CN113897403B - Method for improving oil yield of oleaginous microorganism and preparation method of microbial oil - Google Patents

Method for improving oil yield of oleaginous microorganism and preparation method of microbial oil Download PDF

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CN113897403B
CN113897403B CN202111174795.4A CN202111174795A CN113897403B CN 113897403 B CN113897403 B CN 113897403B CN 202111174795 A CN202111174795 A CN 202111174795A CN 113897403 B CN113897403 B CN 113897403B
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hypophosphite
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cadmium
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黄和
刘孟珍
郭东升
李郡娅
杜远航
杨清昊
王玥
佟令令
袁莉
魏士翔
陈子雷
张怡丹
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Nanjing Normal University
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Abstract

The invention relates to microbial oil, and discloses a method for improving the oil yield of oil-producing microorganisms and a preparation method of microbial oil. The method for improving the grease yield of oleaginous microorganisms comprises the following steps: under the illumination condition, quantum dots, an electron sacrificial agent and an electron transfer agent are added in the fermentation process of oleaginous microorganisms. The preparation method of the microbial oil comprises the following steps: fermenting oleaginous microorganisms by adopting the method to obtain fermentation liquor, and breaking the wall and extracting the fermentation liquor. The method provided by the invention can effectively promote the utilization of nutrient substances by oleaginous microorganisms and improve biomass and grease yield.

Description

Method for improving oil yield of oleaginous microorganism and preparation method of microbial oil
Technical Field
The invention relates to microbial oil, in particular to a method for improving the oil yield of oleaginous microorganisms and a preparation method of microbial oil.
Background
Along with the consumption of petroleum resources, petrochemical products are gradually transferred to oleochemical products, and microbial oil resources belong to resources which can be reproduced indefinitely, and have wide development and utilization values. In recent years, lipid-derived biodiesel has received attention because of its high energy density, clean combustion, non-toxicity, and biodegradability. The waste produced in the paper industry and produced in the agricultural and sideline products and the food industry can be utilized by microbial fermentation, and the environmental protection is realized. The microbial oil can be continuously produced throughout the year except for equipment maintenance without being influenced by the field, the season and the raw material production.
In addition, with the increase of population and the improvement of living standard, the demand of people for functional grease is increasing year by year, and the microbial grease can effectively make up for the deficiency of the supply of the functional grease, for example, the health-care microbial grease rich in polyunsaturated fatty acids (such as linolenic acid, arachidonic acid, EPA, DHA and the like) is extracted from filamentous fungi. The fatty acid synthase in microorganisms uses NADPH as a reducing cofactor, and two NADPH molecules are consumed per step of chain extension of the acyl-CoA, and the chain length of the naturally synthesized acyl-CoA is usually 16 or 18 carbon atoms, so that a great energy supply is required for synthesizing long-chain fatty acids. However, in the existing oleaginous microorganism culture technology, the problems of low nutrient utilization rate, insufficient energy supply, low oil yield and the like still exist, and further improvement is needed.
Therefore, it is of great importance to find a production way to increase the oil yield of oleaginous microorganisms.
Disclosure of Invention
The invention aims to solve the problems of low nutrient utilization rate and low oil yield of oleaginous microorganisms in the prior art, and provides a method for improving the oil yield of oleaginous microorganisms and a preparation method of microbial oil, which can promote the oleaginous microorganisms to utilize nutrient substances and improve the oil yield.
To achieve the above object, a first aspect of the present invention provides a method for increasing the oil yield of an oleaginous microorganism, the method comprising: under the illumination condition, quantum dots, an electron sacrificial agent and an electron transfer agent are added in the fermentation process of oleaginous microorganisms.
Preferably, the wavelength of the light is 425-600nm, the quantum dots are cadmium sulfide quantum dots doped with indium phosphide, the electron sacrificial agent is cysteine, and the electron transfer agent is methyl viologen;
preferably, the addition amount of the quantum dots is 0.1-0.6g/L, the addition amount of the electron sacrificial agent is 0.05-0.5g/L, and the addition amount of the electron transfer agent is 0.05-0.5g/L.
Preferably, the preparation method of the cadmium sulfide quantum dot doped with indium phosphide comprises the following steps:
(a) The method comprises the steps of (1) contacting indium oxide with a hypophosphite solution, and drying to obtain a precursor, and carrying out heat treatment on the precursor to obtain indium phosphide;
(b) And (c) mixing the indium phosphide obtained in the step (a), cadmium salt and sulfur-containing compound in the presence of a reaction solvent.
Preferably, the process of contacting the indium oxide with the hypophosphite solution in step (a) comprises: immersing the indium oxide in the hypophosphite solution;
preferably, the soaking time is 1-4h, the hypophosphite is at least one selected from sodium hypophosphite, potassium hypophosphite, calcium hypophosphite and ferric hypophosphite, and the concentration of the hypophosphite solution is 0.03-0.08mg/mL;
preferably, the conditions of the heat treatment in step (a) include: the temperature is 25-40deg.C, and the time is 8-15min.
Preferably, the reaction solvent in the step (b) is a lower organic alcohol, the cadmium salt is at least one selected from cadmium chloride, cadmium nitrate and cadmium acetate, and the sulfur-containing compound is thioacetamide and/or thiourea;
preferably, the molar ratio of the cadmium element in the indium phosphide and the cadmium salt to the sulfur element in the sulfur-containing compound is 1:0.8-1.2:0.8-1.2;
preferably, the conditions of the mixing reaction include: the ultrasonic power is 180-220W and the time is 8-15h.
Preferably, the indium oxide has an average particle diameter of 0.9 to 1.3 μm;
preferably, the preparation method of the indium oxide comprises the following steps: mixing indium salt solution with alkali solution to form precipitate, drying, calcining and pulverizing the precipitate;
preferably, the process of mixing the indium salt solution with the alkali solution includes: firstly, the alkali solution is dripped into the indium salt solution, and then the reaction is carried out for 20 to 40 minutes, wherein the pH value in the dripping process is 3.5 to 4.5, and the end point pH value is 4.5 to 5.5;
preferably, the drying process includes: sequentially performing primary drying, crushing and secondary drying on the precipitate, wherein the primary drying conditions comprise: the temperature is 80-100 ℃ and the time is 4-10h, and the secondary drying conditions comprise: the temperature is 140-180 ℃ and the time is 3-8h;
preferably, the calcination temperature is 350-450 ℃ and the time is 3-8h.
