Method for regulating environment to promote astaxanthin synthesis of microcystis aeruginosa

Abstract

The application discloses a method for adjusting environment to promote astaxanthin synthesis of Microcystis aeruginosa, and specifically comprises the following steps: culturing Microcystis aeruginosa, determining optimal light quality, determining optimal light intensity and determining optimal temperature and the like. Under the same light intensity and temperature, different light qualities are replaced to culture, white light is taken as a control group, and the optimal light quality for astaxanthin output is determined; under the same light quality and temperature, different light intensities are replaced to culture, and the optimal light intensity for astaxanthin output is determined; under the same light intensity and light quality, different temperatures are replaced to culture, and the optimal temperature for astaxanthin output is determined. In the application, the control variable method is adopted to determine the optimal light quality condition, the optimal light intensity and the optimal temperature for astaxanthin synthesis of Microcystis aeruginosa, so as to provide a theoretical basis for promoting astaxanthin production and resource utilization of Microcystis aeruginosa.

Description

Technical Field

[0001] This invention relates to the field of astaxanthin synthesis technology from Microcystis aeruginosa, and particularly to a method for regulating the environment to promote astaxanthin synthesis from Microcystis aeruginosa. Background Technology

[0002] Astaxanthin (3,3'-dihydroxy-β,β'-carotene-4,4'-dione) is a high-value ketone carotenoid with extremely strong antioxidant capabilities. Its antioxidant capacity is more than 10 times that of other carotenoids and 550 times that of vitamin E. Therefore, astaxanthin is known as a "super antioxidant" or "king of antioxidants" and has been widely used in food, cosmetics, medicine, and aquaculture.

[0003] The market mainly offers chemically synthesized astaxanthin, but its effects on human health are still unclear. Compared to chemically synthesized astaxanthin, natural astaxanthin is safer and has stronger antioxidant capabilities; however, it can only be synthesized in a few bacteria, yeasts, certain microalgae, crabs, shrimps, trout, salmon, and other organisms that feed on microalgae.

[0004] Cyanobacteria are prokaryotic algae that can inhibit the growth of aquatic organisms by releasing algal toxins and volatile organic compounds, leading to a series of environmental and ecological problems. *Microcystis aeruginosa* is a major algal species forming cyanobacterial blooms. Its extracts mainly contain astaxanthin and phytoene, exhibiting strong antioxidant and anti-inflammatory activities, indicating that *Microcystis aeruginosa* has the potential to produce astaxanthin. The astaxanthin synthesis process is highly sensitive to light quality, light intensity, and temperature, and these environmental conditions can be used to regulate astaxanthin synthesis. Therefore, determining the optimal light quality, light intensity, and temperature for astaxanthin synthesis in *Microcystis aeruginosa* is crucial for the large-scale production of astaxanthin using this algae. Summary of the Invention

[0005] (a) Technical problems to be solved

[0006] This invention can solve the problem that the lack of relevant research on the synthesis of astaxanthin by Microcystis aeruginosa makes it difficult to provide a theoretical basis for the production of astaxanthin from Microcystis aeruginosa and the resource utilization of Microcystis aeruginosa.

[0007] (II) Technical Solution

[0008] To achieve the above objectives, the present invention adopts the following technical solution: a method for regulating the environment to promote the synthesis of astaxanthin from Microcystis aeruginosa, specifically including the following steps:

[0009] S1, Cultivation of Microcystis aeruginosa: At a temperature of 25℃, a light exposure of 16h, and a light intensity of 30μmol·m⁻¹ -2 ·s -1The algae were cultured in darkness for 8 hours and at a shaking speed of 90 rpm until the algal cell density reached the logarithmic growth phase, at which point they were used for subsequent treatment.

[0010] S2, Determine the optimal light quality: Under the same light intensity and temperature, culture with different light qualities, and use white light as a control group to determine the optimal light quality for astaxanthin production;

[0011] S3, Determine the optimal light intensity: Under the same light quality and temperature, cultivate with different light intensities to determine the optimal light intensity for astaxanthin production;

[0012] S4, Determine the optimal temperature: Under the same light intensity and quality, cultivate at different temperatures to determine the optimal temperature for astaxanthin production.

[0013] As a preferred embodiment of the present invention, the specific steps for determining the optimal light quality are as follows:

[0014] S21 was cultured at 1×10⁻⁶ ℃ under white, purple, blue, and red light respectively. 7 cells / ml -1 A concentration of Microcystis aeruginosa maintained at a light intensity of 30 μmol·m -2 ·s -1 The temperature was 25℃, and several repetitions were set for each light quality treatment.

[0015] S22, during treatment period 1-5 days, algal cell density was measured;

[0016] S23, on days 1, 3 and 5, the content of photosynthetic pigments, photosynthetic rate, astaxanthin and its precursors were measured;

[0017] S24, day 5, analyze the expression of relevant genes under white light and purple light culture.

