A method for promoting microalgae carbon fixation and oil production by utilizing plant hormones and saline wastewater

Abstract

The present invention belongs to the field of microalgae cultivation technology, and discloses a method for promoting microalgae carbon fixation and oil production by using plant hormones and saline wastewater. Microalgae are cultivated in a wastewater culture medium to which sodium chloride and plant hormones are added. The plant hormones include at least abscisic acid, which can effectively alleviate salt stress and promote microalgae oil accumulation and growth. Plant hormones also include one of auxin and gibberellin. When abscisic acid acts synergistically with auxin or gibberellin, it is more helpful to improve the oil yield of microalgae. In addition, sodium chloride, abscisic acid, 4-amino-2-methylthiopyrimidine-5-carboxamide and 4-methyl-5-thiazoleethanol can also be added to the wastewater to synergistically enhance microalgae biomass and oil accumulation. The present invention provides an effective way to reduce the cost of microalgae cultivation and realize the industrial production of microalgae biodiesel, with both environmental and energy benefits.

Description

Technical Field

[0001] The invention belongs to the technical field of microalgae cultivation, and particularly relates to a method for promoting microalgae carbon fixation and oil production by utilizing plant hormones and saline wastewater. Background Art

[0002] With the acceleration of global industrialization, energy demand continues to rise. The extensive use of fossil fuels has caused a series of serious environmental problems. Seeking sustainable clean energy alternatives has become a top priority.

[0003] As a renewable energy source, biodiesel has attracted much attention in recent years due to its biodegradability and low pollution. Microalgae, a type of tiny single-celled algae, are highly efficient in carbon fixation and oil production. Their oil can be used to produce biodiesel and is considered an ideal third-generation biofuel feedstock. However, despite the many advantages of microalgae, the large-scale production of their lipids still faces severe cost challenges. The high cost of culture medium, harvesting costs, and subsequent oil extraction during the microalgae cultivation process have severely restricted the large-scale industrial production and application of microalgae biodiesel.

[0004] To reduce the cost of microalgae cultivation, wastewater resources are seen as a potential solution. Wastewater comes from a wide range of sources, including industry, agriculture, and urban life. It is rich in nutrients such as nitrogen and phosphorus, which are essential for microalgae growth. Using wastewater as a culture medium for microalgae cultivation not only provides nutrients for the microalgae, realizing waste resource utilization and reducing cultivation costs, but also removes pollutants such as nutrients from the wastewater during growth and captures carbon dioxide, achieving the dual benefits of environmental governance and energy production.

[0005] Previous studies have found that while salt stress can significantly increase lipid accumulation in microalgae, it also inhibits their growth rate, resulting in an ineffective increase in overall lipid yield. Therefore, how to increase microalgae lipid content while accelerating their growth rate to achieve high overall efficiency in microalgae lipid production has become a pressing challenge in microalgae research. Summary of the Invention

[0006] The object of the present invention is to provide a method for promoting microalgae carbon fixation and oil production by utilizing plant hormones and saline wastewater.

[0007] The technical solutions adopted by the present invention to achieve the above-mentioned purpose are:

[0008] A method for promoting microalgae carbon fixation and oil production by utilizing plant hormones and saline wastewater comprises: adding sodium chloride and plant hormones to wastewater, sterilizing the wastewater to obtain a wastewater culture medium, and culturing microalgae in the wastewater culture medium; the plant hormones comprise at least abscisic acid, and further comprise one of auxin and gibberellin.

[0009] Preferably, the mass volume ratio of sodium chloride to wastewater is 1-10g:1-50L.

[0010] Preferably, the mass volume ratio of abscisic acid to wastewater is 1-20 mg:100-2000 mL.

[0011] Preferably, the sterilization temperature is 120-125°C.

[0012] Preferably, the mass volume ratio of auxin to wastewater is 1-20 mg:100-2000 mL.

[0013] Preferably, the mass volume ratio of gibberellin to wastewater is 1-20 mg:100-2000 mL.

[0014] Preferably, the microalgae is Chlorella pyrenoidosa.

[0015] A wastewater culture medium is provided, comprising: adding sodium chloride, abscisic acid, 4-amino-2-methylthiopyrimidine-5-carboxamide, and 4-methyl-5-thiazoleethanol to wastewater, followed by sterilization to obtain the wastewater culture medium. 4-amino-2-methylthiopyrimidine-5-carboxamide and 4-methyl-5-thiazoleethanol synergistically play a key role in the energy metabolism and material transport of microalgae, enhancing the stress resistance of microalgae cells, reducing damage to cells caused by environmental stresses such as salt stress, maintaining normal physiological functions of the cells, and promoting fatty acid synthesis.

