Method for synthesizing long-chain fatty acid and ultra-long-chain fatty acid by biotransformation of carbon dioxide

Abstract

The invention relates to the technical field of bioengineering, in particular to a method for synthesizing long-chain fatty acid and ultra-long-chain fatty acid through biotransformation of carbon dioxide. According to the present invention, the long-chain fatty acid and the ultra-long-chain fatty acid are produced by using the clostridium endophyllum and the Yarrowia lipolytica through the two-step bioconversion cascade carbon sequestration method and using the carbon dioxide as the carbon source through the fermentation, and the long-chain fatty acid and the ultra-long-chain fatty acid are produced, wherein the long-chain fatty acid and the ultra-long-chain fatty acid are used as the carbon source, such that the long-chain fatty acid and the ultra-long-chain fatty acid are produced, according to the method disclosed by the invention, the dependence of the traditional microorganism synthesis of long-chain fatty acid and ultra-long-chain fatty acid on a carbohydrate carbon source is eliminated, the bioconversion from carbon dioxide to the long-chain fatty acid and the ultra-long-chain fatty acid is realized, and a new carbon source selection and method are provided for the synthesis of the long-chain fatty acid and the ultra-long-chain fatty acid.

Description

Technical Field

[0001] The present invention relates to the field of bioengineering technology, and in particular to a method for bioconverting carbon dioxide to synthesize long-chain fatty acids and very-long-chain fatty acids. Background Art

[0002] Fatty acids (FA) are composed of a hydrocarbon chain and a carboxyl group at the end of the hydrocarbon chain, and are widely present in nature. According to the carbon chain length of fatty acids, they can be divided into short-chain fatty acids (C2-C4), medium-chain fatty acids (C6-C12), long-chain fatty acids (C14-C18), and very-long-chain fatty acids (≥C20). Long-chain fatty acids are not only energy storage substances in organisms, but also important components of biological membranes, playing an important role in life activities; in addition, long-chain fatty acids can be synthesized into biodiesel through bioconversion. Compared with traditional fossil diesel, biodiesel has the characteristics of being renewable, non-toxic, and biodegradable, and is an excellent alternative to chemical diesel; very-long-chain polyunsaturated fatty acids, such as docosahexaenoic acid (DHA, C22:6n-4,7,10,13,19), nervonic acid (DHA, C24:1n-9), etc., play an important role in the medical fields such as preventing human cardiovascular diseases and nerve tissue repair and regeneration. Therefore, long-chain fatty acids and very-long-chain fatty acids play an important role in biological metabolic activities and the energy field.

[0003] Plants, microorganisms, and animals can all synthesize long-chain fatty acids and very-long-chain fatty acids, and form diverse fatty acid synthesis pathways according to their respective metabolic characteristics. According to the sources of energy and carbon sources, there are mainly three synthesis routes for long-chain fatty acids and very-long-chain fatty acids: plants and algae use sunlight as energy and CO 2 as a carbon source to synthesize long-chain fatty acids, and soybean oil, peanut oil, rapeseed oil, palm oil, etc. are representative products. The fatty acids produced by these oil crops have become indispensable foods in human life and can be used as raw materials for bioenergy in the transportation field; microorganisms and animals use sugars as energy and carbon sources for fatty acid synthesis, and animal (such as pigs and cows) fats and functional oils (such as DHA and nervonic acid) are their representative products. Since a large amount of land is required when sunlight is used as energy for the synthesis of long-chain fatty acids and very-long-chain fatty acids, and the supply of sugars (mainly starch sugar) is also limited to a certain extent, it is necessary to find alternative energy sources to achieve the biosynthesis of long-chain fatty acids and very-long-chain fatty acids. Using CO 2 as a carbon source and H 2 as an energy source for biosynthesis is a preferred technical route. Especially with the development of electrolytic water hydrogen production technology and the decline of the cost of green hydrogen, this technical route has attracted more and more attention. Therefore, it is urgent to develop a method that uses CO 2 as a carbon source and H 2Biosynthesis of long-chain fatty acids and very long-chain fatty acids as energy sources. Summary of the invention

[0004] The invention provides a method for synthesizing long-chain fatty acids and ultra-long-chain fatty acids by bioconverting carbon dioxide.

