Method for promoting microalgae carbon sequestration and oil production by using plant hormones and salt-containing wastewater

Abstract

The invention belongs to the technical field of microalgae culture, and discloses a method for promoting microalgae carbon sequestration and oil production by using plant hormones and salt-containing wastewater, microalgae are cultured in a wastewater culture medium added with sodium chloride and the plant hormones, the plant hormones at least comprise abscisic acid, and the abscisic acid can effectively relieve salt stress and promote microalgae oil accumulation and growth. The plant hormone further comprises one of auxin and gibberellin, and the abscisic acid and the auxin or the gibberellin have a synergistic effect, so that the grease yield of the microalgae is more favorably improved. In addition, sodium chloride, abscisic acid, 4-amino-2-methylthiopyrimidine-5-carboxamide and 4-methyl-5-thiazoleethanol can also be added into the wastewater, so that the biological yield of the microalgae and the grease accumulation can be synergistically enhanced. The invention provides an effective way for reducing the microalgae culture cost and realizing the industrial production of microalgae biodiesel, and has both environmental and energy benefits.

Description

Technical Field

[0001] The present invention belongs to the technical field of microalgae culture, and particularly relates to a method for promoting carbon fixation and oil production of microalgae by using phytohormones and saline wastewater. Background Art

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

[0003] As a renewable energy source, biodiesel has attracted much attention in recent years due to its advantages such as biodegradability and low pollution. Microalgae, as a type of tiny single-celled algae organisms, have high carbon fixation and oil production capabilities. Their oils can be used to produce biodiesel and are regarded as ideal raw materials for the third-generation biofuels. However, despite the many advantages of microalgae, the large-scale production of their lipids still faces severe cost challenges. The costs of culture media, harvesting, and subsequent oil extraction in the microalgae culture process remain high, seriously restricting the large-scale industrial production and application of microalgae biodiesel.

[0004] To reduce the cost of microalgae culture, wastewater resources are regarded as a potential solution. Wastewater has a wide range of sources, covering industrial, agricultural, and urban domestic fields, and contains abundant nutrients such as nitrogen and phosphorus, which are exactly what microalgae need for growth. Using wastewater as a culture medium to cultivate microalgae can not only provide nutrients for microalgae, realize the recycling of waste resources, and reduce the culture cost, but also microalgae can remove pollutants such as nutrients in the wastewater and capture carbon dioxide during the growth process, with the dual benefits of environmental governance and energy production.

[0005] Previous studies have found that although salt stress can significantly increase the lipid accumulation in microalgae, it also inhibits the growth rate of microalgae, resulting in the overall lipid productivity not being effectively improved. Therefore, how to increase the growth rate of microalgae while increasing their lipid content and achieve the overall high efficiency of microalgae lipid production has become a difficult problem to be solved urgently in the current microalgae research field. Summary of the Invention

[0006] The purpose of the present invention is to provide a method for promoting carbon fixation and oil production of microalgae by using phytohormones and saline wastewater.

[0007] The technical solution adopted by the present invention to achieve the above purpose is as follows: A method for promoting carbon fixation and oil production of microalgae by using phytohormones and saline wastewater, comprising: adding sodium chloride and phytohormones to the wastewater, sterilizing to obtain a wastewater culture medium, and culturing microalgae in the wastewater culture medium; the phytohormones at least include abscisic acid, and the phytohormones also include one of auxin and gibberellin.

[0008] Preferably, the mass - volume ratio of sodium chloride to the wastewater is 1 - 10 g:1 - 50 L.

[0009] Preferably, the mass - volume ratio of abscisic acid to the wastewater is 1 - 20 mg:100 - 2000 mL.

[0010] Preferably, the sterilization temperature is 120 - 125 °C.

[0011] Preferably, the mass - volume ratio of auxin to the wastewater is 1 - 20 mg:100 - 2000 mL.

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

[0013] Preferably, the microalgae is Chlorella pyrenoidosa.