[0016] Preferably, the mass volume ratio of sodium chloride to wastewater is 1-10g:1-50L.

[0017] Preferably, the mass volume ratio of abscisic acid to wastewater is 1-20 mg:100-2000 mL.

[0018] Preferably, the mass volume ratio of 4-amino-2-methylthiopyrimidine-5-carboxamide to wastewater is 1-20 mg:100-2000 mL.

[0019] Preferably, the mass volume ratio of 4-methyl-5-thiazoleethanol to wastewater is 1-20 mg:100-2000 mL.

[0020] More preferably, N-phenylphthalimide can also be added to the wastewater, with the mass volume ratio of N-phenylphthalimide to wastewater being 1-20 mg:100-2000 mL. In a saline wastewater environment, the synergistic effect of N-phenylphthalimide, 4-amino-2-methylthiopyrimidine-5-carboxamide, and 4-methyl-5-thiazoleethanol helps enhance the stress resistance of microalgae cells, ensures the normal physiological function of cells under salt stress, creates favorable conditions for the growth and oil synthesis of microalgae, promotes the growth and oil accumulation of microalgae, and improves the carbon sequestration and oil production efficiency of microalgae in saline wastewater environments.

[0021] The present invention also provides a method for preparing artificial wastewater, comprising: adding NH4Cl, KH2PO4, MgSO4·H2O, CaCl2, NaHCO3, CH3COONa, FeCl3, H3BO3, MnCl2·4H2O, ZnSO4·7H2O, CuSO4·5H2O, and NaMoO4·2H2O into deionized water, and stirring uniformly to obtain artificial wastewater.

[0022] Preferably, the mass volume ratio of NH4Cl to deionized water is 15-310 mg:100-2000 mL.

[0023] Preferably, the mass volume ratio of KH2PO4 to deionized water is 2-44 mg:100-2000 mL.

[0024] Preferably, the mass volume ratio of MgSO4·H2O to deionized water is 2-55 mg:100-2000 mL.

[0025] Preferably, the mass volume ratio of CaCl2 to deionized water is 1-5 mg:400-2000 mL.

[0026] Preferably, the mass volume ratio of NaHCO3 to deionized water is 12.5-250 mg:100-2000 mL.

[0027] Preferably, the mass volume ratio of CH3COONa to deionized water is 20-400 mg:100-2000 mL.

[0028] Preferably, the mass volume ratio of FeCl3 to deionized water is 2-10 mg:500-2500 mL.

[0029] Preferably, the mass volume ratio of H3BO3 to deionized water is 1.4-7 mg:400-2000 mL.

[0030] Preferably, the mass volume ratio of MnCl2·4H2O to deionized water is 1-4 mg:500-2000 mL.

[0031] Preferably, the mass volume ratio of ZnSO4·7H2O to deionized water is 1.5-6 mg:5-20 L.

[0032] Preferably, the mass volume ratio of CuSO4·5H2O to deionized water is 1-2 mg:10-20 L.

[0033] Preferably, the mass volume ratio of NaMoO4·2H2O to deionized water is 1-10 mg:2-20 L.

[0034] The present invention also provides a method for preparing a wastewater culture medium, comprising: adding sodium chloride, abscisic acid and auxin to artificial wastewater, stirring evenly and then sterilizing to obtain the wastewater culture medium.

[0035] Preferably, the mass volume ratio of sodium chloride to artificial wastewater is 1-4g:500-2000mL.

[0036] Preferably, the mass volume ratio of abscisic acid to artificial wastewater is 1-20 mg:100-2000 mL.

[0037] Preferably, the mass volume ratio of auxin to artificial wastewater is 1-20 mg:100-2000 mL.

[0038] Preferably, the sterilization temperature is 120-125°C.

[0039] The present invention also provides a method for cultivating microalgae, comprising: selecting Chlorella pyrenoidosa ( Chlorella pyrenoidosa FACHB-5), the microalgae were pre-cultured in BG11 medium and then inoculated into wastewater medium for cultivation.

[0040] Preferably, the biomass volume ratio of microalgae to wastewater culture medium is 9-72 mg:50-400 mL.

