A photosensitive high-astaxanthin-yielding Haematococcus pluvialis and its application

Abstract

The embodiment of the present invention discloses a Haematococcus pluvialis LT4012, which has a collection number of GDMCC No. 65798 in the Guangdong Provincial Microbial Culture Collection Center. The Haematococcus pluvialis LT4012 can be used for any of the following applications: (A1) to increase the yield of astaxanthin; (A2) to prepare a product for producing astaxanthin; (A3) to produce astaxanthin under weak light; (A4) to prepare a product for inducing astaxanthin gene expression. The light-sensitive, high-yield astaxanthin Haematococcus pluvialis of the present invention has stable performance and is sensitive to strong and weak light signals. Compared with the original algae strain, the algae cells have strong stress resistance, significantly improved astaxanthin production rate, and stable yield.

Description

Technical Field

[0001] The invention relates to light-sensitive high-astaxanthin-yielding Haematococcus pluvialis and applications thereof. Background Art

[0002] Haematococcus pluvialis is a freshwater, single-celled microalgae belonging to the Chlorophyta, Curculiomorpha, and Rhodophyceae families. It is widely distributed in rivers and lakes, temporary blisters formed by rainwater accumulation, and even on rock surfaces. The name Haematococcus pluvialis comes from its red coloration, which stems from its discovery after heavy rainfall and its ability to accumulate large amounts of astaxanthin.

[0003] Haematococcus pluvialis possesses remarkable environmental adaptability, exhibiting four typical cell morphologies throughout its life cycle: zoospores, microspores, colloid colonies, and amoenospores. Under favorable conditions (such as adequate nutrition, appropriate light intensity, and salinity), algal cells undergo vegetative proliferation, primarily in the zoospore and microspore morphologies, accumulating biomass. Under unfavorable conditions (such as nutrient deficiency, high light intensity, and high salinity), the cells form colloid colonies and transform into amoenospores, accumulating large amounts of astaxanthin, with astaxanthin content reaching up to 5% of the cell dry weight. Therefore, Haematococcus pluvialis is considered an excellent source for producing natural astaxanthin. Normally, Haematococcus pluvialis does not accumulate astaxanthin under favorable conditions for cell growth; it only accumulates in significant quantities under unfavorable conditions, where nutrient proliferation is restricted. Therefore, balancing algal cell vegetative growth and astaxanthin accumulation is crucial for the industrial production of natural astaxanthin from microalgae.

[0004] Currently, Haematococcus pluvialis has the following defects: defects in the culture stage, weak stress resistance, poor stress resistance of cells in the zoospore stage, and susceptibility to contamination by other algae and protozoa, resulting in difficulties in expansion, low yield and high culture costs.

[0005] There is a contradiction between biomass accumulation and astaxanthin synthesis. Under favorable growth conditions (such as sufficient nutrition, appropriate light and salinity), algal cells prioritize nutrient proliferation (zoospore reproduction), but do not accumulate astaxanthin at this time; and astaxanthin accumulation is only triggered under adverse stress conditions (such as nutrient deficiency, high light, and high salt), at the expense of biomass growth.

[0006] Defects in the astaxanthin induction stage: High stress conditions lead to cell damage. When strong light is combined with other stress conditions (such as high salt), the tolerance of motile cells is insufficient and photooxidative damage occurs easily, leading to the death of a large number of algal cells and seriously affecting the final astaxanthin production.

[0007] High costs limit large-scale production. The application of high-intensity light and multiple stress conditions significantly increases cultivation energy consumption and equipment costs, restricting the feasibility of commercial large-scale production.

[0008] Existing breeding technologies are unable to efficiently cultivate strains that are resistant to stress, sensitive to both strong and weak light signals, and capable of producing high and stable astaxanthin. These issues lead to high costs and unstable yields for large-scale production, necessitating technological innovation to overcome these bottlenecks. Summary of the Invention

[0009] To this end, the embodiments of the present invention provide a light-sensitive, high-astaxanthin-yielding Haematococcus pluvialis and its application to address the defects of the prior art Haematococcus pluvialis, such as poor stress resistance, insensitivity to strong and weak light signals, and low astaxanthin production.

