Extraction method of microalgae

Abstract

The invention discloses an extraction method of microalgae, and belongs to the technical field of microorganisms, and the extraction method comprises the three steps of microalgae culture, enzymolysis reaction and compounding aftertreatment. When a microalgae extract is applied to a microalgae fertilizer, the yield and quality of crops can be effectively improved; and the schizochytrium limacinum culture medium is treated by using p-hydroxybenzoic acid and diammonium phosphate, so that the cell membrane permeability of the schizochytrium limacinum is changed, other nutrient substances can enter the schizochytrium limacinum conveniently, and meanwhile, cell wall breaking of the schizochytrium limacinum is facilitated. Under a proper ratio of an enzymolysis mixture, pyrolytic biochar, bentonite and biomass tar, the prepared microalgae-biochar simultaneously has abundant pore structures, a large amount of microalgae loads and strong combination structural performance, the activity performance of the microalgae-biochar can be effectively maintained, and the application range of the microalgae-biochar is widened. The method has a wide market prospect when being used for preparing a microalgae fertilizer.

Description

Technical Field

[0001] The invention belongs to the technical field of microorganisms, and in particular relates to a method for extracting microalgae. Background Art

[0003] Microalgae fertilizer is one of the substitutes for chemical fertilizers. It is an effective biologically active substance extracted from microalgae. It can promote plant growth, improve the appearance and taste of agricultural products, and eliminate the damage to plants caused by adverse natural conditions or chemical agents. Compared with traditional chemical fertilizers, microalgae fertilizers are rich in active ingredients such as amino acids, peptides, microalgae polysaccharides and natural plant hormones. They can regulate the nutritional balance of crops, enhance their resistance to stress and disease, and effectively improve the yield and quality of crops. It will not pollute water sources, damage soil and other environmental problems. It is one of the popular products of green and environmentally friendly fertilizers. Based on the above advantages, the extraction of microalgae has always been a key technology in the microalgae bioenergy industry. The extraction technologies of microalgae include: Soxhlet extraction, organic solvent extraction, ultrasonic extraction, osmotic pressure wall breaking extraction, mechanical wall breaking extraction, supercritical carbon dioxide extraction, etc. At present, most of these extraction technologies remain in the laboratory stage, with cumbersome process routes, low extraction rates, high costs, and high energy consumption, which limits their industrial application.

[0004] Therefore, if a new method for effectively extracting microalgae is developed and applied in biofertilizer, it will have broad market application prospects. Summary of the invention

[0005] The purpose of this section is to summarize some aspects of embodiments of the present invention and briefly introduce some preferred embodiments. Some simplifications or omissions may be made in this section and the specification abstract and the invention title of this application to avoid blurring the purpose of this section, the specification abstract and the invention title, and such simplifications or omissions cannot be used to limit the scope of the present invention.

[0006] In view of the above problems and / or the problems existing in the prior art, the present invention is proposed.

[0007] Therefore, the purpose of the present invention is to overcome the deficiencies in the prior art and provide a method for extracting microalgae.

[0008] In order to solve the above technical problems, the present invention provides the following technical solutions: S1. Cultivation of microalgae: adding glucose, magnesium sulfate, potassium dihydrogen phosphate, ammonium ferric citrate, ascorbic acid, sodium silicate, vitamin B12, sodium bicarbonate, and ammonium bicarbonate to deionized water, sterilizing under the condition of pH 8.0, inoculating with the seed liquid of Schizochytrium fungus, culturing under light conditions, filtering after treatment with a treatment agent, washing, collecting algae cells, freeze-drying, and obtaining algae powder; S2. Enzymatic hydrolysis reaction: adding the algae powder obtained in step S1 to deionized water, adding a composite enzyme to the water for reaction, and coarse filtration after the reaction to obtain an enzymatic hydrolysis mixture; S3. Post-compounding treatment: The enzymatic hydrolysis mixture obtained in step S2 is added to pyrolysis biochar, bentonite and biomass tar, mixed, crushed and then dried to obtain microalgae-biochar.

[0009] As a preferred embodiment of the microalgae extraction method of the present invention, wherein: the mass ratio of glucose, magnesium sulfate, potassium dihydrogen phosphate, ammonium ferric citrate, ascorbic acid, sodium silicate, vitamin B12, sodium bicarbonate, ammonium bicarbonate and deionized water in step S1 is 10-25:1-5:1-2:0.1-2:0.1-2:1-3:0.0001-0.001:2-5:0.1-2:1000; the inoculation amount of the Schizochytrium spawn seed liquid is 12-15 v / v%, the intensity of the light is 2500-3000 lx, the culture conditions are O2 ventilation of 2-4 L / min, 22-24 ° C, 100-200 r / min, and culture for 3-5 days, and the treatment agent treatment is treated under the culture conditions for 10-24 hours.

[0010] As a preferred embodiment of the microalgae extraction method of the present invention, the treatment agent in step S1 includes p-hydroxybenzoic acid and diammonium phosphate, wherein the mass ratio of p-hydroxybenzoic acid to diammonium phosphate is 1-5:20-30.

[0011] As a preferred embodiment of the microalgae extraction method of the present invention, the mass ratio of the treatment agent to deionized water in step S1 is 20-30:1000.

[0012] As a preferred embodiment of the method for extracting microalgae of the present invention, the complex enzyme in step S2 comprises lipase and lysozyme, wherein the mass ratio of lipase to lysozyme is 2-7:5-10.

[0013] As a preferred embodiment of the microalgae extraction method of the present invention, the enzymatic hydrolysis reaction in step S2 is carried out at 40-60° C. for 1-5 hours.

[0014] As a preferred embodiment of the method for extracting microalgae according to the present invention, the mass ratio of the enzymatic hydrolysis mixture, pyrolysis biochar, bentonite and biomass tar in step S3 is 6:2:1:1.

[0015] As a preferred embodiment of the microalgae extraction method of the present invention, the pulverization treatment in step S3 is to use a pulverizer to pulverize the mixed material at 20-45° C. for 20-40 minutes, so that the mesh size of the pulverized material is 120-200 meshes.

[0016] As a preferred embodiment of the microalgae extraction method of the present invention, the drying in step S3 is to heat the temperature to 260° C. and dry to constant weight at a heating rate of 8-12° C. / min.

