fermentation culture medium, wherein the initial pH is 6.0, the temperature is 27 ℃, the ventilation volume is 1:1, the rotation speed of the fermentation tank is 120 revolutions per minute, and n-dodecane is added continuously or intermittently to ensure that the n-dodecane concentration in the fermentation liquid is always more than or equal to 20% (v/v), the acid is produced for 24 hours and 89g/L, the acid is produced for 130g/L when the culture is carried out for 36 hours, and the acid yield is kept unchanged after 36 hours. After 40h of fermentation, the acid production rate is 3.25 g/h.L.
Wherein, the recovery method of DC12 is an organic solvent recovery method, after fermentation, the PH is regulated to 2-3 by 6N HCl, after extraction by 120ml diethyl ether, diethyl ether is distilled off to obtain DC12 white crystals, after dissolution by neutral ethanol, titration by standard NaOH solution is carried out, and the purity of DC12 in fermentation broth is calculated to be 99.61%, wherein, the content of monobasic acid is 0.0002%.
Example 2:
the process of fermenting and synthesizing long chain dibasic acid (DC 12) in the fermenter (50L) is as follows:
1. preparation of culture medium
(1) Slant culture medium: wort agar medium;
(2) the fermentation medium comprises 35g/L glucose, 8g/L disodium hydrogen phosphate, 3g/L yeast extract, 5g/L corn steep liquor, 1.5g/L urea, 0.5g/L NaCl, 1g/L KCl and 1.5g/L Tween.
2. Seed culture stage
A Candida cyclovirosa (Candida viswanathii CCTCC M2021097) was spread on a solid slant medium of a large tube of 20X 180mm and cultured at 27℃for 10 hours. The seed culture activated on the inclined plane was collected.
3. Fermentation and acid production stage:
inoculating 2% of the seed culture into a 50L fermentation tank filled with 35L liquid fermentation medium, wherein the initial pH is 7.0, the temperature is 28 ℃, the ventilation rate is 1:0.7, the rotation speed of the fermentation tank is 120 revolutions per minute, and n-dodecane is continuously or intermittently added to ensure that the n-dodecane concentration in the fermentation liquid is always more than or equal to 20% (v/v), culturing is carried out for 24 hours to produce 99.1g/L of acid, and the acid is produced for 162g/L when culturing is carried out for 36 hours. And after fermentation is finished for 40 hours, the acid production rate is 4.05 g/h.L.
After fermentation, demulsification and layering are carried out, the residual nC12 on the upper layer is recovered, the bacterial cells on the lower layer are removed by filter pressing, bacterial cells are combined, 0.6-0.7% of active carbon is added for decolorization at 90 ℃ for 20 minutes, the active carbon is removed by filter pressing, and concentrated H is added after the decolorized clear liquid is heated 2 SO 4 And (3) cooling to the pH value of 3, cooling to the room temperature, press-filtering, drying by air, and drying the solid by a dryer to obtain a white crystalline DC12 product. The purity of DC12 reaches 99.87%, and the content of monoacid is 0.0001%.
Example 3:
the process of fermentative synthesis of long chain dibasic acid (DC 13) is as follows:
1. preparation of culture medium
(1) Slant culture medium: wort agar medium;
(2) the fermentation medium comprises 35g/L glucose, 8g/L disodium hydrogen phosphate, 3g/L yeast extract, 5g/L corn steep liquor, 1.5g/L urea, 0.5g/L NaCl, 1g/L KCl and 1.5g/L Tween.
2. Seed culture stage
A Candida cyclovirosa (Candida viswanathii CCTCC M2021097) was spread on a solid slant medium of a large tube of 20X 180mm and cultured at 27℃for 10 hours.
3. Fermentation:
the obtained culture was inoculated in an inoculum size of 2% by weight into a 500L fermenter containing 189L of a fermentation medium, the initial pH was 7.0, the temperature was 28 ℃, the aeration was 1:0.7, the rotation speed of the fermenter was 120 rpm, and n-tridecane was fed continuously or intermittently so that the n-tridecane concentration in the fermentation broth was always 20% (v/v) or more, and the culture was carried out for 24 hours to produce 90.5g/L of acid and 36 hours to produce 152g/L of acid. And after fermentation is finished for 40 hours, the acid production rate is 3.8 g/h.L.
