Method for improving heat resistance of microalgae

Abstract

The invention relates to a method for improving the heat resistance of microalgae by adding phospholipid or alcohol amine solvents, which comprises the following steps: selecting separated and purified microalgae, inoculating a single algae colony of the microalgae to a BG11 liquid culture medium, respectively preparing phospholipid or alcohol amine solvents of different types and different concentrations, and selecting the optimal treatment concentration and the appropriate solvent type; grouping the algae liquid, adding phospholipid or alcohol amine solvents with the corresponding optimal concentration into an experimental group, and not adding phospholipid or alcohol amine solvents into a control group; after high-temperature stress, comparing the two groups of growth rates, cell survival rates and various physiological indexes; results show that the heat resistance of the algal strain treated by the phospholipid or alcamines solvent is obviously improved, and the heat resistance shows that the cell survival rate is improved, lipid peroxidation is reduced, the antioxidant enzyme activity is enhanced, and the proline content is increased; the method is simple to operate and low in cost, and an effective solution is provided for large-scale culture and application of microalgae in a high-temperature environment.

Description

Technical Field

[0001] The present invention belongs to the field of algal biology and relates to a method for improving the heat tolerance of microalgae. Background Art

[0002] Due to the characteristics of high protein content, rapid growth, and easy cultivation, microalgae play an important role in many fields such as food, feed, biofuels, and wastewater treatment. For example: in the food industry, it is used as a nutritional supplement; in the feed industry, it is used as an additive for animal feed; in the biofuel field, microalgae are regarded as potential biodiesel raw materials due to their high lipid content; in wastewater treatment, it can also effectively remove nutrients such as nitrogen and phosphorus in water. Thus, microalgae have high nutritional and economic value.

[0003] The persecution of microalgae in high-temperature environments is manifested as multiple physiological damages: in high-temperature environments, the activity of key enzymes in photosynthesis is inhibited, resulting in a decrease in carbon fixation efficiency, which in turn leads to the accumulation of reactive oxygen species and oxidative stress, damaging biological macromolecules; the change in cell membrane fluidity triggers ion imbalance and membrane lipid peroxidation, ultimately leading to cell lysis. High temperature also inhibits the growth and reproduction of microalgae by prolonging the cell cycle, promotes the extinction of sensitive species, and changes the community structure, posing a dual threat to ecosystem functions and the application of microalgae biotechnology. With the continuous warming of the environment under the background of global warming, the sustainable utilization of microalgae resources in high-temperature environments still faces severe challenges.

[0004] Currently, the methods for improving the heat tolerance of microalgae mainly include genetic engineering modification and optimization of culture conditions. Although the genetic engineering method has significant effects, it has problems such as complex technology, high cost, and safety disputes; while traditional methods for optimizing culture conditions, such as adjusting light, temperature, nutrients, etc., often have limited improvement effects and are difficult to meet the actual production needs. Therefore, it is of great practical significance to develop a simple, efficient, low-cost, and obvious improvement method to improve the heat tolerance of microalgae.

[0005] Based on the above background, the present invention proposes a method for improving the heat tolerance of microalgae by adding phospholipids or alkanolamines. By adding phospholipids or alkanolamines externally, tolerance is established during the growth process of microalgae, enabling microalgae to still grow and reproduce normally under high-temperature conditions. This method improves the heat tolerance of microalgae and provides a new idea for future research on the resistance ability of microalgae. Summary of the Invention

[0006] Aiming at the shortcomings of the prior art, the present invention provides a method for improving the heat tolerance of microalgae by adding phospholipids or alkanolamines. By adding an appropriate amount of phospholipid solvent or alkanolamine solvent to the ordinary microalgae culture medium, and relying on the roles played by the above two reagents in plant stress resistance, the heat tolerance of microalgae is improved.

[0007] To achieve the above object, the present invention adopts the following technical solutions:

[0008] A method for improving the heat tolerance of microalgae, comprising the following steps:

[0009] 1) Inoculate the purified microalgae into BG11 liquid medium and culture them to the logarithmic growth phase under the conditions of 28°C and 2000 lx light;

[0010] 2) Weigh an appropriate amount of phospholipid, dissolve it in chloroform to prepare a high-concentration stock solution, and set different concentration gradients for the working solution; prepare working solutions with different concentration gradients using pure amine solvents;

[0011] 3) Optimize the concentrations of the phospholipid solvent and the amine solvent: After treating the algal strain with phospholipid and amine solvents at different concentrations respectively, observe whether there is any toxicity to the algal strain, and then determine the optimal treatment concentration.

