Method for producing grease from lignocellulose and application

Abstract

The invention relates to the technical field of renewable energy sources and bioengineering, and discloses a method for producing grease from lignocellulose and application. The method comprises the following steps: S1, performing enzymolysis treatment on raw materials for preparing lignocellulose to obtain enzymatic hydrolysate; s2, mixing the enzymatic hydrolysate with an adsorbent I to obtain a mixed solution; and S3, mixing the mixed solution, a nitrogen source and metal ions II to obtain a fermentation solution, and mixing the fermentation solution with a fermentation strain for microbial fermentation. The method has the advantages of high lignocellulose conversion rate and high grease yield.

Description

Technical Field

[0001] The invention relates to the technical field of renewable energy and bioengineering, and in particular to a method for producing oil from lignocellulose and its application. Background Art

[0002] The existing production cost of functional fatty acids is high and the output is low, which cannot meet the needs of more ordinary residents. In order to reduce the production cost of functional fatty acids, microbial fermentation to produce oils is a commonly used and very promising method. As a promising renewable resource, lignocellulose contains rich carbon sources and is often used as a raw material for various biorefineries. So far, it has been successfully used to produce furfural, fuel ethanol, ferulic acid, various furan compounds and oils. The use of lignocellulose can provide the carbon source required for fermentation for oil-producing microorganisms, but the existing raw material processing methods have low utilization rates and the cost of producing lignocellulose is high. Summary of the invention

[0003] The purpose of the present invention is to overcome the problems of low conversion rate of lignocellulose raw materials and low oil yield in the prior art, and to provide a method and application of producing oil from lignocellulose, which has the advantages of high conversion rate of lignocellulose and high oil yield.

[0004] In order to achieve the above object, the present invention provides a method for producing oil from lignocellulose in a first aspect, comprising the following steps:

[0005] S1, subjecting the raw material for preparing lignocellulose to enzymatic hydrolysis to obtain an enzymatic hydrolyzate;

[0006] S2, the enzymatic hydrolyzate is mixed with an adsorbent to obtain a mixed solution;

[0007] S3, mixing the mixed solution, nitrogen source and metal ions to obtain fermentation solution, and mixing the fermentation solution with fermentation strains to perform microbial fermentation.

[0008] Preferably, in step S2, the adsorbent is selected from at least one of activated carbon, calcium hydroxide and ion exchange resin.

[0009] Further preferably, the mass ratio of the adsorbent to the enzymatic hydrolysate is 0.005-0.06:1.

[0010] Further preferably, the concentration of monosaccharides in the enzymatic hydrolysate is 55-62 g / L, preferably 60-62 g / L.

[0011] Preferably, the mixing conditions I at least include: temperature of 30-100° C., rotation speed of 100-300 rpm, and time of 0.5-11 h.

[0012] Further preferably, the raw material for preparing lignocellulose is selected from at least one of corn stalks, corn cobs and corn starch.

[0013] Preferably, the fermentation strain is selected from at least one of Mucor, Rhizopus and Thraustochytrid.

[0014] Further preferably, the nitrogen source is selected from at least one of peptone, yeast powder, urea, ammonium sulfate, sodium nitrate and ammonium nitrate.

[0015] Further preferably, the inorganic salt providing the metal ion is at least one selected from magnesium salt, sodium salt, copper salt and potassium salt.

[0016] Preferably, in 100 mL of fermentation broth, the amount of the mixed liquid is 30-90 mL, the amount of the nitrogen source is 0.1-0.5 g, and the amount of the inorganic salt providing the metal ions is 0.01-0.15 g.

[0017] More preferably, the inoculation amount of the fermentation strain is 1×10 7 -1×10 9 cfu / mL.

[0018] Further preferably, the conditions for the microbial fermentation include at least: a temperature of 15-35°C, a rotation speed of 120-300 rpm, and a time of 60-240 h.

[0019] Preferably, the method further comprises: before step S1, mixing and contacting the lignocellulosic raw material with an alkaline solution.

[0020] Further preferably, the mass ratio of the raw material for preparing lignocellulose to the alkaline solution is 1:5-19.

[0021] Further preferably, the alkaline solution is a sodium hydroxide alkaline solution and / or a potassium hydroxide alkaline solution.

[0022] Further preferably, the concentration of the solute in the alkaline solution is 0.5-2 wt %.

[0023] Further preferably, the mixed contact conditions at least include: a temperature of 80-140° C. and a time of 10-60 min.

[0024] Preferably, the enzyme used in the enzymatic treatment is cellulase and / or xylanase.

[0025] Further preferably, the mass ratio of the enzyme to the raw material for preparing lignocellulose is 0.01-1:1.

[0026] Further preferably, the conditions for the enzymatic hydrolysis at least include: pH 3.5-5.5, temperature 30-60°C, rotation speed 150-250 rpm, and time 20-72 h.

[0027] Preferably, the method further comprises, after the microbial fermentation is completed, cell disruption and oil extraction are performed.

[0028] Preferably, the solvent for oil extraction is selected from at least one of chloroform, ether, n-hexane and ethanol.

[0029] Further preferably, the solvent is a mixed solvent of n-hexane and ethanol, and the volume ratio of the n-hexane to the ethanol in the mixed solvent is 1-10:1.

[0030] The second aspect of the present invention provides the use of the method for producing fats and oils described in the first aspect in the production of GLA and / or DHA.