[0018] As a preferred embodiment of the present invention, the specific steps for determining the optimal light intensity are as follows:

[0019] S31, using 30, 200, 500 and 1000 μmol·m⁻¹ respectively. -2 ·s -1 Light intensity processing 1×10 7 cells / ml -1 The concentration of Microcystis aeruginosa was controlled at 25°C under white light. Several replicates were performed for each light intensity treatment, with an illumination of 30 μmol·m⁻¹. -2 ·s -1 The treatment was used as a control;

[0020] S32, algal cell density was measured on days 1-5;

[0021] S33, on days 1, 3 and 5, measured the content of photosynthetic pigments, photosynthetic performance, astaxanthin and its precursors.

[0022] As a preferred embodiment of the present invention, the specific steps for determining the optimal temperature are as follows:

[0023] S41 was treated at 25℃, 30℃, and 35℃ respectively, with 1×10 7 cells / ml -1 A concentration of Microcystis aeruginosa at a light intensity of 30 μmol·m -2 ·s -1 The light quality was white light, and several replicates were set for each temperature treatment, with 25℃ treatment as a control.

[0024] S42, algal cell density was measured on days 1-5;

[0025] S43, on days 1, 3 and 5, measured the content of photosynthetic pigments, photosynthetic performance, astaxanthin and its precursors in algal cells.

[0026] As a preferred technical solution of the present invention, the specific steps for determining the photosynthetic pigment content are as follows: take 3 ml of Microcystis aeruginosa culture medium and centrifuge at 6000 rpm for 8 min; extract photosynthetic pigments with 3 ml of 80% acetone; after the photosynthetic pigments are extracted, determine the chlorophyll and carotenoid content using a spectrophotometer.

[0027] As a preferred technical solution of the present invention, the specific steps for determining the photosynthetic rate are as follows: the photosynthetic rate of Microcystis aeruginosa is determined by using an oxygen electrode; 20 ml of Microcystis aeruginosa culture medium is injected into the measurement chamber, LED lamps are used to provide illumination and the temperature is maintained at 25°C, and the release rate is recorded after the O2 release amount stabilizes, and the photosynthetic rate of the algal cells is calculated.

[0028] As a preferred embodiment of the present invention, the specific steps for determining photosynthetic performance are as follows: Take a certain amount of Microcystis aeruginosa culture medium, centrifuge at 8000 rpm for 5 min, resuspend the algal cells in 10 μl of culture medium, and use a pipette to aspirate and drop the cells onto filter paper to form a shape approximately 1 cm in size. 2 Spots were dark-adapted for 15 min; chlorophyll fluorescence induction kinetics were measured using an unmodulated chlorophyll fluorometer; the maximum quantum yield of photosystem II, electron transport efficiency due to trapped exciton energy, quantum yield of electron transport, maximum quantum yield of non-photochemical quenching, and photosynthetic performance index were calculated using Rohácek's method.

[0029] As a preferred embodiment of the present invention, the specific steps for determining the content of astaxanthin and its precursors are as follows: Astaxanthin, β-carotene, and zeaxanthin from *Microcystis aeruginosa* are extracted with 80% acetone; the analysis is performed using high-performance liquid chromatography (HPLC), with the following specific steps: Analysis is performed using a 25cm × 4.6mm, 5μm reversed-phase C18 column at 25℃, with an injection volume of 10μL, a mobile phase of dichloromethane:acetonitrile:methanol at a ratio of 20:70:10, and a flow rate of 1ml·min. -1 The three compounds in the extract were quantitatively analyzed using astaxanthin, β-carotene, and zeaxanthin standards.

[0030] As a preferred embodiment of the present invention, the specific steps for analyzing the expression of relevant genes are as follows: Total RNA is extracted from Microcystis aeruginosa cultured under white and purple light using a total RNA extraction kit. After purification, it is reverse transcribed into cDNA, and then a polyA is ligated to the 3' end of the cDNA. The cDNA is then amplified by PCR, and transcriptome analysis is performed to obtain high-quality reads. After obtaining the high-quality reads, sequence alignment is performed. Gene expression analysis is conducted based on the expected number of fragments per thousand base pairs of transcripts obtained from sequencing per million base pairs. Differentially expressed genes (DEGs) are analyzed using software, and KEGG functional annotation is performed.

[0031] (III) Beneficial Effects

[0032] 1. The method for regulating the environment to promote astaxanthin synthesis in Microcystis aeruginosa provided by this invention uses the controlled variable method to determine the optimal light quality conditions, optimal light intensity, and optimal temperature for astaxanthin synthesis in Microcystis aeruginosa, thereby providing a theoretical basis for promoting astaxanthin production and the resource utilization of Microcystis aeruginosa. Attached Figure Description

[0033] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of the present invention and should not be regarded as a limitation of the scope. For those skilled in the art, other related drawings can be obtained from these drawings without creative effort.

[0034] Figure 1 This is a schematic diagram illustrating the effect of different light qualities on the growth of Microcystis aeruginosa cells according to the present invention;

[0035] Figure 2 This is a schematic diagram illustrating the effect of different light qualities on the photosynthetic pigment content of Microcystis aeruginosa according to the present invention.

[0036] Figure 3 This is a schematic diagram illustrating the effect of different light qualities on the photosynthetic rate of Microcystis aeruginosa according to the present invention.

[0037] Figure 4 This is a schematic diagram illustrating the effect of different light qualities on the content of β-carotene and its precursors in Microcystis aeruginosa.