[0017] Another object of the present invention is to overcome the deficiencies in the prior art and provide an application of microalgae-biochar in the preparation of microalgae fertilizer.

[0018] Beneficial effects of the present invention: When the present invention applies microalgae-biochar to microalgae fertilizer, the yield and quality of crops can be effectively improved. In addition, the present invention uses p-hydroxybenzoic acid and diammonium phosphate to treat the culture solution obtained by culturing Schizochytrium, so as to change the cell membrane permeability of Schizochytrium, facilitate the entry of other nutrients, and help the cell wall of Schizochytrium to be broken. By combining glucose, ascorbic acid and vitamin B12 in the culture medium, the loss of intracellular substances and oxidative stress caused by increased active oxygen content are avoided. By adding glucose as a carbon source and ammonium bicarbonate as a nitrogen source, the content of active substances (polysaccharides, oils, etc.) of Schizochytrium is significantly increased. The active substances of Schizochytrium can directly provide the required nutrition for plants and promote plant growth. They can also improve the nonspecific immunity of crops and enhance the resistance of crops to adverse stress. At the same time, they can also improve the soil, regulate and promote the absorption and utilization of water and nutrients by plants, and help improve soil structure and soil fertility.

[0019] Pyrolysis biochar is a solid product obtained by high-temperature pyrolysis of biomass raw materials under completely or partially anoxic conditions. The raw materials for preparation are very extensive and the cost is low. It has the characteristics of rich pore structure and strong adsorption capacity. In the present invention, by adding pyrolysis biochar to the enzymatic hydrolysis mixture, the microalgae can be embedded in the pore structure of the pyrolysis biochar, so that the microalgae and the pyrolysis biochar form a strong bond. At the same time, bentonite and biomass tar are added to improve the adhesion of the material while also providing additional organic nutrition. Moreover, biomass tar and pyrolysis biochar are both products after the pyrolysis of biomass raw materials, which improves The utilization rate of biomass raw materials is high and it will not pollute the environment. The complex enzymes used in the enzymatic hydrolysis process include lipase and lysozyme. Under the synergistic effect of the two, they can adsorb and rupture the cell wall very well, thereby improving the extraction efficiency of active substances. Under the appropriate ratio of enzymatic hydrolysis mixture, pyrolytic biochar, bentonite and biomass tar, the prepared microalgae-biochar has rich pore structure, a large amount of microalgae load, and strongly combined structural properties, which can effectively maintain the activity performance of microalgae-biochar and has broad market prospects when it is prepared into microalgae fertilizer. DETAILED DESCRIPTION

[0020] In order to make the above-mentioned objects, features and advantages of the present invention more obvious and easy to understand, the specific implementation methods of the present invention are described in detail below in conjunction with the embodiments of the specification.

[0021] In the following description, many specific details are set forth to facilitate a full understanding of the present invention, but the present invention may also be implemented in other ways different from those described herein, and those skilled in the art may make similar generalizations without violating the connotation of the present invention. Therefore, the present invention is not limited to the specific embodiments disclosed below.

[0022] Secondly, the term "one embodiment" or "embodiment" as used herein refers to a specific feature, structure, or characteristic that may be included in at least one implementation of the present invention. The term "in one embodiment" that appears in different places in this specification does not necessarily refer to the same embodiment, nor does it refer to a separate or selective embodiment that is mutually exclusive with other embodiments.

[0023] The Schizochytrium sp. used in the present invention was purchased from the General Microbiological Center of China National Committee for the Preservation and Administration of Microbiological Cultures, and its number is CCTCC No. M209059. For detailed information on the cultivation method of the algae, please refer to Chinese Patent Application No.: 200910033869.5; Schizochytrium spawn seed solution: Schizochytrium was activated by EMMG medium (containing sodium glutamate) (purchased from Xi'an Qiyue Biotechnology Co., Ltd.) and cultured in a shake flask at 30°C for 3 days to obtain Schizochytrium spawn seed solution with a bacterial count of 10 7 ~10 8 cfu / mL; The present invention calculates the oil recovery rate extracted from microalgae with reference to "Process Optimization of Extracting Oil, Protein and Polysaccharide from Schizochytrium by Three-Phase Separation Method".

[0024] The apple test base selected in the present invention is provided by the Weifang Dongli Rural Ecological Fruit and Vegetable Professional Cooperative, and is located in Qianquezhuang Village, Beicheng Street, Weifang Economic Development Zone.

[0025] The traditional fertilizer used in the present invention is Austrian green fertilizer A2 9-14-19+MgO+Fe, which is purchased from Guangzhou Runjie Agricultural Technology Co., Ltd.

[0026] The pyrolysis biochar used in the present invention is a solid product produced by pyrolysis and gasification of straw.

[0027] The biomass tar used in the present invention is a waste product from the process of pyrolysis and gasification of straw, the coking value of the tar is 10% to 56%, and the dynamic viscosity is 147 MPa*s to 8650 MPa*s.

[0028] The bentonite used in the present invention is sodium-based bentonite.

[0029] The present invention refers to Hu Haojie, Tian Shuangqi, Zhao Renyong, et al. Research progress on extraction of active substances from new resource edible microalgae and their application in food [J]. Food Industry Science and Technology, 2022, 43 (2): 390−396. doi: 10.13386 / j.issn1002-0306.2020060048. The proportion of active ingredients in microalgae-biochar was determined.

[0030] Unless otherwise specified, the raw materials used in the present invention are commonly available in the market.

