Immobilized cell material and application thereof in biological production of long-chain dicarboxylic acid

Abstract

The invention provides an immobilized cell material and application thereof in production of long-chain dicarboxylic acid by a biological method. The immobilized material is formed by polymerizing the following monomers: a first monomer: 5-15 parts by weight of a vinyl aromatic compound; 0.2-3 parts by weight of a second monomer which is a compound containing double bonds and at least one epoxy group; and a third monomer: 1-5 parts by weight of a divinyl aromatic compound. The immobilized cell material is prepared by contacting a cell seed solution with a seed culture medium containing the immobilized material at 27-31 DEG C by taking the immobilized material as a carrier of cells. And converting the fermentation substrate into the long-chain dicarboxylic acid by using the immobilized cell material. The immobilized cell material can be recycled for more than four times. The method is high in cell immobilization efficiency and good in use stability, the use efficiency of the cells can be improved, and a substrate can be efficiently converted to synthesize the long-chain binary acid. And the subsequent separation steps of the long-chain binary acid solution and the fermentation cells can be simplified.

Description

Technical Field

[0001] It relates to a solidified material of cells, a immobilization method, and the application of the solidified material of cells in the preparation of long-chain dibasic acids. Background Art

[0002] Long-chain dibasic acids refer to aliphatic dibasic carboxylic acids containing more than 10 carbon atoms (abbreviated as DCn, n≧10), which are fine chemical products with important and extensive industrial uses. They are also important raw materials for synthesizing high-grade fragrances, high-performance engineering plastics, high-temperature dielectrics, high-grade hot-melt adhesives, cold-resistant plasticizers, high-grade lubricating oils, high-grade paints and coatings, etc. in the chemical industry.

[0003] The chemical synthesis method of sebacic acid is mainly to hydrolyze castor oil to produce ricinoleic acid under the action of a catalyst, and then obtain it through high-temperature alkali cracking. The production of sebacic acid by biological fermentation can use petroleum by-products such as wax oil, alkane, fatty acid or its derivatives as raw materials, and ferment and produce sebacic acid by using the unique oxidation ability of microorganisms and the action of intracellular enzymes of microorganisms at low temperature and low pressure. Therefore, it has the advantages of wide raw material sources, simple production process, and mild production conditions, and has obvious advantages over the traditional chemical synthesis method. At present, some studies have been carried out on the preparation of sebacic acid by biological methods, especially in the 1970s and 1980s, and there were relatively many related studies. Yu Zhihua of the Institute of Microbiology, Chinese Academy of Sciences obtained an excellent strain through mutagenesis screening and optimized the conditions for the strain to produce sebacic acid. In a 16-liter fermenter, the sebacic acid in the fermentation broth reached more than 71 g / L, and the sebacic acid finished product was obtained by aqueous phase crystallization, with the product purity reaching more than 99.6% and the extraction yield of 85%. Liu Zutong et al. screened strains that can produce sebacic acid, and the concentration of sebacic acid produced was 2.9%. The Candida lipolytica screened by the Institute of Forestry and Soil Sciences, Chinese Academy of Sciences can produce sebacic acid, with an acid production of 30-40 g / L and a conversion rate of 43-55%. Chinese Patent CN107326051B discloses a sebacic acid produced by a microbial fermentation method and its preparation method. First, Candida tropicalis is subjected to seed culture, then the obtained seeds are inoculated into a fermentation medium, and decane is added to start fermentation conversion to obtain a fermentation broth. Finally, the fermentation broth is purified to obtain a sebacic acid product; all the above reports use free cells for fermentation, and there is no report on immobilized cell fermentation or catalytic conversion.