[0038] Figure 5 This is a schematic diagram illustrating the effect of ultraviolet light on the expression of genes related to photosynthesis and astaxanthin biosynthesis according to the present invention;

[0039] Figure 6 This is a schematic diagram illustrating the effect of light intensity on the growth of Microcystis aeruginosa cells according to the present invention;

[0040] Figure 7 This is a schematic diagram illustrating the effect of light intensity on photosynthetic pigment content according to the present invention;

[0041] Figure 8 This is a schematic diagram illustrating the effect of light intensity on the fluorescence-induced kinetics curve of Microcystis aeruginosa chlorophyll.

[0042] Figure 9 This is a schematic diagram illustrating the effect of light intensity on the chlorophyll fluorescence dynamics parameters of Microcystis aeruginosa according to the present invention;

[0043] Figure 10 This is a schematic diagram illustrating the effect of light intensity on the content of astaxanthin precursor from Microcystis aeruginosa according to the present invention.

[0044] Figure 11 This is a schematic diagram illustrating the effect of light intensity on the astaxanthin content of Microcystis aeruginosa according to the present invention.

[0045] Figure 12 This is a schematic diagram illustrating the effect of high temperature on the growth of Microcystis aeruginosa cells according to the present invention;

[0046] Figure 13 This is a schematic diagram illustrating the effect of high temperature on photosynthetic pigment content according to the present invention;

[0047] Figure 14 This is a schematic diagram illustrating the effect of high temperature on the chlorophyll fluorescence induction kinetics curve of Microcystis aeruginosa according to the present invention;

[0048] Figure 15 This is a schematic diagram illustrating the effect of high temperature on the chlorophyll fluorescence kinetic parameters of Microcystis aeruginosa according to the present invention;

[0049] Figure 16 This is a schematic diagram illustrating the effect of high temperature on the content of astaxanthin precursor from Microcystis aeruginosa according to the present invention.

[0050] Figure 17 This invention aims to demonstrate the effect of high temperature on the astaxanthin content of Microcystis aeruginosa. Detailed Implementation

[0051] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0052] Example 1

[0053] like Figures 1 to 14 As shown, a method for regulating the environment to promote astaxanthin synthesis in Microcystis aeruginosa specifically includes the following steps:

[0054] S1, Cultivation of Microcystis aeruginosa: At a temperature of 25℃, a light exposure of 16h, and a light intensity of 30μmol·m⁻¹ -2 ·s -1 The algae were cultured in darkness for 8 hours and on a shaker at 90 rpm until the cell density reached the logarithmic growth phase, at which point they were used for subsequent treatment.

[0055] S2, Determine the optimal light quality: Under the same light intensity and temperature, culture with different light qualities, and use white light as a control group to determine the optimal light quality for astaxanthin production.

[0056] In this embodiment, the specific steps for determining the optimal light quality are as follows:

[0057] S21 was cultured at 1×10⁻⁶ ℃ under white, purple, blue, and red light respectively. 7 cells / ml -1 A concentration of Microcystis aeruginosa maintained at a light intensity of 30 μmol·m -2 ·s -1 The temperature was 25℃, and several repetitions were set for each light quality treatment.

[0058] S22, during treatment period 1-5 days, algal cell density was measured;

[0059] S23, on days 1, 3, and 5, the content of photosynthetic pigments, photosynthetic rate, astaxanthin, and its precursors were measured. Specifically, the photosynthetic rate measurement procedure was as follows: The photosynthetic rate of *Microcystis aeruginosa* was measured using an oxygen electrode; 20 ml of *Microcystis aeruginosa* culture medium was injected into the measurement chamber, LED light was used to provide illumination, and the temperature was maintained at 25℃. After the O2 release stabilized, its release rate was recorded, and the photosynthetic rate of the algal cells was calculated. The oxygen electrode used was model YZQ-201A, manufactured by Yizongqi Technology Co., Ltd.

[0060] On day 5 (S24), the expression of relevant genes under white and purple light cultivation was analyzed. The specific steps for gene expression analysis were as follows: Total RNA was extracted from *Microcystis aeruginosa* under white and purple light cultivation using a total RNA extraction kit. After purification, it was reverse transcribed into cDNA, and then a polyA ligated to the 3' end of the cDNA. PCR amplification was performed, followed by transcriptome analysis to obtain high-quality reads. Sequence alignment was then performed on the high-quality reads. Gene expression analysis was conducted based on the expected number of fragments per thousand base pairs of transcripts obtained from sequencing per million base pairs.

[0061] S3, Determine the optimal light intensity: Under the same light quality and temperature, cultivate with different light intensities to determine the optimal light intensity for astaxanthin production.

[0062] In this implementation, the specific steps for determining the optimal light intensity are as follows:

[0063] S31, using 30, 200, 500 and 1000 μmol·m⁻¹ respectively. -2 ·s -1 Light intensity processing 1×10 7 cells / ml -1 The concentration of Microcystis aeruginosa was controlled at 25°C under white light. Several replicates were performed for each light intensity treatment, with an illumination of 30 μmol·m⁻¹. -2 ·s -1 The treatment was used as a control;

[0064] S32, algal cell density was measured on days 1-5;

[0065] S33, on days 1, 3 and 5, measured the content of photosynthetic pigments, photosynthetic performance, astaxanthin and its precursors.