[0041] Preferably, the shaking speed is 180-220 rpm.

[0042] Preferably, the temperature is 24-26°C.

[0043] Preferably, the light intensity is 80-100 μmol photon·m -2 ·s -1

[0044] Preferably, the culture period is 8-12 days.

[0045] The present invention also provides a method for preparing a wastewater culture medium, comprising: adding sodium chloride, abscisic acid and gibberellin into artificial wastewater, stirring evenly and then sterilizing to obtain the wastewater culture medium.

[0046] Preferably, the mass volume ratio of sodium chloride to artificial wastewater is 1-4g:500-2000mL.

[0047] Preferably, the mass volume ratio of abscisic acid to artificial wastewater is 1-20 mg:100-2000 mL.

[0048] Preferably, the mass volume ratio of gibberellin to artificial wastewater is 1-20 mg:100-2000 mL.

[0049] Preferably, the sterilization temperature is 120-125°C.

[0050] The present invention also provides a method for preparing a wastewater culture medium, comprising: adding sodium chloride, abscisic acid, 4-amino-2-methylthiopyrimidine-5-carboxamide, and 4-methyl-5-thiazoleethanol to artificial wastewater, stirring evenly, and then sterilizing to obtain the wastewater culture medium.

[0051] Preferably, the mass volume ratio of sodium chloride to artificial wastewater is 1-4g:500-2000mL.

[0052] Preferably, the mass volume ratio of abscisic acid to artificial wastewater is 1-20 mg:100-2000 mL.

[0053] Preferably, the mass volume ratio of 4-amino-2-methylthiopyrimidine-5-carboxamide to artificial wastewater is 1-20 mg:100-2000 mL.

[0054] Preferably, the mass volume ratio of 4-methyl-5-thiazoleethanol to artificial wastewater is 1-20 mg:100-2000 mL.

[0055] Preferably, the sterilization temperature is 120-125°C.

[0056] The present invention utilizes a wastewater culture medium obtained by adding sodium chloride and plant hormones to wastewater, or by adding 4-amino-2-methylthiopyrimidine-5-carboxamide and 4-methyl-5-thiazoleethanol to wastewater to obtain a wastewater culture medium, and cultivates microalgae in the wastewater culture medium. This advantageously results in high biomass, high oil content, and high lipid yield of the cultured microalgae, as well as increased C16-C18 fatty acid content and enhanced chemical stability of biodiesel produced using the microalgae. Therefore, the present invention provides a highly efficient method for promoting carbon sequestration and oil production in microalgae. BRIEF DESCRIPTION OF THE DRAWINGS

[0057] Figure 1 Schematic diagram of the test results of differentially expressed genes in microalgae cultured in Comparative Example 3.

[0058] Figure 2 Schematic diagram of the test results of differentially expressed genes in microalgae cultured in Example 1.

[0059] Figure 3 Schematic diagram of the test results of differentially expressed genes in microalgae cultured in Example 2.

[0060] Figure 4 Schematic diagram of the GO analysis test results of microalgae cultured in Comparative Example 3.

[0061] Figure 5 Schematic diagram of the GO analysis test results of the microalgae cultured in Example 1.

[0062] Figure 6Schematic diagram of the GO analysis test results of the microalgae cultured in Example 2.

[0063] Figure 7 Schematic diagram of the KEGG analysis test results of the microalgae cultured in Comparative Example 3.

[0064] Figure 8 Schematic diagram of the KEGG analysis test results of the microalgae cultured in Example 1.

[0065] Figure 9 This is a schematic diagram of the KEGG analysis test results of the microalgae cultured in Example 2.

[0066] Figure 10 Schematic diagram of microalgae biomass test results.

[0067] Figure 11 Schematic diagram of microalgae oil yield test results. DETAILED DESCRIPTION

[0068] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0069] The following first describes the concepts involved in this application with reference to the accompanying drawings. It should be noted that the following description of each concept is intended only to make the content of this application easier to understand and does not limit the scope of protection of this application. At the same time, the embodiments and features in the embodiments of this application can be combined with each other unless there is a conflict. The following detailed description of this application will be made with reference to the accompanying drawings and in conjunction with the embodiments.