[0010] In order to achieve the above objectives, the embodiments of the present invention provide the following technical solutions:

[0011] According to a first aspect of an embodiment of the present invention, there is provided Haematococcus pluvialis LT4012, whose deposit number in Guangdong Provincial Microbiological Culture Collection Center is GDMCC No. 65798.

[0012] The present invention also provides any of the following applications of Haematococcus pluvialis LT4012:

[0013] (A1) for increasing the yield of astaxanthin;

[0014] (A2) for preparing a product for producing astaxanthin;

[0015] (A3) for producing astaxanthin under low light conditions;

[0016] (A4) for preparing a product for inducing astaxanthin gene expression;

[0017] The weak light intensity is 150 μmolphotons / (m 2 ·s).

[0018] Another aspect of the present invention provides an algae culture, the active ingredient of which is the Haematococcus pluvialis LT4012 described in the claims.

[0019] In one embodiment of the present invention, the product has the following uses 1) or 2): 1) for producing astaxanthin under weak light; 2) for increasing the yield of astaxanthin.

[0020] In one embodiment of the present invention, the algae culture described above is used for any of the following applications: (A1) for increasing the production of astaxanthin; (A2) for preparing a product for producing astaxanthin; (A3) for producing astaxanthin under weak light; (A4) for preparing a product for inducing astaxanthin gene expression.

[0021] The present invention also provides a method for preparing astaxanthin, comprising: culturing the Haematococcus pluvialis LT4012 according to claim 1 to prepare astaxanthin.

[0022] In one embodiment of the present invention, the culture comprises inoculating the above-mentioned Haematococcus pluvialis LT4012 into BBM medium for culture, wherein the BBM medium is: NaNO3 0.25g / L, CaCl2·2H2O 0.025g / L, MgSO4·7H2O 0.075g / L, K2HPO40.075 g / L, KH2PO40.175 g / L, NaCl0.025g / L, trace elements PIV 6ml / L, vitamin B11.2 mg / L, vitamin B 12 10μg / L.

[0023] In one embodiment of the present invention, the trace element PIV is EDTA-Na20.75 g / L, FeCl3·6H2O0.097 g / L, MnCl2·4H2O, 0.041 g / L, ZnCl2·7H2O 0.005 g / L, CoCl2·6H2O 0.002 g / L, and Na2MoO4·2H2O 0.004 g / L.

[0024] Another aspect of the present invention provides a product for preparing astaxanthin, comprising the aforementioned Haematococcus pluvialis LT4012 and the aforementioned BBM culture medium.

[0025] Finally, the present invention also provides the use of the above-mentioned product in the preparation of astaxanthin.

[0026] The embodiments of the present invention have the following advantages:

[0027] The light-sensitive, high-astaxanthin-producing Haematococcus pluvialis algae of the present invention is deposited in the Guangdong Provincial Microbial Culture Collection Center with a deposit number of GDMCC No. 65798 and a deposit date of January 15, 2025. The algae strain has stable performance and is sensitive to strong and weak light signals. Compared with the original algae strain, the algae cells have strong stress resistance, significantly improved astaxanthin production rate, and stable yield.

[0028] The light-sensitive and high-yielding astaxanthin domesticated Haematococcus pluvialis strain of the present invention has a fast growth rate and strong stress resistance. 2·s) light can quickly transform zoospores into red amoespores, eliminating the reliance on strong light during the traditional red algal cell transformation process. This results in a high algal cell survival rate and a short culture cycle. The product has stable performance in both indoor and outdoor culture systems, with an astaxanthin yield exceeding 3.4 mg / L / d and an astaxanthin content as high as 4.86% CDW. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] To more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the figures required for the embodiments or the description of the prior art. Obviously, the figures described below are merely exemplary, and those skilled in the art can derive other implementation diagrams based on the provided figures without inventive effort.

[0030] The structures, proportions, sizes, etc. illustrated in this specification are intended solely to complement the contents disclosed herein and to facilitate understanding and reading by persons skilled in the art. They are not intended to limit the conditions under which the present invention may be implemented and therefore have no substantive technical significance. Any structural modifications, changes in proportions, or adjustments in sizes, without affecting the efficacy and objectives of the present invention, shall remain within the scope of the technical contents disclosed herein.