[0031] Through the above technical solution, the beneficial effects of the present invention are:

[0032] The method for producing oil from lignocellulose provided by the present invention comprises the following steps: firstly, the raw material for preparing lignocellulose is subjected to enzymatic hydrolysis to obtain an enzymatic hydrolyzate; then the enzymatic hydrolyzate is mixed with an adsorbent I to obtain a mixed solution; finally, the mixed solution, a nitrogen source and a metal ion are mixed II to obtain a fermentation solution, and then the fermentation solution and a fermentation strain are mixed for microbial fermentation, which can effectively improve the conversion rate of lignocellulose and the yield of oil. The method can also effectively save the cost of the oil production process, improve the economy of the entire oil production process, and be beneficial to the production and development of downstream products of the oil production process. It is further found that the method can increase the yield of GLA and DHA. DETAILED DESCRIPTION

[0033] The endpoints and any values ​​of the ranges disclosed in this article are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of each range, the endpoint values ​​of each range and the individual point values, and the individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed in this article.

[0034] The first aspect of the present invention provides a method for producing oil from lignocellulose, comprising the following steps:

[0035] S1, subjecting the raw material for preparing lignocellulose to enzymatic hydrolysis to obtain an enzymatic hydrolyzate;

[0036] S2, the enzymatic hydrolyzate is mixed with an adsorbent to obtain a mixed solution;

[0037] S3, mixing the mixed solution, nitrogen source and metal ions to obtain fermentation solution, and mixing the fermentation solution with fermentation strains to perform microbial fermentation.

[0038] The inventors of the present invention unexpectedly discovered during the process of studying the production of oil from lignocellulose that the synergistic effect between the above-mentioned processing steps can make the conversion rate of lignocellulose in the raw material higher, which is conducive to the fermentation strain to use the enzymatic hydrolysis product with lignocellulose as raw material in the enzymatic hydrolysis solution as a carbon source, and to more efficiently ferment and produce oil. The method can effectively improve the conversion rate of lignocellulose and the yield of oil. The method can also effectively save the cost of the oil production process, improve the economy of the entire oil production process, and is conducive to the production and development of downstream products of the oil production process.

[0039] In the present invention, the method for producing oil from lignocellulose can be applied to the fermentation production of oil, and the oil can be a high-nutrition oil or an oil composed of specific fatty acids. The polyunsaturated fatty acids in the high-nutrition oil contain high-value fermentation products such as eicosapentaenoic acid (EPA), γ-linolenic acid (GLA), docosahexaenoic acid (DHA), or other polyunsaturated fatty acid products that can be fermented and produced using lignocellulose as raw material.

[0040] According to the present invention, preferably, step S1 and step S2 also include solid-liquid separation. The solid-liquid separation method can be solid-liquid separation using conventional solid-liquid separation methods in the art; illustratively, the solid-liquid separation method can be at least one of filtration, sedimentation and centrifugation. Specifically, the enzymatic hydrolyzate in step S1 is a filtrate obtained after solid-liquid separation treatment; the mixed solution in step S2 is also a filtrate obtained after solid-liquid separation treatment. The enzymatic hydrolyzate after solid-liquid separation treatment can better contact with the adsorbent and improve the adsorption effect. The mixed solution after solid-liquid separation treatment can be better mixed with nitrogen sources and metal ions, which is more conducive to the growth of fermentation strains and increases the yield of oil.

[0041] According to the present invention, preferably, in step S2, the adsorbent is selected from at least one of activated carbon, calcium hydroxide and ion exchange resin. Specifically, activated carbon alone can be used as an adsorbent, or calcium hydroxide alone can be used as an adsorbent, or ion exchange resin alone can be used as an adsorbent, or activated carbon and calcium hydroxide can be used as an adsorbent, or activated carbon and ion exchange resin can be used as an adsorbent, or calcium hydroxide and ion exchange resin can be used as an adsorbent, or activated carbon, calcium hydroxide and ion exchange resin can be used as an adsorbent. The inventors have found that when the above preferred embodiment is adopted, a relatively pure clear liquid can be obtained after adsorption treatment by the adsorbent, which provides a more suitable environment for the fermentation production of oils and fats, and can improve the conversion rate of lignocellulose and the yield of oils and fats.

[0042] According to the present invention, further preferably, the mass ratio of the adsorbent to the enzymatic hydrolysate is 0.005-0.06:1, specifically 0.005:1, 0.01:1, 0.03:1, 0.06:1, or any value between the above two values. The inventors have found that when the above preferred embodiment is adopted, the lignocellulose conversion rate and oil yield can be further improved.

[0043] According to the present invention, further preferably, the concentration of monosaccharides in the enzymatic hydrolyzate is 55-62g / L, specifically 55g / L, 57g / L, 59g / L, 62g / L, or any value between the above two values; preferably 60-62g / L, specifically 60g / L, 61g / L, 62g / L, or any value between the above two values. Monosaccharides are usually polyhydroxy aldehydes or polyhydroxy ketones containing 3-6 carbon atoms. They cannot be further decomposed into simpler sugars by hydrolysis. More preferably, monosaccharides are selected from at least one of glucose, galactose, mannose, fructose and glucosamine. The detection method of monosaccharide concentration can be detected by conventional detection equipment and methods in the art, for example: copper reduction-direct titration method, glucose oxidase-peroxidase coupling method, fluorescence method, liquid chromatography, ion exchange chromatography, etc. Exemplarily, liquid chromatography (HPLC) can be used to detect the monosaccharide concentration in the hydrolyzate, and the specific method is as follows: the liquid chromatography (HPLC) analysis conditions are HPX-87H chromatographic column, differential detector, mobile phase is 0.0005-0.001mol / L H2SO4 aqueous phase, flow rate is 0.6-0.8mL / min, column temperature is 65-70°C, and time is 15-20min. The inventors found that the monosaccharide concentration in the hydrolyzate obtained by this method is high, which can meet the supply of carbon sources required in the production of oil by the fermentation strain, thereby increasing the yield of oil.