[0005] The present invention adopts a two-step bioconversion cascade carbon fixation method, uses carbon dioxide as a carbon source, and utilizes Clostridium ljungdahlii anaerobic fermentation and Yarrowia lipolytica aerobic fermentation to achieve bioconversion of carbon dioxide to synthesize long-chain fatty acids and very-long-chain fatty acids, eliminates the dependence of traditional microbial synthesis of long-chain fatty acids and very-long-chain fatty acids on carbohydrate carbon sources, and achieves bioconversion from carbon dioxide to long-chain fatty acids and very-long-chain fatty acids.

[0006] In a first aspect, the present invention provides a fermentation bacteria composition, wherein the fermentation bacteria composition comprises Clostridium ljungdahlii and Yarrowia lipolytica.

[0007] The fermented bacteria composition can convert carbon dioxide into long-chain fatty acids and very long-chain fatty acids through two-step fermentation.

[0008] Preferably, the Clostridium ljungdahlii is Clostridium ljungdahlii DSM13528.

[0009] Preferably, the Yarrowia lipolytica is a strain with a deposit number of CGMCC No.15309.

[0010] The present invention discovers that, compared with other compounding methods of bacterial strains, using Clostridium ljungdahlii DSM13528 and Yarrowia lipolytica with the preservation number of CGMCC No. 15309 as the fermentation bacterial strains for two-step fermentation in sequence can enable the two-step fermentation to cooperate better. Using Clostridium ljungdahlii DSM13528 as the bacterial strain for the first-step fermentation can efficiently synthesize acetic acid, and at the same time, its fermentation supernatant contains fewer components that are unfavorable for the growth and acid production of Yarrowia lipolytica. The mash can be directly used as a carbon source and energy source to culture Yarrowia lipolytica. The second-step fermentation uses Yarrowia lipolytica with the preservation number of CGMCC No. 15309 as the bacterial strain, and can further efficiently synthesize long-chain fatty acids and very-long-chain fatty acids by using the fermentation supernatant produced by Clostridium ljungdahlii DSM13528.

[0011] In a second aspect, the present invention provides the application of the above-mentioned fermentation bacterial strain composition in the production of long-chain fatty acids and / or very-long-chain fatty acids.

[0012] Preferably, the production of long-chain fatty acids and / or very-long-chain fatty acids is to synthesize long-chain fatty acids and / or very-long-chain fatty acids by bioconverting carbon dioxide.

[0013] Preferably, the production of long-chain fatty acids and / or very-long-chain fatty acids is carried out using carbon dioxide as a carbon source and hydrogen as an energy source.

[0014] In a third aspect, the present invention provides a method for producing long-chain fatty acids and / or very-long-chain fatty acids. The method is to synthesize long-chain fatty acids by bioconverting carbon dioxide;

[0015] The method includes a first fermentation stage and a second fermentation stage; the first fermentation stage is: using Clostridium ljungdahlii to ferment and produce acetic acid with syngas as a carbon source; the second fermentation stage is: using Yarrowia lipolytica to ferment and produce long-chain fatty acids and / or very-long-chain fatty acids with the fermentation supernatant of the first fermentation stage as a carbon source.

[0016] Preferably, the volume ratio of H 2 and CO 2 in the syngas is (1 - 3):1. More preferably, it is (1 - 2):1.

[0017] Preferably, in the first fermentation stage, CO 2 is used as the carbon source and hydrogen is used as the energy source.

[0018] Preferably, the Clostridium ljungdahlii is Clostridium ljungdahlii DSM13528.

[0019] Preferably, the Yarrowia lipolytica is the strain with the preservation number of CGMCC No. 15309.

[0020] The present invention discovers that, compared with other compounding methods of bacterial strains, using Clostridium ljungdahlii DSM13528 and Yarrowia lipolytica with the preservation number of CGMCC No. 15309 as the fermentation bacterial strains for the two-step fermentation in sequence can enable the two-step fermentation to cooperate better, convert carbon dioxide into long-chain fatty acids and / or ultra-long-chain fatty acids more efficiently, and significantly improve the yield of long-chain fatty acids and / or ultra-long-chain fatty acids.

[0021] In the method described above, the first fermentation medium used in the first fermentation stage comprises the following components: macronutrients, nitrilotriacetic acid, calcium chloride, trace metal elements, sodium tungstate, sodium molybdate, sodium selenate, ferrous sulfate, vitamins, cysteine hydrochloride, sodium bicarbonate, yeast powder and cysteine.

