Method for cleanly producing biodiesel by using esterase compound enzyme
Through the method of esterase complex enzyme, enzyme extraction is extracted using kitchen waste oil and engineering bacterial strains to achieve efficient and environmentally friendly biodiesel production, solving the problems of insufficient tolerance and low yield of bioenzymes, and improving production efficiency and product quality.
Patent Information
- Application Number
- CN202411876873.9
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-12-19
- Publication Date
- 2025-03-28
AI Technical Summary
The prior art is difficult to biocatalyze the production of biodiesel through efficient and environmentally friendly methods, and the biological enzymes are insufficient tolerate the working environment, resulting in high production costs and low yields.
The method of clean production of biodiesel using esterase composite enzymes, including filtration and heating of waste oil in kitchens, fermentation and extraction of enzymes in engineered bacterial strains, configuration and mixing of enzymes, methanol catalytic reaction and carbon dioxide supercritical extraction to obtain biodiesel.
By using engineered bacterial strains and multiple biological enzymes to compound, the tolerance and service life of biological enzymes are improved, production time is shortened, and the yield and quality of biodiesel are improved.
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Abstract
Description
Technical Field
[0001] The invention relates to the technical field of biodiesel preparation, and in particular to a method for cleanly producing biodiesel using an esterase complex enzyme. Background Art
[0002] The current new energy sources mainly include solar energy, biomass energy, hydropower, wind energy, hydrogen energy, geothermal energy, wave energy, ocean current energy and tidal energy, as well as thermal circulation between the ocean surface and deep layers, etc. However, these new energy sources have problems in terms of competing with people for food, cost, environmental pollution, stability, etc., which cannot be overcome in the short term.
[0003] In view of the above-mentioned defects, the inventors of the present invention finally obtained the present invention after a long period of research and practice. Summary of the invention
[0004] The purpose of the present invention is to solve the problem of how to produce biodiesel by biocatalysis in an efficient and environmentally friendly manner and improve the tolerance of biological enzymes to the working environment, and to provide a method for cleanly producing biodiesel by using an esterase complex enzyme.
[0005] In order to achieve the above object, the present invention discloses a method for cleanly producing biodiesel using an esterase complex enzyme, comprising the following steps:
[0006] S1, filter the waste cooking oil to remove solid impurities, and heat it at a temperature greater than 100°C for 30 minutes to achieve the purpose of dehydration;
[0007] S2, inoculating and fermenting the engineered bacteria, extracting and concentrating the fermentation products to obtain esterase, protease and cutinase;
[0008] S3, preparing a composite esterase by mixing the esterase, protease and cutinase obtained in step S2;
[0009] S4, adding the composite esterase obtained in step S3 to the kitchen waste oil treated in step S1 and mixing evenly, adding methanol and reacting to obtain crude biodiesel;
[0010] S5, the crude biodiesel is precipitated, the oil phase is separated from the water phase, and the separated oil phase is subjected to supercritical extraction of carbon dioxide to obtain biodiesel.
[0011] In the step S2, esterase is produced by fermentation with Pichia pastoris, cutinase is produced by fermentation with Pichia pastoris, and protease is produced by fermentation with wood-rotting fungi.
[0012] In step S4, the amount of the composite enzyme ester added is 2 / 10000 of the mass of the waste cooking oil.
[0013] In step S4, the reaction temperature is 40° C. and the reaction time is 12 to 24 hours.
[0014] In step S5, the sedimentation time is 4 to 6 hours.
[0015] Compared with the prior art, the beneficial effects of the present invention are:
[0016] 1. The enzyme-producing strains used in the present invention are all engineered strains, which reduces the subsequent production difficulty and production cost;
[0017] 2. The present invention uses a combination of multiple biological enzymes to improve the tolerance of the biological enzymes to the working environment and extend the service life of the biological enzymes;
[0018] 3. The present invention uses a combination of multiple biological enzymes to treat waste cooking oil, shortening the production time of biodiesel and increasing the yield of biodiesel. DETAILED DESCRIPTION
[0019] The above and other technical features and advantages of the present invention are described in more detail below in conjunction with embodiments.
[0020] Example 1
[0021] (1) Raw material preparation: The waste cooking oil is filtered to remove solid impurities, and heated at a temperature greater than 100°C for 30 minutes to achieve the purpose of dehydration.
[0022] (2) Bio-enzyme fermentation production: Esterase engineering bacteria are inoculated and fermented, and the fermentation products are extracted and concentrated to obtain the required enzyme.
[0023] (3) Preparation of composite esterase: Esterase, protease and cutinase are prepared in a ratio of 75:10:15 to form a composite esterase.
[0024] (4) Biocatalysis: The composite esterase obtained in step (3) is added to the kitchen waste oil treated in step (1) at a ratio of 2 / 10000 and mixed evenly. An appropriate amount of methanol is added and reacted at 40° C. for 24 hours to obtain crude biodiesel.
[0025] (5) Supercritical extraction: The crude biodiesel is allowed to settle for 4 to 6 hours to separate the oil phase from the water phase, and the separated oil phase is subjected to supercritical extraction of carbon dioxide. The test data of the crude biodiesel after catalysis is shown in Table 1.
[0026] Table 1 Detection data of crude biodiesel after catalysis
[0027] Waste cooking oil Initial acid value 173.16 After esterification, the acid value 5.47 Fatty acid methyl ester content 87.31％ Esterification yield 95.83％
[0028] After the reaction, the acid value of the crude biodiesel was 5.47, the yield of crude biodiesel was 95.83%, and the fatty acid methyl ester content of the crude biodiesel was 87.31%.