Preferably, the oleaginous microorganism is selected from at least one of schizochytrium, mortierella alpina and chlorella, preferably schizochytrium.
Preferably, the fermentation process of the oleaginous microorganism comprises: inoculating the oleaginous microorganism into a first culture medium for seed culture to obtain seed liquid, and inoculating the seed liquid into a second culture medium for fermentation culture according to the volume ratio of 8-12%, wherein the quantum dots, the electron sacrificial agent and the electron transfer agent are added in the seed culture stage and/or in the fermentation culture stage;
preferably, the first medium contains: glucose 70-100g/L, KH 2 PO 4 1-5g/L、Na 2 SO 4 10-14g/L、MgSO 4 2-5g/L、KCl 1-3g/L、(NH 4 ) 2 SO 4 2-5g/L、CaCl 2 0.06-1g/L、K 2 SO 4 0.1-1g/L, 8-12g/L monosodium glutamate and 9-15g/L yeast powder;
the second medium contains: glucose 70-100g/L, KH 2 PO 4 1-5g/L、Na 2 SO 4 10-14g/L、MgSO 4 2-5g/L、KCl 1-3g/L、(NH 4 ) 2 SO 4 2-5g/L、CaCl 2 0.06-1g/L、K 2 SO 4 0.1-1g/L, 1-5g/L malic acid, 8-12g/L monosodium glutamate, 9-15g/L yeast extract, 3-7g/L corn steep liquor dry powder, 0.5-1g/L metal ion solution, wherein the metal ion solution contains ZnSO 4 .7H 2 O 1.5-2.5g/L、CuSO 4 1-2g/L；
Preferably, the conditions of the seed culture and the fermentation culture include: the initial pH is 4.5-5.5, the rotating speed is 150-200rpm, and the temperature is 25-35 ℃.
The second aspect of the present invention provides a method for producing microbial oil, comprising: fermenting oleaginous microorganisms by adopting the method to obtain fermentation liquor, and breaking the wall and extracting the fermentation liquor.
Preferably, the wall breaking is carried out by adopting a wall breaking enzyme enzymolysis method;
preferably, the conditions for breaking the wall include: the dosage of the wall breaking enzyme is 2-4g/L, the pH is 10-12, the rotating speed is 150-200rpm, the temperature is 25-35 ℃ and the time is 3-6h;
preferably, the extraction solvent is n-hexane and/or ethanol.
Through the technical scheme, the invention has the beneficial effects that:
according to the method provided by the invention, the quantum dots, the electron sacrificial agent and the electron transfer agent are added in the fermentation process of the oleaginous microorganism under the illumination condition, the oleaginous microorganism is promoted to generate NADPH in the growth process by the quantum dots, so that sufficient reducing force is provided for grease synthesis, and meanwhile, the oleaginous microorganism can be promoted to fully utilize nutrient substances in a culture medium, so that the yield of microbial grease is effectively improved; under the same culture medium condition, the method provided by the invention can improve the oil yield by 206%, further effectively improve the utilization rate of nutrient substances, reduce the production cost of microbial oil, provide a new way for the large-scale production of the microbial oil, and have a great application prospect.
Drawings
FIG. 1 is a graph showing the variation of biomass with fermentation time during fermentation culture of schizochytrium in example 1 and comparative example 1;
FIG. 2 is a graph showing the variation of carbon source consumption with fermentation time during fermentation culture of schizochytrium in example 1 and comparative example 1;
FIG. 3 shows the oil content of the fermentation broth at various amounts of quantum dots added in test example 2.
Detailed Description
The endpoints and any values of the ranges disclosed herein are not limited to the precise range or value, and are understood to encompass values approaching those ranges or values. For numerical ranges, one or more new numerical ranges may be found between the endpoints of each range, between the endpoint of each range and the individual point value, and between the individual point value, in combination with each other, and are to be considered as specifically disclosed herein.
In a first aspect the present invention provides a method for increasing the oil yield of an oleaginous microorganism, the method comprising: under the illumination condition, quantum dots, an electron sacrificial agent and an electron transfer agent are added in the fermentation process of oleaginous microorganisms.
In research and development, the inventor of the invention surprisingly finds that in the fermentation process of oleaginous microorganisms, under the condition of combining illumination, quantum dots, an electron sacrificial agent and an electron transfer agent are added, so that the oleaginous microorganisms can be promoted to generate NADPH in the growth process by the action of the quantum dots, the electron sacrificial agent and the electron transfer agent, sufficient reducing power is provided for the synthesis of long-chain fatty acids in grease, and simultaneously, the oleaginous microorganisms can be promoted to fully utilize nutrient substances in a culture medium, so that the yield of the grease of the microorganisms is effectively improved; under the same culture medium condition, the method provided by the invention has the advantages that the oil yield is obviously improved, the utilization rate of nutrient substances is effectively improved, the production cost of microbial oil is reduced, a new way is provided for the large-scale production of the microbial oil, and the method has a great application prospect.
According to the invention, in order to improve the promotion effect of quantum dots, an electron sacrificial agent and an electron transfer agent on the generation of NADPH and further improve the yield of grease in microorganisms, preferably, the wavelength of light of illumination is 425-600nm, the quantum dots are cadmium sulfide quantum dots doped with indium phosphide, the electron sacrificial agent is cysteine, and the electron transfer agent is methyl viologen.
According to the present invention, in order to improve the efficiency of quantum dots, electron sacrificers and electron transfer agents for promoting NADPH production, it is preferable that the quantum dots are added in an amount of 0.1 to 0.6g/L, the electron sacrificers are added in an amount of 0.05 to 0.5g/L, and the electron transfer agents are added in an amount of 0.05 to 0.5g/L.
As a preferred embodiment of the invention, the preparation method of the indium phosphide-doped cadmium sulfide quantum dot comprises the following steps:
(a) The method comprises the steps of (1) contacting indium oxide with a hypophosphite solution, and drying to obtain a precursor, and carrying out heat treatment on the precursor to obtain indium phosphide;
(b) And (c) mixing the indium phosphide obtained in the step (a), cadmium salt and sulfur-containing compound in the presence of a reaction solvent.