[0022] Preferably, the macronutrients include ammonium chloride, potassium chloride, magnesium sulfate heptahydrate, sodium chloride and potassium dihydrogen phosphate.

[0023] Preferably, the trace metal elements include manganese sulfate, cobalt chloride, zinc sulfate and nickel chloride.

[0024] Preferably, the vitamins include biotin, folic acid, VB6, thiamine, riboflavin, nicotine, calcium pantothenate, cyanocobalamin, p-aminobenzoic acid and lipoic acid.

[0025] Preferably, the first fermentation medium comprises the following components: 8 - 12 mL / L of macroelement solution, 150 - 250 μL / L of nitrilotriacetic acid solution, 0.8 - 1.2 mL / L of calcium chloride solution, 0.8 - 1.2 mL / L of trace metal element stock solution, 150 - 250 μL / L of sodium tungstate stock solution, 150 - 250 μL / L of sodium molybdate and sodium selenite stock solution, 150 - 250 μL / L of ferrous sulfate stock solution, 0.8 - 1.2 mL / L of vitamin stock solution, 8 - 12 mL / L of cysteine hydrochloride stock solution, 8 - 12 mL / L of sodium bicarbonate stock solution, 0.2 - 0.5 g / L of yeast powder and 0.2 - 0.5 g / L of cysteine.

[0026] Among them, the macroelement solution comprises: 100 g / L of ammonium chloride, 10 g / L of potassium chloride, 20 g / L of magnesium sulfate heptahydrate, 80 g / L of sodium chloride, 10 g / L of potassium dihydrogen phosphate.

[0027] The concentration of the nitrilotriacetic acid solution is 0.1 g / mL.

[0028] The concentration of the calcium chloride solution is 0.023 g / mL.

[0029] 100 mL of the trace metal element stock solution comprises: 1.1 g of manganese sulfate tetrahydrate, 0.2 g of cobalt chloride dihydrate, 0.2 g of zinc sulfate heptahydrate, 0.02 g of nickel chloride hexahydrate.

[0030] The concentration of sodium tungstate dihydrate in the sodium tungstate stock solution is 0.025 g / mL. The concentrations of sodium molybdate dihydrate and sodium selenite in the sodium molybdate and sodium selenite stock solution are 0.002 g / mL and 0.001 g / mL respectively.

[0031] The concentration of ferrous sulfate heptahydrate in the ferrous sulfate stock solution is 0.02 g / mL.

[0032] 100 mL of the vitamin stock solution comprises: 2 mg of biotin, 2 mg of folic acid, VB 6 1 mg, 5 mg of thiamine, 5 mg of riboflavin, 5 mg of nicotine, 5 mg of calcium pantothenate, 5 mg of cyanocobalamin, 5 mg of p - aminobenzoic acid, 5 mg of lipoic acid.

[0033] The concentration of the cysteine hydrochloride stock solution is 0.15 g / mL.

[0034] The concentration of the sodium bicarbonate stock solution is 0.1 g / mL.

[0035] In the above - mentioned method, the second fermentation medium used in the second fermentation stage comprises the following components: the fermentation supernatant of the first fermentation stage, yeast powder and ammonium sulfate;

[0036] Preferably, the second fermentation medium uses the fermentation supernatant of the first fermentation stage as a solvent, contains 1-5 g / L of yeast powder and 2-8 g / L of ammonium sulfate, and preferably contains 1-3 g / L of yeast powder and 2-4 g / L of ammonium sulfate.

[0037] In the method described above, the fermentation conditions of the first fermentation stage are: the fermentation temperature is 34-38 °C, and the pH is 5.5-6.5.

[0038] Preferably, the rotation speed in the first fermentation stage is 200-400 rpm, and the inlet gas flow rate is 10 mL / min - 50 mL / min.

[0039] Preferably, in the first fermentation stage, when the OD of Clostridium ljungdahlii 600 <0.5, the inlet gas flow rate is 10-12 mL / min, and when OD 600 ≥0.5, the ventilation volume is adjusted to 38-42 mL / min.

[0040] Preferably, in the first fermentation stage, the pH is controlled to be 5.8-6.2. The pH control can be carried out using potassium hydroxide solution.

[0041] In the method described above, the fermentation conditions of the second fermentation stage are: the fermentation temperature is 26-30 °C, and the pH is 5.0-7.5.

[0042] Preferably, the rotation speed in the second fermentation stage is 200-400 rpm.