[0012] During the concentration optimization process, screen and determine the appropriate treatment concentration, add the phospholipid or amine solution with the appropriate treatment concentration to the algal liquid in the logarithmic growth phase, and continue to culture for 24 h under the conditions of 28°C and 1500 lx light, with the light-dark cycle being 18 h / 6 h respectively;

[0013] 4) Subject the algal liquid cultured in step 3) to stress treatment at high temperature for 2 - 4 h;

[0014] 5) Measure physiological indicators: Use proline (Proline, PRO), malondialdehyde (Malondialdehyde, MDA), catalase (Catalase, CAT), superoxide dismutase (Superoxide dismutase, SOD), peroxidase (Peroxidase, POD) assay kits to detect the physiological and biochemical indicators (including cell survival rate, antioxidant enzyme activity, and proline content) of the algal liquid after stress treatment under different treatments. The determination of chlorophyll content refers to the relevant standard methods to evaluate the improvement effect of the heat tolerance of microalgae.

[0015] Preferably, in step 1), the purified microalgae are a single purified algal species, and the algal species includes one of Chlorella, Scenedesmus, Chlamydomonas, or Coelastrum.

[0016] Further, the algal strain in step 1) is Chlorella sp. obtained by isolation and purification.

[0017] Preferably, in step 2), the phospholipid solvent is one of phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, or phosphatidylglycerol.

[0018] Further, the phospholipid solvent described in step 2) is phosphatidylethanolamine (PE).

[0019] Preferably, the alkanolamine solvent described in step 2) is one of ethanolamine, diethanolamine, methyldiethanolamine or diisopropanolamine.

[0020] Further, the alkanolamine solvent described in step 3) is ethanolamine (MEA).

[0021] Preferably, the different concentration gradient ranges of the phospholipid solvent described in step 2) are set at 0 - 50 mg / L; the different concentration gradient ranges of the alkanolamine solvent are set at 0 - 3 mmol / L; the concentrations of the phospholipid solvent and the alkanolamine solvent are not both 0 at the same time.

[0022] Preferably, the suitable treatment concentration of the phospholipid solvent in step 3) is 5 - 15 mg / L; the suitable treatment concentration of the alkanolamine solvent is 1.6×10 -7 ~1.6×10 -3 mmol / L.

[0023] Further, the suitable treatment concentration of the lipid solvent is 10 mg / L; the suitable treatment concentration of the alkanolamine solvent is 1.6×10 -5 mmol / L.

[0024] Preferably, the high - temperature condition range in step 4) is 25°C - 50°C.

[0025] Preferably, in step 5), after adding the phospholipid solvent or the alkanolamine solvent, the ranges of the improvement of the heat resistance of microalgae are respectively: 20.02 - 41.21%, 21.31 - 42.15%.

[0026] Advantages of the present invention:

[0027] 1) By separately preparing phospholipid solvents or alkanolamine solvents of different types and different concentrations, determining the optimal treatment concentration with less influence on the growth of microalgae through preliminary experiments; grouping the algal liquid in the logarithmic growth phase, adding the corresponding optimal - concentration phospholipid or alkanolamine solvent to the experimental group and not adding it to the control group; after high - temperature stress (45°C, 3 h), comparing the growth rates, cell survival rates and various physiological indexes of the two groups; the obtained results show that the cell survival rate of the treatment group increases, lipid peroxidation decreases, antioxidant enzyme activity enhances, and the proline content increases, indicating that the heat resistance of microalgae is improved;

[0028] 2) By utilizing the regulatory effects of monoethanolamine (MEA) and phosphatidylethanolamine (PE) on lipid metabolism and cell membrane stability, the expression level of serine decarboxylase is increased, the content of ethanolamine is increased, thereby promoting the synthesis of phosphatidylethanolamine, and ultimately achieving the improvement of the heat resistance of microalgae;

[0029] 3) This method is simple to operate and low in cost, providing an effective solution for the large-scale cultivation and application of microalgae in high-temperature environments. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 It is a schematic diagram of the growth of algal groups treated with different concentrations of PE and MEA;

[0031] Figure 2 It is a schematic diagram of cell survival rate data and the effect diagram of methylene blue staining after heat treatment of algal strains treated with MEA and PE at 28 °C and 45 °C for 3 h. Among them, the blue algal cells are dead cells and the green algal cells are live cells;