[0044] According to the present invention, preferably, the conditions of the mixing I at least include: a temperature of 30-100°C, specifically 30°C, 50°C, 80°C, 100°C, or any value between the above two values; a rotation speed of 100-300rpm, specifically 100rpm, 200rpm, 300rpm, or any value between the above two values; a time of 0.5-11h, specifically 0.5h, 1h, 3h, 5h, 7h, 9h, 11h, or any value between the above two values. The inventors found that when the above preferred embodiment is adopted, the lignocellulose conversion rate and oil yield can be further improved.

[0045] According to the present invention, further preferably, the raw material for preparing lignocellulose is selected from at least one of corn stalks, corn cobs and corn starch. More preferably, the fermentation strain is selected from at least one of Mucor, Rhizopus and Thraustochytrium. Even more preferably, Mucor is selected from at least one of Mucor rouxii, Mucor circinelloides and Cunninghamella; Rhizopus is selected from at least one of Rhizopus stolonifer, Rhizopus microsporus and Rhizopus oryzae; and the Thraustochytrium is Schizochytrium. The inventors have found that when the above preferred embodiment is adopted, the lignocellulose conversion rate and oil yield can be further improved.

[0046] According to the present invention, further preferably, the nitrogen source is selected from at least one of peptone, yeast powder, urea, ammonium sulfate, sodium nitrate and ammonium nitrate. More preferably, the inorganic salt providing the metal ion is selected from at least one of magnesium salt, sodium salt, copper salt and potassium salt. Magnesium salt is selected from at least one of magnesium sulfate, magnesium chloride and magnesium nitrate; sodium salt is selected from at least one of sodium sulfate, sodium chloride and sodium nitrate; copper salt is selected from at least one of copper sulfate, copper chloride and copper nitrate; potassium salt is selected from at least one of potassium chloride, potassium sulfate and potassium nitrate. The inventors have found that the cooperation of multiple elements can promote the accumulation of oil in the fermentation strain, further improving the conversion rate of lignocellulose and oil yield.

[0047] According to the present invention, preferably, in 100 mL of fermentation broth, the amount of the mixed solution is 30-90 mL, specifically 30 mL, 60 mL, 90 mL, or any value between the above two values; the amount of the nitrogen source is 0.1-0.5 g, specifically 0.1 g, 0.2 g, 0.3 g, 0.4 g, 0.5 g, or any value between the above two values; the amount of the inorganic salt providing the metal ion is 0.01-0.15 g, specifically 0.01 g, 0.05 g, 0.1 g, 0.15 g, or any value between the above two values; further preferably, the inoculation amount of the fermentation strain is 1×10 7 -1×10 9 cfu / mL, specifically 1×10 7 cfu / mL, 1×10 8 cfu / mL, 2×10 8 cfu / mL, 4×10 8 cfu / mL, 6×10 7 cfu / mL, 8×10 8 cfu / mL, 1×10 9 cfu / mL, or any value between the above two values. The inventors found that when the above preferred embodiment is adopted, the conversion rate of lignocellulose and the oil yield can be further improved.

[0048] According to the present invention, further preferably, the conditions of the microbial fermentation at least include: a temperature of 15-35°C, specifically 15°C, 20°C, 25°C, 30°C, 35°C, or any value between the above two values; a rotation speed of 120-300rpm, specifically 120rpm, 160rpm, 200rpm, 240rpm, 280rpm, 300rpm, or any value between the above two values; a time of 60-240h, specifically 60h, 120h, 180h, 240h, or any value between the above two values. The inventors found that when the above preferred embodiment is adopted, the lignocellulose conversion rate and oil yield can be further improved.

[0049] According to the present invention, preferably, the method further comprises: before step S1, mixing and contacting the raw material of the lignocellulose with an alkaline solution; further preferably, the mass ratio of the raw material for preparing lignocellulose to the alkaline solution is 1:5-19, specifically 1:5, 1:10, 1:15, 1:19, or any value between the above two ratios; more preferably, the alkaline solution is a sodium hydroxide alkaline solution and / or a potassium hydroxide alkaline solution. The inventors have found that the raw material for preparing lignocellulose treated with an alkaline solution can break the complex structure of the fibers in the raw material under the action of the alkaline solution, promote the conversion and release of lignocellulose into monosaccharides during enzymatic hydrolysis, and further improve the lignocellulose conversion rate and oil yield.

[0050] In the present invention, the lignocellulose raw material needs to be disinfected and sterilized before enzymatic hydrolysis. The disinfection and sterilization method can adopt the sterilization method commonly used in the art, such as high temperature and ultraviolet sterilization; high temperature sterilization can be carried out in an autoclave, and the conditions used can adopt the conventional setting parameters in the art.