[0043] The beneficial effects of the present invention at least include: The present invention uses Clostridium ljungdahlii and Yarrowia lipolytica to ferment and produce long-chain fatty acids and very-long-chain fatty acids through a two-step bioconversion cascade carbon fixation method with carbon dioxide as a carbon source. The method of the present invention relieves the dependence of traditional microbial synthesis of long-chain fatty acids and very-long-chain fatty acids on sugar carbon sources, realizes the bioconversion from carbon dioxide to long-chain fatty acids and very-long-chain fatty acids, provides new carbon source options and methods for the synthesis of long-chain fatty acids and very-long-chain fatty acids, and lays a foundation for the development of cell factories for long-chain fatty acids and very-long-chain fatty acids using CO 2 as a carbon source. BRIEF DESCRIPTION OF THE DRAWINGS

[0044] In order to more clearly illustrate the technical solutions in the present invention or the prior art, the following will briefly introduce the drawings required for the description of the embodiments or the prior art. Obviously, the drawings in the following description are some embodiments of the present invention. For those of ordinary skill in the art, other drawings can be obtained based on these drawings without creative efforts.

[0045] Figure 1 For the growth of Clostridium ljungdahlii in Example 1 of the present invention under the conditions of H 2 / CO 2 as the carbon source and energy source.

[0046] Figure 2 For the product accumulation of Clostridium ljungdahlii in Example 1 of the present invention under the conditions of H 2 / CO 2 as the carbon source and energy source.

[0047] Figure 3 For the growth of Yarrowia lipolytica in Example 1 of the present invention under the fermentation conditions of two different carbon sources.

[0048] Figure 4 For the detection results of dry weight and oil content of Yarrowia lipolytica in Example 1 of the present invention under the fermentation conditions of two different carbon sources.

[0049] Figure 5 For the percentage content results of each component of the oil of Yarrowia lipolytica in Example 1 of the present invention under the fermentation conditions of two different carbon sources. Detailed implementation manners

[0050] To make the objectives, technical solutions and advantages of the present invention clearer, the technical solutions in the present invention will be clearly and completely described below with reference to the accompanying drawings in the present invention. Obviously, the described embodiments are some, but not all, of the embodiments of the present invention. All other embodiments obtained by those of ordinary skill in the art based on the embodiments in the present invention without making creative efforts fall within the protection scope of the present invention.

[0051] Example 1 Method for biologically converting carbon dioxide to synthesize long-chain fatty acids and very-long-chain fatty acids

[0052] This example provides a method for biologically converting carbon dioxide to synthesize long-chain fatty acids and very-long-chain fatty acids, including the following steps:

[0053] 1. Fermentation of Clostridium ljungdahlii to produce acetic acid using syngas (H 2 / CO 2 )

[0054] (1) Fermentation strain used: Clostridium ljungdahlii DSM13528, which can be obtained through commercial channels.

[0055] (2) Fermentation medium for Clostridium ljungdahlii: Adjusted DSM 879 medium, containing in 1 L of medium: 10 mL of macroelement solution, 200 μL of nitrilotriacetic acid solution, 1 mL of calcium chloride solution, 1 mL of trace metal stock solution, 100 μL of sodium tungstate stock solution, 100 μL of sodium molybdate and sodium selenite stock solution, 200 μL of ferrous sulfate stock solution, 1 mL of vitamin stock solution, 10 mL of cysteine hydrochloride stock solution, 10 mL of sodium bicarbonate stock solution, 0.25 g of yeast powder and 0.25 g of cysteine, prepared under anaerobic conditions.

[0056] Among them, the preparation method of the macroelement stock solution is as follows: 100 g of ammonium chloride, 10 g of potassium chloride, 20 g of magnesium sulfate heptahydrate, 80 g of sodium chloride, 10 g of potassium dihydrogen phosphate, dissolved with boiled pure water, made up to 1 L, filtered and sterilized with a 0.22 μm filter membrane, and stored at room temperature.

[0057] The preparation method of the nitrilotriacetic acid stock solution is as follows: Take 10 g of nitrilotriacetic acid, dissolve it with sterilized pure water while it is hot. If it cannot be dissolved, add an appropriate amount of sodium hydroxide pellets and stir until dissolved, make up to 100 mL, and store at room temperature.

[0058] The preparation method of the calcium chloride stock solution is as follows: Take 2.3 g of calcium chloride, dissolve it with sterilized pure water while it is hot and make up to 100 mL, filter and sterilize with a 0.22 μm filter membrane, and store at room temperature.

