CN112795610A - Microalgae efficient co-production method based on compound protease enzymolysis technology and product - Google Patents
Microalgae efficient co-production method based on compound protease enzymolysis technology and product Download PDFInfo
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- CN112795610A CN112795610A CN202110108005.6A CN202110108005A CN112795610A CN 112795610 A CN112795610 A CN 112795610A CN 202110108005 A CN202110108005 A CN 202110108005A CN 112795610 A CN112795610 A CN 112795610A
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- RKJUIXBNRJVNHR-UHFFFAOYSA-N indolenine Natural products C1=CC=C2CC=NC2=C1 RKJUIXBNRJVNHR-UHFFFAOYSA-N 0.000 description 1
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Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P21/00—Preparation of peptides or proteins
- C12P21/06—Preparation of peptides or proteins produced by the hydrolysis of a peptide bond, e.g. hydrolysate products
-
- C—CHEMISTRY; METALLURGY
- C10—PETROLEUM, GAS OR COKE INDUSTRIES; TECHNICAL GASES CONTAINING CARBON MONOXIDE; FUELS; LUBRICANTS; PEAT
- C10G—CRACKING HYDROCARBON OILS; PRODUCTION OF LIQUID HYDROCARBON MIXTURES, e.g. BY DESTRUCTIVE HYDROGENATION, OLIGOMERISATION, POLYMERISATION; RECOVERY OF HYDROCARBON OILS FROM OIL-SHALE, OIL-SAND, OR GASES; REFINING MIXTURES MAINLY CONSISTING OF HYDROCARBONS; REFORMING OF NAPHTHA; MINERAL WAXES
- C10G1/00—Production of liquid hydrocarbon mixtures from oil-shale, oil-sand, or non-melting solid carbonaceous or similar materials, e.g. wood, coal
-
- C—CHEMISTRY; METALLURGY
- C10—PETROLEUM, GAS OR COKE INDUSTRIES; TECHNICAL GASES CONTAINING CARBON MONOXIDE; FUELS; LUBRICANTS; PEAT
- C10G—CRACKING HYDROCARBON OILS; PRODUCTION OF LIQUID HYDROCARBON MIXTURES, e.g. BY DESTRUCTIVE HYDROGENATION, OLIGOMERISATION, POLYMERISATION; RECOVERY OF HYDROCARBON OILS FROM OIL-SHALE, OIL-SAND, OR GASES; REFINING MIXTURES MAINLY CONSISTING OF HYDROCARBONS; REFORMING OF NAPHTHA; MINERAL WAXES
- C10G3/00—Production of liquid hydrocarbon mixtures from oxygen-containing organic materials, e.g. fatty oils, fatty acids
- C10G3/42—Catalytic treatment
-
- C—CHEMISTRY; METALLURGY
- C10—PETROLEUM, GAS OR COKE INDUSTRIES; TECHNICAL GASES CONTAINING CARBON MONOXIDE; FUELS; LUBRICANTS; PEAT
- C10G—CRACKING HYDROCARBON OILS; PRODUCTION OF LIQUID HYDROCARBON MIXTURES, e.g. BY DESTRUCTIVE HYDROGENATION, OLIGOMERISATION, POLYMERISATION; RECOVERY OF HYDROCARBON OILS FROM OIL-SHALE, OIL-SAND, OR GASES; REFINING MIXTURES MAINLY CONSISTING OF HYDROCARBONS; REFORMING OF NAPHTHA; MINERAL WAXES
- C10G2300/00—Aspects relating to hydrocarbon processing covered by groups C10G1/00 - C10G99/00
- C10G2300/10—Feedstock materials
- C10G2300/1011—Biomass
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P30/00—Technologies relating to oil refining and petrochemical industry
- Y02P30/20—Technologies relating to oil refining and petrochemical industry using bio-feedstock
Abstract
The invention provides a microalgae high-efficiency co-production method based on a composite protease enzymolysis technology and a product, and the method comprises the following steps: mixing alkaline protease with papain or trypsin to prepare a compound protease solution, and processing microalgae into microalgae powder; adding the compound protease solution into an algae solution containing microalgae powder, uniformly mixing, and pretreating microalgae by using a compound protease enzymolysis technology; after enzymolysis is finished, separating the pretreated microalgae from the microalgae liquid by using high-speed centrifugation, drying a solid-phase product to be used as a raw material of pyrolysis reaction, and collecting supernatant containing polypeptide; mixing the dried solid-phase product microalgae with waste vegetable oil, carrying out co-catalytic pyrolysis, collecting condensed gas generated in the pyrolysis and condensing to obtain a liquid-phase product microalgae bio-oil with low nitrogen content and excellent fuel property, wherein the liquid-phase product supernatant rich in small molecular polypeptides can be used as a raw material in the pharmaceutical industry and the food production industry, so that the high-efficiency microalgae co-production based on the compound protease enzymolysis technology is realized.