[0014] A wastewater medium, comprising: adding sodium chloride, abscisic acid, 4 - amino - 2 - methylthio - 5 - carboxamide pyrimidine and 4 - methyl - 5 - thiazole ethanol to the wastewater and then sterilizing to obtain the wastewater medium. 4 - Amino - 2 - methylthio - 5 - carboxamide pyrimidine and 4 - methyl - 5 - thiazole ethanol play a key role synergistically in the energy metabolism and material transport of microalgae, enhancing the stress resistance of microalgae cells, reducing the damage of environmental stresses such as salt stress to cells, maintaining the normal physiological functions of cells, and promoting the synthesis of fatty acids.

[0015] Preferably, the mass - volume ratio of sodium chloride to the wastewater is 1 - 10 g:1 - 50 L.

[0016] Preferably, the mass - volume ratio of abscisic acid to the wastewater is 1 - 20 mg:100 - 2000 mL.

[0017] Preferably, the mass - volume ratio of 4 - amino - 2 - methylthio - 5 - carboxamide pyrimidine to the wastewater is 1 - 20 mg:100 - 2000 mL.

[0018] Preferably, the mass - volume ratio of 4 - methyl - 5 - thiazole ethanol to the wastewater is 1 - 20 mg:100 - 2000 mL.

[0019] More preferably, N - phenylphthalimide can also be added to the wastewater, and the mass - volume ratio of N - phenylphthalimide to the wastewater is 1 - 20 mg:100 - 2000 mL. In a saline wastewater environment, the synergistic effect of N - phenylphthalimide, 4 - amino - 2 - methylthio - 5 - carboxamide pyrimidine and 4 - methyl - 5 - thiazole ethanol helps to enhance the stress resistance of microalgae cells, ensure the normal physiological functions of cells in a salt - stress environment, create favorable conditions for the growth and oil synthesis of microalgae, promote the growth and oil accumulation of microalgae, and improve the carbon fixation and oil production efficiency of microalgae in a saline wastewater environment.

[0020] 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 evenly to obtain artificial wastewater.

[0021] Preferably, the mass-volume ratio of NH4Cl to deionized water is 15 - 310 mg: 100 - 2000 mL.

[0022] Preferably, the mass-volume ratio of KH2PO4 to deionized water is 2 - 44 mg: 100 - 2000 mL.

[0023] Preferably, the mass-volume ratio of MgSO4·H2O to deionized water is 2 - 55 mg: 100 - 2000 mL.

[0024] Preferably, the mass-volume ratio of CaCl2 to deionized water is 1 - 5 mg: 400 - 2000 mL.

[0025] Preferably, the mass-volume ratio of NaHCO3 to deionized water is 12.5 - 250 mg: 100 - 2000 mL.

[0026] Preferably, the mass-volume ratio of CH3COONa to deionized water is 20 - 400 mg: 100 - 2000 mL.

[0027] Preferably, the mass-volume ratio of FeCl3 to deionized water is 2 - 10 mg: 500 - 2500 mL.

[0028] Preferably, the mass-volume ratio of H3BO3 to deionized water is 1.4 - 7 mg: 400 - 2000 mL.

[0029] Preferably, the mass-volume ratio of MnCl2·4H2O to deionized water is 1 - 4 mg: 500 - 2000 mL.

[0030] Preferably, the mass-volume ratio of ZnSO4·7H2O to deionized water is 1.5 - 6 mg: 5 - 20 L.

[0031] Preferably, the mass-volume ratio of CuSO4·5H2O to deionized water is 1 - 2 mg: 10 - 20 L.

[0032] Preferably, the mass-volume ratio of NaMoO4·2H2O to deionized water is 1 - 10 mg: 2 - 20 L.

[0033] 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.

[0034] Preferably, the mass-volume ratio of sodium chloride to artificial wastewater is 1-4 g:500-2000 mL.

[0035] Preferably, the mass-volume ratio of abscisic acid to artificial wastewater is 1-20 mg:100-2000 mL.

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

[0037] Preferably, the sterilization temperature is 120-125 °C.

[0038] The present invention also provides a method for culturing microalgae, comprising: selecting Chlorella pyrenoidosa ( Chlorella pyrenoidosa FACHB-5), pre-culturing the microalgae in BG11 medium and then inoculating them in the wastewater culture medium for culturing.

[0039] Preferably, the biomass-volume ratio of microalgae to the wastewater culture medium is 9-72 mg:50-400 mL.

[0040] Preferably, the shaker speed is 180-220 rpm.

[0041] Preferably, the temperature is 24-26 °C.