[0029] Example 2
[0030] The raw material was replaced with palmitic acid oil, which was filtered to remove solid impurities, and heated at a temperature greater than 100° C. and maintained for 30 minutes to achieve the purpose of dehydration. The other steps were the same as in Example 1. The crude biodiesel test data after the catalysis was completed are shown in Table 2.
[0031] Table 2 Detection data of crude biodiesel after catalysis
[0032]
[0033]
[0034] After the reaction, the acid value of the crude biodiesel was 5.901, the yield of the crude biodiesel was 93.21%, and the fatty acid methyl ester content of the crude biodiesel was 84.47%.
[0035] Example 3
[0036] The raw material was replaced with swill oil, which was filtered to remove solid impurities, and heated at a temperature greater than 100° C. and maintained for 30 minutes to achieve the purpose of dehydration. The other steps were the same as in Example 1. The crude biodiesel test data after the catalysis was completed are shown in Table 3.
[0037] Table 3 Detection data of crude biodiesel after catalysis
[0038] Swill oil Initial acid value 42.36 After esterification, the acid value 4.31 Fatty acid methyl ester content 86.89％ Esterification yield 95.24％
[0039] After the reaction, the acid value of the crude biodiesel was 4.31, the yield of the crude biodiesel was 95.24%, and the fatty acid methyl ester content of the crude biodiesel was 86.89%.
[0040] Example 4
[0041] In the process of preparing the composite enzyme, esterase, protease and cutinase were prepared into composite esterase in a ratio of 70:10:20. The other steps were the same as those in Example 1. The test data of crude biodiesel after the catalysis was completed were shown in Table 4.
[0042] Table 4 Detection data of crude biodiesel after catalysis
[0043] Waste cooking oil Initial acid value 173.16 After esterification, the acid value 4.97 Fatty acid methyl ester content 86.34％ Esterification yield 94.45％
[0044] After the reaction, the acid value of the crude biodiesel was 4.97, the yield of crude biodiesel was 94.45%, and the fatty acid methyl ester content of the crude biodiesel was 86.34%.
[0045] Example 5
[0046] In the process of preparing the composite enzyme, esterase, protease and cutinase were prepared into composite esterase in a ratio of 80:5:15. The other steps were the same as those in Example 1. The test data of crude biodiesel after the catalysis was completed were shown in Table 5.
[0047] Table 5 Crude biodiesel test data after catalysis
[0048] Waste cooking oil Initial acid value 173.16 After esterification, the acid value 4.66 Fatty acid methyl ester content 85.78％ Esterification yield 94.77％
[0049] After the reaction, the acid value of the crude biodiesel was 4.66, the yield of the crude biodiesel was 94.77%, and the fatty acid methyl ester content of the crude biodiesel was 85.78%.
[0050] Example 6
[0051] During the use of the composite esterase, the enzyme water phase can still be separated after the catalytic reaction to measure its high enzyme activity. The test data of the total catalytic capacity of the enzyme after the composite esterase catalysis is shown in Table 6.
[0052] Table 6 Detection data of total catalytic ability of enzyme after catalysis
[0053] Use times 0 1 2 3 4 Enzyme activity (unit / MU) 100 92 77 68 49
[0054] The initial activity of the composite enzyme is one million U. As shown in Table 6, after multiple reactions, the composite esterase can still maintain a relatively high enzyme activity.
[0055] The above description is only a preferred embodiment of the present invention, which is only illustrative and not restrictive of the present invention. Those skilled in the art understand that many changes, modifications, and even equivalences may be made to the present invention within the spirit and scope defined by the claims of the present invention, but all of them will fall within the scope of protection of the present invention.
Claims
1. A method for cleanly producing biodiesel using esterase complex enzymes, characterized in that: The following steps are involved: S1, filter the waste cooking oil to remove solid impurities, and heat it at a temperature greater than 100°C for 30 minutes to achieve the purpose of dehydration; S2, inoculating and fermenting the engineered bacteria, extracting and concentrating the fermentation products to obtain esterase, protease and cutinase; S3, preparing a composite esterase by mixing the esterase, protease and cutinase obtained in step S2; S4, adding the composite esterase obtained in step S3 to the kitchen waste oil treated in step S1 and mixing evenly, adding methanol and reacting to obtain crude biodiesel; S5, the crude biodiesel is precipitated, the oil phase is separated from the water phase, and the separated oil phase is subjected to supercritical extraction of carbon dioxide to obtain biodiesel.
2. The method for cleanly producing biodiesel using an esterase complex enzyme according to claim 1, characterized in that: In the step S2, esterase is produced by fermentation with Pichia pastoris, cutinase is produced by fermentation with Pichia pastoris, and protease is produced by fermentation with wood-rotting fungi.
3. The method for cleanly producing biodiesel using esterase complex enzyme according to claim 1, characterized in that: In step S3, the volume ratio of esterase, protease and cutinase is (70-85): (5-15): (2-15).
4. The method for cleanly producing biodiesel using esterase complex enzyme according to claim 1, characterized in that: In step S4, the amount of the composite enzyme ester added is 2 / 10000 of the mass of the waste cooking oil.
5. The method for cleanly producing biodiesel using esterase complex enzyme according to claim 1, characterized in that: In step S4, the reaction temperature is 40° C. and the reaction time is 12 to 24 hours.
6. The method for cleanly producing biodiesel using esterase complex enzyme according to claim 1, characterized in that: In step S5, the sedimentation time is 4 to 6 hours.