Description
Technical Field
The invention belongs to the field of biomass resource utilization, and particularly relates to a microalgae efficient co-production method based on a compound protease enzymolysis technology and a product.
Background
The microalgae used as one of the algae plants does not occupy the land for agriculture and forestry and can efficiently fix nitrogen due to the high lipid content and short life cycle, thereby becoming a high-quality raw material for preparing the substitute biodiesel. However, microalgae contain large amounts of protein, up to 14.00-65.20%, resulting in higher N content in the microalgae thermochemical conversion products. N element in the microalgae thermochemical conversion product exists in the form of heterocyclic compounds or non-heterocyclic amine compounds such as pyridine, indole, pyrrole, pyrimidine, amine, amide and the like, and Nitrogen Oxides (NO) can be generated when the biological oil is burnt as alternative fuel when the content of nitrogen-containing compounds in the biological oil is highx) In particular, the emission of fuel-type nitrogen oxides is increased, which causes great environmental pollution. At present, two methods of catalyst denitrification and low-temperature hydrothermal denitrification are mainly adopted to reduce the nitrogen content of the microalgae bio-oil product. The catalyst denitrification refers to that a catalyst is used in the thermochemical conversion process to promote the nitrogen-containing functional groups to be broken so that N escapes in the form of ammonia gas and hydrogen cyanide or nitrogen-containing compounds and other chemical components are deposited to a solid-phase product after being polymerized again, so as to reduce the nitrogen content in the bio-oil, currently, a noble metal catalyst (a catalyst loaded with noble metals with stronger catalytic activity such as platinum, palladium, molybdenum, rhodium, cerium and the like) is generally used or a novel catalyst is developed for the denitrification of the microalgae bio-oil in the thermochemical conversion process, the denitrification effect is excellent (more than 50% of N in the bio-oil can be removed when part of the noble metal catalyst is applied), but the application of noble metal catalysts or the development of special new material catalysts to denitrify the bio-oil has higher cost, and the nitrogen-containing compound is easy to cause catalyst poisoning in the upgrading process, and the recycling performance of the catalyst is influenced. Unlike catalyst denitrification, low temperature hydrothermal denitrification refers to low temperature (150-And separating the amido in the water phase from the solid algae residue in a centrifugal product mode to obtain the solid algae residue with lower nitrogen content, and taking the solid algae residue as a high-quality raw material for preparing oil by thermochemical conversion, thereby reducing the nitrogen content of the microalgae before the thermochemical conversion. Compared with a catalyst denitrification method, the low-temperature hydrothermal denitrification method has lower cost and simple process, but lipid and carbohydrate molecules in the microalgae are hydrolyzed while denitrification is carried out, so that the yield of low-nitrogen solid algal residue obtained by low-temperature hydrothermal denitrification is lower, and the feasibility of large-scale industrial application is not realized.
Disclosure of Invention
Aiming at the technical problems, the invention provides a high-efficiency microalgae co-production method and a product thereof in a composite protease enzymolysis technology, aiming at preparing a composite protease by taking alkaline protease as a main enzyme and trypsin or papain as a coenzyme according to a certain enzyme activity ratio through the composite protease enzymolysis technology for carrying out enzymolysis pretreatment on microalgae using the microalgae, hydrolyzing protein molecules in microalgae cells and separating polypeptide rich in micromolecules through a high-speed centrifugation technology based on the principle that the protease can hydrolyze the protein macromolecules to obtain polypeptide products with small molecules by destroying peptide bonds in the protein macromolecules, thereby obtaining solid microalgae residues with lower nitrogen content. And then, by means of a biomass co-catalytic pyrolysis technology, a CaO catalyst is used, waste vegetable oil is used as a co-pyrolysis substance, low-nitrogen microalgae residue is used as a raw material, bio-oil with low nitrogen content and excellent fuel property is obtained, and the enzymolysis supernatant rich in small molecular polypeptides can be used as a raw material in the pharmaceutical industry and the food production industry according to amino acid components of the enzymolysis supernatant, so that the high-efficiency co-production of microalgae based on the composite protease enzymolysis technology is realized.