[0031] Example 1

[0032] This embodiment provides a method for extracting microalgae, specifically: S1. Microalgae culture: 20 parts by weight of glucose, 5 parts by weight of magnesium sulfate, 2 parts by weight of potassium dihydrogen phosphate, 1 part by weight of ammonium ferric citrate, 1 part by weight of ascorbic acid, 2 parts by weight of sodium silicate, 0.0005 parts by weight of vitamin B12, 3 parts by weight of sodium bicarbonate, and 0.15 parts by weight of ammonium bicarbonate are added to 1000 parts by weight of deionized water, sterilized under the condition of pH 8.0, inoculated with Schizochytrium fungus seed liquid, the inoculation amount is 15v / v%, the light intensity is 3000lx, the O2 ventilation volume is 4L / min, 24°C, 200r / min, after culturing for 5 days, 25 parts by weight of a treating agent (including 1 part by weight of p-hydroxybenzoic acid and 24 parts by weight of diammonium phosphate) are added, and the culture is continued for 12 hours, filtered, washed, algal cells are collected, freeze-dried, and algal powder is obtained; S2. Enzymatic hydrolysis reaction: 100 parts by weight of the algae powder prepared in step S1 was added to 1000 parts of deionized water, 2 parts by weight of the composite enzyme was added thereto, and the mixture was reacted at 45°C for 2 hours, and the enzymatic hydrolysis mixture was obtained by filtration. The pore size of the filter mesh was 200 μm, and the mass ratio of lipase to lysozyme in the composite enzyme was 3:7; S3. Post-compounding treatment: The enzymatic hydrolysis mixture obtained in step S2 is mixed with pyrolytic biochar, bentonite and biomass tar in a mass ratio of 6:2:1:1, and then crushed at 40°C for 30 minutes using a pulverizer to make the mesh size of the crushed material 180 mesh. Then, the temperature is increased to 260°C at a heating rate of 10°C / min and dried to constant weight to obtain microalgae-biochar.

[0033] Example 2

[0034] This embodiment provides a method for extracting microalgae, specifically: S1. Microalgae culture: 20 parts by weight of glucose, 5 parts by weight of magnesium sulfate, 2 parts by weight of potassium dihydrogen phosphate, 1 part by weight of ammonium ferric citrate, 1 part by weight of ascorbic acid, 2 parts by weight of sodium silicate, 0.0005 parts by weight of vitamin B12, 3 parts by weight of sodium bicarbonate, and 0.15 parts by weight of ammonium bicarbonate are added to 1000 parts by weight of deionized water, sterilized under the condition of pH 8.0, inoculated with Schizochytrium fungus seed liquid, the inoculation amount is 15v / v%, the light intensity is 3000lx, the O2 ventilation volume is 4L / min, 24°C, 200r / min, after culturing for 5 days, 25 parts by weight of a treating agent (including 2 parts by weight of p-hydroxybenzoic acid and 23 parts by weight of diammonium phosphate) are added, and the culture is continued for 12 hours, filtered, washed, algal cells are collected, freeze-dried, and algal powder is obtained; S2. Enzymatic hydrolysis reaction: 100 parts by weight of the algae powder prepared in step S1 was added to 1000 parts of deionized water, 2 parts by weight of the composite enzyme was added thereto, and the mixture was reacted at 45°C for 2 hours, and the enzymatic hydrolysis mixture was obtained by filtration. The pore size of the filter mesh was 200 μm, and the mass ratio of lipase to lysozyme in the composite enzyme was 3:7; S3. Post-compounding treatment: The enzymatic hydrolysis mixture obtained in step S2 is mixed with pyrolytic biochar, bentonite and biomass tar in a mass ratio of 6:2:1:1, and then crushed at 40°C for 30 minutes using a pulverizer to make the mesh size of the crushed material 180 mesh. Then, the temperature is increased to 260°C at a heating rate of 10°C / min and dried to constant weight to obtain microalgae-biochar.

[0035] Example 3

[0036] This embodiment provides a method for extracting microalgae, specifically: S1. Microalgae culture: 20 parts by weight of glucose, 5 parts by weight of magnesium sulfate, 2 parts by weight of potassium dihydrogen phosphate, 1 part by weight of ammonium ferric citrate, 1 part by weight of ascorbic acid, 2 parts by weight of sodium silicate, 0.0005 parts by weight of vitamin B12, 3 parts by weight of sodium bicarbonate, and 0.15 parts by weight of ammonium bicarbonate are added to 1000 parts by weight of deionized water, sterilized under the condition of pH 8.0, inoculated with Schizochytrium fungus seed liquid, the inoculation amount is 15v / v%, the light intensity is 3000lx, the O2 ventilation volume is 4L / min, 24°C, 200r / min, after culturing for 5 days, 25 parts by weight of a treating agent (including 3 parts by weight of p-hydroxybenzoic acid and 22 parts by weight of diammonium phosphate) are added, and the culture is continued for 12 hours, filtered, washed, algal cells are collected, freeze-dried, and algal powder is obtained; S2. Enzymatic hydrolysis reaction: 100 parts by weight of the algae powder prepared in step S1 was added to 1000 parts of deionized water, 2 parts by weight of the composite enzyme was added thereto, and the mixture was reacted at 45°C for 2 hours, and the enzymatic hydrolysis mixture was obtained by filtration. The pore size of the filter mesh was 200 μm, and the mass ratio of lipase to lysozyme in the composite enzyme was 3:7; S3. Post-compounding treatment: The enzymatic hydrolysis mixture obtained in step S2 is mixed with pyrolytic biochar, bentonite and biomass tar in a mass ratio of 6:2:1:1, and then crushed at 40°C for 30 minutes using a pulverizer to make the mesh size of the crushed material 180 mesh. Then, the temperature is increased to 260°C at a heating rate of 10°C / min and dried to constant weight to obtain microalgae-biochar.

[0037] Example 4

[0038] This embodiment provides a method for extracting microalgae, specifically: S1. Microalgae culture: 20 parts by weight of glucose, 5 parts by weight of magnesium sulfate, 2 parts by weight of potassium dihydrogen phosphate, 1 part by weight of ammonium ferric citrate, 1 part by weight of ascorbic acid, 2 parts by weight of sodium silicate, 0.0005 parts by weight of vitamin B12, 3 parts by weight of sodium bicarbonate, and 0.15 parts by weight of ammonium bicarbonate are added to 1000 parts by weight of deionized water, sterilized under the condition of pH 8.0, inoculated with Schizochytrium fungus seed liquid, the inoculation amount is 15v / v%, the light intensity is 3000lx, the O2 ventilation volume is 4L / min, 24°C, 200r / min, after culturing for 5 days, 25 parts by weight of a treating agent (including 1 part by weight of p-hydroxybenzoic acid and 24 parts by weight of diammonium phosphate) are added, and the culture is continued for 12 hours, filtered, washed, algal cells are collected, freeze-dried, and algal powder is obtained; S2. Enzymatic hydrolysis reaction: 100 parts by weight of the algae powder prepared in step S1 are added to 1000 parts of deionized water, and a composite enzyme is added thereto to react at 45°C for 2 hours, and the enzymatic hydrolysis mixture is obtained by filtration, wherein the pore size of the filter screen is 200 μm, and the mass ratio of lipase to lysozyme in 2 parts by weight of the composite enzyme is 2:8; S3. Post-compounding treatment: The enzymatic hydrolysis mixture obtained in step S2 is mixed with pyrolytic biochar, bentonite and biomass tar in a mass ratio of 6:2:1:1, and then crushed at 40°C for 30 minutes using a pulverizer to make the mesh size of the crushed material 180 mesh. Then, the temperature is increased to 260°C at a heating rate of 10°C / min and dried to constant weight to obtain microalgae-biochar.