[0004] Immobilized cell fermentation refers to a method in which free cells are localized in a limited spatial area by physical or chemical means and maintained with catalytic activity for repeated use. Through the immobilization of microbial strains, the immobilized cells can be reused, improving the genetic stability of the strains, the activity of the strains and the fermentation performance. The fermentation broth and microorganisms can be conveniently separated to obtain and purify the products. Compared with the traditional free cell fermentation method, the use of the immobilization method for fermentation does not require repeated cultivation of seeds and makes continuous fermentation possible. Chen EC et al. used the sodium alginate embedding method to immobilize Cryptococcus neoformans / Pseudomonas aeruginosa for batch fermentation to convert pentadecane into DC15, and the cells could be stably used for 5 generations. Currently, there are few studies on the immobilized fermentation / catalytic conversion of dibasic acids. Therefore, it is very necessary and urgent to develop an efficient method for immobilized fermentation / catalytic conversion of long-chain dibasic acids. Summary of the Invention

[0005] One object of the present invention is to provide an application of an immobilized material in the preparation of immobilized cells.

[0006] Another object of the present invention is to provide an immobilized cell material, wherein the immobilized cell material uses the immobilized material as a carrier for cells.

[0007] The immobilized material is polymerized from the following monomers:

[0008] The first monomer: vinyl aromatic compound, 5 - 15 parts by weight;

[0009] The second monomer: a compound containing a double bond and at least one epoxy group, 0.2 - 3 parts by weight;

[0010] The third monomer: divinyl aromatic compound, 1 - 5 parts by weight.

[0011] In one embodiment, the cells are Candida cells, preferably including Candida versatilis cells, Candida tropicalis cells or Candida sake.

[0012] In one embodiment, the immobilized cell material is prepared by bringing the cell seed solution into contact with the immobilized material. The ratio of the cell seed solution to the immobilized material is preferably 1 ml:(0.5 - 5) g, further 1 ml:(0.8 - 2.5) g, and further 1 ml:(1.5 - 2.5) g. The optical density OD620 of the cell seed solution is 15 - 20.

[0013] In one embodiment, the number of times the immobilized cell material can be recycled is more than 4 times, further 4 - 10 times, and further 4 - 7 times, such as 5 times. The use temperature is 27 - 31 °C. The use method is to bring the immobilized cell material into contact with the fermentation substrate to convert the fermentation substrate into long-chain dibasic acids.

[0014] In one embodiment, the first monomer, the second monomer and the third monomer are different.

[0015] In one embodiment, the vinyl aromatic compound includes styrene, vinyltoluene, α-methylstyrene, p-methylstyrene, α-butylstyrene, 4-n-butylstyrene, and is preferably styrene.

[0016] In one embodiment, the compound containing a double bond and at least one epoxy group includes: glycidyl acrylate, glycidyl methacrylate, glycidyl ethyl acrylate, and is preferably glycidyl methacrylate.

[0017] In one embodiment, the divinyl aromatic compound includes divinylbenzene.

[0018] In one embodiment, the immobilized material is polymerized from the following monomers:

[0019] 5 to 15 parts of styrene, 0.2 to 3 parts of glycidyl methacrylate, 1 to 5 parts of divinylbenzene, and the parts are by weight.

[0020] In one embodiment, the polymerization reaction is carried out in the presence of an initiator. The initiator is preferably azobisisobutyronitrile, and the weight parts of the initiator are preferably 0.1 to 2 parts.

[0021] In one embodiment, the polymerization reaction is carried out in the presence of a surfactant. The surfactant is selected from any one or a combination of two or more of Span-20, Span-40, Span-60, Span-65, Span-80, Span-85. The weight parts of the surfactant are preferably 2 to 10 parts.

[0022] In one embodiment, the polymerization reaction is carried out in the presence of water. The weight parts of the water are preferably 70 to 90 parts.

[0023] In one embodiment, the preparation method of the immobilized material includes the following steps: weighing each raw material according to the formula, mixing the raw materials except water evenly, then adding water, stirring and mixing evenly to obtain an emulsion; transferring the emulsion to a mold, carrying out a polymerization reaction at 70 to 80 °C, the reaction time is 16 to 24 h, and drying to obtain the immobilized material.