According to the present invention, preferably, the process of contacting the indium oxide with the hypophosphite solution in step (a) comprises: the indium oxide is immersed in the hypophosphite solution so that the indium oxide can be fully contacted with the hypophosphite solution, and the conversion rate of the indium oxide into the indium phosphide is improved. Preferably, the time of the impregnation is 1 to 4 hours, and may specifically be 1 hour, 2 hours, 3 hours, 4 hours, or any value in the range formed by any two values.
According to the present invention, the substance type of calcium hypophosphite is not particularly limited as long as it can be formed into a corresponding solution form and is sufficiently contacted with indium oxide. Preferably, the hypophosphite is selected from at least one of sodium hypophosphite, potassium hypophosphite, calcium hypophosphite and iron hypophosphite. The above materials are all routine choices in the art and are commercially available.
According to the present invention, the concentration of the hypophosphite solution is not particularly limited. In order to promote the conversion efficiency of indium oxide, the concentration of the hypophosphite solution is preferably 0.03 to 0.08mg/mL, and specifically may be 0.03mg/mL, 0.05mg/mL, 0.07mg/mL, 0.08mg/mL, or any value in the range constituted by any two of the above values.
According to the present invention, preferably, the conditions of the heat treatment in step (a) include: the temperature is 25-40deg.C, specifically 25 deg.C, 30 deg.C, 35 deg.C, 40 deg.C, or any value in the range of any two values; the time is 8-15min, and can be specifically 8min, 10min, 12min, 14min, 15min, or any value in the range formed by any two values.
According to the present invention, preferably, the reaction solvent in step (b) is a lower organic alcohol, and may be specifically one or more of methanol, ethanol, n-propanol, isopropanol, 1-butanol and 2-butanol. The lower organic alcohol may be used as a reaction solvent in the form of a pure lower organic alcohol or may be used as a reaction solvent in the form of a mixture with water, and preferably a lower organic alcohol-water solution is used as a reaction solvent, for example, an ethanol-water solution in a volume ratio of 3:1.
According to the present invention, preferably, the cadmium salt is selected from at least one of cadmium chloride, cadmium nitrate, and cadmium acetate.
According to the present invention, preferably, the sulfur-containing compound is thioacetamide and/or thiourea.
The above materials are all routine choices in the art and are commercially available.
According to the invention, in order to improve the generation efficiency of the cadmium sulfide quantum dot doped with indium phosphide, preferably, the molar ratio of cadmium element in the indium phosphide and cadmium salt to sulfur element in the sulfur-containing compound is 1:0.8-1.2:0.8-1.2;
according to the present invention, preferably, the conditions of the mixing reaction include: the ultrasonic power is 180-220W, and can be specifically 180W, 190W, 200W, 210W and 220W or any value in a range formed by any two values; the time is 8-15h, and can be specifically 8h, 10h, 12h, 14h, 15h, or any value in a range formed by any two values.
According to the present invention, it is preferable that the average particle diameter of the indium oxide is 0.9 to 1.3 μm.
According to the invention, indium oxide may be obtained commercially or may be prepared according to the preparation methods in the prior art. Preferably, the preparation method of the indium oxide comprises the following steps: mixing indium salt solution with alkali solution to form precipitate, drying, calcining and pulverizing.
According to the present invention, in order to improve the reaction efficiency of the indium salt and the alkali and improve the purity of indium oxide, it is preferable that the process of mixing the indium salt solution with the alkali solution includes: firstly, the alkali solution is dripped into the indium salt solution, then the reaction is carried out for 20 to 40 minutes, the pH value in the dripping process is 3.5 to 4.5, and the end point pH value is 4.5 to 5.5. Specifically, the temperature at which the indium salt solution is mixed with the alkali solution is 80-100 ℃.
According to the present invention, the alkali solution may be one of sodium hydroxide solution, potassium hydroxide solution and aqueous ammonia; the precipitate may be obtained by any means known in the art for solid-liquid separation, such as filtration, centrifugation.
According to the present invention, preferably, the drying process includes: sequentially performing primary drying, crushing and secondary drying on the precipitate, wherein the primary drying conditions comprise: the temperature is 80-100 ℃ and the time is 4-10h, and the secondary drying conditions comprise: the temperature is 140-180deg.C, and the time is 3-8h. Preferably, the precipitate is dried to produce indium hydroxide having an average particle size of 1-2. Mu.m. In the present invention, the pulverization or pulverization is carried out by a pulverization method conventional in the art, for example, a pulverizer, a grinder, etc.
According to the present invention, indium hydroxide obtained by drying the precipitate is calcined to obtain indium oxide. Preferably, the calcination temperature is 350-450 ℃, specifically 350 ℃, 370 ℃, 390 ℃, 410 ℃, 430 ℃, 450 ℃, or any value in the range formed by any two values; the time is 3-8h, and specifically can be 3h, 4h, 5h, 6h, 7h, 8h, or any value in a range formed by any two values.
According to the present invention, preferably, the oleaginous microorganism is selected from at least one of schizochytrium, mortierella alpina and chlorella, preferably schizochytrium.
According to the present invention, in order to increase the fermentation rate and the oil production efficiency of the oil producing microorganism, preferably, the fermentation process of the oil producing microorganism includes: inoculating the oleaginous microorganism into a first culture medium for seed culture to obtain seed liquid, and inoculating the seed liquid into a second culture medium for fermentation culture according to the volume ratio of 8-12%, wherein the quantum dots, the electron sacrificial agent and the electron transfer agent are added in the seed culture stage and/or in the fermentation culture stage.
Preferably, the quantum dot, the electron sacrificial agent and the electron transfer agent are added at the stage of the fermentation culture; further preferably, the quantum dot, the electron sacrificial agent and the electron transfer agent are added at an initial stage of the fermentation culture.