[0070] Example 1:

[0071] Preparation of artificial wastewater: Add NH4Cl, KH2PO4, MgSO4·H2O, CaCl2, NaHCO3, CH3COONa, FeCl3, H3BO3, MnCl2·4H2O, ZnSO4·7H2O, CuSO4·5H2O, and NaMoO4·2H2O to deionized water and stir evenly to obtain artificial wastewater. The mass volume ratio of NH4Cl to deionized water is 154.3 mg:1L, the mass volume ratio of KH2PO4 to deionized water is 22 mg:1L, the mass volume ratio of MgSO4·H2O to deionized water is 27.5 mg:1L, the mass volume ratio of CaCl2 to deionized water is 2.5 mg:1L, the mass volume ratio of NaHCO3 to deionized water is 125 mg:1L, the mass volume ratio of CH3COONa to deionized water is 200 mg:1L, and the mass volume ratio of FeCl3 The mass volume ratio of H3BO3 to deionized water is 4.0 mg:1L, the mass volume ratio of MnCl2·4H2O to deionized water is 3.5 mg:1L, the mass volume ratio of ZnSO4·7H2O to deionized water is 0.3 mg:1L, the mass volume ratio of CuSO4·5H2O to deionized water is 0.1 mg:1L, and the mass volume ratio of NaMoO4·2H2O to deionized water is 0.5 mg:1L.

[0072] Preparation of wastewater culture medium: Add sodium chloride, abscisic acid, and auxin to artificial wastewater, stir well, and sterilize to obtain the wastewater culture medium. The mass-to-volume ratio of sodium chloride to artificial wastewater is 2 g:1 L, the mass-to-volume ratio of abscisic acid to artificial wastewater is 10 mg:1 L, and the mass-to-volume ratio of auxin to artificial wastewater is 10 mg:1 L. Sterilize at 121°C.

[0073] Cultivation of microalgae: Chlorella pyrenoidosa ( Chlorella pyrenoidosa FACHB-5) were pre-cultured in BG11 medium and then inoculated into wastewater medium for 10 days. The biomass volume ratio of microalgae to wastewater medium was 36 mg:200 mL, the shaker speed was 200 rpm, the temperature was 25°C, and the light intensity was 90 μmol photon·m -2 ·s -1 .

[0074] Example 2: Compared with Example 1, the only difference between this example and Example 1 is the preparation of the wastewater culture medium.

[0075] Preparation of wastewater culture medium: Add sodium chloride, abscisic acid, and gibberellin to artificial wastewater, stir well, and sterilize to obtain the wastewater culture medium. The mass-to-volume ratio of sodium chloride to artificial wastewater is 2 g:1 L, the mass-to-volume ratio of abscisic acid to artificial wastewater is 10 mg:1 L, and the mass-to-volume ratio of gibberellin to artificial wastewater is 10 mg:1 L. Sterilize at 121°C.

[0076] Example 3: Compared with Example 1, the only difference between this example and Example 1 is the preparation of the wastewater culture medium.

[0077] Preparation of wastewater culture medium: Sodium chloride, abscisic acid, 4-amino-2-methylthiopyrimidine-5-carboxamide, and 4-methyl-5-thiazoleethanol were added to artificial wastewater, stirred evenly, and sterilized to obtain the wastewater culture medium. The mass-to-volume ratio of sodium chloride to artificial wastewater was 2 g:1 L, the mass-to-volume ratio of abscisic acid to artificial wastewater was 10 mg:1 L, the mass-to-volume ratio of 4-amino-2-methylthiopyrimidine-5-carboxamide to artificial wastewater was 10 mg:1 L, and the mass-to-volume ratio of 4-methyl-5-thiazoleethanol to artificial wastewater was 10 mg:1 L. The sterilization temperature was 121°C.

[0078] Example 4: Compared with Example 1, the only difference between this example and Example 1 is the preparation of the wastewater culture medium.

[0079] Preparation of wastewater culture medium: Sodium chloride, abscisic acid, 4-amino-2-methylthiopyrimidine-5-carboxamide, and 4-methyl-5-thiazoleethanol were added to artificial wastewater, stirred evenly, and sterilized to obtain the wastewater culture medium. The mass-to-volume ratio of sodium chloride to artificial wastewater was 2 g:1 L, the mass-to-volume ratio of abscisic acid to artificial wastewater was 10 mg:1 L, the mass-to-volume ratio of 4-amino-2-methylthiopyrimidine-5-carboxamide to artificial wastewater was 15 mg:1 L, and the mass-to-volume ratio of 4-methyl-5-thiazoleethanol to artificial wastewater was 10 mg:1 L. Sterilization temperature was 121°C.