[0039] Example 5

[0040] This embodiment provides a method for extracting microalgae, specifically: S1. Microalgae culture: 20 parts by weight of glucose, 5 parts by weight of magnesium sulfate, 2 parts by weight of potassium dihydrogen phosphate, 1 part by weight of ammonium ferric citrate, 1 part by weight of ascorbic acid, 2 parts by weight of sodium silicate, 0.0005 parts by weight of vitamin B12, 3 parts by weight of sodium bicarbonate, and 0.15 parts by weight of ammonium bicarbonate are added to 1000 parts by weight of deionized water, sterilized under the condition of pH 8.0, inoculated with Schizochytrium fungus seed liquid, the inoculation amount is 15v / v%, the light intensity is 3000lx, the O2 ventilation volume is 4L / min, 24°C, 200r / min, after culturing for 5 days, 25 parts by weight of a treating agent (including 1 part by weight of p-hydroxybenzoic acid and 24 parts by weight of diammonium phosphate) are added, and the culture is continued for 12 hours, filtered, washed, algal cells are collected, freeze-dried, and algal powder is obtained; S2. Enzymatic hydrolysis reaction: 100 parts by weight of the algae powder prepared in step S1 was added to 1000 parts of deionized water, 2 parts by weight of the composite enzyme was added thereto, and the mixture was reacted at 45°C for 2 hours, and the enzymatic hydrolysis mixture was obtained by filtration. The pore size of the filter mesh was 200 μm, and the mass ratio of lipase to lysozyme in the composite enzyme was 5:5; S3. Post-compounding treatment: The enzymatic hydrolysis mixture obtained in step S2 is mixed with pyrolytic biochar, bentonite and biomass tar in a mass ratio of 6:2:1:1, and then crushed at 40°C for 30 minutes using a pulverizer to make the mesh size of the crushed material 180 mesh. Then, the temperature is increased to 260°C at a heating rate of 10°C / min and dried to constant weight to obtain microalgae-biochar.

[0041] Comparative Example 1

[0042] This comparative example provides a method for extracting microalgae, specifically: S1. Microalgae culture: 20 parts by weight of glucose, 5 parts by weight of magnesium sulfate, 2 parts by weight of potassium dihydrogen phosphate, 1 part by weight of ammonium ferric citrate, 1 part by weight of ascorbic acid, 2 parts by weight of sodium silicate, 0.0005 parts by weight of vitamin B12, 3 parts by weight of sodium bicarbonate, and 0.15 parts by weight of ammonium bicarbonate are added to 1000 parts by weight of deionized water, sterilized under the condition of pH 8.0, inoculated with Schizochytrium fungus seed liquid, the inoculation amount is 15v / v%, the light intensity is 3000lx, the O2 ventilation volume is 4L / min, 24°C, 200r / min, after culturing for 5 days, 25 parts by weight of a treating agent (including 1 part by weight of p-hydroxybenzoic acid and 24 parts by weight of diammonium phosphate) are added, and the culture is continued for 12 hours, filtered, washed, algal cells are collected, freeze-dried, and algal powder is obtained; S2. Enzymatic hydrolysis reaction: 100 parts by weight of the algae powder prepared in step S1 was added to 1000 parts of deionized water, 2 parts by weight of the composite enzyme was added thereto, and the mixture was reacted at 45°C for 2 hours, and the enzymatic hydrolysis mixture was obtained by filtration, the pore size of the filter mesh was 200 μm, and the mass ratio of cellulase to lysozyme in the composite enzyme was 3:7; S3. Post-compounding treatment: The enzymatic hydrolysis mixture obtained in step S2 is mixed with pyrolytic biochar, bentonite and biomass tar in a mass ratio of 6:2:1:1, and then crushed at 40°C for 30 minutes using a pulverizer to make the mesh size of the crushed material 180 mesh. Then, the temperature is increased to 260°C at a heating rate of 10°C / min and dried to constant weight to obtain microalgae-biochar.

[0043] Comparative Example 2

[0044] This comparative example provides a method for extracting microalgae, specifically: S1. Microalgae culture: 20 parts by weight of glucose, 5 parts by weight of magnesium sulfate, 2 parts by weight of potassium dihydrogen phosphate, 1 part by weight of ammonium ferric citrate, 1 part by weight of ascorbic acid, 2 parts by weight of sodium silicate, 0.0005 parts by weight of vitamin B12, 3 parts by weight of sodium bicarbonate, and 0.15 parts by weight of ammonium bicarbonate are added to 1000 parts by weight of deionized water, sterilized under the condition of pH 8.0, inoculated with Schizochytrium fungus seed liquid, the inoculation amount is 15v / v%, the light intensity is 3000lx, the O2 ventilation volume is 4L / min, 24°C, 200r / min, after culturing for 5 days, 25 parts by weight of a treating agent (including 1 part by weight of p-hydroxybenzoic acid and 24 parts by weight of diammonium phosphate) are added, and the culture is continued for 12 hours, filtered, washed, algal cells are collected, freeze-dried, and algal powder is obtained; S2. Enzymatic hydrolysis reaction: 100 parts by weight of the algae powder prepared in step S1 was added to 1000 parts of deionized water, 2 parts by weight of lipase was added thereto, and the mixture was reacted at 45°C for 2h, and the enzymatic hydrolysis mixture was obtained by filtering, and the pore size of the filter mesh was 200 μm; S3. Post-compounding treatment: The enzymatic hydrolysis mixture obtained in step S2 is mixed with pyrolytic biochar, bentonite and biomass tar in a mass ratio of 6:2:1:1, and then crushed at 40°C for 30 minutes using a pulverizer to make the mesh size of the crushed material 180 mesh. Then, the temperature is increased to 260°C at a heating rate of 10°C / min and dried to constant weight to obtain microalgae-biochar.