[0051] According to the present invention, it is further preferred that the concentration of the solute in the alkaline solution is 0.5-2wt%, specifically 0.5wt%, 1wt%, 1.5wt%, 2wt%, or any value between the above two values; more preferably, the mixed contact conditions at least include: the temperature is 80-140°C, specifically 80°C, 100°C, 120°C, 140°C, or any value between the above two values; the time is 10-60min, specifically 10min, 30min, 60min, or any value between the above two values. Specifically, the lignocellulose raw material and the alkaline solution can be mixed and heated in a sterilizing pot, and after the mixed sterilization is completed, the lignocellulose raw material can be cooled to room temperature and washed with water, washed with water, dried and then enzymolyzed. The inventors found that when the above preferred embodiment is adopted, the lignocellulose conversion rate and oil yield can be further improved.

[0052] According to the present invention, the enzyme for the enzymatic treatment can be selected from at least one of saccharifying enzyme, cellulase and xylanase; preferably, the enzyme for the enzymatic treatment is cellulase and / or xylanase. Specifically, it can be a single cellulase, or a single xylanase, or a composite enzyme of cellulase and xylanase; further preferably, the enzyme for the enzymatic treatment is a composite enzyme of cellulase and xylanase. More preferably, the mass ratio of cellulase to xylanase in the composite enzyme is 1:0.9-1.1. The inventors have found that when the above preferred embodiment is adopted, the conversion rate of lignocellulose and the oil yield can be further improved.

[0053] According to the present invention, it is further preferred that the mass ratio of the enzyme to the raw material for preparing lignocellulose is 0.01-1:1, specifically 0.01:1, 0.1:1, 0.5:1, 1:1, or any value between the above two values; more preferably, the conditions for enzymatic hydrolysis include at least: pH 3.5-5.5, specifically 3.5, 4, 4.5, 5, 5.5, or any value between the above two values; the pH of the enzymatic hydrolyzate can be adjusted using an acid solution commonly used in the art, and illustratively, a 36-72wt% sulfuric acid solution can be used, specifically 36wt% sulfuric acid solution, 50wt% sulfuric acid solution, 72wt% sulfuric acid solution, or a sulfuric acid solution of any concentration between the above two concentrations. The temperature is 30-60°C, specifically 30°C, 40°C, 50°C, 60°C, or any value between the above two values; the speed is 150-250rpm, specifically 150rpm, 200rpm, 250rpm, or any value between the above two values; the time is 20-72h, specifically 20h, 30h, 40h, 50h, 60h, 70h, 72h, or any value between the above two values. The inventors found that when the above preferred embodiment is adopted, the lignocellulose conversion rate and oil yield can be further improved.

[0054] According to the present invention, preferably, the fermentation time is 144-199h, specifically 144h, 150h, 160h, 170h, 180h, 190h, 199h, or any value between the above two values. The inventors found that when the above preferred embodiment is adopted, the lignocellulose conversion rate and oil yield can be further improved.

[0055] According to the present invention, preferably, the method further comprises, after the fermentation of the microorganism is finished, cell disruption and oil extraction are performed. There is no particular limitation on the cell disruption method, and the cell disruption method commonly used in the art can be used; further preferably, the cell disruption method is selected from at least one of dry heat disruption, acid heat disruption, alkali heat disruption and enzyme disruption. There is no particular limitation on the method of oil extraction, and the oil extraction method commonly used in the art can be used; further preferably, the oil extraction method is selected from at least one of hydrothermal method, solvent extraction and lipid extraction; the lipid extraction method is Soxhlet extraction or rotary evaporation. Exemplarily, the fermented broth after fermentation can be filtered to obtain mycelium, and then the mycelium can be vacuum dried and then cell disruption can be performed to obtain mycelium powder; then the mycelium powder is mixed and dissolved with water and then mixed with an extraction solvent to obtain an extract rich in oil, and then the extraction solvent is removed by reduced pressure distillation to obtain oil. The inventors have found that when the above preferred embodiment is adopted, the oil yield can be further increased.

[0056] According to the present invention, preferably, the solvent for oil extraction is selected from at least one of chloroform, ether, n-hexane and ethanol. There is no special requirement for the extraction solvent, as long as it can separate the oil from the fermentation broth. Specifically, chloroform and ether can be used as extraction solvents; or n-hexane and ethanol can be used as extraction solvents; or chloroform, ether and n-hexane can be used as extraction solvents; or ether, n-hexane and ethanol can be used as extraction solvents; preferably, the solvent is a mixed solvent of n-hexane and ethanol, and the volume ratio of n-hexane to ethanol in the mixed solvent is 1-10:1, specifically 1:1, 5:1, 10:1, or any value between the above two ratios. The inventors found that when the above preferred embodiment is adopted, the oil yield can be further improved.