According to the present invention, preferably, the first medium contains: glucose 70-100g/L, KH 2 PO 4 1-5g/L、Na 2 SO 4 10-14g/L、MgSO 4 2-5g/L、KCl 1-3g/L、(NH 4 ) 2 SO 4 2-5g/L、CaCl 2 0.06-1g/L、K 2 SO 4 0.1-1g/L, 8-12g/L monosodium glutamate and 9-15g/L yeast powder. Wherein, the inoculation amount of the first culture medium is generally 0.5-2% by volume, and if the microorganism strain stored in the glycerol tube is used for inoculation, the strain in each glycerol tube is inoculated into 100mL of the first culture medium.
According to the present invention, preferably, the second medium contains: glucose 70-100g/L, KH 2 PO 4 1-5g/L、Na 2 SO 4 10-14g/L、MgSO 4 2-5g/L、KCl 1-3g/L、(NH 4 ) 2 SO 4 2-5g/L、CaCl 2 0.06-1g/L、K 2 SO 4 0.1-1g/L, 1-5g/L malic acid, 8-12g/L monosodium glutamate, 9-15g/L yeast extract, 3-7g/L corn steep liquor dry powder, 0.5-1g/L metal ion solution, wherein the metal ion solution contains ZnSO 4 .7H 2 O 1.5-2.5g/L、CuSO 4 1-2g/L. In the present invention, the metal ion solution may contain trace metal ions of other types.
According to the present invention, preferably, the conditions of the seed culture and the fermentation culture include: the initial pH is 4.5-5.5, the rotating speed is 150-200rpm, and the temperature is 25-35 ℃. The culture time of the seed culture and the fermentation culture is respectively adjusted according to different oleaginous microorganisms, for example, the seed culture time of schizochytrium can be 20-30 hours; the fermentation culture time can be 6-8 days, or the carbon source in the fermentation liquid is exhausted as the fermentation end point.
The second aspect of the present invention provides a method for producing microbial oil, comprising: fermenting oleaginous microorganisms by adopting the method to obtain fermentation liquor, and breaking the wall and extracting the fermentation liquor.
According to the invention, the cell wall breaking of the microorganism can be performed in a conventional manner in the art, preferably by a wall breaking enzymatic hydrolysis method, so as to improve the wall breaking efficiency and reduce the damage to metabolites in the microorganism cells.
According to the present invention, preferably, the wall breaking conditions include: the dosage of the wall breaking enzyme is 2-4g/L, and can be specifically 2g/L, 3g/L, 4g/L or any value in the range formed by any two values; the pH is 10-12, and can be specifically 10, 11, 12 or any value in a range formed by any two values; the rotation speed is 150-200rpm, and can be specifically 150rpm, 160rpm, 170rpm, 180rpm, 190rpm, 200rpm, or any value in a range formed by any two values; the temperature is 25-35deg.C, specifically 25 deg.C, 30 deg.C, 35 deg.C, or any value in the range of any two values; the time is 3-6h, and specifically can be 3h, 4h, 5h, 6h, or any value in a range formed by any two values.
According to the present invention, in order to enhance the extraction efficiency of the microbial oil, it is preferable that the extraction solvent is n-hexane and/or ethanol, preferably n-hexane and ethanol, to extract the oil with n-hexane while disrupting the microbial cells with ethanol. The extracted oil contains polyunsaturated fatty acids such as oleic acid, palmitic acid, linoleic acid, eicosapentaenoic acid, and docosahexaenoic acid.
The present invention will be described in detail by examples.
In the following examples and comparative examples, schizochytrium sp.HX-308 was isolated and screened from coastal areas by the laboratory, and was deposited at China center for type culture Collection (CCTCC for short), with a accession number of CCTCC M209059, and other materials and reagents were commercially available, with a room temperature of 25.+ -. 5 ℃.
Preparation example 1
(a) Dropwise adding ammonia water into a solution containing 10mmol of indium nitrate at 90 ℃, wherein the pH value in the dropwise adding process is 4.0, the end point pH value is 5.0, reacting for 30min after the dropwise adding is finished, filtering to obtain a white filter cake, washing the white filter cake with slurry for 3 times and leaching for 2 times, drying the white filter cake at 90 ℃ for 6h in a constant-temperature drying box, cooling to obtain powder, then drying the powder in the constant-temperature drying box at 160 ℃ for 5h, cooling and powdering to obtain indium hydroxide with white appearance and average granularity of 1.12 um; placing indium hydroxide into a calciner with the temperature of 400 ℃ for calcination for 5 hours, cooling and pulverizing to obtain indium oxide with light yellow appearance and average granularity of 1.06 mu m;
(b) Immersing the indium oxide obtained in the step (a) in a sodium hypophosphite solution with the concentration of 0.05mg/ml for 2 hours, then drying for 1 hour to obtain a precursor, and treating the precursor at the temperature of 30 ℃ for 10 minutes to obtain indium phosphide;
(c) And (3) dissolving the indium phosphide obtained in the step (b), 10mmol of cadmium acetate and 10mmol of thioacetamide in an ethanol-water solution (volume ratio is 3:1), reacting for 12 hours under the ultrasonic condition that the ultrasonic power is 200W to obtain a uniform and stable dispersion solution, and filtering the dispersion solution to obtain the indium phosphide doped cadmium sulfide quantum dot.
Preparation example 2
(a) Dropwise adding ammonia water into a solution containing 10mmol of indium nitrate at the temperature of 80 ℃, wherein the pH value in the dropwise adding process is 3.5, the end point pH value is 4.5, reacting for 20min after the dropwise adding is finished, filtering to obtain a white filter cake, pulping and washing the white filter cake for 3 times and leaching for 2 times, drying the white filter cake at the temperature of 80 ℃ for 10h in a constant-temperature drying box, cooling to obtain powder, then drying the powder in the constant-temperature drying box at the temperature of 140 ℃ for 8h, cooling and powdering to obtain the indium hydroxide with white appearance and average granularity of 1.2 mu m; placing indium hydroxide into a calciner with the temperature of 350 ℃ for calcination for 8 hours, cooling and pulverizing to obtain indium oxide with light yellow appearance and average granularity of 1.1 mu m;
(b) Immersing the indium oxide obtained in the step (a) in a potassium hypophosphite solution with the concentration of 0.03mg/ml for 4 hours, drying for 1 hour to obtain a precursor, and treating the precursor at the temperature of 25 ℃ for 15 minutes to obtain indium phosphide;
(c) And (3) dissolving the indium phosphide obtained in the step (b), 12mmol of cadmium acetate and 12mmol of thioacetamide in an ethanol-water solution (volume ratio is 3:1), reacting for 15 hours under the ultrasonic condition that the ultrasonic power is 180W to obtain a uniform and stable dispersion solution, and filtering the dispersion solution to obtain the indium phosphide doped cadmium sulfide quantum dot.