[0042] Preferably, the light intensity is 80-100 μmol photon·m -2 ·s -1 Preferably, the culture period is 8-12 d.

[0043] The present invention also provides a method for preparing a wastewater culture medium, comprising: adding sodium chloride, abscisic acid, and gibberellin to artificial wastewater, stirring evenly, and then sterilizing to obtain the wastewater culture medium.

[0044] Preferably, the mass-volume ratio of sodium chloride to artificial wastewater is 1-4 g:500-2000 mL.

[0045] Preferably, the mass-volume ratio of abscisic acid to artificial wastewater is 1-20 mg:100-2000 mL.

[0046] Preferably, the mass-volume ratio of gibberellin to artificial wastewater is 1-20 mg:100-2000 mL.

[0047] Preferably, the sterilization temperature is 120-125 °C.

[0048] The present invention also provides a method for preparing a wastewater culture medium, comprising: adding sodium chloride, abscisic acid, 4-amino-2-methylthio-pyrimidine-5-carboxamide, and 4-methyl-5-thiazole ethanol to artificial wastewater, stirring evenly, and then sterilizing to obtain the wastewater culture medium.

[0049] Preferably, the mass-volume ratio of sodium chloride to artificial wastewater is 1-4 g:500-2000 mL.

[0050] Preferably, the mass-volume ratio of abscisic acid to artificial wastewater is 1-20 mg:100-2000 mL.

[0051] Preferably, the mass-volume ratio of 4-amino-2-methylthio-pyrimidine-5-carboxamide to artificial wastewater is 1-20 mg:100-2000 mL.

[0052] Preferably, the mass-volume ratio of 4-methyl-5-thiazole ethanol to artificial wastewater is 1-20 mg:100-2000 mL.

[0053] Preferably, the sterilization temperature is 120-125 °C.

[0054] Since the present invention adopts the method of adding sodium chloride and plant hormones to wastewater to obtain the wastewater culture medium, or adding 4-amino-2-methylthio-pyrimidine-5-carboxamide and 4-methyl-5-thiazole ethanol to wastewater to obtain the wastewater culture medium, and culturing microalgae in the wastewater culture medium, the following beneficial effects are achieved: the microalgae obtained by culturing have high biomass, high oil content and high lipid productivity, and the content of C16-C18 fatty acids is increased, and the chemical stability of the biodiesel prepared from microalgae is improved. Therefore, the present invention is a method for efficiently promoting carbon fixation and oil production by microalgae. Description of the Drawings

[0055] Figure 1 Schematic diagram of the test results of differentially expressed genes of microalgae cultured in Comparative Example 3.

[0056] Figure 2 Schematic diagram of the test results of differentially expressed genes of microalgae cultured in Example 1.

[0057] Figure 3 Schematic diagram of the test results of differentially expressed genes of microalgae cultured in Example 2.

[0058] Figure 4 Schematic diagram of the test results of GO analysis of microalgae cultured in Comparative Example 3.

[0059] Figure 5 Schematic diagram of the test results of GO analysis of microalgae cultured in Example 1.

[0060] Figure 6Schematic diagram of GO analysis test results of microalgae cultured in Example 2.

[0061] Figure 7 Schematic diagram of KEGG analysis test results of microalgae cultured in Comparative Example 3.

[0062] Figure 8 Schematic diagram of KEGG analysis test results of microalgae cultured in Example 1.

[0063] Figure 9 Schematic diagram of KEGG analysis test results of microalgae cultured in Example 2.

[0064] Figure 10 Schematic diagram of microalgae biomass test results.

[0065] Figure 11 Schematic diagram of microalgae oil yield test results. Detailed implementation manners

[0066] Next, the technical solutions in the embodiments of the present invention will be clearly and completely described in conjunction with the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the protection scope of the present invention.

[0067] First, the concepts involved in this application will be described in conjunction with the accompanying drawings. It should be noted here that the descriptions of the following concepts are only for making the content of this application easier to understand, and do not represent a limitation on the protection scope of this application; at the same time, without conflict, the embodiments in this application and the features in the embodiments can be combined with each other. Next, this application will be described in detail with reference to the accompanying drawings and in conjunction with the embodiments.