[0080] Example 5: Compared with Example 1, the only difference between this example and Example 1 is the preparation of the wastewater culture medium.

[0081] Preparation of wastewater culture medium: Sodium chloride, abscisic acid, 4-amino-2-methylthiopyrimidine-5-carboxamide, 4-methyl-5-thiazoleethanol, and N-phenylphthalimide are added to artificial wastewater, stirred evenly, and sterilized to obtain the wastewater culture medium. The mass volume ratio of sodium chloride to artificial wastewater is 2 g:1 L, the mass volume ratio of abscisic acid to artificial wastewater is 10 mg:1 L, the mass volume ratio of 4-amino-2-methylthiopyrimidine-5-carboxamide to artificial wastewater is 10 mg:1 L, the mass volume ratio of 4-amino-2-methylthiopyrimidine-5-carboxamide to artificial wastewater is 10 mg:1 L, the mass volume ratio of 4-methyl-5-thiazoleethanol to artificial wastewater is 10 mg:1 L, and the mass volume ratio of N-phenylphthalimide to artificial wastewater is 10 mg:1 L. The sterilization temperature is 121°C.

[0082] Example 6: Compared with Example 1, the only difference between this example and Example 1 is the preparation of the wastewater culture medium.

[0083] Preparation of wastewater culture medium: Sodium chloride, abscisic acid, 4-amino-2-methylthiopyrimidine-5-carboxamide, 4-methyl-5-thiazoleethanol, and N-phenylphthalimide are added to artificial wastewater, stirred evenly, and sterilized to obtain the wastewater culture medium. The mass volume ratio of sodium chloride to artificial wastewater is 2 g:1 L, the mass volume ratio of abscisic acid to artificial wastewater is 10 mg:1 L, the mass volume ratio of 4-amino-2-methylthiopyrimidine-5-carboxamide to artificial wastewater is 10 mg:1 L, the mass volume ratio of 4-amino-2-methylthiopyrimidine-5-carboxamide to artificial wastewater is 10 mg:1 L, the mass volume ratio of 4-methyl-5-thiazoleethanol to artificial wastewater is 10 mg:1 L, and the mass volume ratio of N-phenylphthalimide to artificial wastewater is 15 mg:1 L. The sterilization temperature is 121°C.

[0084] Comparative Example 1: This comparative example differs from Example 1 only in the preparation of the wastewater culture medium.

[0085] Preparation of wastewater culture medium: The wastewater culture medium was obtained by sterilizing artificial wastewater at a sterilization temperature of 121°C.

[0086] Comparative Example 2: This comparative example differs from Example 1 only in the preparation of the wastewater culture medium.

[0087] Preparation of wastewater culture medium: Add sodium chloride to artificial wastewater, stir thoroughly, and sterilize to obtain the wastewater culture medium. The mass volume ratio of sodium chloride to artificial wastewater is 2g:1L. The sterilization temperature is 121°C.

[0088] Comparative Example 3: Compared with Example 1, this comparative example differs only in the preparation of the wastewater culture medium.

[0089] Preparation of wastewater culture medium: Add sodium chloride and abscisic acid to artificial wastewater, stir well, and sterilize to obtain the wastewater culture medium. The mass-to-volume ratio of sodium chloride to artificial wastewater is 2 g:1 L, and the mass-to-volume ratio of abscisic acid to artificial wastewater is 10 mg:1 L. Sterilize at 121°C.

[0090] Comparative Example 4: Compared with Example 1, this comparative example differs only in the preparation of the wastewater culture medium.

[0091] Preparation of wastewater culture medium: Sodium chloride, abscisic acid, and 4-amino-2-methylthiopyrimidine-5-carboxamide are added to artificial wastewater, stirred, and sterilized to obtain the wastewater culture medium. The mass-to-volume ratio of sodium chloride to artificial wastewater is 2 g:1 L, the mass-to-volume ratio of abscisic acid to artificial wastewater is 10 mg:1 L, and the mass-to-volume ratio of 4-amino-2-methylthiopyrimidine-5-carboxamide to artificial wastewater is 10 mg:1 L. Sterilize at 121°C.

[0092] Comparative Example 5: Compared with Example 1, this comparative example differs only in the preparation of the wastewater culture medium.