[0066] S4, Determine the optimal temperature: Under the same light intensity and quality, cultivate at different temperatures to determine the optimal temperature for astaxanthin production.

[0067] In this embodiment, S41, 1×10⁻⁶ cells are treated at 25°C, 30°C, and 35°C respectively. 7 cells / ml -1 A concentration of Microcystis aeruginosa at a light intensity of 30 μmol·m -2 ·s -1 The light quality was white light, and several replicates were set for each temperature treatment, with 25℃ treatment as a control.

[0068] S42, algal cell density was measured on days 1-5;

[0069] S43, on days 1, 3 and 5, measured the content of photosynthetic pigments, photosynthetic performance, astaxanthin and its precursors in algal cells.

[0070] In this embodiment, the specific steps for determining the photosynthetic pigment content in steps S23, S33, and S43 are as follows: take 3 ml of Microcystis aeruginosa culture medium and centrifuge at 6000 rpm for 8 min; extract photosynthetic pigments with 3 ml of 80% acetone; after the photosynthetic pigment extraction is completed, determine the chlorophyll and carotenoid content using a spectrophotometer.

[0071] In this embodiment, the specific steps for determining photosynthetic performance in steps S33 and S43 are as follows: Take a certain amount of Microcystis aeruginosa culture medium, centrifuge at 8000 rpm for 5 min, resuspend the algal cells in 10 μl of culture medium, pipette the cells and drop them onto filter paper to form a spot of about 1 cm2 in size, and dark adapt for 15 min; use a YZQ-500 unmodulated chlorophyll fluorescence meter to measure its chlorophyll fluorescence and determine the chlorophyll fluorescence induction kinetic curve; calculate the maximum quantum yield (φPo), electron transport efficiency (Ψo) caused by the trapped exciton energy, quantum yield of electron transport (φEo), maximum quantum yield of non-photochemical quenching (φDo), and photosynthetic performance index (PIABS) of photosystem II (PSII) using Rohácek's method.

[0072] In this embodiment, the specific steps for determining the content of astaxanthin and its precursors in steps S23, S33, and S43 are as follows: Astaxanthin, β-carotene, and zeaxanthin from *Microcystis aeruginosa* are extracted with 80% acetone; the analysis is performed using high-performance liquid chromatography (HPLC), with the following specific steps: A 25cm × 4.6mm, 5μm reversed-phase C18 column is used for analysis at a column temperature of 25℃, an injection volume of 10μL, and a mobile phase of dichloromethane:acetonitrile:methanol in a ratio of 20:70:10, with a flow rate of 1ml·min. -1 The three compounds in the extract were quantitatively analyzed using astaxanthin, β-carotene, and zeaxanthin standards.

[0073] In summary, by using the controlled variable method, the optimal light quality, optimal light intensity, and optimal temperature for astaxanthin synthesis by Microcystis aeruginosa were determined, thus providing a theoretical basis for promoting astaxanthin production and the resource utilization of Microcystis aeruginosa.

[0074] The following data was obtained during the experiment:

[0075] I. For determining optimal light quality, please refer to [link / reference]. Figures 1-5 .

[0076] Microcystis aeruginosa was cultured under white light (control), purple light, blue light, and red light. Purple light significantly (P<0.05) promoted algal cell growth, increasing cell density by 12.7% after 5 days of culture. Blue light significantly (P<0.05) inhibited Microcystis aeruginosa growth, decreasing cell density by 25.4% on day 5. There was no significant difference between the red light treatment and the control. Figure 1 The effects of different light qualities on the growth of Microcystis aeruginosa cells. CK: control, white light. *: significant difference compared with CK at the P<0.05 level (the same below).

[0077] Effects of different light qualities on photosynthetic pigment content: Red light had little effect on chlorophyll and carotenoid content in Microcystis aeruginosa cells, with no significant differences observed during cultivation. Purple light had no significant effect on chlorophyll content, but significantly (P<0.05) increased carotenoid content on days 3 and 5. From day 3 onwards, blue light significantly (P<0.05) decreased chlorophyll content in algal cells, but had no significant effect on carotenoid content. Figure 2 The effect of different light qualities on the photosynthetic pigment content of Microcystis aeruginosa (A: chlorophyll; B: carotenoids in the figure).

[0078] Effects of different light qualities on photosynthetic rate: From day 1, red and violet light significantly (P<0.05) increased the photosynthetic rate (O2 release rate) of *Microcystis aeruginosa*, with increases of 98.6% and 97.6%, respectively, by day 5. Blue light significantly (P<0.05) decreased the photosynthetic rate of *Microcystis aeruginosa*, with a decrease of 46.8% by day 5. Figure 3 The effect of different light qualities on the photosynthetic rate of Microcystis aeruginosa.