[0057] As a relatively preferred embodiment of the present invention, taking corn stalks as a lignocellulose raw material to ferment and produce GLA-rich oil as an example, the method comprises the following steps:

[0058] S1. Mix corn stalks with a dry weight of 1000-1500 g and a sodium hydroxide solution with a concentration of 0.5-2 wt %, and put them into a sterilizing pot (model GF54DA), wherein the mass ratio of corn stalks to sodium hydroxide solution is 1:5-19; mix them at a temperature of 80-140° C. for 10-60 min, cool the corn stalks to room temperature, take them out and wash them with water, and then dry the corn stalks after washing; mix the dried corn stalks with a mixture of cellulase and xylanase; After the complex enzymes are mixed, the pH of the mixed solution is adjusted to 3.5-5.5 by using a 36-72wt% sulfuric acid solution, and then the mixed enzymes are hydrolyzed for 20-72h at a temperature of 30-60°C and a rotation speed of 150-250rpm, wherein the mass ratio of the complex enzyme to the corn stalk is 0.01-1:1, and the mass ratio of the cellulase to the xylanase in the complex enzyme is 1:0.9-1.1; and an enzymolysis solution with a monosaccharide concentration of 60-62g / L is obtained by centrifugal separation;

[0059] S2, mixing the enzymatic hydrolysate with the adsorbent at a temperature of 30-100° C. and a rotation speed of 100-300 rpm for 0.5-11 h, wherein the mass ratio of the adsorbent to the enzymatic hydrolysate is 0.005-0.06:1, and obtaining a mixed solution by centrifugation;

[0060] S3, after mixing the mixed solution with water, adding a nitrogen source and an inorganic salt providing metal ions, mixing for 60-240 hours at a temperature of 15-35°C and a rotation speed of 120-300 rpm to obtain a fermentation liquid, wherein in 100 mL of the fermentation liquid, the amount of the mixed solution is 30-90 mL, the amount of the nitrogen source is 0.1-0.5 g, the amount of the inorganic salt is 0.01-0.15 g, and the inoculum amount of the fermentation strain is 1×10 7 -1×10 9 cfu / mL, the fermentation broth and the fermentation strain were mixed for microbial fermentation for 144-199h, and the mycelium was obtained by suction filtration.

[0061] The mycelium is vacuum dried and then crushed to obtain mycelium powder, water is added to dissolve the mycelium, and then a mixture of n-hexane and ethanol is added, wherein the volume ratio of water, n-hexane and ethanol is 1-1.5:1-10:1, and the mixture is allowed to stand for 12-14 hours after mixing, and an oil-rich extract is separated, and the extraction solvent is removed by reduced pressure distillation to obtain GLA-rich oil.

[0062] The above materials can all be obtained commercially.

[0063] The second aspect of the present invention provides the use of the method for producing fats and oils described in the first aspect in the production of GLA and / or DHA.

[0064] According to the present invention, preferably, the fermentation process of GLA and DHA can be optimized based on a machine learning algorithm. Further preferably, the machine learning algorithm is a Gaussian algorithm, and the construction process of the Gaussian algorithm is to train through the original data of oil fermentation to construct a lignocellulosic raw material enzymatic hydrolysis and mixed fermentation model, specifically including the enzymatic hydrolysis time of the lignocellulosic raw material, the adsorption time of the adsorbent, the enzyme dosage of the enzymatic hydrolysis treatment and the conditional model of the amount of adsorbent added, and the parameter model of the fermentation time and the composition of the fermentation medium. The optimal fermentation conditions are optimized and predicted by the machine algorithm to achieve quantitative control of the GLA and DHA fermentation process.

[0065] In the present invention, the detection method of GLA or DHA in oil can be carried out by a commonly used test method in the art. For example, gas chromatography can be used for determination. The specific detection process is as follows: gas phase analysis conditions: use DB-23 (60m*0.25mm*0.25μm) chromatographic column; select FID as the detector; use high-purity nitrogen as the carrier gas; use 30-32 / 1 as the split ratio; set the injection port temperature to 245-250°C; set the detector temperature to 275-280°C; the injection volume is 0.5-1μL; heating program: at the beginning of the program, the gas chromatography column temperature is set to 95-100°C, and the program is first raised to 190-196°C at a rate of 20-25°C / min. Subsequently, the program is raised to 220-230°C at a rate of 2-3°C / min, and maintained at 220-230°C for 12-15min. The gas phase column flow rate was set to 3-5 mL / min; the tail blow flow rate was set to 30-35 mL / min; the hydrogen flow rate was 40-45 mL / min, and the air flow rate was set to 400-410 mL / min.

[0066] According to the present invention, preferably, the construction process of the Gaussian algorithm includes: (1) establishing a mathematical model between the enzymatic hydrolysis conditions and the fermentation process of the lignocellulosic raw material based on the original data of oil fermentation and the regression algorithm; (2) using the mathematical model in step (1), calculating and predicting the optimal fermentation parameters under different treatment conditions; (3) optimizing the Gaussian algorithm through machine learning to determine the optimal fermentation conditions.

[0067] According to the present invention, preferably, the kernel function of the Gaussian algorithm is selected from one or more of the RBF kernel, the linear kernel, the polynomial kernel and the Markov kernel. Ensure that the efficiency and effect of entering the next fermentation broth fermentation stage are maximized. The inventor adopts that when the above preferred embodiment is adopted, the lignocellulose conversion rate and oil yield can be further improved.

[0068] The present invention will be described in detail below through examples.

[0069] The detection methods involved in the following examples are detected according to conventional conditions in the art or according to the conditions recommended by the detection equipment manufacturer; the corn stalks and corn cobs used in the examples were purchased from Jiangsu Lianyungang Farm Co., Ltd.; cellulase and xylanase were purchased from Qingdao Weilan Biotechnology Co., Ltd.; Cunninghamella echinulata ATCC 42616 was purchased from the American Type Culture Collection (ATCC); Schizochytrium HX-308, with a preservation number of CCTCC M209059, was purchased from the China Center for Type Culture Collection and stored in 20% (v / v) glycerol at -80°C; other materials and reagents are commercially available.