Preparation example 3
(a) Dropwise adding ammonia water into a solution containing 10mmol of indium nitrate at the temperature of 100 ℃, wherein the pH value in the dropwise adding process is 4.5, the end point pH value is 5.5, reacting for 40min after the dropwise adding is finished, filtering to obtain a white filter cake, pulping and washing the white filter cake for 3 times and leaching for 2 times, drying the white filter cake at the temperature of 100 ℃ for 4h in a constant-temperature drying box, cooling to obtain powder, then placing the powder into the constant-temperature drying box at the temperature of 180 ℃ for drying for 3h, cooling and powdering to obtain the indium hydroxide with white appearance and average granularity of 1.02 mu m; placing indium hydroxide into a calciner with the temperature of 450 ℃ for calcination for 3 hours, cooling and pulverizing to obtain indium oxide with light yellow appearance and average granularity of 0.98 mu m;
(b) Immersing the indium oxide obtained in the step (a) in a sodium hypophosphite solution with the concentration of 0.08mg/ml for 1h, drying for 1h to obtain a precursor, and treating the precursor at the temperature of 40 ℃ for 8min to obtain indium phosphide;
(c) And (3) dissolving the indium phosphide obtained in the step (b), 8mmol of cadmium acetate and 8mmol of thioacetamide in an ethanol-water solution (volume ratio is 3:1), reacting for 8 hours under the ultrasonic condition that the ultrasonic power is 220W to obtain a uniform and stable dispersion solution, and filtering the dispersion solution to obtain the indium phosphide doped cadmium sulfide quantum dot.
Preparation example 4
Cadmium sulfide quantum dots doped with indium phosphide were prepared as in preparation example 1, except that the amounts of cadmium acetate and thioacetamide used in step (c) were replaced with 5mmol, respectively.
Preparation example 5
(a) 10mmol of commercial indium oxide with the average particle diameter of 2 mu m is immersed in a sodium hypophosphite solution with the concentration of 0.08mg/ml for 1h, then dried for 1h to obtain a precursor, and the precursor is treated for 8min at the temperature of 40 ℃ to obtain indium phosphide;
(b) And (3) dissolving the indium phosphide obtained in the step (a), 8mmol of cadmium acetate and 8mmol of thioacetamide in an ethanol-water solution (volume ratio is 3:1), reacting for 8 hours under the ultrasonic condition that the ultrasonic power is 220W to obtain a uniform and stable dispersion solution, and filtering the dispersion solution to obtain the indium phosphide doped cadmium sulfide quantum dot.
Example 1
(1) The formula of the first culture medium is as follows: glucose 85g/L, KH 2 PO 4 3g/L、Na 2 SO 4 12g/L、MgSO 4 3g/L、KCl 2g/L、(NH 4 ) 2 SO 4 3g/L、CaCl 2 0.5g/L、K 2 SO 4 0.5g/L, 10g/L monosodium glutamate, 12g/L yeast powder, and sterilizing at 121deg.C for 20 min; the formula of the second culture medium is as follows: glucose 85g/L, KH 2 PO 4 3g/L、Na 2 SO 4 12g/L、MgSO 4 3g/L、KCl 2g/L、(NH 4 ) 2 SO 4 3g/L、CaCl 2 0.5g/L、K 2 SO 4 0.5g/L, 3g/L malic acid, 10g/L monosodium glutamate, 12g/L yeast extract, 5g/L corn steep liquor dry powder, 0.8g/L metal ion solution (metal ion solution contains ZnSO) 4 .7H 2 O 2g/L、CuSO 4 1.5 g/L), sterilizing at 121deg.C for 20 min;
(2) Inoculating schizochytrium strains into a first culture medium according to an inoculum size of 1 volume percent, culturing for 24 hours under the conditions of an initial pH value of 5 and a temperature of 30 ℃ to obtain first generation seed liquid, inoculating the first generation seed liquid into the first culture medium according to an inoculum size of 1 volume percent, culturing for 24 hours under the conditions of an initial pH value of 5 and a temperature of 30 ℃ to obtain second generation seed liquid, inoculating the second generation seed liquid into the first culture medium according to an inoculum size of 1 volume percent, and culturing for 24 hours under the conditions of an initial pH value of 5 and a temperature of 30 ℃ to obtain third generation seed liquid;
(3) Inoculating the third generation seed solution into a second culture medium according to 10 volume percent of inoculation amount, adding the indium phosphide-doped cadmium sulfide quantum dot, cysteine and methyl viologen (the addition amount of the quantum dot is 0.3g/L, the addition amount of the cysteine is 0.2g/L, and the addition amount of the methyl viologen is 0.2 g/L) obtained in preparation example 1 under the condition of illumination wavelength of 500nm, and culturing for 7 days under the condition that the initial pH value is 5 and the temperature is 30 ℃ to obtain fermentation liquor;
(4) Adding wall breaking enzyme into the fermentation liquor in an adding amount of 3g/L, carrying out enzymolysis for 4 hours at the conditions of pH of 11, rotating speed of 180rpm and temperature of 30 ℃ to obtain wall breaking liquid, mixing and extracting the wall breaking liquid with ethanol and n-hexane in a volume ratio of 1:1:1 to obtain n-hexane phase, and carrying out rotary evaporation on the n-hexane phase to remove the n-hexane to obtain the microbial oil.