[0024] Further, the method includes the following steps: weighing each raw material according to the formula, stirring and mixing the raw materials except water for 5 to 10 min, then adding water and stirring for 5 to 10 min to obtain an emulsion; transferring the obtained emulsion to a mold, carrying out a polymerization reaction at 70 to 80 °C, the reaction time is 16 to 24 h; then drying to obtain the immobilized material.

[0025] In one embodiment, the stirring speed is 200 to 350 rpm, further preferably 200 to 300 rpm.

[0026] In one embodiment, the method for removing water is to use a freeze dryer to remove water.

[0027] In one embodiment, the immobilized cell material is prepared by contacting the cell seed solution with the seed culture medium containing the immobilized material at 27 to 31 °C.

[0028] Preferably, the ratio of the cell seed solution to the immobilized material is 1 ml:(0.5 - 5) g, further preferably 1 ml:(0.8 - 2.5) g, and further preferably 1 ml:(1.5 - 2.5) g.

[0029] Preferably, the ratio of the seed culture medium to the immobilized material is (15 - 50) ml:1 g.

[0030] Preferably, the optical density OD620 of the cell seed solution is 15 to 20.

[0031] In one embodiment, the method for preparing the immobilized cell material includes the following steps:

[0032] Take the cell seed solution, add the seed solution to the seed culture medium containing the immobilized material, and culture it by shaking at a speed of 150 to 300 rpm at 27 to 31 °C for 10 to 24 h, and wash it with physiological saline to obtain the immobilized cell material.

[0033] In one embodiment, the method for preparing the cell seed solution includes the following steps: inoculate the cells into the seed culture medium, and culture it by shaking at a speed of 150 to 350 rpm at 27 to 31 °C for 16 to 48 h to obtain the seed solution.

[0034] In one embodiment, the components of the seed culture medium include: sucrose 10 - 25 g / L, corn steep liquor 2 - 8 g / L, yeast extract 1 - 8 g / L, potassium dihydrogen phosphate 4 - 12 g / L, and urea 0.5 - 4 g / L.

[0035] In one embodiment, the immobilized cell material is washed with physiological saline at a temperature of 27 to 31 °C. Wash it more than twice.

[0036] In one embodiment, washing with physiological saline includes the following steps: shake it at a speed of 70 to 150 rpm in physiological saline for 10 to 20 min, and the temperature of the physiological saline is 27 to 31 °C.

[0037] In one embodiment, the step of washing with physiological saline is repeated, and the number of repetitions is more than once.

[0038] A third object of the present invention is to provide a method for producing a long-chain dibasic acid, the method comprising the following steps: contacting an immobilized cell material with a fermentation substrate and converting the fermentation substrate into a long-chain dibasic acid.

[0039] In one embodiment, the method further comprises the following steps: recovering the immobilized cell material, washing it and reusing it to convert the fermentation substrate into a long-chain dibasic acid.

[0040] In one embodiment, the fermentation substrate is an alkane, preferably including any one of decane, n-undecane, n-dodecane, n-tridecane, n-tetradecane, n-pentadecane or n-hexadecane.

[0041] In one embodiment, the long-chain dibasic acid is an aliphatic dibasic carboxylic acid containing more than 10 carbon atoms. Preferably, the long-chain dibasic acid is an aliphatic dibasic carboxylic acid containing 10 to 16 carbon atoms.

[0042] Preferably, the way of contacting the immobilized cell material with the fermentation substrate is:

[0043] contacting the immobilized cell material with a fermentation medium containing the fermentation substrate; or contacting the immobilized cell material with a buffer solution containing the fermentation substrate.

[0044] In one embodiment, the immobilized cell material is contacted with a fermentation medium containing the fermentation substrate, and the fermentation substrate is used for fermentative production of a long-chain dibasic acid.