[0032] Figure 3 It is a comparison chart of physiological indexes of Chlorella vulgaris before and after treatment with phosphatidylethanolamine (PE) and ethanolamine (MEA) at 45 °C;

[0033] Figure 4 It is a comparison chart of the cell survival amounts of algal strains treated with MEA and PE after heat treatment at different temperatures. DETAILED DESCRIPTION OF THE INVENTION

[0034] The present invention will be further described below through specific examples. In order to make the invention purpose, technical solutions and beneficial technical effects of the present invention clearer, the present invention will be further described in detail below in conjunction with the examples. It should be understood that the examples described in this specification are only for explaining the present invention and not for limiting the present invention.

[0035] Unless otherwise stated, all the film adhesives and reagents used in the examples can be obtained commercially or synthesized according to conventional methods, and can be used directly without further treatment, and the instruments used in the examples can be obtained commercially.

[0036] Example 1:

[0037] The microalgae used in the present invention are sourced as follows: a single pure algal species of Chlorella vulgaris obtained by isolation and purification.

[0038] 1. Obtaining of the experimental algal strain Chlorella vulgaris algal solution

[0039] Under sterile conditions, use an inoculation loop to pick a single colony of Chlorella purified and stored on solid medium and inoculate it into 100 mL of BG11 liquid medium for shaking culture. The culture temperature is 28 °C, the light-dark cycle is 18 h / 6 h respectively, and the light intensity is 2000 lx. When the algal cells grow to the logarithmic growth phase, perform subsequent experimental treatments.

[0040] 2. Preparation of phosphatidylethanolamine (PE) and monoethanolamine (MEA)

[0041] Preparation of phosphatidylethanolamine PE solution: Weigh an appropriate amount of PE and dissolve it in chloroform to prepare a high-concentration stock solution of 100 mg / L. Set the concentration gradient of the working solution in the range of 0 - 40 mg / L.

[0042] Preparation of monoethanolamine (MEA) solution: Purchase high-purity monoethanolamine from a biological reagent company. Set the concentration gradient of the working solution in the range of 0 - 3 mmol / L.

[0043] 3. Concentration optimization of phosphatidylethanolamine (PE) and monoethanolamine (MEA)

[0044] Different algae have different sensitivities to PE and MEA, and the optimal treatment concentration needs to be determined through preliminary experiments.

[0045] PE concentration optimization: Divide the algal solution into a control group and an experimental group. Add different concentrations of PE solution to the experimental group, and do not add PE to the control group. Keep other conditions the same as those of the treatment group. Set 3 biological replicates for each group of experiments to ensure the reliability of the data. After static culture for 7 - 10 days, observe the growth status of the algal strains.

[0046] MEA concentration optimization: Divide the algal solution into a control group and an experimental group. Add MEA treatment solution to the experimental group, and do not add MEA to the control group. Keep other conditions the same as those of the treatment group. Observe the growth status of the algal strains after static culture for 7 - 10 days.

[0047] The results of the growth status of microalgae are as Figure 1 shown. Group A is the algal strains treated with different concentrations of PE, and Group B is the algal strains treated with different concentrations of MEA. It was observed that when the concentration of phosphatidylethanolamine PE was 10 mg / L, the growth of the algal strains was similar to that of the control group, and when the concentration of monoethanolamine MEA was 1.6×10 -5 mmol / L, the growth of the algal strains was not much different from that of the control group.

[0048] 4. Determination of algal cell viability using methylene blue

[0049] After determining the optimal treatment concentrations of MEA and PE, the algal solution in the logarithmic growth phase was aliquoted and treated with MEA and PE respectively. After culturing for 24 h under the same light and temperature conditions, the algal strains were subjected to high-temperature stress treatment at 45 °C for 3 h. Using the principle that methylene blue can penetrate the cell membrane of dead cells and bind to intracellular substances to make them colored, while the cell membrane of live cells is intact and the dye cannot enter and is not colored, the experimental group and the control group after high-temperature stress were immediately stained with methylene blue to obtain the survival rate of algal cells.

[0050] The results are as Figure 2 shown. After observation by methylene blue staining, the number of surviving algal cells at 28 °C in the control group was more than that under the 45 °C stress treatment, indicating that the condition of 28 °C was more suitable for algal growth; however, when comparing the number of surviving algal cells directly heat-treated at 45 °C for 3 h without adding PE and MEA with the number of surviving algal cells of Chlorella treated with PE and MEA under the condition of heat treatment at 45 °C for 3 h, it was found that the treatment groups with PE and MEA respectively had a significant increase compared with the untreated group. The data showed that the cell survival rates of the PE treatment group and the MEA treatment group increased by 33% and 35% respectively compared with the untreated group.