[0068] Example 1: 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:1 L, the mass-volume ratio of KH2PO4 to deionized water is 22 mg:1 L, the mass-volume ratio of MgSO4·H2O to deionized water is 27.5 mg:1 L, the mass-volume ratio of CaCl2 to deionized water is 2.5 mg:1 L, the mass-volume ratio of NaHCO3 to deionized water is 125 mg:1 L, the mass-volume ratio of CH3COONa to deionized water is 200 mg:1 L, the mass-volume ratio of FeCl3 to deionized water is 4.0 mg:1 L, the mass-volume ratio of H3BO3 to deionized water is 3.5 mg:1 L, the mass-volume ratio of MnCl2·4H2O to deionized water is 2.0 mg:1 L, the mass-volume ratio of ZnSO4·7H2O to deionized water is 0.3 mg:1 L, the mass-volume ratio of CuSO4·5H2O to deionized water is 0.1 mg:1 L, and the mass-volume ratio of NaMoO4·2H2O to deionized water is 0.5 mg:1 L.

[0069] Preparation of wastewater medium: Add sodium chloride, abscisic acid, and auxin to the artificial wastewater, stir evenly, and sterilize to obtain the wastewater 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 auxin to artificial wastewater is 10 mg:1 L, and the sterilization temperature is 121 °C.

[0070] Cultivation of microalgae: Chlorella pyrenoidosa ( Chlorella pyrenoidosa FACHB-5) is selected. The microalgae are pre-cultured in BG11 medium and then inoculated into the wastewater medium for 10 days. The biomass-volume ratio of microalgae to wastewater medium is 36 mg:200 mL, the shaker speed is 200 rpm, the temperature is 25 °C, and the light intensity is 90 μmol photon·m -2 ·s -1 .

[0071] Example 2: Compared with Example 1, the only difference in this example lies in the preparation of the wastewater medium.

[0072] Preparation of wastewater culture medium: Sodium chloride, abscisic acid, and gibberellin were added to artificial wastewater, stirred evenly, and then sterilized to obtain the wastewater culture medium. The mass-volume ratio of sodium chloride to artificial wastewater was 2 g:1 L, the mass-volume ratio of abscisic acid to artificial wastewater was 10 mg:1 L, the mass-volume ratio of gibberellin to artificial wastewater was 10 mg:1 L, and the sterilization temperature was 121 °C.

[0073] Example 3: The only difference between this example and Example 1 lies in the preparation of the wastewater culture medium.

[0074] 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 then sterilized to obtain the wastewater culture medium. The mass-volume ratio of sodium chloride to artificial wastewater was 2 g:1 L, the mass-volume ratio of abscisic acid to artificial wastewater was 10 mg:1 L, the mass-volume ratio of 4-amino-2-methylthiopyrimidine-5-carboxamide to artificial wastewater was 10 mg:1 L, the mass-volume ratio of 4-methyl-5-thiazoleethanol to artificial wastewater was 10 mg:1 L, and the sterilization temperature was 121 °C.

[0075] Example 4: The only difference between this example and Example 1 lies in the preparation of the wastewater culture medium.

[0076] 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 then sterilized to obtain the wastewater culture medium. The mass-volume ratio of sodium chloride to artificial wastewater was 2 g:1 L, the mass-volume ratio of abscisic acid to artificial wastewater was 10 mg:1 L, the mass-volume ratio of 4-amino-2-methylthiopyrimidine-5-carboxamide to artificial wastewater was 15 mg:1 L, the mass-volume ratio of 4-methyl-5-thiazoleethanol to artificial wastewater was 10 mg:1 L, and the sterilization temperature was 121 °C.

[0077] Example 5: The only difference between this example and Example 1 lies in the preparation of the wastewater culture medium.

[0078] Preparation of wastewater culture medium: Sodium chloride, abscisic acid, 4-amino-2-methylthiopyrimidine-5-carboxamide, 4-methyl-5-thiazoleethanol, and N-phenylphthalimide were added to artificial wastewater, stirred evenly, and then sterilized to obtain the wastewater culture medium. The mass-volume ratio of sodium chloride to artificial wastewater was 2 g:1 L, the mass-volume ratio of abscisic acid to artificial wastewater was 10 mg:1 L, the mass-volume ratio of 4-amino-2-methylthiopyrimidine-5-carboxamide to artificial wastewater was 10 mg:1 L, the mass-volume ratio of 4-methyl-5-thiazoleethanol to artificial wastewater was 10 mg:1 L, the mass-volume ratio of N-phenylphthalimide to artificial wastewater was 10 mg:1 L, and the sterilization temperature was 121 °C.