[0070] Unless otherwise specified, the room temperature is 25±5℃.

[0071] Example 1

[0072] S1. Mix corn stalks with a dry weight of 1000 g and a sodium hydroxide solution with a concentration of 2 wt %, and put them into a sterilizing pot (model GF54DA), wherein the mass ratio of corn stalks to sodium hydroxide solution is 1:5; mix them at a temperature of 120°C for 20 minutes, cool them to room temperature, take out the corn stalks, wash them with water, and dry them after washing; mix the dried corn stalks with a composite enzyme of cellulase and xylanase, adjust the pH of the mixture to 5.0 with a 36 wt % sulfuric acid solution, and then mix and enzymolyze them at a temperature of 50°C and a rotation speed of 250 rpm for 72 hours, wherein the mass ratio of the composite enzyme to corn stalks is 1:1, and the mass ratio of cellulase and xylanase in the composite enzyme is 1:1; obtain the enzymatic hydrolyzate by centrifugation; and detect them by liquid chromatography, under the detection conditions of HPX-87H chromatographic column, differential detector, and mobile phase of 0.0005 mol / L H2SO4 aqueous phase, flow rate of 0.6mL / min, column temperature of 65℃, elution time of 15min; monosaccharide concentration in the hydrolyzate was 62g / L;

[0073] S2, mixing 100 mL of the enzymatic hydrolysate obtained in step S1 with activated carbon as an adsorbent at a temperature of 80° C. and a rotation speed of 170 rpm for 2 h, wherein the mass ratio of activated carbon to enzymatic hydrolysate is 0.06:1, and obtaining a mixed solution by centrifugation;

[0074] S3, 30 mL of the mixed solution obtained in step S2 and 70 mL of water were mixed, and then nitrogen source peptone and inorganic salt magnesium sulfate providing metal ions were added, and the mixture was mixed at a temperature of 25 ° C and a rotation speed of 180 rpm to obtain a fermentation broth, wherein in 100 mL of the fermentation broth, the amount of peptone was 0.5 g, the amount of magnesium sulfate was 0.15 g, the fermentation strain was Cunninghamella sporeana ATCC 42616, and the inoculum size was 2×10 7cfu / mL, the fermentation liquid and the fermentation strain were mixed for microbial fermentation for 168 hours, and then the mycelium of Cunninghamella spp. was obtained by suction filtration.

[0075] The mycelium of Cunninghamella spinosa is vacuum dried and then crushed to obtain Cunninghamella spinosa mycelium powder, water is added to dissolve the mycelium, and then a mixture of n-hexane and ethanol is added, wherein the volume ratio of water, n-hexane and ethanol is 1:1.5:1, the mixture is allowed to stand for 12 hours, and an extract rich in oil is separated, and the extraction solvent is removed by reduced pressure distillation to obtain oil rich in GLA.

[0076] Example 2

[0077] S1. Mix corn stalks with a dry weight of 1000 g and a sodium hydroxide solution with a concentration of 1 wt %, and put them into a sterilizing pot (model GF54DA), wherein the mass ratio of corn stalks to sodium hydroxide solution is 1:10; mix them at a temperature of 140° C. for 60 min, cool them to room temperature, take out the corn stalks, wash them with water, and dry them after washing; mix the dried corn stalks with a composite enzyme of cellulase and xylanase, adjust the pH of the mixed solution to 5.5 with a 72 wt % sulfuric acid solution, and then mix and enzymolyze them at a temperature of 60° C. and a rotation speed of 200 rpm for 48 h, wherein the mass ratio of the composite enzyme to corn stalks is 0.01:1, and the mass ratio of cellulase and xylanase in the composite enzyme is 1:0.9; obtain an enzymatic solution by centrifugation; and detect them by liquid chromatography, under the detection conditions of HPX-87H chromatographic column, differential detector, and mobile phase of 0.0005 mol / L H2SO4 aqueous phase, flow rate of 0.6mL / min, column temperature of 65℃, elution time of 15min; monosaccharide concentration in the hydrolyzate was 61g / L;

[0078] S2, mixing 100 mL of the enzymatic hydrolysate obtained in step S1 with an adsorbent ion exchange resin at a temperature of 100° C. and a rotation speed of 300 rpm for 0.5 h, wherein the mass ratio of the ion exchange resin to the enzymatic hydrolysate is 0.005:1, and obtaining a mixed solution by centrifugation;

[0079] S3, after mixing 70 mL of the mixed solution obtained in step S2 with 30 mL of water, adding nitrogen source urea and inorganic salt sodium sulfate providing metal ions, mixing at a temperature of 35 ° C and a rotation speed of 300 rpm to obtain a fermentation broth, wherein in 100 mL of the fermentation broth, the amount of urea is 0.1 g, the amount of sodium sulfate is 0.01 g, the fermentation strain is Cunninghamella sporeana ATCC 42616, and the inoculum amount is 1×10 8 cfu / mL, the fermentation liquid and the fermentation strain were mixed for microbial fermentation for 144 hours, and then the mycelium of Cunninghamella spp. was obtained by suction filtration.