[0093] Preparation of wastewater culture medium: Add sodium chloride, abscisic acid, and 4-methyl-5-thiazoleethanol to artificial wastewater, stir well, and sterilize to obtain the wastewater culture medium. The mass-to-volume ratio of sodium chloride to artificial wastewater is 2 g:1 L, the mass-to-volume ratio of abscisic acid to artificial wastewater is 10 mg:1 L, and the mass-to-volume ratio of 4-methyl-5-thiazoleethanol to artificial wastewater is 10 mg:1 L. Sterilize at 121°C.

[0094] Experimental Example 1: Microalgae transcriptome sequencing analysis test.

[0095] Test samples: microalgae cultured in Examples 1-2 and Comparative Examples 2-3.

[0096] Testing method: After extracting total RNA from microalgae, transcriptome sequencing was performed, and differentially expressed genes were analyzed using DESeq2 software. The microalgae cultured in Examples 1-2 and Comparative Example 3 were compared with the microalgae cultured in Comparative Example 2 to screen out genes with significant differential expression under different conditions; GO enrichment analysis was performed on the genes in the gene set using Goatools software to obtain the functions in which the differential genes were more concentrated, and the Fisher method was used for exact test, with the corrected P Padjust < 0.05; KEGG enrichment analysis was performed on the genes in DEGs and up-regulated DEGs, and then compared with the KEGG database to determine the biological processes in which the differentially expressed genes were involved.

[0097] The results of the differentially expressed genes test of microalgae cultured in comparative example 3 are as follows: Figure 1 As shown in Example 1, the test results of differentially expressed genes in microalgae cultured are as follows Figure 2 As shown in Example 2, the test results of differentially expressed genes in microalgae cultured are as follows Figure 3 As shown in the figure, under the synergistic effect of abscisic acid, auxin and gibberellin, the differentially expressed genes of microalgae were mainly up-regulated, indicating that these three exogenous plant hormones increased the expression of certain genes related to the salt tolerance of microalgae in the process of regulating the growth and metabolism of Chlorella, thereby improving the growth and oil accumulation of microalgae.

[0098] The results of GO analysis of microalgae cultured in comparative example 3 are as follows: Figure 4 As shown in Example 1, the GO analysis test results of the microalgae cultured are as follows Figure 5 As shown in Example 2, the GO analysis test results of the microalgae cultured are as follows Figure 6 As shown, it can be seen that under the regulation of plant hormones, the intrinsic components of the cell membrane and the components of the membrane are significantly enriched, and the oxidative damage under salt stress is alleviated mainly by promoting the transport mediated by the endoplasmic reticulum to the Golgi apparatus vesicles; in Example 1, after the addition of abscisic acid and auxin, the catalytic activity of the microalgae and the activity of the protein peptide modification activating enzyme are significantly improved, and the antioxidant capacity of the microalgae is effectively improved.

[0099] The KEGG analysis test results of the microalgae cultured in Comparative Example 3 are as follows: Figure 7 As shown in the figure, the KEGG analysis test results of the microalgae cultured in Example 1 are as follows: Figure 8 As shown in the figure, the KEGG analysis test results of the microalgae cultured in Example 2 are as follows: Figure 9 As shown in the data, abscisic acid mainly affects the degradation of valine, leucine and isoleucine, proteasome and autophagy; the combined application of abscisic acid and gibberellin has a significant effect on carotenoid biosynthesis, sulfur metabolism, photosynthesis, pyrimidine metabolism, etc.; the combined application of abscisic acid and auxin mainly acts on the improvement of the basic signaling pathway of the biosynthesis pathway of the terpenoid skeleton, thereby alleviating oxidative damage under salt stress through more pathways.

[0100] Test Example 2: Microalgae biomass test.

[0101] Test samples: microalgae cultured in various examples and comparative examples.

[0102] Test method: Take 50mL of algae liquid, centrifuge and discard the supernatant, dry at 60°C for 12 hours to reach constant weight, cool to room temperature in a desiccator, and then weigh.