After fermentation, demulsification and layering are carried out, the residual nC13 on the upper layer is recovered, the bacterial cells on the lower layer are removed by filter pressing, bacterial cells are combined, 0.6-0.7% of active carbon is added for decolorization at 90 ℃ for 20 minutes, the active carbon is removed by filter pressing, and concentrated H is added after the decolorized clear liquid is heated 2 SO 4 And (3) cooling to the pH value of 3, cooling to the room temperature, press-filtering, drying by air, and drying the solid by a dryer to obtain a white crystalline DC13 product. The purity of DC13 reaches 99.85%, and the content of monoacid is 0.002%.
Example 4:
the process of fermenting and synthesizing long chain dibasic acid (DC 11) in the fermenter (50L) is as follows:
1. preparation of culture medium
(1) Slant culture medium: wort agar medium;
(2) the fermentation medium comprises 35g/L glucose, 8g/L disodium hydrogen phosphate, 3g/L yeast extract, 5g/L corn steep liquor, 1.5g/L urea, 0.5g/L NaCl, 1g/L KCl and 1.5g/L Tween.
2. Seed culture stage
A Candida cyclovirosa (Candida viswanathii CCTCC M2021097) was spread on a solid slant medium of a large tube of 20X 180mm and cultured at 27℃for 10 hours. The seed culture activated on the inclined plane was collected.
3. Fermentation and acid production stage:
inoculating 2% of the seed culture into a 50L fermentation tank filled with 35L liquid fermentation medium, wherein the initial pH is 7.0, the temperature is 28 ℃, the ventilation rate is 1:0.7, the rotation speed of the fermentation tank is 120 revolutions per minute, and the n-undecane is continuously or intermittently added to ensure that the n-undecane concentration in the fermentation liquid is always more than or equal to 20% (v/v), culturing is carried out for 24 hours to produce 92.1g/L of acid, and the acid production is 168g/L when culturing is carried out for 36 hours. And after fermentation is finished for 40 hours, the acid production rate is 4.2 g/h.L.
After fermentation, demulsification and layering are carried out, the residual nC11 on the upper layer is recovered, the bacterial cells on the lower layer are removed by filter pressing, bacterial cells are combined, 0.6-0.7% of active carbon is added for decolorization at 90 ℃ for 20 minutes, the active carbon is removed by filter pressing, and concentrated H is added after the decolorized clear liquid is heated 2 SO 4 And (3) cooling to the pH value of 3, cooling to the room temperature, press-filtering, drying by air, and drying the solid by a dryer to obtain a white crystalline DC11 product. The purity of DC11 reaches 99.87%, and the content of monoacid is 0.0001%.
Example 5:
the process of fermenting and synthesizing long chain dibasic acid (DC 10) in the fermenter (50L) is as follows:
1. preparation of culture medium
(1) Slant culture medium: wort agar medium;
(2) the fermentation medium comprises 35g/L glucose, 8g/L disodium hydrogen phosphate, 3g/L yeast extract, 5g/L corn steep liquor, 1.5g/L urea, 0.5g/L NaCl, 1g/L KCl and 1.5g/L Tween.
2. Seed culture stage
A Candida cyclovirosa (Candida viswanathii CCTCC M2021097) was spread on a solid slant medium of a large tube of 20X 180mm and cultured at 27℃for 10 hours. The seed culture activated on the inclined plane was collected.
3. Fermentation and acid production stage:
inoculating 2% of the seed culture into a 50L fermentation tank filled with 35L liquid fermentation medium, wherein the initial pH is 7.0, the temperature is 28 ℃, the ventilation rate is 1:0.7, the rotation speed of the fermentation tank is 120 revolutions per minute, and n-decane is added continuously or intermittently to ensure that the n-decane concentration in the fermentation liquid is always more than or equal to 20% (v/v), and the culture is carried out for 24 hours to produce 89.1g/L of acid and 142g/L of acid when the culture is carried out for 36 hours. And after fermentation is finished for 40 hours, the acid production rate is 3.55 g/h.L.
After fermentation, demulsification and layering are carried out, the residual nC10 on the upper layer is recovered, the bacterial cells on the lower layer are removed by filter pressing, bacterial cells are combined, 0.6-0.7% of active carbon is added for decolorization at 90 ℃ for 20 minutes, the active carbon is removed by filter pressing, and concentrated H is added after the decolorized clear liquid is heated 2 SO 4 And (3) cooling to the pH value of 3, cooling to the room temperature, press-filtering, drying by air, and drying the solid by a dryer to obtain a white crystalline DC10 product. DC10 purity up to 99.97%, wherein the content of monoacid is 0.0004%.