In order to reduce the denitrification cost of microalgae and ensure higher denitrification efficiency and low-nitrogen solid algae residue yield, the invention uses protease to carry out enzymolysis on the microalgae so as to reduce the nitrogen content of the microalgae, the invention selects alkaline protease, trypsin and papain as protease for carrying out enzymolysis on the microalgae so as to obtain a low-nitrogen microalgae residue raw material so as to further improve the protein recovery rate and the polypeptide yield, uses the alkaline protease as main enzyme, uses the trypsin or the papain as coenzyme, and prepares the compound protease according to the enzyme activity ratio of the main enzyme to the coenzyme of 2:1 or 3:1, and the compound protease enzymolysis process for the microalgae raw material is used for obtaining the low-nitrogen microalgae raw material for thermochemical conversion.
The product obtained by the enzymolysis of the composite protein of the microalgae powder is low-nitrogen solid algae residue and supernatant liquid which is rich in micromolecular polypeptide and partial unhydrolyzed microalgae protein, the low-nitrogen solid algae residue is used as a high-quality biomass raw material to be subjected to co-catalytic pyrolysis to obtain high-quality bio-oil which can be used for producing high-quality bio-alternative fuel, the polypeptide contained in the supernatant liquid prepared by the enzymolysis of the microalgae by protease has very high nutritive value, and the supernatant liquid which is rich in the polypeptide and is obtained by the enzymolysis of the protease can be used as a high-quality production raw material of health-care food and medicines.
In order to promote the selectivity of paraffin substances in the pyrolysis of low-nitrogen microalgae, make finished bio-oil products closer to high-quality commercial fuels in terms of physicochemical properties and increase the feasibility of the bio-oil products as alternative fuels, the invention uses a CaO catalyst as a catalyst used in the catalytic pyrolysis of the low-nitrogen microalgae, and uses waste vegetable oil as a co-pyrolysis substance to finish the catalytic co-pyrolysis quality improvement of solid products after the enzymatic hydrolysis of the low-nitrogen microalgae, thereby further promoting the decarboxylation reaction of various fatty acid molecules rich in the waste vegetable oil, remarkably improving the contents of paraffin and alcohol substances in the bio-oil products and increasing the content of low-boiling aromatic compounds.
The invention relates to a microalgae high-efficiency co-production method based on a compound protease enzymolysis technology, which comprises the following steps: three typical proteases, namely alkaline protease, papain and trypsin, are selected, and composite protease is compounded according to the characteristics of the three proteases and the enzymolysis strength of the microalgae plant protein according to a certain proportion and is used for enzymolysis and denitrification of the protein in the microalgae. The low-nitrogen solid phase product obtained after the enzymolysis of the microalgae can be converted into a bio-oil product with lower nitrogen content and superior fuel property by a catalytic pyrolysis technology, and the microalgae polypeptide enriched in the supernatant can be used for preparing functional or health-care food according to the actual composition of the amino acid of the microalgae polypeptide. The invention applies the composite protease compounded by alkaline protease, papain and trypsin according to a certain proportion to the denitrification pretreatment of the microalgae, reduces the energy consumption of the microalgae in the denitrification aspect while efficiently removing the nitrogen content in the microalgae, and prepares the bio-oil with lower nitrogen content and excellent fuel property by carrying out co-catalytic pyrolysis on the low-nitrogen solid phase product obtained after the enzyme pretreatment and the waste vegetable oil. In addition, the polypeptide in the supernatant after proteolysis can be used as a raw material for preparing functional or health-care food according to the amino acid components of the polypeptide. The whole process flow has the advantages of few steps, high cyclic utilization rate, low energy consumption, high cleaning degree, higher cost benefit and environmental protection benefit.
The invention is realized by the following technical scheme: a microalgae high-efficiency co-production method based on a compound protease enzymolysis technology comprises the following steps:
step S1, mixing alkaline protease with papain or trypsin to prepare a compound protease solution, and processing microalgae into microalgae powder;
step S2, adding the compound protease liquid into the algae liquid containing the microalgae powder, uniformly mixing, and pretreating the microalgae by using a compound protease enzymolysis technology;
step S3, after the enzymolysis is finished, separating the pretreated microalgae from the algae liquid by using high-speed centrifugation, drying the solid-phase product to be used as a raw material of the pyrolysis reaction, and collecting the supernatant containing the polypeptide;
and step S4, mixing the dried solid-phase product microalgae with the waste vegetable oil, carrying out co-catalytic pyrolysis, collecting condensed gas generated in the pyrolysis and condensing to obtain a liquid-phase product microalgae bio-oil.
In the above embodiment, in step S1, the microalgae species is chlorella, scenedesmus obliquus, or spirulina.
In the scheme, the compound protease is prepared by taking alkaline protease as a main enzyme, taking papain or trypsin as a coenzyme and preparing the main enzyme and the coenzyme according to the enzyme activity ratio of 2:1 or 3: 1.