[0045] In a preferred embodiment, the mass-volume ratio of the immobilized cell material to the fermentation medium is (0.2-6) g: 15 ml, further (0.8-4) g: 15 ml, further (0.8-2.5) g: 15 ml, further (0.8-1.5) g: 15 ml.

[0046] In a preferred embodiment, the fermentation temperature is 27-31 °C.

[0047] In a preferred embodiment, stirring is carried out during the fermentation, and the stirring speed is 150-350 rpm.

[0048] In a preferred embodiment, the fermentation process is carried out in a shake flask or a fermenter.

[0049] In a preferred embodiment, the components of the fermentation medium include: sucrose 10-40 g / L, corn steep liquor 1-5 g / L, yeast extract 1-5 g / L, potassium dihydrogen phosphate 4-12 g / L, potassium nitrate 1-10 g / L, sodium chloride 0-3 g / L, urea 0.5-4 g / L.

[0050] In a preferred embodiment, the content of the fermentation substrate in the fermentation medium is 10% to 40%, and the percentage is by volume.

[0051] In a preferred embodiment, the method further comprises the following steps: after fermentation is completed, the immobilized cell material is washed, and the washed immobilized cell material is used for the next fermentation to produce long-chain dibasic acids. By recycling the immobilized cell material, cyclic fermentation is carried out.

[0052] In a preferred embodiment, after fermentation is completed, the immobilized cell material is washed with physiological saline. The temperature of the physiological saline is 27 to 31 °C. It is preferably washed more than twice.

[0053] In a preferred embodiment, the immobilized cell material is washed by oscillating in physiological saline at 27 to 31 °C at a rotation speed of 70 to 150 rpm for 10 to 20 min. This step is repeated, and the total number of washes is more than twice.

[0054] In a preferred embodiment, the total number of fermentation cycles is more than 4 times, further 4 to 10 times, further 4 to 7 times, for example 5 times.

[0055] In one embodiment, the immobilized cell material is contacted with a buffer solution containing a fermentation substrate to catalytically convert the fermentation substrate to produce long-chain dibasic acids.

[0056] In a preferred embodiment, the pH value of the buffer solution is 5 to 7, further 5.5 to 6.5.

[0057] In a preferred embodiment, the temperature of the catalytic conversion is 27 to 31 °C.

[0058] In a preferred embodiment, the mass-volume ratio of the immobilized cell material to the buffer solution is (0.5 to 3) g: 15 ml, further (0.8 to 1.5) g: 15 ml.

[0059] In a preferred embodiment, the content of the fermentation substrate in the buffer solution is 10% to 40%, and the percentage is by volume.

[0060] In a preferred embodiment, the buffer solution is a PBS buffer solution.

[0061] In a preferred embodiment, stirring is carried out during the catalytic conversion process, and the rotation speed is 200 to 350 rpm.

[0062] In a preferred embodiment, the catalytic conversion process is carried out in a shake flask or a fermenter.

[0063] In a preferred embodiment, after the catalytic conversion process is completed, the immobilized cell material is washed, and the washed immobilized cell material is used for the next catalytic conversion of the fermentation substrate to produce long-chain dibasic acids. By repeatedly using the immobilized cell material for catalytic conversion, cyclic catalytic conversion is carried out.

[0064] In a preferred embodiment, the immobilized cell material is washed with physiological saline at 27-31°C. The number of washing times is more than two.

[0065] In a preferred embodiment, the immobilized cell material is washed by oscillating in physiological saline at 27-31°C at a rotation speed of 70-150 rpm for 10-20 min. This step is repeated, and the total number of washings is more than two.

[0066] In a preferred embodiment, the total number of cyclic catalytic conversions is more than 4 times, further 4-10 times, and further 4-6 times.