[0051] 5. Determination and comparison of the physiological indexes of Chlorella, including the following:

[0052] (1) Determination of malondialdehyde (MDA) content

[0053] Weigh 0.05 - 0.2 g of algal mud, add 0.5 - 2 mL of extraction solution, and perform ice bath homogenization. Centrifuge at about 12000 rpm at 4 °C for 10 min and take the supernatant. Add 300 uL of working solution to 200 uL of the sample, mix well, incubate in a water bath at 90 - 95 °C for 30 min, take it out and cool on ice, centrifuge at 12000 rpm at 25 °C for 10 min, take 200 uL of the supernatant and transfer it to a 96-well plate, and read the absorbance A at 523 nm and 600 nm respectively. ΔA = A532 - A600.

[0054] (2) Determination of superoxide dismutase (SOD) activity

[0055] Weigh 0.05 - 0.2 g of algal mud, add 0.5 - 2 mL of extraction solution, and perform ice bath homogenization. Centrifuge at about 12000 rpm at 4 °C for 10 min and take the supernatant. After adding the reagent at room temperature (25 °C), measure the absorbance value A of each tube at 450 nm.

[0056] (3) Determination of peroxidase (POD) activity

[0057] Weigh 0.05 - 0.2 g of algal sludge, add 0.5 - 2 mL of extraction solution, and perform homogenization in an ice bath. Centrifuge at about 12,000 rpm for 10 min at 4°C, and take the supernatant. Add the reagents in the kit in the order specified in the instruction manual, mix well, immediately measure the absorbance A1 at 470 nm, measure A2 after 1 min, and ΔA = A2 - A1.

[0058] (4) Determination of catalase (CAT) activity

[0059] Weigh 0.05 - 0.2 g of algal sludge, add 0.5 - 2 mL of extraction solution, and perform homogenization in an ice bath. Centrifuge at about 12,000 rpm for 10 min at 4°C, and take the supernatant. Add the reagents in the kit in the order specified in the instruction manual, mix well, react at room temperature (25°C) for 5 min, take 200 μL and transfer it to a 96-well plate, measure the absorbance A at 510 nm, and ΔA = Ablank - Aassay.

[0060] (5) Determination of proline (PRO) content

[0061] Weigh 0.05 - 0.2 g of algal sludge, add 0.5 - 2 mL of extraction solution, and perform homogenization in an ice bath. Centrifuge at about 12,000 rpm for 10 min at 4°C, and take the supernatant. Add the reagents in the kit in the order specified in the instruction manual and mix well, place in a water bath at 95°C for 30 min, and cool to room temperature. Pipette 200 μL of the clear liquid into a 96-well plate, immediately measure the absorbance A at 520 nm, and ΔA = A 测定 -A 空白 .

[0062] The results are as Figure 3 shown. From the perspective of the effect of high temperature on the chlorophyll of Chlorella, the chlorophyll content of the three groups of different treated algal strains gradually decreased with the increase of the high temperature stress degree. The malondialdehyde (MDA) content of the algal strains treated with PE and MEA under the stress of 45°C for 3 h was lower than that of the control group, which indicates that PE and MEA reduced the lipid peroxidation of algal cells to a certain extent. Under the three groups of different high temperature stresses, the activities of SOD, POD, and CAT of the algal strains were generally significantly higher than those of the control group, but there were obvious differences in the increase amplitude. Thus, after the treatment of PE and MEA, the antioxidant enzyme activities of algal cells can be activated to varying degrees under the high temperature stress level of 45°C, so as to scavenge the excessive free radicals in the body and reduce the degree of damage to plants. Under different high temperature stresses, the PRO content of the MEA-treated group of algal strains was higher than that of the untreated group and the PE-treated group. The difference in the PRO content of Chlorella in different treatment groups indirectly affected the adaptability of algal cells to the high temperature stress environment after the treatment of MEA and PE.

[0063] Example 2:

[0064] Replace the following conditions: The cultured algal solution in step 3) is stressed at high temperature for 2 h; the rest is the same as in Example 1.

[0065] Example 3:

[0066] Replace the following conditions: The algal solution after cultivation in step 3) is stress-treated at high temperature for 4 h; the rest is the same as in Example 1.