In the above scheme, in the step S1, the microalgae is processed into microalgae powder with a fineness of 100 meshes.
In the above scheme, the step S2 of performing enzymolysis pretreatment on the microalgae by using the compound protease enzymolysis technology specifically includes:
the enzymolysis time is 5-7h, the enzymolysis pH value is 7.0-9.0, the enzymolysis temperature is 45-55 ℃, the mass fraction of the microalgae in the algae liquid is 4-6%, the usage amount of the compound protease is 3000U/g substrate, and the enzymolysis substrate is microalgae powder.
In the scheme, after the enzymolysis in the step S3 is finished, the pH value of the enzymolysis liquid is adjusted to 4.0, the enzymolysis liquid is placed in a boiling water bath for 10min to inactivate enzyme, a high-speed centrifuge is used for separating a solid-phase product and a liquid-phase product, the centrifugal speed is 5500r/min, and the centrifugal time is 15 min.
In the above embodiment, the collected supernatant containing the polypeptide is subjected to amino acid analysis in step S3, and the amino acid component in the supernatant is determined and then stored at-20 ℃ in the dark.
In the scheme, the mass mixing ratio of the solid-phase product and the waste vegetable oil in the step S4 is 1:3-1: 5; the catalyst is a CaO catalyst, and the addition amount of the catalyst is 25% of the total mass of the solid-phase product and the waste vegetable oil.
In the above scheme, the reaction conditions of the catalytic pyrolysis in the step S4 are as follows: the pyrolysis temperature is set to be 500-600 ℃, the heating rate is 10-100 ℃/s, and the gas phase residence time is 0.5-10 s; and (3) putting the mixed solid-phase product, the waste vegetable oil and the CaO catalyst into a pyrolysis reactor, starting to heat, blowing nitrogen at the rate of 0.8L/min during the heating, and changing the nitrogen rate to 0.2L/min after the set temperature is reached.
Microalgae bio-oil and supernatant containing polypeptide prepared by the method for efficiently coproducing microalgae based on the compound protease enzymolysis technology.
Compared with the prior art, the invention has the beneficial effects that: the invention uses the compound protease which is prepared by taking alkaline protease as a main enzyme, taking trypsin or papain as a coenzyme, and using the main enzyme and the coenzyme according to the enzyme activity ratio of 2:1 or 3:1, and carries out enzymolysis pretreatment on microalgae represented by microalgae based on a compound protease enzymolysis technology according to the principle that the protease can decompose macromolecular protein to form micromolecular polypeptide by breaking peptide bonds, and then separates the micromolecular polypeptide obtained after the protein enzymolysis by virtue of a high-speed centrifugal technology, thereby reducing the nitrogen content in the microalgae, and converts the low-nitrogen microalgae into bio-oil with excellent fuel properties by catalytic pyrolysis. After the protease enzymolysis process of the microalgae is finished, the obtained supernatant rich in the micromolecule polypeptides is subjected to amino acid composition analysis according to an amino acid composition detection method in food, and the microalgae polypeptides are used as production raw materials in the pharmaceutical industry and the food manufacturing industry according to the types and the component contents of the microalgae polypeptides, so that high-value utilization of proteins in the microalgae is realized, and the overall utilization rate of the components in the microalgae is improved. Carrying out co-catalytic pyrolysis and quality improvement on a solid-phase product obtained after enzymolysis of microalgae protease by using a CaO catalyst and waste vegetable oil as a co-catalyst. The CaO catalyst can be utilized to improve the generation of paraffin in the bio-oil product by a decarboxylation reaction and other paths, especially the addition of waste vegetable oil with various fatty acids as main components can further promote the generation of paraffin, improve the quality of the bio-oil product in the aspect of fuel, and enable the solid-phase product after the enzymolysis of microalgae to realize further high-efficiency quality improvement of the microalgae. The addition of the waste vegetable oil can effectively improve the yield of the bio-oil product after the co-catalytic pyrolysis, improve the quality of the bio-oil product and simultaneously improve the yield of the high-quality bio-oil product. Finally, the waste vegetable oil with huge yield and higher treatment difficulty and cost is used as the co-pyrolysis product, so that the method has certain environmental protection value and ecological value. In conclusion, the invention applies the composite protease enzymolysis technology to pretreat chlorella, scenedesmus obliquus, spirulina and the like, microalgae residues after enzymolysis are separated by the high-speed centrifugation technology to be used as biomass raw materials and mixed with waste vegetable oil according to the proportion of 1:3-1:5, the mixture is converted into bio-oil with excellent fuel property by the biomass co-catalytic pyrolysis technology under the action of a CaO catalyst, and the microalgae enzymolysis supernatant which is rich in small molecular polypeptide after amino acid component analysis is used as a production raw material in the industries of food industry, pharmaceutical industry and the like, thereby realizing the high-efficiency poly-generation of microalgae. The system has the advantages of less overall steps, high recycling rate, low pollution, less overall energy consumption, high overall utilization rate of raw materials, and higher cost benefit and environmental protection value.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is further described in detail with reference to the following embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.