[0103] The test results of the microalgae biomass cultured in the present invention are as follows: Figure 10As shown, from Comparative Examples 1-2, it can be seen that the biomass of microalgae in saline wastewater is much lower than that in non-saline wastewater, and salinity greatly inhibits the growth of Chlorella pyrenoidosa; from Comparative Example 3 and Examples 1-2, it can be seen that the presence of abscisic acid can significantly alleviate the adverse effects of salinity stress in saline wastewater on microalgae, enhance the adaptability of microalgae to salt stress, and abscisic acid synergistically acts with auxin or gibberellin to regulate the growth of microalgae in saline wastewater. It can play a greater role; from Examples 3-4 and Comparative Examples 4-5, 4-amino-2-methylthiopyrimidine-5-carboxylic acid The results show that the addition of N-phenylphthalimide to 4-amino-2-methylthiopyrimidine-5-carboxamide can synergistically enhance the biomass of microalgae and improve the growth of microalgae under salt stress. At the same time, the adjustment of 4-amino-2-methylthiopyrimidine-5-carboxamide can actively regulate the growth of microalgae. As can be seen from Examples 5-6, the addition of N-phenylphthalimide to 4-amino-2-methylthiopyrimidine-5-carboxamide and 4-methyl-5-thiazoleethanol can more effectively promote the growth of microalgae in saline wastewater and regulate the physiological metabolism of microalgae cells under salt stress on a larger scale.

[0104] Test Example 3: Microalgae oil yield test.

[0105] Test samples: microalgae cultured in various examples and comparative examples.

[0106] Test method: Take 50mL of algae liquid, discard the supernatant after centrifugation, resuspend with deionized water, add chloroform-methanol solution after cell disruption, so that the final mass ratio of chloroform, methanol and deionized water is 2:1:0.8, shake well and let it stand for 10 minutes, remove the lower chloroform oil layer after centrifugation, and obtain the microalgae oil extract after nitrogen blowing. The microalgae oil content is weighed and the microalgae oil yield is calculated.

[0107] The test results of the oil yield of microalgae cultured in the present invention are as follows: Figure 11As shown, from Comparative Examples 1-2, it can be seen that the addition of sodium chloride to the wastewater significantly increased the lipid content of the microalgae, but due to the significant inhibitory effect of sodium chloride on the growth of microalgae, the oil yield of the microalgae did not increase synchronously but decreased; from Comparative Example 3 and Examples 1-2, it can be seen that abscisic acid significantly promoted the oil accumulation of Chlorella pyrenoidosa in saline wastewater, and thanks to the significant promoting effect of abscisic acid on the growth of microalgae in saline wastewater, the oil yield of the microalgae was further improved. At the same time, the combined application of abscisic acid with auxin and gibberellin can improve the oil yield of the microalgae; From Examples 3-4 and Comparative Examples 4-5, it can be seen that 4-amino-2-methylthiopyrimidine-5-carboxamide and 4-methyl-5-thiazoleethanol can have a positive effect on improving the oil content and yield of microalgae, and the two need to work synergistically; from Examples 5-6, it can be seen that N-phenylphthalimide is added to 4-amino-2-methylthiopyrimidine-5-carboxamide and 4-methyl-5-thiazoleethanol. The combined effect of the three can more effectively promote the growth of microalgae in saline wastewater and the accumulation of oil content, and the lipid yield of the microalgae is also the highest.

[0108] Test Example 4: Microalgae oil fatty acid group test.

[0109] Test samples: microalgae oil extracts of Examples 1-2 and Comparative Examples 1-3 obtained in Experimental Example 2.

[0110] Test method: Dissolve 10 mg of microalgae oil extract in potassium hydroxide-methanol solution, vortex mix, and heat in a 75°C water bath for 15 minutes. After cooling to room temperature, add 2 mL of n-hexane and extract with oscillation for 5 minutes. Centrifuge at 3000 rpm for 5 minutes. The upper n-hexane phase is collected for GC-MS analysis to calculate the content of each fatty acid. Gas chromatography conditions: A DB-5MS capillary column (30 m × 0.25 mm × 0.25 μm) was used as the chromatographic column; high-purity helium was used as the carrier gas at a flow rate of 1.0 mL / min; the inlet temperature was 250°C; the split ratio was 10:1; and the temperature program was as follows: initial temperature 100°C, hold for 2 minutes, increase the temperature at 10°C / min to 200°C, hold for 5 minutes, then increase the temperature at 5°C / min to 280°C, hold for 5 minutes. Mass spectrometry conditions: EI ion source, electron energy 70 eV; ion source temperature 250°C; scan range m / z 40-500; solvent delay time 3 min.