Example 6:
the process of fermenting and synthesizing long chain dibasic acid (DC 14) in the fermenter (50L) is as follows:
1. preparation of culture medium
(1) Slant culture medium: wort agar medium;
(2) the fermentation medium comprises 35g/L glucose, 8g/L disodium hydrogen phosphate, 3g/L yeast extract, 5g/L corn steep liquor, 1.5g/L urea, 0.5g/L NaCl, 1g/L KCl and 1.5g/L Tween.
2. Seed culture stage
A Candida cyclovirosa (Candida viswanathii CCTCC M2021097) was spread on a solid slant medium of a large tube of 20X 180mm and cultured at 27℃for 10 hours. The seed culture activated on the inclined plane was collected.
3. Fermentation and acid production stage:
inoculating 2% of the seed culture into a 50L fermentation tank filled with 35L liquid fermentation medium, wherein the initial pH is 7.0, the temperature is 28 ℃, the ventilation rate is 1:0.7, the rotation speed of the fermentation tank is 120 revolutions per minute, and n-tetradecane is continuously or intermittently added to ensure that the concentration of the n-tetradecane in the fermentation liquid is always more than or equal to 20% (v/v), and the culture is carried out for 24 hours to produce 93.1g/L of acid and 122g/L of acid when the culture is carried out for 36 hours. And after fermentation is finished for 40 hours, the acid production rate is 3.05 g/h.L.
After fermentation, demulsification and layering are carried out, and the residual nC14 at the upper layer is recoveredRemoving thallus from the thallus layer by press filtration, mixing the clear liquid, adding 0.6-0.7% active carbon, decolorizing at 90deg.C for 20 min, press filtering to remove active carbon, heating the decolorized clear liquid, and adding concentrated H 2 SO 4 And (3) cooling to the pH value of 3, cooling to the room temperature, press-filtering, drying by air, and drying the solid by a dryer to obtain a white crystalline DC14 product. The purity of DC14 reaches 99.87%, and the content of monoacid is 0.0001%.
Example 7:
the process of fermenting and synthesizing long chain dibasic acid (DC 18) in the fermenter (5L) is as follows:
1. preparation of culture medium
(1) Slant culture medium: wort agar medium;
(2) the fermentation medium comprises 35g/L glucose, 8g/L disodium hydrogen phosphate, 3g/L yeast extract, 5g/L corn steep liquor, 1.5g/L urea, 0.5g/L NaCl, 1g/L KCl and 1.5g/L Tween.
2. Seed culture stage
A Candida cyclovirosa (Candida viswanathii CCTCC M2021097) was spread on a solid slant medium of a large tube of 20X 180mm and cultured at 27℃for 10 hours. The seed culture activated on the inclined plane was collected.
3. Fermentation and acid production stage:
inoculating 2% of the seed culture into a 5L fermentation tank filled with 3L liquid fermentation medium, wherein the initial pH is 7.0, the temperature is 28 ℃, the ventilation rate is 1:0.7, the rotation speed of the fermentation tank is 120 revolutions per minute, and the n-octadecane is continuously or intermittently added, so that the concentration of the n-octadecane in the fermentation liquid is always more than or equal to 20% (v/v), the culture is carried out for 24 hours to produce 78.1g/L of acid, and the acid production is 150g/L when the culture is carried out for 36 hours. After 40 hours of fermentation, the acid production rate is 3.75 g/h.L.
After fermentation, demulsification and layering are carried out, the residual nC18 on the upper layer is recovered, the bacterial cells on the lower layer are removed by filter pressing, bacterial cells are combined, 0.6-0.7% of active carbon is added for decolorization at 90 ℃ for 20 minutes, the active carbon is removed by filter pressing, and concentrated H is added after the decolorized clear liquid is heated 2 SO 4 And (3) cooling to the pH value of 3, cooling to the room temperature, press-filtering, drying by air, and drying the solid by a dryer to obtain a white crystalline DC18 product. DC18 purity up to 99.67% with monobasic acid contentThe amount was 0.0003%.
Example 8:
the process of fermenting and synthesizing long chain dibasic acid (DC 13) in the fermenter (50L) is as follows:
1. preparation of culture medium
(1) Slant culture medium: wort agar medium;
(2) the fermentation medium comprises 35g/L glucose, 8g/L disodium hydrogen phosphate, 3g/L yeast extract, 5g/L corn steep liquor, 1.5g/L urea, 0.5g/L NaCl, 1g/L KCl and 1.5g/L Tween.