The invention relates to a microalgae high-efficiency co-production method based on a composite protease enzymolysis technology, which comprises the following steps:
step S1, preparing a compound protease solution from alkaline protease and papain or trypsin according to a certain enzyme activity ratio, drying microalgae into algae powder, then screening by using a vibrating screen machine to obtain microalgae algae powder with the fineness of 100 meshes, and drying in a forced air drying oven to constant weight for later use;
step S2, adding the compound protease liquid prepared according to a certain enzyme activity ratio into the microalgae liquid containing the microalgae, uniformly mixing, and pretreating the microalgae by using a compound protease enzymolysis technology;
step S3, after the enzymolysis is finished, separating the pretreated microalgae from the algae liquid by using a high-speed centrifuge, and placing the solid-phase product in a blast box for drying for 24 hours for later use as a raw material for biomass pyrolysis; collecting supernatant, analyzing amino acid components, and storing at-20 deg.C in dark place;
and step S4, mixing the dried solid-phase product microalgae with the pre-treated waste vegetable oil according to a certain mass ratio, carrying out co-catalytic pyrolysis by using a pyrolysis reactor, collecting condensed gas generated in the pyrolysis and condensing by using a condenser, wherein the condensed liquid-phase product is the microalgae bio-oil.
Preferably, the microalgae species are Chlorella (Chlorella vulgaris), Scenedesmus obliquus (Scenedesmus obliquus), Spirulina (Spirulina).
Preferably, the compound protease is a compound protease prepared by using alkaline protease as a main enzyme, papain or trypsin as a coenzyme and using the main enzyme and the coenzyme according to the enzyme activity ratio of 2:1 or 3: 1.
Preferably, the analysis of the components of the polypeptides in the collected supernatant is carried out by detecting the components according to the detection method given in GB 5009.124-2016: determination of amino acids in food, determining the amino acid components in the supernatant, storing the supernatant at-20 ℃ in the absence of light, and further separating the polypeptide components from the amino acid components for use as raw materials for producing food, medicine, cosmetics, and the like.
Preferably, the microalgae is subjected to enzymolysis pretreatment, the enzymolysis process comprises the steps of carrying out enzymolysis for 5-7 hours, carrying out enzymolysis at the pH value of 7.0-9.0 and the enzymolysis temperature of 45-55 ℃, wherein the mass fraction of the microalgae in the microalgae liquid is 4-6%, the using amount of the compound protease is 3000U/g substrate, and the enzymolysis substrate in the compound protein enzymolysis process is microalgae powder. After the enzymolysis is finished, adjusting the pH value of the enzymolysis liquid to 4.0, and putting the enzymolysis liquid in a boiling water bath for 10min to inactivate the enzyme. And separating the solid-phase product from the liquid-phase product by using a high-speed centrifuge, wherein the centrifugal speed is 5500r/min, and the centrifugal time is 15 min.
Preferably, the waste vegetable oil refers to waste vegetable oil which is used for many times in restaurants such as schools, factories and the like and does not meet the requirement of edible vegetable oil, or waste vegetable oil which is stored by vegetable oil manufacturers for food or industry and has exceeded the longest service life. The kind of the waste vegetable oil used includes palm oil, soybean oil, rapeseed oil, etc.
Preferably, the catalytic pyrolysis process is that the pyrolysis temperature is set to be 500-600 ℃, the temperature rise rate is 10-100 ℃/s, the gas phase residence time is 0.5-10s, the mass mixing ratio of the solid phase product to the waste vegetable oil is 1:3-1:5, 5g of the solid phase product is weighed for pyrolysis reaction in each pyrolysis, the catalyst is a CaO catalyst prepared based on a hydration calcination method, the addition amount of the catalyst is 25% of the total mass of the solid phase product and the waste vegetable oil, and the catalytic mode is in-situ catalysis. And putting the solid-phase product, the waste vegetable oil and the CaO catalyst which are mixed in proportion into a pyrolysis reactor, and starting to heat. During the temperature rise, nitrogen gas was blown in at a rate of 0.8L/min to provide an inert gas atmosphere, and after reaching the set temperature, the nitrogen gas rate was changed to 0.2L/min. Preserving the temperature for 1h, cooling to room temperature and collecting the product.