Example 2
(1) The formula of the first culture medium is as follows: glucose 70g/L, KH 2 PO 4 1g/L、Na 2 SO 4 10g/L、MgSO 4 2g/L、KCl 1g/L、(NH 4 ) 2 SO 4 2g/L、CaCl 2 0.06g/L、K 2 SO 4 0.1g/L, 8g/L monosodium glutamate, 9g/L yeast powder, and sterilizing at 121deg.C for 20 min; the formula of the second culture medium is as follows: glucose 70g/L, KH 2 PO 4 1g/L、Na 2 SO 4 10g/L、MgSO 4 2g/L、KCl 1g/L、(NH 4 ) 2 SO 4 2g/L、CaCl 2 0.06g/L、K 2 SO 4 0.1g/L, 1g/L malic acid, 8g/L monosodium glutamate, 9g/L yeast extract, 3g/L corn steep liquor dry powder, 0.5g/L metal ion solution (metal ion solution contains ZnSO) 4 .7H 2 O 2g/L、CuSO 4 1.5 g/L), sterilizing at 121deg.C for 20 min;
(2) Inoculating schizochytrium strains into a first culture medium according to an inoculum size of 1 volume percent, culturing for 24 hours under the conditions of an initial pH value of 4.5 and a temperature of 25 ℃ to obtain first generation seed liquid, inoculating the first generation seed liquid into the first culture medium according to an inoculum size of 1 volume percent, culturing for 24 hours under the conditions of an initial pH value of 4.5 and a temperature of 25 ℃ to obtain second generation seed liquid, inoculating the second generation seed liquid into the first culture medium according to an inoculum size of 1 volume percent, and culturing for 24 hours under the conditions of an initial pH value of 4.5 and a temperature of 25 ℃ to obtain third generation seed liquid;
(3) Inoculating the third generation seed solution into a second culture medium according to the inoculation amount of 12 vol%, adding cadmium sulfide quantum dot doped with indium phosphide, cysteine and methyl viologen obtained in preparation example 2 (the addition amount of the quantum dot is 0.1g/L, the addition amount of the cysteine is 0.05g/L, and the addition amount of the methyl viologen is 0.05 g/L) under the condition that the illumination wavelength is 425nm, and culturing for 8 days under the condition that the initial pH value is 4.5 and the temperature is 25 ℃ to obtain fermentation liquor;
(4) Adding wall breaking enzyme into fermentation liquor in an adding amount of 4g/L, carrying out enzymolysis for 6 hours at the conditions of pH of 10, rotating speed of 150rpm and temperature of 25 ℃ to obtain wall breaking liquid, mixing and extracting the wall breaking liquid with ethanol and n-hexane in a volume ratio of 1:1:1 to obtain n-hexane phase, and carrying out rotary evaporation on the n-hexane phase to remove the n-hexane to obtain microbial oil.
Example 3
(1) The formula of the first culture medium is as follows: glucose 100g/L, KH 2 PO 4 5g/L、Na 2 SO 4 14g/L、MgSO 4 5g/L、KCl 3g/L、(NH 4 ) 2 SO 4 5g/L、CaCl 2 1g/L、K 2 SO 4 1g/L, 12g/L monosodium glutamate, 15g/L yeast powder, and sterilizing at 121 ℃ for 20min for later use; the formula of the second culture medium is as follows: glucose 100g/L, KH 2 PO 4 5g/L、Na 2 SO 4 14g/L、MgSO 4 5g/L、KCl 3g/L、(NH 4 ) 2 SO 4 5g/L、CaCl 2 1g/L、K 2 SO 4 1g/L, 5g/L malic acid, 12g/L monosodium glutamate, 15g/L yeast extract, 7g/L corn steep liquor dry powder, and 1g/L metal ion solution (metal ion solution contains ZnSO) 4 .7H 2 O 2g/L、CuSO 4 1.5 g/L), sterilizing at 121deg.C for 20 min;
(2) Inoculating schizochytrium strains into a first culture medium according to an inoculum size of 1 volume percent, culturing for 20 hours at an initial pH of 5.5 and a temperature of 35 ℃ to obtain first generation seed liquid, inoculating the first generation seed liquid into the first culture medium according to an inoculum size of 1 volume percent, and culturing for 20 hours at an initial pH of 5.5 and a temperature of 35 ℃ to obtain second generation seed liquid;
(3) Inoculating the second generation seed solution into a second culture medium according to the inoculation amount of 8 volume percent, adding the cadmium sulfide quantum dot doped with indium phosphide, cysteine and methyl viologen obtained in preparation example 1 (the addition amount of the quantum dot is 0.6g/L, the addition amount of the cysteine is 0.5g/L, and the addition amount of the methyl viologen is 0.5 g/L) under the condition that the illumination wavelength is 600nm, and culturing for 7 days under the condition that the initial pH is 5.5 and the temperature is 35 ℃ to obtain fermentation liquor;
(4) Adding wall breaking enzyme into the fermentation liquor in an adding amount of 3g/L, carrying out enzymolysis for 3 hours at the conditions of pH of 12, rotating speed of 200rpm and temperature of 35 ℃ to obtain wall breaking liquid, mixing and extracting the wall breaking liquid with ethanol and n-hexane in a volume ratio of 1:1:1 to obtain n-hexane phase, and carrying out rotary evaporation on the n-hexane phase to remove the n-hexane to obtain the microbial oil.
Example 4
A microbial oil was prepared as in example 1, except that the indium phosphide-doped cadmium sulfide quantum dots obtained in preparation example 1 in step (3) were replaced with cadmium sulfide quantum dots.
Example 5
A microbial oil was prepared according to the method of example 1, except that the indium phosphide-doped cadmium sulfide quantum dot obtained in the preparation example 1 in the step (3) was replaced with a zinc selenide quantum dot, wherein the preparation method of the zinc selenide quantum dot is as follows:
s1, adding 34.6 mug of mercaptopropionic acid into 50mL of zinc acetate solution with the concentration of 3.2mM at room temperature, wherein the adding process is carried out under the condition of magnetic stirring, and after the addition of the mercaptopropionic acid is finished, adjusting the pH to 8 by using 6M sodium hydroxide solution to obtain zinc precursor solution;
s2, adding 4.3mg of sodium selenite pentahydrate into the zinc precursor solution obtained in the step S1 under the condition of magnetic stirring;
s3, adding 0.92mL of hydrazine hydrate into the solution obtained in the step S2, and heating and refluxing for 15min at a constant temperature of 100 ℃ to obtain a water-soluble ZnSe quantum dot solution;
s4, mixing the ZnSe quantum dot solution prepared in the step S3 with isopropanol in a volume ratio of 1:1, and centrifuging at a rotating speed of 8000 rpm; removing supernatant, dissolving the obtained precipitate in water, mixing with isopropanol at volume ratio of 1:10, centrifuging at 8000rpm for purification, repeating the above centrifuging and purifying operation for 2-3 times, and dissolving the precipitate in water to obtain zinc selenide quantum dot.