[0067] The technical principle used in the present invention in the above embodiments is: Utilize the regulatory effects of monoethanolamine (MEA) and phosphatidylethanolamine (PE) on lipid metabolism and cell membrane stability; By analyzing the lipid metabolism pathway, it can be seen that the algal strain increases the content of ethanolamine by increasing the expression level of serine decarboxylase, thereby promoting the synthesis of phosphatidylethanolamine, and finally achieving the improvement of the heat tolerance of microalgae.

[0068] The above embodiments show that the present invention provides a simple and convenient method for changing the heat tolerance of microalgae:

[0069] Taking the results in Examples 1 and 2 as a specific illustration, Figure 1 The results intuitively show the effects of different concentration treatments on the growth of algal strains, providing a basis for determining the optimal treatment concentration; Figure 2 The results clearly show the differences in cell survival between the treatment group and the control group at different temperatures, strongly proving the improvement of the heat tolerance of the algal strain after treatment; Figure 3 Details show the changes in physiological indexes such as MDA, SOD, POD, CAT, and PRO before and after reagent treatment, revealing the mechanism of PE and MEA in improving the heat tolerance of microalgae from a physiological level, and verifying the effect of the reagents (ethanolamine and phosphatidylethanolamine) on establishing tolerance during the growth process of microalgae to improve heat tolerance performance; Figure 4 It shows that the conditions of high temperature stress are preferably 25 - 50. Beyond this range, the number of surviving cells is small.

[0070] The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. Any form of modification, equivalent replacement, improvement, etc. within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A method for improving the heat resistance of microalgae, characterized in that, It includes the following steps: 1) Inoculate the purified microalgae into BG11 liquid medium and culture them under the conditions of 28°C and 2000 lx light until the logarithmic growth phase; 2) Prepare phospholipid solvents and alkanolamine solvents with different concentration gradients and optimize their concentrations; 3) During the process of optimizing the concentration, screen and determine the appropriate treatment concentration, add the phospholipid or alkanolamine solution with the appropriate treatment concentration to the algal solution in the logarithmic growth phase, and continue to culture it for 24 h under the conditions of 28°C, 1500 lx light, and a light-dark ratio of 16 / 8 h; 4) Subject the algal solution cultured in step 3) to stress treatment at high temperature for 2 - 4 h; 5) Detect the cell viability, antioxidant enzyme activity, and proline content of the algal solution after the stress treatment in step 4) to evaluate the improvement effect of the heat tolerance of the microalgae.

2. The method for improving the heat resistance of microalgae according to claim 1, characterized in that In step 1), the purified microalgae are a single purified algal species obtained by separation and purification, and the algal species includes one of Chlorella, Scenedesmus, Chlamydomonas, or Coelastrum.

3. A method for improving the heat resistance of microalgae according to claim 1, characterized in that, In step 2), the phospholipid solvent is one of phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, or phosphatidylglycerol.

4. A method for improving the heat resistance of microalgae according to claim 1, characterized in that, In step 2), the alkanolamine solvent is one of monoethanolamine, diethanolamine, methyldiethanolamine, or diisopropanolamine.

5. A method for improving the heat resistance of microalgae according to claim 1, characterized in that, In step 2), the different concentration gradient ranges of the phospholipid solvent are set at 0 - 50 mg / L.

6. A method for improving the heat resistance of microalgae according to claim 5, characterized in that, In step 2), the different concentration gradient ranges of the alkanolamine solvent are set at 0 - 3 mmol / L; The concentrations of the phospholipid solvent and the alkanolamine solvent are not both 0 at the same time.

7. A method for improving the heat resistance of microalgae according to claim 6, characterized in that, In step 3), the appropriate treatment concentration of the phospholipid solvent is 5 - 15 mg / L.

8. A method for improving the heat resistance of microalgae according to claim 7, characterized in that, Step 3) The appropriate treatment concentration of the alkanolamine solvent is 1.6×10 -7 ~1.6×10 -3 mmol / L.

9. A method for improving the heat resistance of microalgae according to claim 1, characterized in that, In step 4), the high temperature condition range is 25°C - 50°C.

10. A method for improving the heat resistance of microalgae according to claim 1, characterized in that, In step 5), the range of the improvement in the heat tolerance of the microalgae after adding the phospholipid solvent is: 20.02 - 41.21%; the range of the improvement in the heat tolerance of the microalgae after adding the alkanolamine solvent is 21.31 - 42.15%.

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