Example 1
(1) Preparation of compound protease and preparation of chlorella raw material
The alkaline protease and the papain are purchased from Beijing Soilebao science and technology Limited, and the enzyme activity is obtained from a commodity label of the purchased protease, wherein the enzyme activity of the alkaline protease is 200000U/g, and the enzyme activity of the papain is 100000U/g. Preparing the alkaline protease and the papain into the compound protease according to the enzyme activity ratio of 2:1, and drying and storing the compound protease at the temperature of 2-6 ℃ in a dark place. Drying Chlorella to obtain Chlorella powder, sieving with a sieving machine to 100 mesh fineness, and drying in a forced air drying oven at 105 deg.C for 24 hr.
(2) Enzymolysis pretreatment of chlorella
Taking 8g of chlorella powder ground to 100 meshes, putting the chlorella powder into a 500ml beaker, adding 192ml of deionized water, adding 16000U of compound protease according to the addition amount of the protease of 2000U/g of substrate, adjusting the pH value to 7.0, and incubating the mixed solution at the constant temperature of 45 ℃ for 5 hours by using a constant-temperature water bath heating device. Adjusting the pH value of the algae solution to 4.0 after the specified time is reached, and putting the algae solution in a boiling water bath for 10min for inactivation. The mixture was centrifuged for 15min at 5500r/min using a high speed centrifuge and the supernatant was separated from the solid phase product. The supernatant is stored at-20 deg.C in dark place, and a small amount of sample is taken for quantitative analysis of amino acids according to determination method of amino acids in food. The solid phase product was dried in a forced air drying cabinet at 105 ℃ for 24 h. The yield of the low-nitrogen solid algae residue of the solid phase product can reach more than 50 percent, and the nitrogen removal can reach 30 to 35 percent.
(3) Pyrolytic conversion of chlorella
5g of chlorella solid-phase product pretreated by enzyme is uniformly mixed with 15g of waste vegetable oil after treatment, the mass ratio of the chlorella solid-phase product subjected to enzymolysis to the waste vegetable oil is 1:3, then 5g of CaO catalyst is added, the using amount of the catalyst is 25 percent of the sum of the mass of the waste vegetable oil and the chlorella solid-phase product, and the mixture is uniformly mixed and then placed in a pyrolysis reactor. Setting the pyrolysis reaction temperature to be 500 ℃, setting the temperature rise rate to be 100 ℃/s, setting the gas phase residence time to be 5s, keeping the temperature for 1h after the set temperature is reached, and carrying out pyrolysis under the protection of a nitrogen atmosphere, wherein the nitrogen flow rate is set to be 0.8L/min during the temperature rise period, and the nitrogen flow rate is set to be 0.2L/min during the temperature keeping period. After the temperature of the pyrolysis reactor is reduced to room temperature, firstly collecting condensed liquid as chlorella biological oil, collecting non-condensable gas in the gas collection bag to obtain chlorella biological gas, and finally opening the pyrolysis reactor to collect the residual solid-phase product to obtain chlorella biochar. The chlorella biological oil is sealed in a brown reagent bottle and then stored in a dark and dry place at the temperature of 2-6 ℃.
Example 2
(1) Preparation of compound protease and preparation of scenedesmus obliquus raw material
The alkaline protease and the trypsin are purchased from Beijing Soilebao science and technology Limited, and the enzyme activity is obtained from a commodity label of the purchased protease, wherein the enzyme activity of the alkaline protease is 200000U/g, and the enzyme activity of the trypsin is 250000U/g. Preparing the alkaline protease and the trypsin into the compound protease according to the enzyme activity ratio of 3:1, and drying and storing the compound protease at the temperature of 2-6 ℃ in a dark place. Drying Scenedesmus obliquus to obtain Scenedesmus obliquus powder, sieving to 100 mesh fineness with a sieving machine, and drying in a forced air drying oven at 105 deg.C for 24 hr.
(2) Enzymolysis pretreatment of Scenedesmus obliquus
Taking 10g of scenedesmus obliquus powder ground to 100 meshes, putting the scenedesmus obliquus powder into a 500ml beaker, adding 190ml of deionized water, adding 25000U of compound protease with enzyme activity according to the addition amount of the protease of 2500U/g of substrate, adjusting the pH value to 8.0, and incubating the mixed solution at the constant temperature of 50 ℃ for 6 hours by using a constant-temperature water bath heating device. Adjusting the pH value of the algae solution to 4.0 after the specified time is reached, and putting the algae solution in a boiling water bath for 10min for inactivation. The mixture was centrifuged for 15min at 5500r/min using a high speed centrifuge and the supernatant was separated from the solid phase product. The supernatant is stored at-20 deg.C in dark place, and a small amount of sample is taken for quantitative analysis of amino acids according to determination method of amino acids in food. The solid phase product was dried in a forced air drying cabinet at 105 ℃ for 24 h.