Example 6
A microbial oil was prepared as in example 1, except that the amount of indium phosphide-doped cadmium sulfide quantum dots added in step (3) was replaced with 1g/L.
Example 7
A microbial oil was prepared as in example 1, except that the indium phosphide-doped cadmium sulfide quantum dot in step (3) was replaced with the indium phosphide-doped cadmium sulfide quantum dot prepared in preparation example 4.
Example 8
A microbial oil was prepared as in example 1, except that the indium phosphide-doped cadmium sulfide quantum dot in step (3) was replaced with the indium phosphide-doped cadmium sulfide quantum dot prepared in preparation example 5.
Example 9
A microbial oil was prepared as in example 1, except that step (3) was replaced with:
(3) Inoculating the third generation seed solution into a second culture medium according to 10 volume percent inoculum size, culturing for 3 days under the conditions of initial pH of 5 and temperature of 30 ℃, adding the cadmium sulfide quantum dot doped with indium phosphide, cysteine and methyl viologen (the addition amount of the quantum dot is 0.3g/L, the addition amount of the cysteine is 0.2g/L, and the addition amount of the methyl viologen) obtained in preparation example 1 under the condition of illumination wavelength of 500nm, and continuously culturing for 7 days to obtain a fermentation broth.
Comparative example 1
(1) The formula of the first culture medium is as follows: glucose 85g/L, KH 2 PO 4 3g/L、Na 2 SO 4 12g/L、MgSO 4 3g/L、KCl 2g/L、(NH 4 ) 2 SO 4 3g/L、CaCl 2 0.5g/L、K 2 SO 4 0.5g/L, 10g/L monosodium glutamate, 12g/L yeast powder, and sterilizing at 121deg.C for 20 min; the formula of the second culture medium is as follows: glucose 85g/L, KH 2 PO 4 3g/L、Na 2 SO 4 12g/L、MgSO 4 3g/L、KCl 2g/L、(NH 4 ) 2 SO 4 3g/L、CaCl 2 0.5g/L、K 2 SO 4 0.5g/L, 3g/L malic acid, 10g/L monosodium glutamate, 12g/L yeast extract, 5g/L corn steep liquor dry powder, 0.8g/L metal ion solution (metal ion solution contains ZnSO) 4 .7H 2 O 2g/L、CuSO 4 1.5 g/L), sterilizing at 121deg.C for 20 min;
(2) Inoculating schizochytrium strains into a first culture medium according to an inoculum size of 1 volume percent, culturing for 24 hours under the conditions of an initial pH value of 5 and a temperature of 30 ℃ to obtain first generation seed liquid, inoculating the first generation seed liquid into the first culture medium according to an inoculum size of 1 volume percent, culturing for 24 hours under the conditions of an initial pH value of 5 and a temperature of 30 ℃ to obtain second generation seed liquid, inoculating the second generation seed liquid into the first culture medium according to an inoculum size of 1 volume percent, and culturing for 24 hours under the conditions of an initial pH value of 5 and a temperature of 30 ℃ to obtain third generation seed liquid;
(3) Inoculating the third generation seed liquid into a second culture medium according to 10 volume percent of inoculation amount, and culturing for 7 days under the conditions that the initial pH is 5 and the temperature is 30 ℃ to obtain fermentation liquor;
(4) Adding wall breaking enzyme into the fermentation liquor in an adding amount of 3g/L, carrying out enzymolysis for 4 hours at the conditions of pH of 11, rotating speed of 180rpm and temperature of 30 ℃ to obtain wall breaking liquid, mixing and extracting the wall breaking liquid with ethanol and n-hexane in a volume ratio of 1:1:1 to obtain n-hexane phase, and carrying out rotary evaporation on the n-hexane phase to remove the n-hexane to obtain the microbial oil.
Comparative example 2
A microbial oil was prepared as in example 1, except that step (3) was replaced with:
(3) The third generation seed solution was inoculated into the second medium at an inoculum size of 10 vol%, and under the condition of an illumination wavelength of 500nm, the indium phosphide-doped cadmium sulfide quantum dot (the addition amount of the quantum dot is 0.3 g/L) obtained in preparation example 1 was added, and the mixture was cultured for 7 days at an initial pH of 5 and a temperature of 30℃to obtain a fermentation broth.
Test example 1
The fermentation broths obtained in examples 1-9 and comparative examples 1-2 were tested for biomass, lipid content and lipid composition, and the results are shown in tables 1 and 2.
TABLE 1
TABLE 2
Numbering device C14:0(％) C16:0(％) C20:5(％) DPA(％) C22:6(％)
Example 1 11.21 20.89 4.13 12.975 51.01
Example 2 10.53 18.71 3.21 14.25 50.30
Example 3 9.54 19.65 2.36 15.31 48.21
Example 4 11.21 17.32 1.21 14.65 49.05
Example 5 12.51 16.51 0.96 13.21 47.32
Example 6 12.02 19.32 0.88 14.26 49.51
Example 7 13.30 17.54 1.30 12.83 50.11
Example 8 12.54 18.43 0.53 15.90 47.76
Example 9 11.71 20.43 0.97 13.71 48.55
Comparative example 1 9.80 18.32 0.72 12.23 50.08
Comparative example 2 10.51 19.42 0.96 12.39 49.37
In table 2,% represents the mass percentage of each fatty acid based on the total amount of the oil.
In the fermentation culture process of example 1 and comparative example 1, the change of biomass and the consumption of carbon source were recorded every 24 hours, and as a result, as shown in fig. 1 and 2, it was found that after adding the cadmium sulfide quantum dot doped with indium phosphide, the biomass of schizochytrium was significantly improved, and the utilization rate of the carbon source in the culture medium was also improved.