[0079] Example 6: The difference between this example and Example 1 lies only in the preparation of the wastewater culture medium.

[0080] Preparation of the wastewater culture medium: Sodium chloride, abscisic acid, 4-amino-2-methylthio-pyrimidine-5-carboxamide, 4-methyl-5-thiazole ethanol, and N-phenylphthalimide were added to artificial wastewater. After stirring evenly, it was sterilized to obtain the wastewater culture medium. The mass-volume ratio of sodium chloride to artificial wastewater was 2 g:1 L, the mass-volume ratio of abscisic acid to artificial wastewater was 10 mg:1 L, the mass-volume ratio of 4-amino-2-methylthio-pyrimidine-5-carboxamide to artificial wastewater was 10 mg:1 L, the mass-volume ratio of 4-methyl-5-thiazole ethanol to artificial wastewater was 10 mg:1 L, the mass-volume ratio of N-phenylphthalimide to artificial wastewater was 15 mg:1 L, and the sterilization temperature was 121 °C.

[0081] Comparative Example 1: The difference between this comparative example and Example 1 lies only in the preparation of the wastewater culture medium.

[0082] Preparation of the wastewater culture medium: The artificial wastewater was sterilized to obtain the wastewater culture medium, and the sterilization temperature was 121 °C.

[0083] Comparative Example 2: The difference between this comparative example and Example 1 lies only in the preparation of the wastewater culture medium.

[0084] Preparation of the wastewater culture medium: Sodium chloride was added to artificial wastewater. After stirring evenly, it was sterilized to obtain the wastewater culture medium. The mass-volume ratio of sodium chloride to artificial wastewater was 2 g:1 L, and the sterilization temperature was 121 °C.

[0085] Comparative Example 3: The difference between this comparative example and Example 1 lies only in the preparation of the wastewater culture medium.

[0086] Preparation of the wastewater culture medium: Sodium chloride and abscisic acid were added to artificial wastewater. After stirring evenly, it was sterilized to obtain the wastewater culture medium. The mass-volume ratio of sodium chloride to artificial wastewater was 2 g:1 L, the mass-volume ratio of abscisic acid to artificial wastewater was 10 mg:1 L, and the sterilization temperature was 121 °C.

[0087] Comparative Example 4: The difference between this comparative example and Example 1 lies only in the preparation of the wastewater culture medium.

[0088] Preparation of wastewater medium: Sodium chloride, abscisic acid, and 4-amino-2-methylthio-pyrimidine-5-carboxamide were added to artificial wastewater. After stirring evenly, it was sterilized to obtain the wastewater medium. The mass-volume ratio of sodium chloride to artificial wastewater was 2 g:1 L, the mass-volume ratio of abscisic acid to artificial wastewater was 10 mg:1 L, the mass-volume ratio of 4-amino-2-methylthio-pyrimidine-5-carboxamide to artificial wastewater was 10 mg:1 L, and the sterilization temperature was 121 °C.

[0089] Comparative Example 5: This comparative example is the same as Example 1, except for the preparation of the wastewater medium.

[0090] Preparation of wastewater medium: Sodium chloride, abscisic acid, and 4-methyl-5-thiazoleethanol were added to artificial wastewater. After stirring evenly, it was sterilized to obtain the wastewater medium. The mass-volume ratio of sodium chloride to artificial wastewater was 2 g:1 L, the mass-volume ratio of abscisic acid to artificial wastewater was 10 mg:1 L, the mass-volume ratio of 4-methyl-5-thiazoleethanol to artificial wastewater was 10 mg:1 L, and the sterilization temperature was 121 °C.

[0091] Test Example 1: Microalgae transcriptome sequencing analysis test.

[0092] Test samples: Microalgae cultured in Examples 1-2 and Comparative Examples 2-3.