(3) Pyrolytic conversion of Scenedesmus obliquus
5g of solid phase product of Scenedesmus obliquus pretreated by enzyme is uniformly mixed with 20g of treated waste vegetable oil, the mass ratio of the solid phase product of Scenedesmus obliquus to the waste vegetable oil after enzymolysis is 1:4, 6.25g of CaO catalyst is added, the using amount of the catalyst is 25 percent of the sum of the mass of the waste vegetable oil and the solid phase product of Scenedesmus obliquus, and the mixture is placed in a pyrolysis reactor after uniform mixing. Setting the pyrolysis reaction temperature to be 550 ℃, setting the temperature rise rate to be 50 ℃/s, setting the gas phase residence time to be 10s, keeping the temperature for 1h after the set temperature is reached, and carrying out pyrolysis under the protection of a nitrogen atmosphere, wherein the nitrogen flow rate is set to be 0.8L/min during the temperature rise period, and the nitrogen flow rate is set to be 0.2L/min during the temperature keeping period. After the temperature of the pyrolysis reactor is reduced to room temperature, firstly, the condensed liquid is collected as scenedesmus obliquus bio-oil, the non-condensable gas is collected in the gas collection bag to be scenedesmus obliquus bio-gas, and finally, the pyrolysis reactor is opened to collect the residual solid-phase product to be scenedesmus obliquus biochar. The scenedesmus obliquus bio-oil is sealed in a brown reagent bottle and then stored in a dark and dry place at the temperature of 2-6 ℃.
Example 3
(1) Preparation of compound protease and preparation of spirulina raw material
The alkaline protease and the papain are purchased from Beijing Soilebao science and technology Limited, and the enzyme activity is obtained from a commodity label of the purchased protease, wherein the enzyme activity of the alkaline protease is 200000U/g, and the enzyme activity of the papain is 250000U/g. Preparing the alkaline protease and the papain into the compound protease according to the enzyme activity ratio of 3:1, and drying and storing the compound protease at the temperature of 2-6 ℃ in a dark place. Drying Spirulina to obtain Spirulina powder, sieving with a sieving machine to 100 mesh fineness, and drying in a forced air drying oven at 105 deg.C for 24 hr.
(2) Enzymolysis pretreatment of spirulina
Taking 12g of spirulina powder ground to 100 meshes, putting the spirulina powder into a 500ml beaker, adding 188ml of deionized water, adding compound protease with enzyme activity of 36000U according to the addition amount of the protease of 3000U/g of substrate, adjusting the pH value to 9.0, and incubating the mixed solution at the constant temperature of 55 ℃ for 7 hours by using a constant-temperature water bath heating device. Adjusting the pH value of the algae solution to 4.0 after the specified time is reached, and putting the algae solution in a boiling water bath for 10min for inactivation. The mixture was centrifuged for 15min at 5500r/min using a high speed centrifuge and the supernatant was separated from the solid phase product. The supernatant is stored at-20 deg.C in dark place, and a small amount of sample is taken for quantitative analysis of amino acids according to determination method of amino acids in food. The solid phase product was dried in a forced air drying cabinet at 105 ℃ for 24 h.
(3) Pyrolytic conversion of spirulina
5g of the spirulina solid-phase product subjected to enzyme pretreatment is uniformly mixed with 25g of the treated waste vegetable oil, the mass ratio of the spirulina solid-phase product subjected to enzyme hydrolysis to the waste vegetable oil is 1:5, 7.5g of CaO catalyst is added, the using amount of the catalyst is 25 percent of the sum of the mass of the waste vegetable oil and the spirulina solid-phase product, and the mixture is uniformly mixed and then placed in a pyrolysis reactor. Setting the pyrolysis reaction temperature to be 600 ℃, setting the temperature rise rate to be 100 ℃/s, setting the gas phase residence time to be 5s, keeping the temperature for 1h after the set temperature is reached, and carrying out pyrolysis under the protection of a nitrogen atmosphere, wherein the nitrogen flow rate is set to be 0.8L/min during the temperature rise period, and the nitrogen flow rate is set to be 0.2L/min during the temperature keeping period. And after the temperature of the pyrolysis reactor is reduced to room temperature, firstly collecting condensed liquid as the spirulina biological oil, collecting non-condensable gas in the gas collection bag to obtain spirulina biological gas, and finally opening the pyrolysis reactor to collect the residual solid-phase product to obtain the spirulina biochar. The spirulina biological oil is sealed in a brown reagent bottle and then stored in a dark and dry place at the temperature of 2-6 ℃.