[0045] Comparative Example 3

[0046] This comparative example provides a method for extracting microalgae, specifically: S1. Microalgae culture: 20 parts by weight of glucose, 5 parts by weight of magnesium sulfate, 2 parts by weight of potassium dihydrogen phosphate, 1 part by weight of ammonium ferric citrate, 1 part by weight of ascorbic acid, 2 parts by weight of sodium silicate, 0.0005 parts by weight of vitamin B12, 3 parts by weight of sodium bicarbonate, and 0.15 parts by weight of ammonium bicarbonate are added to 1000 parts by weight of deionized water, sterilized under the condition of pH 8.0, inoculated with Schizochytrium fungus seed liquid, the inoculation amount is 15v / v%, the light intensity is 3000lx, the O2 ventilation volume is 4L / min, 24°C, 200r / min, after culturing for 5 days, 25 parts by weight of a treating agent (including 1 part by weight of p-hydroxybenzoic acid and 24 parts by weight of diammonium phosphate) are added, and the culture is continued for 12 hours, filtered, washed, algal cells are collected, freeze-dried, and algal powder is obtained; S2. Enzymatic hydrolysis reaction: 100 parts by weight of the algae powder obtained in step S1 was added to 1000 parts of deionized water, 2 parts by weight of lysozyme was added thereto, and the mixture was reacted at 45°C for 2h, and the enzymatic hydrolysis mixture was obtained by filtering, and the pore size of the filter mesh was 200 μm; S3. Post-compounding treatment: The enzymatic hydrolysis mixture obtained in step S2 is mixed with pyrolytic biochar, bentonite and biomass tar in a mass ratio of 6:2:1:1, and then crushed at 40°C for 30 minutes using a pulverizer to make the mesh size of the crushed material 180 mesh. Then, the temperature is increased to 260°C at a heating rate of 10°C / min and dried to constant weight to obtain microalgae-biochar.

[0047] Comparative Example 4

[0048] This comparative example provides a method for extracting microalgae, specifically: S1. Microalgae culture: 20 parts by weight of glucose, 5 parts by weight of magnesium sulfate, 2 parts by weight of potassium dihydrogen phosphate, 1 part by weight of ammonium ferric citrate, 1 part by weight of ascorbic acid, 2 parts by weight of sodium silicate, 0.0005 parts by weight of vitamin B12, 3 parts by weight of sodium bicarbonate, and 0.15 parts by weight of ammonium bicarbonate are added to 1000 parts by weight of deionized water, sterilized under the condition of pH 8.0, inoculated with Schizochytrium fungus seed liquid, the inoculation amount is 15v / v%, the light intensity is 3000lx, the O2 ventilation volume is 4L / min, 24°C, 200r / min, after culturing for 5 days, 25 parts by weight of a treating agent (including 1 part by weight of p-hydroxybenzoic acid and 24 parts by weight of diammonium phosphate) are added, and the culture is continued for 12 hours, filtered, washed, algal cells are collected, freeze-dried, and algal powder is obtained; S2. Enzymatic hydrolysis reaction: 100 parts by weight of the algae powder prepared in step S1 was added to 1000 parts of deionized water, 2 parts by weight of the composite enzyme was added thereto, and the mixture was reacted at 45°C for 2 hours, and the enzymatic hydrolysis mixture was obtained by filtration. The pore size of the filter mesh was 200 μm, and the mass ratio of lipase to lysozyme in the composite enzyme was 7:3; S3. Post-compounding treatment: The enzymatic hydrolysis mixture obtained in step S2 is mixed with pyrolytic biochar, bentonite and biomass tar in a mass ratio of 6:2:1:1, and then crushed at 40°C for 30 minutes using a pulverizer to make the mesh size of the crushed material 180 mesh. Then, the temperature is increased to 260°C at a heating rate of 10°C / min and dried to constant weight to obtain microalgae-biochar.

[0049] Comparative Example 5

[0050] This comparative example provides a method for extracting microalgae, specifically: S1. Microalgae culture: 20 parts by weight of glucose, 5 parts by weight of magnesium sulfate, 2 parts by weight of potassium dihydrogen phosphate, 1 part by weight of ammonium ferric citrate, 1 part by weight of ascorbic acid, 2 parts by weight of sodium silicate, 0.0005 parts by weight of vitamin B12, 3 parts by weight of sodium bicarbonate, and 0.15 parts by weight of ammonium bicarbonate are added to 1000 parts by weight of deionized water, sterilized under the condition of pH 8.0, inoculated with Schizochytrium fungus seed liquid, the inoculation amount is 15v / v%, the light intensity is 3000lx, the O2 ventilation volume is 4L / min, 24°C, 200r / min, after culturing for 5 days, 25 parts by weight of a treating agent (including 1 part by weight of p-hydroxybenzoic acid and 24 parts by weight of diammonium phosphate) are added, and the culture is continued for 12 hours, filtered, washed, algal cells are collected, freeze-dried, and algal powder is obtained; S2. Enzymatic hydrolysis reaction: 100 parts by weight of the algae powder prepared in step S1 was added to 1000 parts of deionized water, 2 parts by weight of the composite enzyme was added thereto, and the mixture was reacted at 45°C for 2 hours, and the enzymatic hydrolysis mixture was obtained by filtration. The pore size of the filter mesh was 200 μm, and the mass ratio of lipase to lysozyme in the composite enzyme was 3:7; S3. Post-compounding treatment: The enzymatic hydrolysis mixture obtained in step S2 is mixed with pyrolyzed biochar and bentonite in a mass ratio of 6:2:2, and then crushed at 40°C for 30 minutes using a crusher to make the mesh size of the crushed material 180 mesh. Then, the temperature is increased to 260°C at a heating rate of 10°C / min and dried to constant weight to obtain microalgae-biochar.