[0079] Effects of different light qualities on the content of astaxanthin and its precursors: Compared with white light treatment, β-carotene content increased by 7.6% (P<0.05) after 5 days of purple light treatment, while it decreased by 11.9% (P<0.05) and 17.8% (P<0.05) after blue light and red light treatment, respectively. Figure 4 A). The xanthine content of maize increased by 14.3% (P<0.05) after 5 days of purple light treatment, and decreased by 31.2% (P<0.05) and 35.7% (P<0.05) after 3 and 5 days of blue light treatment, respectively. Figure 4 B). β-Carotene and zeaxanthin are precursors to astaxanthin. Their high content under ultraviolet light is beneficial for the formation of the final product, astaxanthin. Furthermore, only ultraviolet light promotes astaxanthin synthesis in *Microcystis aeruginosa*, increasing its content by 31.1% (P<0.05) and 1.4 times (P<0.05) after 3 and 5 days of treatment, respectively. Figure 4 C)( Figure 4The effect of different light qualities on the content of astaxanthin and its precursors in Microcystis aeruginosa (A: β-carotene; B: zeaxanthin; C: astaxanthin).

[0080] Effects of ultraviolet light on the expression of related genes: Compared with white light treatment, ultraviolet light treatment upregulated the expression of eight genes related to photosynthetic electron transport (psbD, psbM, psbP, psbU, psbY, psb28-2, petF, and petJ), four genes involved in carbon fixation (rpiA, PRK, FBP, and xfp), three genes in the MEP pathway (dxr, ispD, and gds), and three genes in the astaxanthin synthesis pathway (crtISO, lcy, and bkt1). Figure 5 The effect of violet light on the expression of genes related to photosynthesis and astaxanthin biosynthesis. In this figure, W: control, white light; P: violet light.

[0081] Summary of optimal light quality: Red and violet light did not affect the chlorophyll content of *Microcystis aeruginosa*, while blue light treatment for 3 days significantly reduced chlorophyll content; violet light treatment for 3 days significantly increased carotenoid content. Figure 2 ).

[0082] Photosynthesis is the main process of material and energy metabolism in algae. After one day of treatment with red and purple light, the photosynthetic rate (O2 release rate) of Microcystis aeruginosa significantly increased, while blue light showed an inhibitory effect. Figure 3 Under ultraviolet light treatment, the content of carotenoids increased significantly, which is beneficial to the capture and transfer of light energy by Microcystis aeruginosa; at the same time, eight genes related to photosynthetic electron transport and four genes related to carbon fixation were significantly upregulated. Figure 5 This promotes the photosynthetic performance of algal cells. Under red light treatment, the photosynthetic products generated by the increased photosynthetic rate may be used for storage rather than cell growth, resulting in no significant increase in cell density; under violet light treatment, the photosynthetic products generated by the high photosynthetic rate may be used for algal cell growth, thereby promoting an increase in algal cell density. Figure 1 ).

[0083] White, purple, blue, and red light had different effects on the synthesis of β-carotene, zeaxanthin, and astaxanthin in Microcystis aeruginosa. Only purple light significantly increased the contents of β-carotene, zeaxanthin, and astaxanthin. Figure 4 ). Purple light can upregulate the expression of three genes in the MEP pathway and three genes in the astaxanthin synthesis pathway. Figure 5 This promotes the formation of β-carotene, zeaxanthin, and astaxanthin.

[0084] II. For determining the optimal light intensity, please refer to [link / reference]. Figures 6-11 .

[0085] Effect of light intensity on algal cell growth: The inhibitory effect on *Microcystis aeruginosa* cell growth gradually increased with increasing light intensity. Compared with the control (30 μmol·m⁻¹), the inhibition effect on *Microcystis aeruginosa* cell growth was significantly reduced. -2 ·s -1 Compared to 200 and 500 μmol·m⁻², algal cell densities were significantly lower. -2 ·s -1 The light intensity decreased significantly (P<0.05) after 3 days of light treatment, at 1000 μmol·m -2 ·s -1 After 1 day of light intensity treatment, the intensity significantly decreased (P<0.05). Figure 6 Effect of light intensity on the growth of Microcystis aeruginosa cells, CK: control, 25℃, 30 μmol·m -2 ·s -1 Treatment. *: The difference was statistically significant at the P<0.05 level compared with the control. (The same applies below)

[0086] Effect of light intensity on photosynthetic pigment content: The content of photosynthetic pigments in *Microcystis aeruginosa* cells gradually decreased with increasing light intensity. At light intensities of 200, 500, and 1000 μmol·m⁻¹, the content of photosynthetic pigments decreased. -2 ·s -1 Under light intensity treatment, the contents of chlorophyll and carotenoids decreased significantly (P<0.05) from day 1 onwards. Figure 7 The effect of light intensity on photosynthetic pigment content (A: chlorophyll content; B: carotenoid content in the figure).