[0080] The mycelium of Cunninghamella spinosa is vacuum dried and then crushed to obtain Cunninghamella spinosa mycelium powder, water is added to dissolve the mycelium, and then a mixture of n-hexane and ethanol is added, wherein the volume ratio of water, n-hexane and ethanol is 1.5:10:1, the mixture is allowed to stand for 14 hours, and an extract rich in oil is separated, and the extraction solvent is removed by reduced pressure distillation to obtain oil rich in GLA.

[0081] Example 3

[0082] S1. Mix corn cobs with a dry weight of 1000 g and a potassium hydroxide solution with a concentration of 0.5 wt %, and put them into a sterilizing pot (model GF54DA), wherein the mass ratio of corn cobs to potassium hydroxide solution is 1:19; mix them at a temperature of 80°C for 10 minutes, cool them to room temperature, take out the corn cobs, wash them with water, and then dry them; mix the dried corn cobs with a composite enzyme of cellulase and xylanase, adjust the pH of the mixture to 3.5 with a 50 wt % sulfuric acid solution, and then mix and enzymolyze them at a temperature of 30°C and a rotation speed of 150 rpm for 20 hours, wherein the mass ratio of the composite enzyme to corn cobs is 0.5:100, and the mass ratio of cellulase and xylanase in the composite enzyme is 1:1.1; obtain an enzymatic solution by centrifugation; and detect them by liquid chromatography, under the detection conditions of HPX-87H chromatographic column, differential detector, and mobile phase of 0.0005 mol / L H2SO4 aqueous phase, flow rate of 0.6mL / min, column temperature of 65℃, elution time of 15min; monosaccharide concentration in the hydrolyzate was 58.5g / L;

[0083] S2, mixing 100 mL of the enzymatic hydrolyzate obtained in step S1 with adsorbent calcium hydroxide at a temperature of 30° C. and a rotation speed of 100 rpm for 11 hours, wherein the mass ratio of calcium hydroxide to the enzymatic hydrolyzate is 0.02:1, and obtaining a mixed solution by centrifugation;

[0084] S3, after mixing 90 mL of the mixed solution obtained in step S2 with 10 mL of water, adding nitrogen source ammonium sulfate and inorganic salt copper sulfate providing metal ions, mixing at a temperature of 15 ° C and a rotation speed of 120 rpm to obtain a fermentation broth, wherein in 100 mL of the fermentation broth, the amount of ammonium sulfate is 0.3 g, the amount of copper sulfate is 0.1 g, the fermentation strain is Cunninghamella sporeana ATCC 42616, and the inoculum size is 1×10 9 cfu / mL, the fermentation liquid and the fermentation strain were mixed for microbial fermentation for 199 hours, and then the mycelium of Cunninghamella spp. was obtained by suction filtration.

[0085] The mycelium of Cunninghamella spinosa is vacuum dried and then crushed to obtain Cunninghamella spinosa mycelium powder, water is added to dissolve the mycelium, and then a mixture of n-hexane and ethanol is added, wherein the volume ratio of water, n-hexane and ethanol is 1.2:5:1, the mixture is allowed to stand for 13 hours, and an extract rich in oil is separated, and the extraction solvent is removed by reduced pressure distillation to obtain oil rich in GLA.

[0086] Example 4

[0087] The fermentation production of oil was carried out according to the method of Example 1, except that in step S3, Cunninghamella echinops ATCC 42616 was replaced with Schizochytrium HX-308 (deposit number CCTCC M209059), to obtain oil rich in DHA.

[0088] Example 5

[0089] The fermentation production and detection of oil were carried out according to the method of Example 1, except that in step S1, the complex enzyme was replaced by a single lignocellulase. The monosaccharide concentration in the enzymatic hydrolyzate was 55 g / L as detected by liquid chromatography.

[0090] Example 6

[0091] The fermentation production of oil was carried out according to the method of Example 1, except that in step S2, the mass ratio of the adsorbent to the enzymatic hydrolysate was 0.08:1.

[0092] Example 7

[0093] The fermentation production of oil is carried out according to the method of Example 1, except that in step S2, the activated carbon is replaced by organic modified bentonite, and the organic modified bentonite is prepared according to the method in CN102719490A.

[0094] Example 8

[0095] The fermentation production and detection of oil and fat were carried out according to the method of Example 1, except that in step S1, the complex enzyme was replaced with saccharifying enzyme. The monosaccharide concentration in the enzymatic hydrolyzate was 45 g / L as detected by liquid chromatography.

[0096] Example 9

[0097] The fermentation production of oil was carried out according to the method of Example 1, except that in step S3, Cunninghamella echinops was replaced with Yarrowia lipolytica NRRL Y-1095 (from the research group of Professor Jin Mingjie of Nanjing University of Science and Technology) to obtain oil containing oleic acid and linoleic acid.

[0098] Example 10

[0099] The fermentation production and detection of oil and fat were carried out according to the method of Example 3, except that in step S1, the pH of the mixed solution was adjusted to 6.5. The monosaccharide concentration in the enzymatic hydrolyzate was 54 g / L as detected by liquid chromatography.

[0100] Comparative Example 1

[0101] The fermentation production of oil is carried out according to the method of Example 4, except that step S2 is omitted, and the enzymatic hydrolyzate in step S1 is directly mixed with the nitrogen source and the inorganic salt to obtain oil rich in DHA.

[0102] Comparative Example 2

[0103] The fermentation production of oil is carried out according to the method of Example 1, except that step S2 is omitted, and the enzymatic hydrolyzate in step S1 is directly mixed with the nitrogen source and the inorganic salt to obtain the GLA-rich oil.