[0031] Figure 1 This is a comparison of the number of differentially expressed genes between the algae strain of Example 1 of the present invention and the original algae strain, the treatment group is LT4012, and the control group is the original strain;

[0032] Figure 2 This is a volcano plot of differentially expressed genes between the algae strain of Example 1 of the present invention and the original algae strain. The blue dots on the left represent down-regulated genes, the brown dots in the middle represent genes with no differential expression, and the yellow dots on the right represent up-regulated genes. A larger value on the horizontal axis indicates a higher differential expression fold, and a larger value on the vertical axis indicates a more significant differential expression.

[0033] Figure 3 This is a GO function enrichment analysis diagram of the up-regulated genes of the algae strain in Example 1 of the present invention and the original algae strain, wherein the enrichment of differentially expressed genes between the two algae strains in biological process (BP), cell composition (CC), and molecular function (MF) is shown; the abscissa represents the number of genes enriched in this entry, with p.adjust < 0.05 being set as the significance threshold, and the color of the bar corresponds to the p.adjust value, from small to large, corresponding to blue to red, with the redder the color, the more significant it is, indicating that the function corresponding to the differentially expressed gene in this entry is more important;

[0034] Figure 4This is a GO function enrichment analysis diagram of the down-regulated genes of the algae strain in Example 1 of the present invention and the original algae strain, wherein the enrichment of differentially expressed genes between the two algae strains in biological process (BP), cell composition (CC), and molecular function (MF) is shown; the horizontal axis represents the number of genes enriched in the entry, with p.adjust < 0.05 being set as the significance threshold, and the color of the bar corresponding to the p.adjust value, from small to large, corresponding to blue to red, with the redder the color, the more significant it is, indicating that the function corresponding to the differentially expressed gene in the entry is more important;

[0035] Figure 5 This is a KEGG metabolic pathway enrichment analysis diagram of differentially expressed genes between the algae strain of Example 1 and the original strain. It shows the enrichment of differentially expressed genes between the two strains in gene-related functions and pathways. The vertical axis represents the enriched pathway description, while the horizontal axis represents the number of differentially expressed genes in the pathway. The figure shows the 10 most significantly enriched pathways.

[0036] Figure 6 This is a flowchart of the acclimation of photosensitive high-yielding astaxanthin Haematococcus pluvialis under drought stress in Example 2 of the present invention, which shows a round of acclimation screening process under drought combined with high light stress. The intensity of the high light stress set in each round is different and is set to 500 μmolphotons / (m 2 ·s)、300μmolphotons / (m 2 ·s)、150μmolphotons / (m 2 s) are adjusted downwards in sequence;

[0037] Figure 7 These are the laboratory shake flask culture results of the domesticated strain of Example 2 of the present invention. Figure A shows the dry weight and cell number of algae cells at the end of each microalgae growth period; Figure B shows the dry weight and cell number of algae cells after strong light induction; Figure C shows the astaxanthin content and astaxanthin yield in algae cells after strong light induction;

[0038] Figure 8 Figure 2 is the result of genetic stability evaluation of the domesticated strains in Example 2 of the present invention. Figure A shows the percentage increase or decrease in dry cell weight of each domesticated strain compared to the wild type; Figure B shows the percentage increase or decrease in astaxanthin yield of each domesticated strain compared to the wild type.

[0039] Figure 9 Table 4 provides a summary of excellent photosensitive Haematococcus pluvialis strains under drought stress provided by the embodiments of the present invention. DETAILED DESCRIPTION

[0040] The following describes the implementation of the present invention using specific embodiments. Those skilled in the art will readily understand the other advantages and benefits of the present invention from the disclosure herein. Obviously, the embodiments described are only a portion of the present invention, not all of it. All other embodiments derived by persons of ordinary skill in the art based on the embodiments of the present invention without inventive effort are intended to fall within the scope of protection of the present invention.