[0087] Effects of light intensity on photosynthetic performance: at 200, 500, and 1000 μmol·m -2 ·s -1 After 5 days of light intensity treatment, the chlorophyll fluorescence intensity of Microcystis aeruginosa gradually decreased from point O to point P with increasing light intensity. Figure 8 The effect of light intensity on the chlorophyll fluorescence induction kinetics of Microcystis aeruginosa. -2 ·s -1 The light intensity decreased significantly after 5 days of treatment (P<0.05), while it decreased at 500 and 1000 μmol·m⁻¹. -2 ·s -1 The light intensity decreased significantly (P<0.05) after 1 day of treatment. On day 5, φP O The levels decreased by 11.8% (P<0.05), 14.9% (P<0.05), and 20.7% (P<0.05) under the three light intensities, respectively. Figure 9 A). Similar to φPo, Ψo, φEo, and PI ABS All three light intensity treatments showed a decreasing trend. Figure 9(B, C, E). Under the three light intensities, the trend of φDo was opposite to that of φPo, increasing by 21.38% (P<0.05), 27.1% (P<0.05), and 37.9% (P<0.05) respectively on day 5. Figure 9 D)( Figure 9 The effect of light intensity on the chlorophyll fluorescence kinetic parameters of Microcystis aeruginosa is shown in the figure. A: φPo; B: Ψo; C: φEo; D: φDo; E: PI ABS )

[0088] Effect of light intensity on astaxanthin precursor content: at 200, 500, and 1000 μmol·m -2 ·s -1 Under light intensity treatment, the β-carotene content decreased significantly (P<0.05) after 1 day of treatment; after 5 days of treatment, the decreases were 18.3% (P<0.05), 43.7% (P<0.05), and 66.1% (P<0.05), respectively. Figure 10 A). 200 μmol·m -2 ·s -1 While light intensity reduced the xanthine content of zeaxanthin, there was no significant difference compared to the control, whereas the other two light intensities significantly (P<0.05) reduced the xanthine content of zeaxanthin. Figure 10 B)( Figure 10 The effect of light intensity on the content of astaxanthin precursor in Microcystis aeruginosa (in the figure, A: β-carotene content; B: zeaxanthin content).

[0089] Effects of light intensity on astaxanthin content: All three light intensity treatments increased the astaxanthin content of Microcystis aeruginosa, with 200 μmol·m⁻¹ being the most effective. -2 ·s -1 After 3 days of light intensity treatment, at 500 and 1000 μmol·m -2 ·s -1 One day after light intensity treatment, the astaxanthin content was significantly higher than the control (P<0.05). On day 5, the astaxanthin content increased by 30.9% (P<0.05), 56.1% (P<0.05), and 70.8% (P<0.05) under the three light intensity treatments, respectively. Figure 11 (Effect of light intensity on astaxanthin content in Microcystis aeruginosa).

[0090] Conclusion on optimal light intensity: 200, 500, and 1000 μmol·m⁻¹ -2 ·s -1 Light intensity treatment of Microcystis aeruginosa showed that the inhibitory effect on algal cell growth gradually increased with increasing light intensity. Figure 6Chlorophyll and carotenoids are the main photosynthetic pigments and play important roles in light energy absorption and conversion. The photosynthetic pigment content of *Microcystis aeruginosa* cells significantly decreased after high light intensity treatment. Figure 7 ).

[0091] After high light intensity treatment of Microcystis aeruginosa, its φPo, Ψo, φEo and PI were... ABS All showed a gradual decreasing trend. Figure 9 (A, B, C, E) This indicates that both photosynthetic electron production and transfer in algal cells are inhibited under high light intensity conditions. As light intensity increases, φDo gradually increases ( Figure 9 D). This may be because the absorbed light energy exceeds the amount required for the formation of downstream assimilation forces (NADPH and ATP), and the excess light energy is dissipated as heat, thus avoiding photo-oxidative damage. The chlorophyll fluorescence induction kinetics curve of *Microcystis aeruginosa* gradually decreases with increasing light intensity. Figure 8 The reason for this may be due to the obstruction of electron transfer on the PSII donor side, thus affecting P680. + This is due to accumulation. Under high light intensity treatment, the content of photosynthetic pigments in Microcystis aeruginosa decreases, electron production and transfer decrease, and heat dissipation increases, thereby reducing the photosynthetic performance of algal cells and affecting their growth.

[0092] As light intensity increases, the contents of both β-carotene and zeaxanthin gradually decrease. Figure 10 The astaxanthin content gradually increases. Figure 11 This may be because high light intensity promotes the conversion of β-carotene and zeaxanthin into astaxanthin, thereby leading to a decrease in the content of β-carotene and zeaxanthin and an increase in the content of astaxanthin.

[0093] III. For determining the optimal temperature, please refer to [link / reference]. Figures 12-17 .

[0094] Effects of high temperature on algal cell growth: The inhibitory effect on Microcystis aeruginosa cell growth gradually increased with increasing temperature. Compared with the 25℃ treatment (control), algal cell density decreased significantly (P<0.05) after 3 days of treatment at 30℃ and significantly (P<0.05) after 2 days of treatment at 35℃; by day 5, the decreases were 11.0% (P<0.05) and 19.0% (P<0.05), respectively. Figure 12 The effect of high temperature on the growth of Microcystis aeruginosa cells, CK: control, treated at 25℃. *: The difference compared with the control was statistically significant at the P<0.05 level, the same below).

[0095] Effects of High Temperature on Photosynthetic Pigment Content: The content of photosynthetic pigments in Microcystis aeruginosa cells gradually decreased with increasing temperature. Chlorophyll and carotenoid contents decreased significantly (P<0.05) after 3 days of treatment at 30℃ and significantly (P<0.05) after 1 day of treatment at 35℃. On day 5, chlorophyll content decreased by 43.0% (P<0.05) and 56.4% (P<0.05), respectively, and carotenoid content decreased by 21.7% (P<0.05) and 30.5% (P<0.05), respectively. Figure 13 The effect of high temperature on photosynthetic pigment content (in the figure, A: chlorophyll content; B: carotenoid content).