[0067] Compared with the prior art, the beneficial effects of the present invention at least include:

[0068] The present invention uses a preferred immobilized material to prepare immobilized cells and uses them to convert the fermentation substrate into long-chain dibasic acids. The method of the present invention has high immobilization efficiency for cells, good use stability, can improve the use efficiency of cells, and can efficiently convert the substrate to synthesize long-chain dibasic acids. It can also simplify the subsequent separation steps of the long-chain dibasic acid solution and the fermentation cells. Specific Embodiments

[0069] Example 1-A Production of Sebacic Acid by Cyclic Fermentation of Immobilized Cell Material

[0070] I. Preparation of Immobilized Material

[0071] 1. Formula: Styrene 8%, GMA (glycidyl methacrylate) 1%, DVB (divinylbenzene) 2%, Span 80 5%, AIBN (azobisisobutyronitrile) 0.5%, and the rest is water. The percentages are all mass-volume ratios.

[0072] 2. Preparation of the immobilized material: Weigh each raw material according to the formula, mix the raw materials except water, stir at room temperature for 8 min at a stirring speed of 200 rpm to dissolve the solid AIBN, then add water dropwise, continue stirring at room temperature for 10 min at a stirring speed of 300 rpm, transfer the obtained emulsion to a mold, carry out a polymerization reaction at 73°C for 22 h, and then place it in a freeze dryer for drying to obtain the immobilized material.

[0073] II. Cell Immobilization and Fermentation Using the Immobilized Cell Material

[0074] 1. Formula:

[0075] Seed culture medium: sucrose 20 g / L, corn steep liquor 4 g / L, yeast extract 3 g / L, potassium dihydrogen phosphate 10 g / L, urea 3 g / L.

[0076] Fermentation medium: sucrose 20 g / L, corn steep liquor 4 g / L, yeast extract 3 g / L, potassium dihydrogen phosphate 10 g / L, potassium nitrate 4 g / L, urea 1 g / L. The content of decane in the fermentation culture is 20% (v / v).

[0077] 2. Cell immobilization: Inoculate Candida in the seed culture medium, shake-culture at 30 °C and 250 rpm for 24 h to prepare a cell seed solution, and the optical density OD620 of the cell seed solution is 18. Take 1 mL of the cultured cell seed solution and add it to 30 mL of the seed culture medium containing 1 g of the immobilization material, and shake-culture at 30 °C and 250 rpm for 16 h. Then transfer the immobilized cell material to 30 mL of physiological saline, shake at 30 °C and 100 rpm for 15 min for washing, and repeat this step twice to obtain the immobilized cell material.

[0078] 3. Use the immobilized cell material for cyclic fermentation

[0079] First fermentation: Transfer 1 g of the immobilized cell material to 15 mL of the fermentation medium, and perform shake-flask fermentation at 29 °C and 250 rpm. After the fermentation is completed, repeat the washing of the immobilized cell material with physiological saline, and the washing process is the same as in step 2.

[0080] Cyclic fermentation: Transfer the washed immobilized cell material to 15 mL of the fermentation medium for the second fermentation: perform shake-flask fermentation at 29 °C and 250 rpm. After the fermentation is completed, repeat the washing of the immobilized cell material with physiological saline, and the washing process is the same as in step 2. And so on for the next fermentation, and ferment a total of 5 times.

[0081] Example 1-B

[0082] It is basically the same as Example 1-A, except that: the amount of the immobilized cell material used in step 3 is 0.5 g.

[0083] Example 1-C

[0084] It is basically the same as Example 1-A, except that: the amount of the immobilized cell material used in step 3 is 1.5 g.

[0085] Example 1-D

[0086] It is basically the same as Example 1-A, except that: the amount of the immobilization material used in step 2 is 2 g.

[0087] Example 1-E

[0088] It is basically the same as Example 1-A, except that: the amount of the immobilized material used in Step 2 is 3 g.

[0089] The acid production concentrations in each fermentation of Examples 1-A to 1-E are shown in Table 1.