2. Seed culture stage
A Candida cyclovirosa (Candida viswanathii CCTCC M2021097) was spread on a solid slant medium of a large tube of 20X 180mm and cultured at 27℃for 10 hours. The seed culture activated on the inclined plane was collected.
3. Fermentation and acid production stage:
inoculating 2% of the seed culture into a 50L fermentation tank filled with 35L liquid fermentation medium, wherein the initial pH is 7.0, the temperature is 28 ℃, the ventilation rate is 1:0.7, the rotation speed of the fermentation tank is 120 revolutions per minute, and n-tridecane is added continuously or intermittently to ensure that the concentration of the n-tridecane in the fermentation liquid is always more than or equal to 20% (v/v), culturing is carried out for 24 hours to produce 96.7g/L of acid, and the acid is produced for 138g/L when culturing is carried out for 36 hours. And after fermentation is finished for 40 hours, the acid production rate is 3.45 g/h.L.
After fermentation, demulsification and layering are carried out, the residual nC13 on the upper layer is recovered, the bacterial cells on the lower layer are removed by filter pressing, bacterial cells are combined, 0.6-0.7% of active carbon is added for decolorization at 90 ℃ for 20 minutes, the active carbon is removed by filter pressing, and concentrated H is added after the decolorized clear liquid is heated 2 SO 4 And (3) cooling to the pH value of 3, cooling to the room temperature, press-filtering, drying by air, and drying the solid by a dryer to obtain a white crystalline DC13 product. The purity of DC13 reaches 99.76%, and the content of monoacid therein is 0.0002%.
Comparative example 1:
the comparative example uses candida tropicalis UH-2-48 with the collection number of CGMCC No.0239 strain (see patent ZL 95117436.3), and other methods are the same as in example 1:
(1) A loop of Candida tropicalis UH-2-48 cells was applied to a solid slant of wort in 15X 180 large tubes and incubated at 28℃for 2 days.
(2) One strain is taken, and the strain is inoculated into a 250ml triangular flask filled with 30ml of liquid seed culture medium and cultured on a rotary shaking table at the temperature of 28-30 ℃ for 40 hours. The liquid seed culture medium contains KH 2 PO 4 8g/l, 5g/l yeast extract, 3g/l corn steep liquor, 30g/l sucrose, 3g/l urea and tap water, and the pH is 5.0.
(3) 3.5ml of the above cultured seed liquid was introduced into a 500ml Erlenmeyer flask containing 15ml of the fermentation medium, and fermentation was performed on a 220 rpm rotary shaker at 28-30℃for 4 days, with pH adjusted to 7.5-8.0 with 6N NaOH every 24 hours. KH is contained in the mixed liquid of the fermentation medium 2 PO 4 10g/l, sucrose 20/l, yeast extract 3g/l, corn steep liquor 3.5g/l, urea 1.5g/l, and nC12,200 ml/l, tap water configuration, pH7.2. Sterilizing at 110deg.C for 30 min.
After fermentation, pH was adjusted to 2-3 with 6N HCl, extracted with 120ml of diethyl ether, the diethyl ether was distilled off to give DC12 white powder, which was dissolved in 15ml of medium-sized ethanol, titrated with a standard NaOH solution, and the content of DC12 in the fermentation broth was calculated to be 67.7g/l, and the purity was calculated to be 98.11%, wherein the content of monoacid was more than 2.2%.
By comparison, it was found that example 1 of the present invention resulted in a significantly shorter product cycle.
Comparative example 2:
the comparative example uses candida tropicalis UH-2-48 with the collection number of CGMCC No.0239 strain (see patent ZL 95117436.3), and other methods are the same as in example 2:
(1) Seed medium and culture method, and fermentation medium and fermentation method are the same as in example 2.
(2) Culturing 3000mL for two days, with OD (x 30, 620 nm) of 0.81pH 3.8, strengthening, inoculating strain liquid of Candida tropicalis UH-2-48 without impurities into a primary seed tank filled with 700L seed culture medium, sterilizing at 121deg.C for 40 min, 29 deg.C for 350 r/min, and tank pressure of 0.8kg/cm 2 Aeration rate was 1:0.8, and cultured for 36 hours as seeds of secondary seed stocks.