[0059] The preparation method of the trace metal stock solution is as follows: 1.1 g of manganese sulfate tetrahydrate, 0.2 g of cobalt chloride dihydrate, 0.2 g of zinc sulfate heptahydrate, 0.02 g of nickel chloride hexahydrate, dissolved with sterilized pure water while it is hot and made up to 100 mL, filtered and sterilized with a 0.22 μm filter membrane, and stored at room temperature.

[0060] The preparation method of the sodium tungstate stock solution is as follows: Take 2.5 g of sodium tungstate dihydrate, dissolve it with sterilized pure water while it is hot and make up to 100 mL, filter and sterilize with a 0.22 μm filter membrane, and store at room temperature.

[0061] The preparation method of the sodium molybdate and sodium selenite stock solution is as follows: Take 0.2 g of sodium molybdate dihydrate and 0.1 g of sodium selenite, dissolve it with sterilized pure water while it is hot and make up to 100 mL, filter and sterilize with a 0.22 μm filter membrane, and store at room temperature.

[0062] The preparation method of the ferrous sulfate stock solution is as follows: Take 2 g of ferrous sulfate heptahydrate, dissolve it with sterilized pure water while it is hot and make up to 100 mL, aliquot into 1.5 mL centrifuge tubes, and store at -20 °C.

[0063] The preparation method of the vitamin stock solution is as follows: 2 mg of biotin, 2 mg of folic acid, VB 6Dissolve 1 mg of thiamine, 5 mg of riboflavin, 5 mg of niacin, 5 mg of calcium pantothenate, 5 mg of cyanocobalamin, 5 mg of p-aminobenzoic acid, and 5 mg of lipoic acid in boiling pure water while it is hot, make up the volume to 100 mL, filter and sterilize with a 0.22-μm filter membrane into a sterile gas bottle, replace the air with high-purity nitrogen while it is hot, and store in the dark at room temperature.

[0064] The preparation method of the cysteine hydrochloride stock solution is as follows: Take 15 g of cysteine hydrochloride, make up the volume to 100 mL with boiling pure water, filter and sterilize with a 0.22-μm filter membrane into a sterile gas bottle, replace the air with high-purity nitrogen while it is hot, and store at room temperature.

[0065] The preparation method of the sodium bicarbonate stock solution is as follows: Take 10 g of sodium bicarbonate, make up the volume to 100 mL with boiling pure water, filter and sterilize with a 0.22-μm filter membrane into a sterile gas bottle, replace the air with high-purity nitrogen while it is hot, and store at room temperature.

[0066] (3) Fermentation conditions: Use a 5-L Guoqiang fermenter, load 3 L of liquid for fermentation, keep the temperature at 36 °C, the rotation speed at 300 rpm, the inoculation amount at 5% (150 mL), and the carbon source composition is H 2 / CO 2 (v / v, 60:40). During the fermentation process, the OD of Clostridium is 600 Keep the inlet gas flow rate at 10 mL / min before it reaches 0.5, and adjust the ventilation volume to 40 mL / min after it reaches 0.5. Control the pH value with a 4-M potassium hydroxide solution throughout the fermentation process and keep it at 6.0.

[0067] (4) Fermentation results

[0068] Growth: The growth of Clostridium ljungdahlii under the conditions of using H 2 / CO 2 as the carbon source and energy source is represented by the optical density (OD 600 ) of the fermentation broth at 600 nm. It is measured that 1 OD of Clostridium cells is equivalent to 0.2 g / L of dry biomass weight. The results are as Figure 1 shown.

[0069] Product accumulation: The main product of Clostridium ljungdahlii under the conditions of using H 2 / CO 2 as the carbon source and energy source is acetic acid, and the yield reaches 34 g / L. A small amount of ethanol is synthesized, and the yield is 1.6 g / L ( Figure 2 ). After centrifuging the fermentation broth mainly containing acetic acid, the supernatant is directly used for the fermentation of Yarrowia lipolytica.

[0070] 2. Fermentation of acetic acid by Yarrowia lipolytica to produce long-chain fatty acids and very-long-chain fatty acids

[0071] (1) Strains used for fermentation: The strain with the preservation number of CGMCC No. 15309, which was preserved in the China General Microbiological Culture Collection Center (CGMCC) on January 25, 2018. The address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing. The preservation number is CGMCC No. 15309 and it has been disclosed in the patent application CN110358692A.