[0090] Table 1

[0091]

[0092] Example 2-A: Production of Sebacic Acid by Catalytic Conversion of Immobilized Cell Materials

[0093] I. Preparation of Immobilized Materials

[0094] 1. Formula: Styrene 8%, GMA (Glycidyl Methacrylate) 1%, DVB (Divinylbenzene) 2%, Span 80 5%, AIBN (Azobisisobutyronitrile) 0.5%, and the rest is water. The percentages are all mass-volume ratios.

[0095] 2. Preparation of immobilized cell materials: Weigh each raw material according to the formula, mix the raw materials except water, stir at room temperature for 10 min with a stirring speed of 200 rpm to dissolve the solid AIBN, then add water dropwise and continue stirring at room temperature for 10 min with a stirring speed of 350 rpm; transfer the obtained emulsion to a mold, carry out a polymerization reaction at 72 °C for 20 h, and then place it in a freeze dryer for drying to obtain the immobilized material.

[0096] II. Cell Immobilization and Catalytic Conversion of Sebacic Acid Using Immobilized Cells

[0097] 1. Seed medium formula: Sucrose 20 g / L, Corn Steep Liquor 4 g / L, Yeast Extract 3 g / L, Potassium Dihydrogen Phosphate 10 g / L, Urea 3 g / L.

[0098] 2. Immobilization of cells: Inoculate Candida sp. into the seed medium, shake culture at 30 °C with a rotation speed of 250 rpm for 24 h to prepare a cell seed solution, and the optical density OD620 of the cell seed solution is 18. Take 1 mL of the cultured cell seed solution and add it to 30 mL of the seed medium containing 1 g of the immobilized material, shake culture at 30 °C with a rotation speed of 250 rpm for 16 h. Then transfer the immobilized cell material to 30 mL of physiological saline, shake at 30 °C with a rotation speed of 100 rpm for 15 min for washing, and repeat this step twice to obtain the immobilized cell material.

[0099] 3. Catalytic conversion using immobilized cell materials:

[0100] First catalysis: Transfer 1 g of the immobilized cell material to 15 mL of PBS buffer with a pH of 6 containing 20% (v / v) decane, and conduct shake flask catalysis at 29 °C with a rotation speed of 250 rpm. After the catalysis is completed, repeatedly wash the immobilized cell material with physiological saline, and the washing process is the same as in Step 2.

[0101] Circular catalysis: Transfer 1 g of the immobilized cell material to 15 mL of PBS buffer containing 20% (v / v) decane for the second catalytic conversion: Conduct shake flask catalysis at a temperature of 29 °C and a rotation speed of 250 rpm. After the catalytic conversion is completed, repeatedly wash the immobilized cell material with physiological saline, and the washing process is the same as in Step 2. And so on for the next catalytic conversion, with a total of 5 cycles.

[0102] Example 2-B

[0103] It is basically the same as Example 2-A, except that: the amount of the immobilized cell material used in Step 3 is 0.5 g.

[0104] Example 2-C

[0105] It is basically the same as Example 2-A, except that: the amount of the immobilized cell material used in Step 3 is 2 g.

[0106] The acid production concentrations of each catalytic conversion in Examples 2-A to 2-C are shown in Table 2.

[0107] Table 2

[0108]

[0109] Example 3-A

[0110] It is basically the same as Example 1-A, except that: the fermentation substrate is n-dodecane, and dodecanedioic acid is fermented and produced.

[0111] Example 3-B

[0112] It is basically the same as Example 3-A, except that: the amount of the immobilized cell material used in Step 3 is 0.5 g.

[0113] Example 3-C

[0114] It is basically the same as Example 3-A, except that: the amount of the immobilized cell material used in Step 3 is 2 g.

[0115] The acid production concentrations of each fermentation in Examples 3-A to 3-C are shown in Table 3.