As can be seen from the data in table 1, the method provided by the invention is adopted in example 1-example 9, compared with comparative examples 1 and 2, not only the biomass after fermentation of schizochytrium is improved, but also the content of grease is obviously improved, and meanwhile, the obtained grease contains rich polyunsaturated fatty acids; when the addition amount of the cadmium sulfide quantum dot doped with indium phosphide was 0.3g/L, the grease yield of example 1 was improved by 206% compared with comparative example 1.
Test example 2
The microbial oil was prepared according to the method of example 1, and the addition amounts of the quantum dots in step (3) were set to 0g/L, 0.1g/L, 0.2g/L, 0.3g/L, 0.4g/L, 0.5g/L, 0.6g/L, respectively, and the oil content in the obtained fermentation broth was tested, as shown in fig. 3, which also further revealed that the oil yield of schizochytrium fermentation could be significantly improved in the case of adding the quantum dots.
The preferred embodiments of the present invention have been described in detail above, but the present invention is not limited thereto. Within the scope of the technical idea of the invention, a number of simple variants of the technical solution of the invention are possible, including combinations of the individual technical features in any other suitable way, which simple variants and combinations should likewise be regarded as being disclosed by the invention, all falling within the scope of protection of the invention.

Claims (7)

1. A method for increasing the oil production of oleaginous microorganisms, comprising: under the illumination condition, quantum dots, an electron sacrificial agent and an electron transfer agent are added in the fermentation process of oleaginous microorganisms; the wavelength of light is 425-600nm, the quantum dot is cadmium sulfide quantum dot doped with indium phosphide, the electron sacrificial agent is cysteine, the electron transfer agent is methyl viologen, the addition amount of the quantum dot is 0.1-0.6g/L, the addition amount of the electron sacrificial agent is 0.05-0.5g/L, the addition amount of the electron transfer agent is 0.05-0.5g/L, and the oleaginous microorganism is schizochytrium;
the preparation method of the cadmium sulfide quantum dot doped with indium phosphide comprises the following steps:
(a) The indium oxide is contacted with hypophosphite solution, then dried to obtain a precursor, and the precursor is subjected to heat treatment to obtain indium phosphide; the process of contacting indium oxide with a hypophosphite solution includes: dipping indium oxide in hypophosphite solution for 1-4h, wherein the hypophosphite is at least one selected from sodium hypophosphite, potassium hypophosphite, calcium hypophosphite and ferric hypophosphite, the concentration of the hypophosphite solution is 0.03-0.08mg/mL, and the heat treatment conditions comprise: the temperature is 25-40deg.C, and the time is 8-15min;
(b) Mixing indium phosphide obtained in the step (a) with cadmium salt and sulfur-containing compound in the presence of a reaction solvent; the reaction solvent is lower organic alcohol, the cadmium salt is at least one of cadmium chloride, cadmium nitrate and cadmium acetate, and the sulfur-containing compound is thioacetamide and/or thiourea; the molar ratio of the cadmium element in the indium phosphide and the cadmium salt to the sulfur element in the sulfur-containing compound is 1:0.8-1.2:0.8-1.2, and the conditions of the mixing reaction include: carrying out under ultrasonic conditions for 8-15h;
the average particle diameter of the indium oxide is 0.9-1.3 mu m, and the preparation method of the indium oxide comprises the following steps: mixing indium salt solution with alkali solution to form precipitate, drying, calcining and pulverizing the precipitate; the process of mixing the indium salt solution with the alkali solution comprises the following steps: firstly, alkali solution is dripped into indium salt solution, then the reaction is carried out for 20 to 40 minutes, the pH value in the dripping process is 3.5 to 4.5, and the end point pH value is 4.5 to 5.5; the drying process comprises the following steps: the precipitate is subjected to primary drying, crushing and secondary drying in sequence, and the primary drying conditions comprise: the temperature is 80-100 ℃, the time is 4-10h, and the secondary drying conditions comprise: the temperature is 140-180 ℃ and the time is 3-8h; the calcination temperature is 350-450 ℃ and the calcination time is 3-8h.
2. The method of claim 1, wherein the fermentation process of the oleaginous microorganism comprises: inoculating the oleaginous microorganism into a first culture medium for seed culture to obtain seed liquid, and inoculating the seed liquid into a second culture medium for fermentation culture according to the volume ratio of 8-12%, wherein the quantum dots, the electron sacrificial agent and the electron transfer agent are added in the seed culture stage and/or in the fermentation culture stage.
3. The method of claim 2, wherein the first medium comprises: glucose 70-100g/L, KH 2 PO 4 1-5g/L、Na 2 SO 4 10-14g/L、MgSO 4 2-5g/L、KCl 1-3g/L、(NH 4 ) 2 SO 4 2-5g/L、CaCl 2 0.06-1g/L、K 2 SO 4 0.1-1g/L, 8-12g/L monosodium glutamate and 9-15g/L yeast powder;
the second medium contains: glucose 70-100g/L, KH 2 PO 4 1-5g/L、Na 2 SO 4 10-14g/L、MgSO 4 2-5g/L、KCl 1-3g/L、(NH 4 ) 2 SO 4 2-5g/L、CaCl 2 0.06-1g/L、K 2 SO 4 0.1-1g/L, 1-5g/L malic acid, 8-12g/L monosodium glutamate, 9-15g/L yeast extract, 3-7g/L corn steep liquor dry powder and 0.5-1g/L metal ion solution, wherein the metal ion solution contains ZnSO 4 .7H 2 O 1.5-2.5g/L、CuSO 4 1-2g/L。
4. The method of claim 2, wherein the conditions of the seed culture and the fermentation culture comprise: the initial pH is 4.5-5.5, the rotating speed is 150-200rpm, and the temperature is 25-35 ℃.
5. The preparation method of the microbial oil is characterized by comprising the following steps: fermenting oleaginous microorganisms by adopting the method of any one of claims 1-4 to obtain fermentation liquor, and breaking wall and extracting the fermentation liquor.
6. The method according to claim 5, wherein the wall breaking is performed by a wall breaking enzymatic hydrolysis method.
7. The method of claim 6, wherein the wall breaking conditions comprise: the dosage of the wall breaking enzyme is 2-4g/L, the pH is 10-12, the rotating speed is 150-200rpm, the temperature is 25-35 ℃ and the time is 3-6h;
the extraction solvent is n-hexane and/or ethanol.
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