(3) Inoculating 700L of the sterile seed solution cultured in step (2) into the culture medium containing 6.5m 3 Seed cultureRadical, 10m sterilized at 121℃for 40 min 3 In a secondary seed mother tank, the temperature is 30 ℃, the rotation speed is 200 r/min, and the tank pressure is 1kg/cm 2 The aeration rate is 1:0.7, and the culture is carried out for 40 to 24 hours, and the culture is used as the seed of fermentation.
(4) Inoculating the robust and sterile strain solution with no impurities cultured in step (3) into a strain solution with no impurities and 33m 3 Fermentation medium, 50m sterilized at 121℃for 40 min 3 In a fermentation tank, at 30 ℃,200 revolutions per minute and a tank pressure of 1kg/cm 2 Ventilation 1:0.5, nC at the beginning 12 4m 3 Within 30 hours, the pH of the system is controlled below 7.0, and the thalli grow rapidly and simultaneously generate 20.1g/L DC 12 Then the pH of the system is 7.0-8.0, fermentation is continued until the acid yield reaches 108g/L for 67 hours, and a certain amount of nC is added every day after 70 hours 12 The concentration of n-alkane in the fermentation liquor is always more than 5% (v/v). Fermenting for 139 hours, wherein the acid yield reaches 133g/L, fermenting is finished until 163 hours, the acid yield reaches 143g/L, and the fermentation rate is 0.88 g/h.L.
After fermentation, demulsification layering is carried out, and the upper layer residual nC is recovered 12 Filtering the lower thallus layer to remove thallus, mixing the clear liquid, adding 0.6-0.7% active carbon, decolorizing at 90deg.C for 20 min, filtering to remove active carbon, heating the decolorized clear liquid, and adding concentrated H 2 SO 4 Cooling to room temperature, press-filtering, air drying, drying the solid substance by a dryer to obtain white powdery DC 12 And (5) a product. DC (direct current) 12 The purity reaches 96.35%, wherein the content of monoacid is more than 1.5%.
The comparison shows that the product obtained in the embodiment 2 of the invention has larger crystal size and smaller product volume, and the acid production rate is obviously improved.
It should be noted that in this document, relational terms such as "first" and "second" and the like are used solely to distinguish one entity or action from another entity or action without necessarily requiring or implying any actual such relationship or order between such entities or actions. Moreover, the terms "comprises," "comprising," or any other variation thereof, are intended to cover a non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements does not include only those elements but may include other elements not expressly listed or inherent to such process, method, article, or apparatus. Without further limitation, an element defined by the phrase "comprising one … …" does not exclude the presence of other like elements in a process, method, article, or apparatus that comprises the element.
The foregoing is merely a specific embodiment of the disclosure to enable one skilled in the art to understand or practice the disclosure. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the generic principles defined herein may be applied to other embodiments without departing from the spirit or scope of the disclosure. Thus, the present disclosure is not intended to be limited to the embodiments shown herein but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.
Claims (5)
1. A method for synthesizing and producing long-chain dibasic acid by using biological method comprises the following steps:
(1) Fermentation and culture stage of candida viscidosa: inoculating candida viscidosa into a liquid fermentation medium for fermentation culture;
(2) A stage of separating and recovering long-chain dibasic acid in the fermentation liquor, wherein,
the preservation number of the candida viscidosa is CCTCC NO: M2021097;
the long-chain dicarboxylic acid is one of straight-chain dicarboxylic acids containing 10-14 and 18 carbon atoms.
2. The method of claim 1, wherein the liquid fermentation medium comprises: 10-90g/L of carbon source, 4-15g/L of sodium phosphate, 3-8 g/L of yeast extract, 3-8 g/L of corn steep liquor, 0.5-1.5g/L of urea, 0.5-1g/L of NaCl, 1-10g/L of KCl and 0.5-1.5g/L of defoamer.
3. The method according to claim 2, wherein n-alkane is fed continuously or intermittently in the fermentation culture stage of candida viscidosa so that the n-alkane concentration in the fermentation broth is always equal to or more than 20% (v/v); the n-alkane is one of n-alkanes containing 10-14 and 18 carbon atoms.
4. The method according to any one of claims 1 to 3, wherein the fermentation and culture stage of candida viscidosa is performed at a pH of 6.0 to 7.5 and a temperature of 24 to 29 ℃ for 20 to 40 hours.
5. The method according to claim 4, wherein the fermentation and cultivation stage of candida viscidosa is carried out at a pH of 6.0-7.5 and a temperature of 24-29 ℃ for 24-36 hours.
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