[0111] The test results of the lipid fatty acid group of the microalgae cultured in the present invention are shown in Table 1:

[0112] Table 1 Test results of microalgae oil fatty acid group

[0113]

[0114] The result shows that several fatty acids with higher content in the pyrenoid chlorella cultivated by the present invention are respectively linoleic acid (C18:2n6c) and α-linolenic acid (C18:3n3) and palmitic acid (C16:0). C16-C18 fatty acid is considered to be the raw material of ideal biodiesel. Therefore, the lipid produced by the pyrenoid chlorella cultivated by the present invention has a good prospect of being developed into biodiesel. As can be seen from Comparative Examples 1-3, sodium chloride and abscisic acid do not significantly change the ratio of C16-C18 fatty acids, but change the saturation of fatty acids to a certain extent. Under 20g / L salt stress, the ratio of saturated fatty acids in microalgae lipids decreases, the ratio of monounsaturated fatty acids significantly increases, and the ratio of polyunsaturated fatty acids slightly decreases. Under the effect of abscisic acid, the ratio of saturated fatty acids and monounsaturated fatty acids in microalgae fatty acids slightly increases, and the ratio of polyunsaturated fatty acids slightly decreases. As can be seen from Examples 1-2, the combined use of abscisic acid with auxin and gibberellins further increases the ratio of saturated fatty acids and monounsaturated fatty acids in microalgae fatty acids, and further reduces the ratio of polyunsaturated fatty acids in fatty acids, indicating that under the regulation of plant hormones, the stress pressure suffered by microalgae is reduced. The change in the saturation of microalgae fatty acids also has a certain impact on the quality of the biodiesel produced. Unsaturated fatty acids, especially polyunsaturated fatty acids, easily react with oxygen under storage or high temperature, resulting in the degradation of biodiesel. The rise in the saturation of fatty acids is conducive to improving the antioxidant properties and chemical stability of the biodiesel produced. Therefore, the combined use of abscisic acid with auxin and gibberellins in the present invention is conducive to promoting the chemical stability of microalgae biodiesel.

[0115] The embodiments and / or implementation methods described above are only used to illustrate the preferred embodiments and / or implementation methods for realizing the technology of the present invention, and do not impose any form of limitation on the implementation methods of the technology of the present invention. Any person skilled in the art may make slight changes or modifications to other equivalent embodiments without departing from the scope of the technical means disclosed in the content of the present invention, but they should still be regarded as technologies or embodiments that are essentially the same as the present invention.

[0116] This article uses specific examples to illustrate the principles and implementation methods of this application. The description of the above embodiments is only used to help understand the method and core ideas of this application. The above is only the preferred implementation method of this application. It should be pointed out that due to the limitations of textual expression, there are objectively infinite specific structures. For ordinary technicians in this technical field, without departing from the principles of this application, they can also make several improvements, modifications or changes, and can also combine the above technical features in an appropriate manner; these improvements, modifications, changes or combinations, or the direct application of the inventive concept and technical solution to other occasions without improvement, should be regarded as the scope of protection of this application.

Claims

1. A method for promoting carbon sequestration and oil production in microalgae using plant hormones and saline wastewater, comprising: Sodium chloride, plant hormones, 4-amino-2-methylthiopyrimidine-5-carboxamide and 4-methyl-5-thiazoleethanol are added to wastewater and then sterilized to obtain a wastewater culture medium, and microalgae are cultured in the wastewater culture medium; the plant hormones include at least abscisic acid, the mass volume ratio of the sodium chloride to the wastewater is 2g:1L, the mass volume ratio of the abscisic acid to the wastewater is 10mg:1L, the mass volume ratio of the 4-amino-2-methylthiopyrimidine-5-carboxamide to the wastewater is 10-15mg:1L, and the mass volume ratio of the 4-methyl-5-thiazoleethanol to the wastewater is 10mg:1L.

2. The method of promoting microalgae carbon fixation and oil production by utilizing plant hormones and saline wastewater according to claim 1, characterized in that: The sterilization temperature is 120-125°C.

3. A wastewater culture medium comprising: Sodium chloride, abscisic acid, 4-amino-2-methylthiopyrimidine-5-carboxamide and 4-methyl-5-thiazoleethanol are added to wastewater and then sterilized to obtain a wastewater culture medium, wherein the mass volume ratio of the sodium chloride to the wastewater is 2 g:1L, the mass volume ratio of the abscisic acid to the wastewater is 10 mg:1L, the mass volume ratio of the 4-amino-2-methylthiopyrimidine-5-carboxamide to the wastewater is 10-15 mg:1L, and the mass volume ratio of the 4-methyl-5-thiazoleethanol to the wastewater is 10 mg:1L.

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