[0051] Comparative Example 6

[0052] This comparative example provides a method for extracting microalgae, specifically: S1. Microalgae culture: 20 parts by weight of glucose, 5 parts by weight of magnesium sulfate, 2 parts by weight of potassium dihydrogen phosphate, 1 part by weight of ammonium ferric citrate, 1 part by weight of ascorbic acid, 2 parts by weight of sodium silicate, 0.0005 parts by weight of vitamin B12, 3 parts by weight of sodium bicarbonate, and 0.15 parts by weight of ammonium bicarbonate are added to 1000 parts by weight of deionized water, sterilized under the condition of pH 8.0, inoculated with Schizochytrium fungus seed liquid, the inoculation amount is 15v / v%, the light intensity is 3000lx, the O2 ventilation volume is 4L / min, 24°C, 200r / min, after culturing for 5 days, 25 parts by weight of a treating agent (including 8 parts by weight of p-hydroxybenzoic acid and 17 parts by weight of diammonium phosphate) are added, and the culture is continued for 12 hours, filtered, washed, algal cells are collected, freeze-dried, and algal powder is obtained; S2. Enzymatic hydrolysis reaction: 100 parts by weight of the algae powder prepared in step S1 was added to 1000 parts of deionized water, 2 parts by weight of the composite enzyme was added thereto, and the mixture was reacted at 45°C for 2 hours, and the enzymatic hydrolysis mixture was obtained by filtration. The pore size of the filter mesh was 200 μm, and the mass ratio of lipase to lysozyme in the composite enzyme was 3:7; S3. Post-compounding treatment: The enzymatic hydrolysis mixture obtained in step S2 is mixed with pyrolysis biochar and biomass tar in a mass ratio of 6:2:2, and then crushed in a pulverizer at 40°C for 30 minutes to make the mesh size of the crushed material 180 mesh, and then heated to 260°C at a heating rate of 10°C / min and dried to constant weight to obtain microalgae-biochar.

[0053] Comparative Example 7

[0054] This comparative example provides a method for extracting microalgae, specifically: S1. Microalgae culture: 20 parts by weight of glucose, 5 parts by weight of magnesium sulfate, 2 parts by weight of potassium dihydrogen phosphate, 1 part by weight of ammonium ferric citrate, 1 part by weight of ascorbic acid, 2 parts by weight of sodium silicate, 0.0005 parts by weight of vitamin B12, 3 parts by weight of sodium bicarbonate, and 0.15 parts by weight of ammonium bicarbonate are added to 1000 parts by weight of deionized water, sterilized under the condition of pH 8.0, inoculated with Schizochytrium fungus seed liquid, the inoculation amount is 15v / v%, the light intensity is 3000lx, the O2 ventilation volume is 4L / min, 24°C, 200r / min, after culturing for 5 days, 25 parts by weight of a treating agent (including 10 parts by weight of p-hydroxybenzoic acid and 15 parts by weight of diammonium phosphate) are added, and the culture is continued for 12 hours, filtered, washed, algal cells are collected, freeze-dried, and algal powder is obtained; S2. Enzymatic hydrolysis reaction: 100 parts by weight of the algae powder prepared in step S1 was added to 1000 parts of deionized water, 2 parts by weight of the composite enzyme was added thereto, and the mixture was reacted at 45°C for 2 hours, and the enzymatic hydrolysis mixture was obtained by filtration. The pore size of the filter mesh was 200 μm, and the mass ratio of lipase to lysozyme in the composite enzyme was 3:7; S3. Post-compounding treatment: The enzymatic hydrolysis mixture obtained in step S2 is mixed with pyrolytic biochar, bentonite and biomass tar in a mass ratio of 6:2:1:1, and then crushed at 40°C for 30 minutes using a pulverizer to make the mesh size of the crushed material 180 mesh. Then, the temperature is increased to 260°C at a heating rate of 10°C / min and dried to constant weight to obtain microalgae-biochar.

[0055] Comparative Example 8

[0056] This comparative example provides a method for extracting microalgae, specifically: S1. Microalgae culture: 20 parts by weight of glucose, 5 parts by weight of magnesium sulfate, 2 parts by weight of potassium dihydrogen phosphate, 1 part by weight of ammonium ferric citrate, 1 part by weight of ascorbic acid, 2 parts by weight of sodium silicate, 0.0005 parts by weight of vitamin B12, 3 parts by weight of sodium bicarbonate, and 0.15 parts by weight of ammonium bicarbonate are added to 1000 parts by weight of deionized water, sterilized under the condition of pH 8.0, inoculated with Schizochytrium fungus seed liquid, the inoculation amount is 15v / v%, the light intensity is 3000lx, the O2 ventilation volume is 4L / min, 24°C, 200r / min, after culturing for 5 days, 25 parts by weight of a treating agent (including 15 parts by weight of p-hydroxybenzoic acid and 10 parts by weight of diammonium phosphate) are added, and the culture is continued for 12 hours, filtered, washed, algal cells are collected, freeze-dried, and algal powder is obtained; S2. Enzymatic hydrolysis reaction: 100 parts by weight of the algae powder prepared in step S1 was added to 1000 parts of deionized water, 2 parts by weight of the composite enzyme was added thereto, and the mixture was reacted at 45°C for 2 hours, and the enzymatic hydrolysis mixture was obtained by filtration. The pore size of the filter mesh was 200 μm, and the mass ratio of lipase to lysozyme in the composite enzyme was 3:7; S3. Post-compounding treatment: The enzymatic hydrolysis mixture obtained in step S2 is mixed with pyrolytic biochar, bentonite and biomass tar in a mass ratio of 6:2:1:1, and then crushed at 40°C for 30 minutes using a pulverizer to make the mesh size of the crushed material 180 mesh. Then, the temperature is increased to 260°C at a heating rate of 10°C / min and dried to constant weight to obtain microalgae-biochar.