It should be understood that although the present description has been described in terms of various embodiments, not every embodiment includes only a single embodiment, and such description is for clarity purposes only, and those skilled in the art will recognize that the embodiments described herein may be combined as suitable to form other embodiments, as will be appreciated by those skilled in the art.
The above-listed detailed description is only a specific description of a possible embodiment of the present invention, and they are not intended to limit the scope of the present invention, and equivalent embodiments or modifications made without departing from the technical spirit of the present invention should be included in the scope of the present invention.
Claims (10)
1. A microalgae high-efficiency co-production method based on a compound protease enzymolysis technology is characterized by comprising the following steps:
step S1, mixing alkaline protease with papain or trypsin to prepare a compound protease solution, and processing microalgae into microalgae powder;
step S2, adding the compound protease liquid into the algae liquid containing the microalgae powder, uniformly mixing, and pretreating the microalgae by using a compound protease enzymolysis technology;
step S3, after the enzymolysis is finished, separating the pretreated microalgae from the algae liquid by using high-speed centrifugation, drying the solid-phase product to be used as a raw material of the pyrolysis reaction, and collecting the supernatant containing the polypeptide;
and step S4, mixing the dried solid-phase product microalgae with the waste vegetable oil, carrying out co-catalytic pyrolysis, collecting condensed gas generated in the pyrolysis and condensing to obtain a liquid-phase product microalgae bio-oil.
2. The method for efficiently coproducing microalgae according to claim 1, wherein the microalgae species in step S1 is chlorella, scenedesmus obliquus or spirulina.
3. The microalgae high-efficiency co-production method based on the composite protease enzymolysis technology as claimed in claim 1, characterized in that the composite protease is prepared by using alkaline protease as a main enzyme, papain or trypsin as a coenzyme, and the main enzyme and the coenzyme according to an enzyme activity ratio of 2:1 or 3: 1.
4. The method for efficiently coproducing microalgae based on the composite protease enzymolysis technology as claimed in claim 1, wherein the microalgae in the step S1 is processed into microalgae powder with a fineness of 100 meshes.
5. The method for efficiently coproducing microalgae according to claim 1, wherein the step S2 of performing enzymolysis pretreatment on the microalgae by using the composite protease enzymolysis technology specifically comprises the following steps:
the enzymolysis time is 5-7h, the enzymolysis pH value is 7.0-9.0, the enzymolysis temperature is 45-55 ℃, the mass fraction of the microalgae in the algae liquid is 4-6%, the usage amount of the compound protease is 3000U/g substrate, and the enzymolysis substrate is microalgae powder.
6. The microalgae high-efficiency co-production method based on the compound protease enzymolysis technology as claimed in claim 1, wherein after the enzymolysis in step S3 is finished, the pH of the enzymolysis liquid is adjusted to 4.0, the enzymolysis liquid is placed in a boiling water bath for 10min to inactivate the enzyme, a solid-phase product and a liquid-phase product are separated by a high-speed centrifuge, the centrifugation speed is 5500r/min, and the centrifugation time is 15 min.
7. The method for efficiently coproducing microalgae according to the claim 1 and the step S3, wherein the collected supernatant containing the polypeptides is subjected to amino acid analysis, and amino acid components in the supernatant are determined and then stored at-20 ℃ in a dark place.
8. The microalgae high-efficiency co-production method based on the compound protease enzymolysis technology is characterized in that the mass mixing ratio of the solid-phase product to the waste vegetable oil in the step S4 is 1:3-1: 5; the catalyst is a CaO catalyst, and the addition amount of the catalyst is 25% of the total mass of the solid-phase product and the waste vegetable oil.
9. The method for efficiently coproducing microalgae based on the composite protease enzymolysis technology as claimed in claim 1, wherein the catalytic pyrolysis in the step S4 is performed under the following reaction conditions: the pyrolysis temperature is set to be 500-600 ℃, the heating rate is 10-100 ℃/s, and the gas phase residence time is 0.5-10 s; and (3) putting the mixed solid-phase product, the waste vegetable oil and the CaO catalyst into a pyrolysis reactor, starting to heat, blowing nitrogen at the rate of 0.8L/min during the heating, and changing the nitrogen rate to 0.2L/min after the set temperature is reached.
10. The microalgae bio-oil and the supernatant containing the polypeptide prepared by the microalgae high-efficiency co-production method based on the compound protease enzymolysis technology according to any one of claims 1 to 9.
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