[0093] Test method: After extracting the total RNA of microalgae, transcriptome sequencing was performed. DESeq2 software was used for differential expression gene analysis. The microalgae cultured in Examples 1-2 and Comparative Example 3 were compared pairwise with the microalgae cultured in Comparative Example 2 to screen out genes with significant differential expression under different conditions. Goatools software was used for GO enrichment analysis of the genes in the gene set to obtain the functions where the differential genes were concentrated, and Fisher's exact test was used with adjusted P Padjust < 0.05. KEGG enrichment analysis was performed on the genes in DEGs and upregulated DEGs, and then compared with the KEGG database to determine the biological processes involved in the differential expression genes.

[0094] The test results of differential expression genes of the microalgae cultured in Comparative Example 3 are as Figure 1 shown, and the test results of differential expression genes of the microalgae cultured in Example 1 are as Figure 2 shown, and the test results of differential expression genes of the microalgae cultured in Example 2 are as Figure 3 shown. Under the synergistic action of abscisic acid with auxin and gibberellin, the differential expression genes of microalgae were mainly upregulated, indicating that these three exogenous plant hormones increased the expression of certain genes related to the salt tolerance ability of microalgae during the regulation of the growth and metabolism of Chlorella, thereby improving the growth and lipid accumulation of microalgae.

[0095] The GO analysis test results of the microalgae cultured in Comparative Example 3 are as Figure 4 shown. The GO analysis test results of the microalgae cultured in Example 1 are as Figure 5 shown. The GO analysis test results of the microalgae cultured in Example 2 are as Figure 6 shown. It can be seen that under the regulation of plant hormones, the inherent components and components of the cell membrane are significantly enriched, and mainly relieve oxidative damage under salt stress by promoting the vesicle-mediated transport from the endoplasmic reticulum to the Golgi apparatus; in Example 1, after adding abscisic acid and auxin, the catalytic activity of the microalgae and the activity of protein peptide modification activating enzymes are significantly improved, effectively improving the antioxidant capacity of the microalgae.

[0096] The KEGG analysis test results of the microalgae cultured in Comparative Example 3 are as Figure 7 shown. The KEGG analysis test results of the microalgae cultured in Example 1 are as Figure 8 shown. The KEGG analysis test results of the microalgae cultured in Example 2 are as Figure 9 shown. It can be seen that abscisic acid mainly affects the degradation proteasome autophagy of valine, leucine and isoleucine; the combined application of abscisic acid and gibberellin has a significant impact on carotenoid biosynthesis, sulfur metabolism, photosynthesis, pyrimidine metabolism, etc.; the combined application of abscisic acid and auxin mainly acts on the improvement of the biosynthetic pathway of the terpene skeleton basic signaling pathway, so as to relieve oxidative damage under salt stress through more pathways.

[0097] Test Example 2: Microalgae biomass test.

[0098] Test samples: Microalgae cultured in each example and comparative example.

[0099] Test method: Take 50 mL of algal liquid, centrifuge and discard the supernatant, dry it at 60 °C for 12 h until constant weight, and weigh it after cooling to room temperature in a desiccator.

[0100] The test results of the microalgae biomass cultured by the present invention are as Figure 10As shown, it can be seen from Comparative Examples 1-2 that the biological yield of microalgae in saline wastewater is much lower than that in non-saline wastewater, and the salinity greatly inhibits the growth of Chlorella pyrenoidosa; it can be seen from Comparative Example 3 and Examples 1-2 that the presence of abscisic acid can significantly alleviate the adverse effects caused by the salinity stress of saline wastewater on microalgae, enhance the adaptability of microalgae to salt stress, and abscisic acid and auxin or gibberellin act synergistically to play a greater advantage in regulating the growth of microalgae in saline wastewater; it can be seen from Examples 3-4 and Comparative Examples 4-5 that 4-amino-2-methylthio-pyrimidine-5-carboxamide and 4-methyl-5-thiazole ethanol can synergistically enhance the biological yield of microalgae and increase the growth of microalgae in a salt stress environment. At the same time, the adjustment of 4-amino-2-methylthio-pyrimidine-5-carboxamide can play a positive regulatory role in the growth of microalgae; it can be seen from Examples 5-6 that adding N-phenylphthalimide on the basis of 4-amino-2-methylthio-pyrimidine-5-carboxamide and 4-methyl-5-thiazole ethanol, the combined action of the three can more effectively promote the growth of microalgae in saline wastewater and regulate the physiological metabolism of microalgae cells under salt stress in a larger range.