[0041] Example 1: Expression of genes related to astaxanthin accumulation induced by the Haematococcus pluvialis strain of the present invention

[0042] This embodiment provides a light-sensitive, acclimated Haematococcus pluvialis strain (named LT4012), Guangdong Provincial Microbiological Culture Collection Center, with a deposit number of GDMCC No. 65798 and a deposit date of January 15, 2025.

[0043] This algae strain (LT4012) can generate 2 ·s) light to rapidly proliferate algae cells and transfer 150μmolphotons / (m 2 ·s) light can induce the rapid transformation of zoospores into red amoespores, with a transformation efficiency of over 90% within 4 days. At the harvest time, the dry weight of algal cells reaches over 1.32g / L, the astaxanthin content reaches over 4.5%, and the astaxanthin yield reaches over 3.14mg / L / d.

[0044] The LT4012 algae strain was obtained by cultivating the Haematococcus pluvialis strain FACHB797 (from the Chinese Freshwater Algae Seed Bank) through drought combined with step-by-step weak light induction.

[0045] The LT4012 algae strain that had grown to the logarithmic phase and the original strain FACHB797 were simultaneously transferred with 150 μmol photons / (m 2 After culturing under light for 4 days, RNA was extracted and transcriptome sequencing was performed. The number of differentially expressed genes was compared. Figure 1 ; Volcano plot of differential gene expression ( Figure 2 ); GO function enrichment analysis of up-regulated genes ( Figure 3 ); GO function enrichment analysis of down-regulated genes ( Figure 4 ); KEGG metabolic pathway enrichment analysis of differentially expressed genes ( Figure 5 ).pass Figure 1-5 It can be seen that: after 150μmolphotons / (m 2After light induction (s), the acclimated strain LT4012 showed significant differences in gene expression compared to the original strain FACHB797, with 2,738 genes upregulated and 1,655 downregulated. GO functional enrichment analysis clearly revealed significant upregulation of the cell wall, cell membrane, and fatty acid synthesis pathways in LT4012. During the transition from zoospores to amoespores to synthesize astaxanthin, the cell wall thickened significantly, astaxanthin-encapsulating lipid bodies formed abundantly within the cells, and fatty acid content increased significantly. In contrast to LT4012, the original strain FACHB797 maintained active chlorophyll synthesis and photosynthesis at this light intensity, demonstrating that green zoospores were present in the acclimated strain. This suggests that the acclimated strain LT4012 rapidly responds to light intensity changes, inducing the expression of genes involved in astaxanthin accumulation.

[0046] Example 2: Domestication and cultivation of Haematococcus pluvialis strains of the present invention

[0047] In the present embodiment, it is divided into laboratory culture and outdoor culture. Laboratory culture is completed in a clean and sterile environment, and static culture is carried out in an artificial climate box. Except for the 96-well plate acclimation screening process (using 3N BBM culture medium), the laboratory 250ml triangular flask culture uses standard BBM culture medium. Greenhouse and outdoor expansion culture are carried out in 1L column reactors, and standard BBM culture medium is used in each stage of expansion culture.

[0048] Table 1. Elements and concentrations of BBM and 3N BBM culture media

[0049]

[0050]

[0051] PIV ingredient list:

[0052] Serial number Components concentration 1 <![CDATA[EDTA-Na2]]> 0.75g / L 2 <![CDATA[FeCl3·6H2O]]> 0.097g / L 3 <![CDATA[MnCl2·4H2O]]> 0.041g / L 4 <![CDATA[ZnCl2·7H2O]]> 0.005g / L 5 <![CDATA[CoCl2·6H2O]]> 0.002g / L 6 <![CDATA[Na2MoO4·2H2O]]> 0.004g / L

[0053] The method for cultivating photosensitive, high-yielding astaxanthin Haematococcus pluvialis under drought stress includes the following steps: using FACHB797, FACHB717 (China Freshwater Algae Seed Bank), and UMN874 (University of Minnesota Algae Seed Bank, USA) as the original algae species for cultivation, and using BBM medium with a nitrogen concentration of 3 times for cultivation. The basic cultivation process is as follows:

[0054] (1) Strong light stress

[0055] The algae strains in the vegetative growth stage (algae cell concentration is about 4.0×10 5 cells / ml) were transferred into 800 μmol photons / (m 2·s) strong light stress, with a light-dark ratio of 24:0 and a culture temperature of 23-25°C. After culturing for 2 days, the algal cells gradually turned red.