[0104] Test Case

[0105] Take the oils and fats prepared in Examples 1 to 10 and Comparative Examples 1 to 2, and perform a methyl esterification reaction. The specific test method is: take 20 mg of oil and fat, add 500 μL of 1 mol / L sodium hydroxide methanol solution, react for 2 hours, then add 50 μL of concentrated sulfuric acid to terminate the reaction, add n-hexane for extraction, and take the supernatant for detection. Use a gas chromatograph (model Shimadzu GC 2030) to determine the content of GLA and DHA in the oil and fat, and the test results are shown in Table 1.

[0106] Table 1

[0107]

[0108] Note: “ / ” in Table 1 means no data involved.

[0109] It can be seen from the results in Table 1 that, compared with the method of comparative example 1-comparative example 2, the content of GLA and DHA in the fermented oils and fats obtained by Examples 1-10 of the method of producing oils and fats from lignocellulose of the present invention is higher, and the technical effect is significantly better.

[0110] The method for producing oil from lignocellulose provided by the present invention can effectively improve the conversion rate of lignocellulose and the yield of oil. The method can also effectively save the cost of the oil production process, improve the economy of the entire oil production process, and is conducive to the production and development of downstream products of the oil production process.

[0111] The preferred embodiments of the present invention are described in detail above, but the present invention is not limited thereto. Within the technical concept of the present invention, the technical solution of the present invention can be subjected to a variety of simple modifications, including the combination of various technical features in any other suitable manner, and these simple modifications and combinations should also be regarded as the contents disclosed by the present invention and belong to the protection scope of the present invention.

Claims

1. A method for producing oil from lignocellulose, characterized in that: The following steps are involved: S1, subjecting the raw material for preparing lignocellulose to enzymatic hydrolysis to obtain an enzymatic hydrolyzate; S2, the enzymatic hydrolyzate is mixed with an adsorbent to obtain a mixed solution; S3, mixing the mixed solution, nitrogen source and metal ions to obtain fermentation solution, and mixing the fermentation solution with fermentation strains to perform microbial fermentation.

2. The method for producing fats and oils according to claim 1, characterized in that: In step S2, the adsorbent is selected from at least one of activated carbon, calcium hydroxide and ion exchange resin; Preferably, the mass ratio of the adsorbent to the enzymatic hydrolysate is 0.005-0.06:1; preferably, in step S1, the concentration of monosaccharides in the enzymatic hydrolysate is 55-62 g / L, preferably 60-62 g / L.

3. The method for producing fats and oils according to claim 1, characterized in that: The mixing conditions of the mixing I include at least: a temperature of 30-100° C., a rotation speed of 100-300 rpm, and a time of 0.5-11 h; Preferably, the raw material for preparing lignocellulose is selected from at least one of corn stalks, corn cobs and corn starch.

4. The method for producing fats and oils according to claim 1, characterized in that: The fermentation strain is selected from at least one of Mucor, Rhizopus and Thraustochytrid; Preferably, the nitrogen source is selected from at least one of peptone, yeast powder, urea, ammonium sulfate, sodium nitrate and ammonium nitrate; Preferably, the inorganic salt providing the metal ion is selected from at least one of magnesium salt, sodium salt, copper salt and potassium salt.

5. The method for producing fat according to any one of claims 1 to 4, characterized in that: In 100 mL of fermentation broth, the amount of the mixed solution is 30-90 mL, the amount of the nitrogen source is 0.1-0.5 g, and the amount of the inorganic salt providing the metal ion is 0.01-0.15 g; Preferably, the inoculation amount of the fermentation strain is 1×10 7 -1×10 9 cfu / mL; Preferably, the conditions for the microbial fermentation include at least: a temperature of 15-35°C, a rotation speed of 120-300 rpm, and a time of 60-240 h.

6. The method for producing fat according to any one of claims 1 to 4, characterized in that: The method further comprises: before step S1, mixing and contacting the lignocellulose raw material with an alkaline solution; Preferably, the mass ratio of the raw material for preparing lignocellulose to the alkaline solution is 1:5-19; Preferably, the alkaline solution is a sodium hydroxide alkaline solution and / or a potassium hydroxide alkaline solution; Preferably, the concentration of the solute in the alkaline solution is 0.5-2wt%; Preferably, the mixed contact conditions at least include: a temperature of 80-140° C. and a time of 10-60 min.

7. The method for producing fat according to any one of claims 1 to 4, characterized in that: The enzyme for enzymatic treatment is cellulase and / or xylanase; Preferably, the mass ratio of the enzyme to the raw material for preparing lignocellulose is 0.01-1:1; Preferably, the conditions for the enzymatic hydrolysis include at least: pH 3.5-5.5, temperature 30-60° C., rotation speed 150-250 rpm, and time 20-72 h.

8. The method for producing fats and oils according to any one of claims 1 to 4, characterized in that: The method further comprises performing cell disruption and oil extraction after the microbial fermentation is completed.

9. The method for producing fats and oils according to claim 8, characterized in that: The solvent for oil extraction is selected from at least one of chloroform, ether, n-hexane and ethanol; Preferably, the solvent is a mixed solvent of n-hexane and ethanol, and the volume ratio of the n-hexane to the ethanol in the mixed solvent is 1-10:

1.

10. Use of the method for producing fats and oils according to any one of claims 1 to 9 in producing GLA and / or DHA.

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