[0096] Effects of high temperature on chlorophyll fluorescence: Under high temperature treatments of 30℃ and 35℃, the chlorophyll fluorescence intensity of Microcystis aeruginosa from point O to point P gradually decreased with increasing light intensity. Figure 14 The effect of high temperature on the chlorophyll fluorescence induction kinetics of *Microcystis aeruginosa* was investigated. (Figure A: Day 1; B: Day 3; C: Day 5). Compared with the control, the φPo of *Microcystis aeruginosa* was significantly reduced (P<0.05) after 3 days of high-temperature treatment at 30℃ and 35℃. On day 5, φPo decreased by 10.1% (P<0.05) and 13.57% (P<0.05), respectively. Figure 15 A). Similar to φPo, Ψo, φEo, and PI ABS Both high-temperature treatments showed a decreasing trend. Figure 15 (B, C, E). Conversely, φDo increased significantly under high-temperature treatments at 30℃ and 35℃, increasing by 18.4% (P<0.05) and 24.6% (P<0.05) respectively on day 5. Figure 15 D)( Figure 15 The effect of high temperature on the chlorophyll fluorescence kinetic parameters of Microcystis aeruginosa. In this figure, A: φPo; B: Ψo; C: φEo; D: φDo; E: PI ABS ).

[0097] Effects of high temperature on astaxanthin precursor content: Under high temperature treatments of 30℃ and 35℃, the contents of both β-carotene and zeaxanthin decreased significantly (P<0.05) after 1 day of treatment; on day 5, the contents of β-carotene decreased by 53.9% (P<0.05) and 68.6% (P<0.05), respectively. Figure 16 A) The xanthine content of maize decreased by 33.3% (P<0.05) and 62.0% (P<0.05), respectively. Figure 16 B)( Figure 16 The effect of high temperature on the content of astaxanthin and its precursors in Microcystis aeruginosa (in the figure, A: β-carotene content; B: zeaxanthin content).

[0098] Effect of high temperature on astaxanthin content: Both 30℃ and 35℃ high-temperature treatments increased the astaxanthin content of *Microcystis aeruginosa*, and the astaxanthin content was significantly higher than the control after one day of treatment (P<0.05). On day 5, the astaxanthin content increased by 72.7% (P<0.05) and 94.3% (P<0.05), respectively. Figure 17 The effect of high temperature on astaxanthin content in Microcystis aeruginosa.

[0099] Summary of optimal temperatures: High temperatures of 30℃ and 35℃ significantly inhibit the growth of Microcystis aeruginosa cells. Figure 12 Under high-temperature treatment at 30℃ and 35℃, the contents of chlorophyll and carotenoids in Microcystis aeruginosa cells were significantly reduced. Figure 13 ).

[0100] The fluorescence induction kinetics curve of chlorophyll in Microcystis aeruginosa gradually decreased with increasing temperature. Figure 14 The reason for this may be due to the obstruction of electron transfer on the PSII donor side, thus affecting P680. + This is due to accumulation. Under high temperature conditions, φPo, Ψo, φEo, and PI... ABS Both show a gradual decreasing trend with increasing temperature, while φDo gradually increases. Figure 15 This indicates that high-temperature stress leads to a decrease in the absorption and conversion of light energy in algal cells, as well as the efficiency of PSII electron production and transfer, and promotes the heat dissipation of absorbed light energy to avoid photo-oxidative damage. Under high-temperature conditions, the photosynthetic pigment content of Microcystis aeruginosa decreases, the efficiency of electron production and transfer decreases, and heat dissipation increases, thereby reducing the photosynthetic performance of algal cells and inhibiting algal cell growth. Figure 12 ).

[0101] As temperature increases, the contents of β-carotene and zeaxanthin in Microcystis aeruginosa gradually decrease. Figure 16 The astaxanthin content gradually increases. Figure 17 This may be because high temperatures promote the conversion of β-carotene and zeaxanthin into astaxanthin, resulting in a decrease in the content of β-carotene and zeaxanthin and an increase in the content of astaxanthin.