[0056] (2) Drought stress

[0057] Take 3 ml of the algae cell suspension from step (1) and evenly drip it onto a sterilized and dried circular tin foil tray (bottom diameter: 4.5 cm; top diameter: 5 cm; height: 1.3 cm) to form a water film. Subsequently, the tin foil tray is placed in a sterile drying chamber (highly transparent PE material, length × width × height = 11 cm × 8 cm × 3 cm). The two ends of the drying chamber are equipped with air inlet and outlet holes, connected to the air pipe, and a disc gas sterilization filter is installed at the interface. Sterile air is introduced into the drying chamber at a ventilation rate of 100 ml / min. After about 24 hours, the ventilation is stopped. At this time, the algae cell culture liquid is blown dry, and a layer of red dry algae can be seen at the bottom of the tin foil tray. The dry algae in the tin foil tray is maintained for different drying times (24 hours, 48 ​​hours, 72 hours, 96 hours, 120 hours) under the same culture conditions as step (1).

[0058] (3) Weak light rehydration plate screening

[0059] Add 5 ml of 3N BBM culture medium to each tin foil tray in step (2) and soak for 2 hours. Then, use a pipette to gently pipette to resuspend the algae cells that have dried on the wall. Then, dilute the resuspended algae cells 16 times with 3N BBM culture medium and distribute them into four 96-well transparent microplates. Add 200 μl of sample to each well and transfer them to different weak light conditions (100 μmol photons / (m 2 ·s)、50μmolphotons / (m 2 ·s)、15μmol photons / (m 2 ·s)) to resume growth, and the ratio of the number of green wells in the 96-well plate under different drought-weak light rehydration conditions was shown in Table 2.

[0060] Table 2. Exploration of rehydration culture conditions under drought combined with strong light stress (FACHB797)

[0061]

[0062] (4) High light stress plate rescreening

[0063] 100 μl of algal cell suspension was drawn from each ELISA plate after the recovery of growth in step (3) and added to the corresponding new ELISA plate. The new ELISA plate with the sample was then transferred to a 600 μmol photons / (m 2Table 3 shows the ratio of red to dead cells in the well plates that recovered from greening after the second high light stress, with a light-dark ratio of 24:0 and a culture temperature of 23-25°C.

[0064] Table 3. Exploration of drought-induced acclimation conditions (FACHB797)

[0065]

[0066] (5) Selection of excellent algae strains and photosensitivity acclimation

[0067] The ratio of greening in the well plate after different drought treatment times in step (3) and the ratio of redding after secondary strong light stress in step (4) were compared to determine the optimal drought treatment time (72-96h, under which the ratio of greening in the well plate was 31%, which basically ensured that the algal cells in the well were monoclonal and convenient for the next round of screening) and rehydration light intensity (50μmolphotons / (m 2 s). Algal cells that resume growth under this light intensity are more sensitive to changes in light intensity and have a higher reddening rate. At the same time, microalgae with a fast greening rate and a high reddening rate were selected for the next round of strong light-drought stress-weak light rehydration acclimation, and the strong light stress intensity was gradually reduced (set to 500 μmolphotons / (m 2 ·s)、300μmolphotons / (m 2 ·s)、150μmolphotons / (m 2 Finally, by comparing the greening speed of algal cells after rehydration and the redding ratio and mortality rate under strong light, we can obtain better domesticated strains such as Figure 9 As shown in Table 4, the numbers were recorded and the plants were selected to be stored on 3NBBM plates. After the algal colonies were formed, the photosensitivity evaluation experiment was carried out.