[0057] Comparative Example 9

[0058] This comparative example provides a method for extracting microalgae, specifically: S1. Microalgae culture: 20 parts by weight of glucose, 5 parts by weight of magnesium sulfate, 2 parts by weight of potassium dihydrogen phosphate, 1 part by weight of ammonium ferric citrate, 1 part by weight of ascorbic acid, 2 parts by weight of sodium silicate, 0.0005 parts by weight of vitamin B12, 3 parts by weight of sodium bicarbonate, and 0.15 parts by weight of ammonium bicarbonate are added to 1000 parts by weight of deionized water, sterilized under the condition of pH value of 8.0, inoculated with Schizochytrium fungus seed liquid, the inoculation amount is 15v / v%, the light intensity is 3000lx, the O2 ventilation volume is 4L / min, 24°C, 200r / min, cultured for 5 days, filtered, washed, algal cells are collected, freeze-dried, and algal powder is obtained; S2. Enzymatic hydrolysis reaction: 100 parts by weight of the algae powder prepared in step S1 was added to 1000 parts of deionized water, 2 parts by weight of the composite enzyme was added thereto, and the mixture was reacted at 45°C for 2 hours, and the enzymatic hydrolysis mixture was obtained by filtration. The pore size of the filter mesh was 200 μm, and the mass ratio of lipase to lysozyme in the composite enzyme was 3:7; S3. Post-compounding treatment: The enzymatic hydrolysis mixture obtained in step S2 is mixed with pyrolytic biochar, bentonite and biomass tar in a mass ratio of 6:2:1:1, and then crushed at 40°C for 30 minutes using a pulverizer to make the mesh size of the crushed material 180 mesh. Then, the temperature is increased to 260°C at a heating rate of 10°C / min and dried to constant weight to obtain microalgae-biochar.

[0059] Comparative Example 10

[0060] This comparative example provides a method for extracting microalgae, specifically: S1. Microalgae culture: 20 parts by weight of glucose, 5 parts by weight of magnesium sulfate, 2 parts by weight of potassium dihydrogen phosphate, 1 part by weight of ammonium ferric citrate, 1 part by weight of ascorbic acid, 2 parts by weight of sodium silicate, 0.0005 parts by weight of vitamin B12, 3 parts by weight of sodium bicarbonate, and 0.15 parts by weight of ammonium bicarbonate are added to 1000 parts by weight of deionized water, sterilized under the condition of pH 8.0, inoculated with Schizochytrium fungus seed liquid, the inoculation amount is 15v / v%, the light intensity is 3000lx, the O2 ventilation volume is 4L / min, 24°C, 200r / min, after culturing for 5 days, 25 parts by weight of a treating agent (including 1 part by weight of p-hydroxybenzoic acid and 24 parts by weight of diammonium phosphate) are added, and the culture is continued for 12 hours, filtered, washed, algal cells are collected, freeze-dried, and algal powder is obtained; S2. Enzymatic hydrolysis reaction: add 100 parts by weight of the algae powder obtained in step S1 to 1000 parts of deionized water, add 2 parts by weight of complex enzyme thereto, react at 45°C for 2 hours, filter to obtain an enzymatic hydrolysis mixture, the pore size of the filter mesh is 200 μm, and the mass ratio of lipase to lysozyme in the complex enzyme is 3:7.

[0061] Comparative Example 11

[0062] This comparative example provides a method for extracting microalgae, specifically: S1. Microalgae culture: 20 parts by weight of glucose, 5 parts by weight of magnesium sulfate, 2 parts by weight of potassium dihydrogen phosphate, 1 part by weight of ammonium ferric citrate, 1 part by weight of ascorbic acid, 2 parts by weight of sodium silicate, 0.0005 parts by weight of vitamin B12, 3 parts by weight of sodium bicarbonate, and 0.15 parts by weight of ammonium bicarbonate are added to 1000 parts by weight of deionized water, sterilized under the condition of pH 8.0, inoculated with Schizochytrium fungus seed liquid, the inoculation amount is 15v / v%, the light intensity is 3000lx, the O2 ventilation volume is 4L / min, 24°C, 200r / min, after culturing for 5 days, 25 parts by weight of a treating agent (including 1 part by weight of p-hydroxybenzoic acid and 24 parts by weight of diammonium phosphate) are added, and the culture is continued for 12 hours, filtered, washed, algal cells are collected, freeze-dried, and algal powder is obtained; S2. Enzymatic hydrolysis reaction: 100 parts by weight of the algae powder prepared in step S1 was added to 1000 parts of deionized water, 2 parts by weight of the composite enzyme was added thereto, and the mixture was reacted at 45°C for 2 hours, and the enzymatic hydrolysis mixture was obtained by filtration. The pore size of the filter mesh was 200 μm, and the mass ratio of lipase to lysozyme in the composite enzyme was 3:7; S3. Post-compounding treatment: The enzymatic hydrolysis mixture obtained in step S2 was mixed with the pyrolysis biochar in a mass ratio of 6:4, and then crushed at 40°C for 30 minutes using a crusher to make the mesh size of the crushed material 180 mesh. The mixture was then heated to 260°C at a heating rate of 10°C / min and dried to constant weight to obtain microalgae-biochar.

[0063] Example 6

[0064] This embodiment provides an application of a microalgae extract in preparing a microalgae fertilizer, specifically: The prepared microalgae-biochar extracted from Examples 1 to 5 and Comparative Examples 1 to 11 is used for later use; Seventeen plots of one mu (666.7 m2) were demarcated in the apple orchard. 2 ) were used for comparative experiments in the selected areas, where the growth of apple trees was comparable and representative of the entire apple orchard.

[0065] The experiment set up two treatment methods: 1) Only traditional fertilizer was applied as the control group; 2) While applying traditional fertilizers, the same mass of microalgae-biochar was applied three times.

[0066] Fertilization method: Use root spraying method and apply fertilizer at the same time.

[0067] Fertilization cycle: The test object is Yantai Red Fuji apple. This variety of apple usually starts to sprout in April, blooms in May, and the fruiting period is generally from July to August. According to the growth cycle of apples, fertilization was carried out on April 7, June 7, and August 7 in this test. On August 28, 2024, three apple trees with similar growth were selected in different areas, and 5 apples were randomly picked from each direction on each apple tree. The various indicators of the obtained apples were tested and compared, and the proportion of active ingredients in the obtained microalgae-biochar was determined. The results are shown in Table 1.