[0072] (2) Fermentation medium:

[0073] Experimental group: Using the fermentation supernatant of Clostridium ljungdahlii in step 1 as the fermentation medium, and adding yeast powder with a final concentration of 1.32 g / L and ammonium sulfate with a final concentration of 2.64 g / L to the fermentation medium;

[0074] Control group: 1 L of the fermentation medium contains 33 g of glucose, 1.32 g of yeast powder and 2.64 g of ammonium sulfate.

[0075] (3) Fermentation conditions: The experimental group and the control group were resuscitated in their respective fermentation media. After 24 h, they were inoculated into 500 mL shake flasks containing 30 mL of fermentation medium at a final OD 600 = 0.1, cultured at 28 °C with a rotation speed of 200 rpm for 120 h.

[0076] (4) Fermentation results

[0077] ① Growth

[0078] The growth of Yarrowia lipolytica under the fermentation conditions of two different carbon sources in the experimental group and the control group was represented by the optical density (OD 600 ) of the fermentation broth at 600 nm, and the dry weight was measured by weighing. The results are as Figure 3 shown.

[0079] ② Dry weight and oil production

[0080] Dry weight determination: Take 5 mL of the fermentation broth, centrifuge to remove the supernatant, wash the precipitated cells once with pure water, centrifuge to remove the supernatant to obtain a cell sample without medium components. The sample was placed in a -80 °C refrigerator and frozen overnight, and then the water was removed by freeze-drying and the dry weight was weighed. Through calculation, the yeast dry weight in the experimental group (using the fermentation supernatant of Clostridium as the carbon source, that is, acetic acid as the carbon source) was 4.2 ± 0.2 g / L, and the yeast dry weight in the control group (using glucose as the carbon source) was 7.3 ± 0.1 g / L ( Figure 4 ), that is, the biomass under the condition of using glucose as the carbon source was significantly higher than that under the condition of using acetic acid as the carbon source.

[0081] Oil extraction and production determination:

[0082] Oil extraction: Sampling, mixing 1 g of wet bacterial cells with 5 mL of deionized water, aspirating and pipetting repeatedly until dispersed in water. Transfer the mixture into a glass centrifuge tube, add 3 mL of 4 mol / L hydrochloric acid for cell disruption, tighten the lid and place it in a shaker for horizontal oscillation for 1 h. Then take it out and boil it in boiling water for 8 min, and then freeze it in a -20 °C refrigerator for 30 min. Take it out of the refrigerator, after restoring to room temperature, add 6 mL of chloroform:methanol = 1:1 extraction agent, shake well, and then centrifuge at 4000 rpm for 10 min using a centrifuge. After taking it out of the centrifuge, transfer the lower chloroform layer to another glass tube, then add 3 mL of 0.05% sodium chloride solution to the new glass tube, shake well and centrifuge at 4000 rpm for 10 min. Again, use a pipette to take out the lower chloroform layer and transfer it to a pre-weighed glass tube. Use a nitrogen blower to heat and dry the chloroform to leave the oil. Then place it in an oven at 65 °C for drying for 1 h, take it out and weigh it.

[0083] Fatty acid methylation: Add 2.6 mL of methanol:sulfuric acid = 98:2 (V / V) solvent to the weighed oil. Tighten the lid and place it in an oven at 85 °C for methylation reaction for 3 h. During the reaction, shake it every half hour to ensure full reaction. Take it out of the oven, after cooling to room temperature, add 1 mL of saturated sodium chloride solution, then add 1 mL of n-hexane, shake well. After centrifuging at 4000 rpm for 10 min using a centrifuge, take the upper n-hexane layer and transfer it to a 2 mL EP tube. Centrifuge the EP tube at 12000 rpm for 5 min, then take 200 μL and transfer it into the inner insert tube of a gas chromatography sample vial, and analyze the proportion of each component in the oil by gas chromatography to calculate the yield of each component.

[0084] Through measurement, under the experimental group (using the supernatant of Clostridium fermentation as the carbon source, that is, acetic acid as the carbon source), the yeast oil yield was 1.5 ± 0.1 g / L, and under the control group (using glucose as the carbon source), the yeast oil yield was 2.9 ± 0.5 g / L ( Figure 4 ). The results of fatty acid composition analysis showed ( Figure 5 ) that the ratio of very long-chain fatty acids (C24 fatty acids) in the total oil under the experimental group (using the supernatant of Clostridium fermentation as the carbon source, that is, acetic acid as the carbon source) was higher than that under the control group (using glucose as the carbon source).