[0101] Test Example 3: Measurement of microalgae oil yield.

[0102] Test samples: Microalgae cultured in each example and comparative example.

[0103] Test method: Take 50 mL of algal solution, 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 stand for 10 min, take the lower chloroform oil layer after centrifugation, and obtain the microalgae oil extract after nitrogen blowing, weigh to obtain the microalgae oil content, and calculate the microalgae oil yield.

[0104] The test results of the microalgae oil yield cultured by the present invention are as Figure 11As shown, it can be seen from Comparative Examples 1-2 that adding sodium chloride to the wastewater significantly increased the lipid content of microalgae. However, due to the significant inhibitory effect of sodium chloride on the growth of microalgae, the oil yield of microalgae did not increase synchronously but decreased. It can be seen from Comparative Example 3 and Examples 1-2 that abscisic acid significantly promoted the oil accumulation of Chlorella pyrenoidosa in saline wastewater. And due to the significant promoting effect of abscisic acid on the growth of microalgae in saline wastewater, the oil yield of microalgae was further increased at this time. At the same time, when abscisic acid was combined with auxin and gibberellin, the oil yield of microalgae could be increased. It can be seen from Examples 3-4 and Comparative Examples 4-5 that 4-amino-2-methylthio-pyrimidine-5-carboxamide and 4-methyl-5-thiazole ethanol had a positive impact on improving the oil content and yield of microalgae, and the two needed to work synergistically. It can be seen from Examples 5-6 that adding N-phenylphthalimide based on 4-amino-2-methylthio-pyrimidine-5-carboxamide and 4-methyl-5-thiazole ethanol, the combined effect of the three could more effectively promote the growth of microalgae and the accumulation of oil content in saline wastewater, and the lipid yield of microalgae also reached the highest.

[0105] Test Example 4: Testing of the fatty acid composition of microalgae oil.

[0106] Test samples: Microalgae oil extracts from Examples 1-2 and Comparative Examples 1-3 obtained in Test Example 2.

[0107] Test method: Dissolve 10 mg of microalgae oil extract in potassium hydroxide-methanol solution, vortex and mix well, then heat in a water bath at 75 °C for 15 min. After cooling to room temperature, add 2 mL of n-hexane, shake and extract for 5 min, centrifuge at 3000 rpm for 5 min, and take the upper n-hexane phase for GC-MS analysis to calculate the content of each fatty acid. Gas chromatography conditions: The chromatographic column is selected as a DB-5MS capillary column (30 m × 0.25 mm × 0.25 μm); the carrier gas is high-purity helium, with a flow rate of 1.0 mL / min; the inlet temperature is 250 °C; the split ratio is 10:1; the programmed temperature rise: the initial temperature is 100 °C, hold for 2 minutes, rise to 200 °C at a rate of 10 °C / min, hold for 5 min, then rise to 280 °C at a rate of 5 °C / min, and hold for 5 min. Mass spectrometry conditions: EI ion source, electron energy 70 eV; ion source temperature 250 °C; scanning range m / z 40 - 500; solvent delay time 3 min.

[0108] The test results of the fatty acid composition of the microalgae lipid cultured in the present invention are shown in Table 1: Table 1 Test results of the fatty acid composition of microalgae oil