[0068] Yield per mu (jin) Single fruit weight (g) Active ingredient percentage (%) Example 1 8786 218.7 89.32 Example 2 8706 217.1 88.73 Example 3 8652 208.6 86.38 Example 4 8573 199.4 84.55 Example 5 8566 197.8 81.38 Comparative Example 1 8572 198.6 75.31 Comparative Example 2 8678 205.1 74.63 Comparative Example 3 8693 206.8 71.57 Comparative Example 4 8510 196.5 63.33 Comparative Example 5 8482 192.2 65.59 Comparative Example 6 8489 194.8 61.34 Comparative Example 7 8307 193.5 67.65 Comparative Example 8 8234 192.1 63.24 Comparative Example 9 8281 192.9 62.16 Comparative Example 10 8229 189.6 - Comparative Example 11 8250 191.9 63.06 Control group 8002 187.3 - According to Table 1, it can be seen that, with the control group of simply applying conventional fertilizers, applying microalgae-biochar to apples at the same time can effectively improve the yield and quality of apples: the yield is increased by 9.8%; the weight of a single fruit is increased by 11%, and the microalgae extraction method provided by the embodiment of the present invention can make the proportion of effective active ingredients in microalgae-biochar reach up to 96.3%. This is because the cell membrane permeability of Schizochytrium is changed by treating the culture medium of Schizochytrium with p-hydroxybenzoic acid and diammonium phosphate, which facilitates the entry of other nutrients and helps the cell wall of Schizochytrium. However, when the content of p-hydroxybenzoic acid is higher than that of diammonium phosphate, its autotoxicity will inhibit the growth of Schizochytrium, thereby reducing the effective active ingredients.

[0069] When using different ratios or types of complex enzymes, it will also have a negative impact on the extraction rate of microalgae. This is because the synergistic effect of lipase and lysozyme can well adsorb and break the cell wall. When a single enzyme is used or cellulase is used to replace lipase, the effective active ingredients in the microalgae-biochar also decrease. This is because the synergistic effect is lost, and cellulase mainly acts on cellulose and cannot replace lipase and lysozyme to work synergistically to extract oils from cells.

[0070] At the same time, different admixtures will also affect the effective active ingredients in microalgae-biochar. This is because under the appropriate ratio of enzymatic hydrolysis mixture, pyrolysis biochar, bentonite and biomass tar, the prepared microalgae-biochar has a rich pore structure, a large amount of microalgae load, and a strong combination of structural properties, which can effectively maintain the activity of microalgae-biochar. Adjusting the ratio will change the structure of microalgae-biochar and reduce the microalgae load, thereby reducing the active substances in the microalgae-biochar and reducing its growth-promoting effect on plants.

[0071] Therefore, taking into account various factors such as economic cost and extraction efficiency, the microalgae extraction method provided by the embodiment of the present invention has broad market application prospects, and when it is applied to microalgae fertilizer, it also has a significant positive effect on the growth of crops, and the microalgae itself will not have a negative impact on the ecological environment. When the extract is applied to biological fertilizer, there is no need to worry about the negative impact on the ecological environment or soil. Therefore, the microalgae extract extracted by the present invention can also be used to develop new biological pesticides, bioplastics, etc., further broadening its market application scope.

[0072] It should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention rather than to limit it. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solutions of the present invention may be modified or replaced by equivalents without departing from the spirit and scope of the technical solutions of the present invention, which should all be included in the scope of the claims of the present invention.

Claims

1. A method for extracting microalgae, characterized in that: The following steps are involved: S1. Microalgae culture: Glucose, magnesium sulfate, potassium dihydrogen phosphate, ammonium ferric citrate, ascorbic acid, sodium silicate, vitamin B12, sodium bicarbonate, and ammonium bicarbonate are added to deionized water, sterilized at a pH of 8.0, inoculated with Schizochytrium spore seed liquid, cultured under light conditions, treated with a treatment agent, filtered, washed, and algae cells were collected, freeze-dried, and algae powder was obtained; S2. Enzymatic hydrolysis reaction: adding the algae powder obtained in step S1 to deionized water, adding a composite enzyme to the water for reaction, and coarse-filtration after the reaction to obtain an enzymatic hydrolysis mixture; S3. Post-compounding treatment: The enzymatic hydrolysis mixture obtained in step S2 is added to pyrolysis biochar, bentonite and biomass tar, mixed, crushed and then dried to obtain microalgae-biochar.

2. The method for extracting microalgae according to claim 1, characterized in that: The mass ratio of glucose, magnesium sulfate, potassium dihydrogen phosphate, ammonium ferric citrate, ascorbic acid, sodium silicate, vitamin B12, sodium bicarbonate, ammonium bicarbonate and deionized water in step S1 is 10-25:1-5:1-2:0.1-2:0.1-2:1-3:0.0001-0.001:2-5:0.1-2:1000; the inoculation amount of the schizochytrium fungus seed liquid is 12-15 v / v%, the intensity of the light is 2500-3000 lx, the culture conditions are O2 ventilation of 2-4 L / min, 22-24 ° C, 100-200 r / min, and culture for 3-5 days, and the treatment agent treatment is treated under the culture conditions for 10-24 hours.

3. The method for extracting microalgae according to claim 1, characterized in that: The treatment agent in step S1 includes p-hydroxybenzoic acid and diammonium phosphate, wherein the mass ratio of p-hydroxybenzoic acid to diammonium phosphate is 1-5:20-30.

4. The method for extracting microalgae according to claim 1, characterized in that: In step S1, the mass ratio of the treatment agent to deionized water is 20-30:1000.

5. The method for extracting microalgae according to claim 1, characterized in that: The complex enzyme in step S2 includes lipase and lysozyme, wherein the mass ratio of lipase to lysozyme is 2-7:5-10.

6. The method for extracting microalgae according to claim 1, characterized in that: The enzymatic hydrolysis reaction in step S2 is carried out at 40-60° C. for 1-5 hours.

7. The method for extracting microalgae according to claim 1, characterized in that: The mass ratio of the enzymatic hydrolysis mixture, pyrolysis biochar, bentonite and biomass tar in step S3 is 6:2:1:

1.

8. The method for extracting microalgae according to claim 1, characterized in that: The pulverization treatment in step S3 is to use a pulverizer to pulverize at 20-45° C. for 20-40 minutes, so that the mesh size of the pulverized material is 120-200 meshes.

9. The method for extracting microalgae according to claim 1, characterized in that: The drying in step S3 is to increase the temperature to 260° C. at a heating rate of 8-12° C. / min and dry to constant weight.

10. Use of microalgae-biochar obtained by the microalgae extraction method according to any one of claims 1 to 9 in preparing microalgae fertilizer.