[0102] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A method of modulating environmental conditions to promote astaxanthin synthesis in Microcystis aeruginosa, comprising specifically the steps of: S1, Cultivation of Microcystis aeruginosa: At a temperature of 25℃, a light exposure of 16h, and a light intensity of 30μmol·m⁻¹ -2 ·s -1 The algae were cultured in darkness for 8 hours at a shaking speed of 90 rpm until the cell density reached the logarithmic growth phase, at which point they were used for subsequent treatments. S2: Determining the optimal light quality: Under the same light intensity and temperature, different light qualities were used for cultivation, with white light as a control group, to determine the optimal light quality for astaxanthin production. S3: Determining the optimal light intensity: Under the same light quality and temperature, different light intensities were used for cultivation to determine the optimal light intensity for astaxanthin production. S4: Determining the optimal temperature: Under the same light intensity and light quality, different temperatures were used for cultivation to determine the optimal temperature for astaxanthin production. The specific steps to determine the optimal light quality are as follows: S21, culture 1×10⁶ cells / years of plants under white, purple, blue, and red light respectively. 7 cells / ml -1 A concentration of Microcystis aeruginosa maintained at a light intensity of 30 μmol·m -2 ·s -1 The temperature was 25℃, and several replicates were set for each light quality treatment; S22, algal cell density was measured during treatment 1-5 days; S23, photosynthetic pigment content, photosynthetic rate, astaxanthin and its precursor content were measured on days 1, 3 and 5; S24, on day 5, the expression of relevant genes under white light and purple light culture was analyzed. The specific steps for determining the optimal light intensity are: S31, respectively using 30, 200, 500 and 1000 μmol·m -2 ·s -1 Light intensity treatment 1×10 7 Cell·ml -1 Cupricola microcystis at a concentration of 1×10 -2 ·s -1 Treatment as control; S32, on the 1st-5th day, the algal cell density is determined; S33, on the 1st, 3rd and 5th day, the photosynthetic pigment content, photosynthetic performance, astaxanthin and its precursor content are determined; The specific steps for determining the optimal temperature are as follows: S41, treat 1×10⁻⁶ cells at 25℃, 30℃, and 35℃ respectively. 7 cells / ml -1 A concentration of Microcystis aeruginosa at a light intensity of 30 μmol·m -2 ·s -1 The light quality was white light, and several replicates were set up for each temperature treatment, with 25℃ treatment as a control; S42, algal cell density was measured on days 1-5; S43, algal cell photosynthetic pigment content, photosynthetic performance, astaxanthin and its precursor content were measured on days 1, 3 and 5. The optimal light intensity was determined to be 1000 pmol m -2 ·s -1 The optimal light quality was determined to be purple light, and the optimal temperature was determined to be 35°C.

2. The method for regulating environmental factors to promote astaxanthin synthesis in Microcystis aeruginosa according to claim 1, characterized in that: The specific steps of the determination of the photosynthetic pigment content are as follows: 3 ml of Microcystis aeruginosa culture solution is centrifuged at 6000 rpm for 8 min; 3 ml of 80% acetone is used to extract the photosynthetic pigment; after the extraction of the photosynthetic pigment is completed, the contents of chlorophyll and carotenoid are determined by using a spectrophotometer.

3. The method for regulating environmental factors to promote astaxanthin synthesis in Microcystis aeruginosa according to claim 1, characterized in that: The specific steps of the determination of the photosynthetic rate are as follows: the photosynthetic rate of Microcystis aeruginosa is determined by using an oxygen electrode; 20 ml of Microcystis aeruginosa culture solution is sucked and injected into a measuring chamber, LED light is used to provide illumination and the temperature is kept at 25°C; after the O2 release amount is stable, the release rate is recorded, and the photosynthetic rate of the algal cells is calculated.

4. The method for regulating environmental factors to promote astaxanthin synthesis in Microcystis aeruginosa according to claim 1, characterized in that: The specific steps of photosynthetic performance determination are as follows: a certain amount of Microcystis aeruginosa culture solution is centrifuged at 8000 rpm for 5 min, and the algal cells are resuspended in 10 μl of culture medium, sucked by a pipette gun, and dropped on filter paper to form a size of about 1 cm 2 Spots, dark adaptation for 15 min; the chlorophyll fluorescence induction kinetics curve is determined by using a non-modulated chlorophyll fluorescence meter; and the maximum quantum yield of photosystem II photochemistry, the electron transfer efficiency caused by the captured exciton energy, the quantum yield of electron transfer, the maximum quantum yield of non-photochemical quenching, and the photosynthetic performance index are calculated by Rohácek's method.

5. The method of claim 1, wherein the environmental condition is selected from the group consisting of temperature, pH, light, and nitrogen availability. The specific steps for determining the content of astaxanthin and its precursors are as follows: astaxanthin, β-carotene and zeaxanthin in Microcystis aeruginosa are extracted by using 80% acetone; high performance liquid chromatography is used for determination and analysis, and the specific steps are as follows: a reversed-phase C18 column with a size of 25 cm*4.6 mm and a thickness of 5 μm is used for analysis, the column temperature is 25 ℃, the injection amount is 10 μl, the mobile phase is a solution of dichloromethane:acetonitrile:methanol=20:70:10, and the flow rate is 1 ml·min -1 ; astaxanthin, β-carotene and zeaxanthin standard samples are used for quantitative analysis of the three compounds in the extract.

6. The method of claim 1, wherein the environmental condition is selected from the group consisting of temperature, pH, light, and nitrogen availability. The specific steps of the analysis of the expression of related genes are as follows: total RNA is extracted from Microcystis aeruginosa cultured under white light and purple light by using a total RNA extraction kit; after purification, the total RNA is reversely transcribed into cDNA; then polyA is connected to the 3' end of the cDNA, and the cDNA is amplified by PCR to obtain high-quality reads by transcriptome analysis; after the high-quality reads are obtained, sequence alignment is performed; gene expression analysis is performed according to the expected fragment number of each thousand base transcript sequence obtained by sequencing each million base pairs; software is used to analyze differentially expressed genes (DEGs) and perform KEGG functional annotation.

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