[0109] The results show that several fatty acids with relatively high contents in the Chlorella pyrenoidosa cultured by the present invention are linoleic acid (C18:2n6c), α-linolenic acid (C18:3n3) and palmitic acid (C16:0) respectively. C16-C18 fatty acids are considered as ideal raw materials for biodiesel. Therefore, the lipids produced by the Chlorella pyrenoidosa cultured by the present invention have good prospects for being developed into biodiesel. It can be seen from Comparative Examples 1-3 that sodium chloride and abscisic acid do not significantly change the ratio of C16-C18 fatty acids, but to a certain extent change the saturation degree of fatty acids. Under 20 g / L salt stress, the proportion of saturated fatty acids in microalgae lipids decreases, the proportion of monounsaturated fatty acids increases significantly, and at the same time the proportion of polyunsaturated fatty acids decreases slightly. Under the action of abscisic acid, the proportions of saturated fatty acids and monounsaturated fatty acids in microalgae fatty acids increase slightly, and at the same time the proportion of polyunsaturated fatty acids decreases slightly. It can be seen from Examples 1-2 that the combined use of abscisic acid with auxin and gibberellin further increases the proportions of saturated fatty acids and monounsaturated fatty acids in microalgae fatty acids, and further down-regulates the proportion of polyunsaturated fatty acids in fatty acids, indicating that under the regulation of plant hormones, the stress pressure on microalgae decreases. The change in the saturation degree of microalgae fatty acids also has a certain impact on the quality of the produced biodiesel. Unsaturated fatty acids, especially polyunsaturated fatty acids, are prone to react with oxygen during storage or at high temperatures, resulting in the degradation of biodiesel. The increase in the saturation degree of fatty acids is beneficial to improving the antioxidant performance and chemical stability of the produced biodiesel. Therefore, the combined use of abscisic acid with auxin and gibberellin in the present invention is beneficial to enhancing the chemical stability of microalgae biodiesel.

[0110] The above-described embodiments and / or implementation manners are only used to illustrate the preferred embodiments and / or implementation manners for realizing the technology of the present invention, and do not impose any formal restrictions on the implementation manners of the technology of the present invention. Any person skilled in the art, without departing from the scope of the technical means disclosed in the content of the present invention, may make some modifications or changes to other equivalent embodiments, but should still be regarded as the same technology or embodiment as the present invention in essence.

[0111] Specific examples are used in this article to elaborate on the principles and implementation manners of the present application. The description of the above embodiments is only used to help understand the method and its core idea of the present application. The above is only the preferred implementation manner of the present application. It should be noted that due to the limitation of literal expression and objectively existing infinite specific structures, for those of ordinary skill in the art, without departing from the principle of the present application, several improvements, refinements or changes can be made, or the above technical features can be combined in an appropriate manner; these improvements, refinements, changes or combinations, or directly applying the concept and technical solution of the invention to other occasions without improvement, should all be regarded as the protection scope of the present application.

Claims

1. A method for promoting carbon fixation and oil production of microalgae by using phytohormones and saline wastewater, comprising: Add sodium chloride and plant hormones to the wastewater, and then sterilize it to obtain a wastewater culture medium. Cultivate microalgae in the wastewater culture medium; the plant hormones include at least abscisic acid, and the mass-volume ratio of sodium chloride to wastewater is 1-10 g: 1-50 L, and the mass-volume ratio of abscisic acid to wastewater is 1-20 mg: 100-2000 mL.

2. The method for promoting microalgae carbon fixation and oil production by using phytohormones and saline wastewater according to claim 1, wherein The sterilization temperature is 120-125 °C.

3. A method for promoting microalgae carbon fixation and oil production by using phytohormones and saline wastewater according to claim 1, characterized in that, The plant hormones also include one of auxin and gibberellin.

4. A method for promoting microalgae carbon fixation and oil production by using phytohormones and saline wastewater according to claim 3, characterized in that, The mass-volume ratio of auxin to wastewater is 1-20 mg: 100-2000 mL.

5. A method for promoting microalgae carbon fixation and oil production by using phytohormones and saline wastewater according to claim 3, characterized in that, The mass-volume ratio of gibberellin to wastewater is 1-20 mg: 100-2000 mL.

6. A wastewater culture medium, comprising: Add sodium chloride, abscisic acid, 4-amino-2-methylthio-pyrimidine-5-carboxamide, and 4-methyl-5-thiazole ethanol to the wastewater, and then sterilize it to obtain a wastewater culture medium.

7. The wastewater culture medium according to claim 6, characterized in that, The mass-volume ratio of sodium chloride to wastewater is 1-10 g: 1-50 L.

8. A wastewater culture medium according to claim 6, characterized in that, The mass-volume ratio of abscisic acid to wastewater is 1-20 mg: 100-2000 mL.

9. A wastewater culture medium according to claim 6, wherein The mass-volume ratio of 4-amino-2-methylthio-pyrimidine-5-carboxamide to wastewater is 1-20 mg: 100-2000 mL.

10. A wastewater culture medium according to claim 6, characterized in that, The mass-volume ratio of 4-methyl-5-thiazole ethanol to wastewater is 1-20 mg: 100-2000 mL.

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