[0116] Table 3

[0117]

[0118] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it; although the present invention has been described in detail with reference to the foregoing embodiments, those of ordinary skill in the art should understand that they can still modify the technical solutions recorded in the foregoing embodiments, or perform equivalent replacements on some or all of the technical features, and these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. Use of an immobilized material in the preparation of immobilized cells, wherein the immobilized material is polymerized from the following monomers: The first monomer: vinyl aromatic compound, 5 - 15 parts by weight; The second monomer: a compound containing a double bond and at least one epoxy group, 0.2 - 3 parts by weight; The third monomer: divinyl aromatic compound, 1 - 5 parts by weight.

2. An immobilized cell material, characterized in that, Using the immobilized material according to claim 1 as a carrier for cells.

3. The immobilized cell material according to claim 2, wherein The cells are Candida cells, preferably including Candida versatilis cells, Candida tropicalis cells or Candida sake.

4. The immobilized cell material according to claim 2, wherein The number of times the immobilized cell material can be recycled is more than 4 times.

5. The immobilized cell material according to claim 2, wherein Preparing the immobilized cell material by bringing the cell seed solution into contact with the immobilized material.

6. The immobilized cell material according to claim 1 or 5, characterized in that, The method for preparing the immobilized cell material includes the following steps: bringing the cell seed solution into contact with a seed culture medium containing the immobilized material at 27 - 31 °C to prepare the immobilized cell material.

7. The immobilized cell material according to claim 5 or 6, characterized in that, The ratio of the cell seed solution to the immobilized material is 1 ml : (0.5 - 5) g, further 1 ml : (0.8 - 2.5) g, further 1 ml : (1.5 - 2.5) g; and / or, The ratio of the seed culture medium to the immobilized material is (15 - 50) ml : 1 g; and / or, The optical density OD620 of the cell seed solution is 15 - 20.

8. A method for producing long-chain dibasic acid, characterized in that, Including the following steps: bringing the immobilized cell material according to claim 2 into contact with a fermentation substrate to convert the fermentation substrate into a long-chain dicarboxylic acid.

9. The method according to claim 8, wherein The way of bringing the immobilized cell material into contact with the fermentation substrate is: Bringing the immobilized cell material into contact with a fermentation culture medium containing the fermentation substrate; or, Bringing the immobilized cell material into contact with a buffer solution containing the fermentation substrate.

10. The method according to claim 8 or 9, characterized in that The method further includes the following steps: recovering the immobilized cell material, washing it and then reusing it to convert the fermentation substrate into a long-chain dicarboxylic acid.

11. The method according to claim 8 or 9, characterized in that, The fermentation substrate is an alkane, preferably including any one of decane, n-undecane, n-dodecane, n-tridecane, n-tetradecane, n-pentadecane or n-hexadecane.

12. The method according to claim 9, wherein The mass-volume ratio of the immobilized cell material to the fermentation culture medium is (0.2 - 6) g : 15 ml, further (0.8 - 2.5) g : 15 ml, further (0.8 - 1.5) g : 15 ml; and / or, The fermentation temperature is 27 - 31 °C; and / or, Stirring is carried out during the fermentation process, and the stirring speed is 150 - 350 rpm; and / or, The components of the fermentation culture medium include: sucrose 10 - 40 g / L, corn steep liquor 1 - 5 g / L, yeast extract 1 - 5 g / L, potassium dihydrogen phosphate 4 - 12 g / L, potassium nitrate 1 - 10 g / L, sodium chloride 0 - 3 g / L, urea 0.5 - 4 g / L; and / or, The content of the fermentation substrate in the fermentation culture medium is 10% - 40%, and the percentage is by volume.

13. The method according to claim 9, wherein The pH value of the buffer solution is 5 - 7; and / or, The temperature for catalytic conversion is 27 - 31 °C; and / or, The mass-volume ratio of the immobilized cell material to the buffer solution is (0.5 - 3) g : 15 ml, further (0.8 - 1.5) g : 15 ml; and / or, The content of the fermentation substrate in the buffer solution is 10% to 40%, and the percentage is by volume.

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