[0085] In summary, the method of using Clostridium ljungdahlii to ferment acetic acid by using H 2 / CO 2 and using the fermentation broth for the fermentation of Yarrowia lipolytica to produce oil in the present invention realizes the biological fixation of CO 2Biotransformation for synthesizing long-chain fatty acids and very-long-chain fatty acids. Compared with the production of oils and fats using glucose as a carbon source, using the method of the present invention, Yarrowia lipolytica is more inclined to produce a high proportion of very-long-chain fatty acids (C24:1 and C24:0), providing an effective method for synthesizing long-chain fatty acids and very-long-chain fatty acids using CO 2 as a carbon source.

[0086] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit them; 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 described in the foregoing embodiments, or perform equivalent replacements for some of the technical features; and these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A fermentation strain composition, characterized in that, the fermentation strain composition comprises Clostridium ljungdahlii and Yarrowia lipolytica.

2. The fermentation strain composition according to claim 1, characterized in that, the Clostridium ljungdahlii is Clostridium ljungdahlii DSM13528; and / or, the Yarrowia lipolytica is the strain with the preservation number of CGMCC No. 15309.

3. Use of the fermentation strain composition according to claim 1 or 2 in the production of long-chain fatty acids and / or very long-chain fatty acids.

4. The use according to claim 3, characterized in that, the production of long-chain fatty acids and / or very long-chain fatty acids is to synthesize long-chain fatty acids and / or very long-chain fatty acids by biological conversion of carbon dioxide.

5. A method for producing long-chain fatty acids and / or very long-chain fatty acids, characterized in that, the method is to synthesize long-chain fatty acids by biological conversion of carbon dioxide; the method comprises a first fermentation stage and a second fermentation stage; the first fermentation stage is: using Clostridium ljungdahlii to ferment and produce acetic acid with syngas as the carbon source; the second fermentation stage is: using Yarrowia lipolytica to ferment and produce long-chain fatty acids and / or very long-chain fatty acids with the fermentation supernatant of the first fermentation stage as the carbon source; Preferably, the volume ratio of H 2 and CO 2 in the syngas is (1-3):

1.

6. The method according to claim 5, characterized in that, the Clostridium ljungdahlii is Clostridium ljungdahlii DSM13528; and / or, the Yarrowia lipolytica is the strain with the preservation number of CGMCC No. 15309.

7. The method according to claim 5 or 6, characterized in that, the first fermentation medium used in the first fermentation stage comprises the following components: macronutrients, nitrilotriacetic acid, calcium chloride, trace metal elements, sodium tungstate, sodium molybdate, sodium selenate, ferrous sulfate, vitamins, cysteine hydrochloride, sodium bicarbonate, yeast powder and cysteine; preferably, the macronutrients include ammonium chloride, potassium chloride, magnesium sulfate heptahydrate, sodium chloride and potassium dihydrogen phosphate; and / or, the trace metal elements include manganese sulfate, cobalt chloride, zinc sulfate and nickel chloride; and / or, the vitamins include biotin, folic acid, VB6, thiamine, riboflavin, nicotine, calcium pantothenate, cyanocobalamin, p-aminobenzoic acid and lipoic acid.

8. The method according to any one of claims 5 to 7, characterized in that, The second fermentation medium used in the second fermentation stage comprises the following components: the fermentation supernatant of the first fermentation stage, yeast powder, and ammonium sulfate; Preferably, the second fermentation medium uses the fermentation supernatant of the first fermentation stage as a solvent and contains 1-5 g / L of yeast powder and 2-8 g / L of ammonium sulfate.

9. The method according to any one of claims 5 to 8, characterized in that, the fermentation conditions of the first fermentation stage are: the fermentation temperature is 34-38 °C, and the pH is 5.5-6.5; Preferably, the rotation speed in the first fermentation stage is 200-400 rpm, and the intake air flow rate is 10 mL / min-50 mL / min.

10. The method according to any one of claims 5 to 9, characterized in that, the fermentation conditions of the second fermentation stage are: the fermentation temperature is 26-30 °C, and the pH is 5.0-7.5; Preferably, the rotation speed in the second fermentation stage is 200-400 rpm.

Images