[0068] (6) Evaluation experiment of photosensitivity-adapted strains

[0069] The better domesticated strains were selected to evaluate their growth and astaxanthin production. The experimental period was 21 days and they were cultured in 250 mL Erlenmeyer flasks in the laboratory. The initial cell concentration was 2.5×10 4 cells / mL, light-dark ratio 16:8, culture temperature 23-25°C, light intensity 50 μmolphotons / (m 2 ·s), and shake the flask 4 times a day. After an average of 15 days of culture, when the algal cells reached the logarithmic phase, the light intensity was increased to 150-500 μmolphotons / (m 2 ·s) stress for 4 to 6 days, and finally harvested for testing (test results see Figure 7The excellent domesticated strains are divided into two categories: one is LT4012, LT4003, LT1503, and LT1517, which have relatively small cells and proliferate rapidly. The dry weight of LT1517 is as high as 1.30g / L in the late logarithmic phase. Although the dry weight accumulation of LT4012 during the growth period is not as good as that of LT1517, the dry weight increase of LT4012 after light stress induction is the largest among all domesticated strains, reaching 1.32g / L. Its astaxanthin content is also as high as 4.52%, and the yield reaches 3.14mg / (L·d). The other category is LT2112 and LT2126, which proliferate slowly, have relatively large cells, and are easy to transform into amoenospores. Although the number of cells at the end of the proliferation period is only 2×10 5 cells / mL, but more than half of its cells have transformed into green amoenospores. Its cell resistance is better than that of zoospores with higher activity, and it will have a higher survival advantage in subsequent outdoor cultivation.

[0070] (7) Genetic stability evaluation of excellent domesticated strains: In order to ensure the scalability of domesticated strains in later large-scale production generations and the stability of astaxanthin production, the genetic stability evaluation of excellent domesticated strains was carried out. The strains were continuously subcultured in 250ml triangular shake flasks in the laboratory. The 1st, 3rd, 5th and 10th generation domesticated strains were selected and cultured for 21 days under the same culture conditions as described in step (6). The wild type was cultured as a control at the same time, and the microalgae were harvested for dry weight and astaxanthin content determination. The increase or decrease in the dry weight of the harvested algae cells and the astaxanthin content compared with the wild type was used as the evaluation index of domestication stability ( Figure 8 A comprehensive evaluation revealed the genetic stability of the mutant strains as follows: LT4003 ≈ LT4056 > LT4012 > LT2126 ≈ LT2112 > LT1503 ≈ LT1517. LT1503 and LT1517 were discarded because their photosensitivity and astaxanthin accumulation decreased with increasing generations, essentially returning to wild-type levels after five generations. While LT4003 and LT4056 were genetically stable, they exhibited low photosensitivity, remaining actively motile after light induction and exhibiting low astaxanthin accumulation. Therefore, the domesticated strains LT4012, LT2126, and LT2112 were ultimately selected for outdoor screening and evaluation.

[0071] (8) Outdoor evaluation experiment on the growth of photosensitivity, high-yield astaxanthin-producing strains and astaxanthin accumulation

[0072] The three better domesticated strains, LT4012, LT2126 and LT2112, were further expanded indoors for greenhouse and outdoor cultivation screening and evaluation. First, in a greenhouse (natural light, temperature 25°C), a 1L column reactor was used to maintain the initial concentration of the three algal cells at approximately 2.5×10 4cells / mL, the algal cells were cultured for 14 days and samples were taken to evaluate their growth. Then, LT4012 was placed in a 150 μmolphotons / (m 2 ·s) light, LT2126 and LT2112 were placed under 300μmolphotons / (m 2 s) light for 7 days, and algal cells were harvested to evaluate astaxanthin accumulation. Because LT2126 and LT2112 turned red more slowly, the light stress period was increased to 12 days, and cells were harvested for analysis.

[0073] In order to further increase the biomass of algal cells and enhance their stability during outdoor cultivation, the cultivation mode was optimized and divided into three stages (cultivation stage, transformation stage, and accumulation stage). During the cultivation stage, 3N-BBM culture medium was used to promote algal cell growth, and 1g / L NaCl was added to promote the transformation of motile cells into immobile cells. Finally, the cells were placed under continuous light stress to complete the accumulation of astaxanthin. After the proliferation culture, the addition of 1g / L NaCl for 3 days of osmotic stress promoted the transformation of motile cells into immobile cells. At the same time, during the early drought acclimation process, the algal cells also developed a certain adaptability to osmotic stress, which ensured the survival rate of the algal cells during the transformation process.

[0074] A comprehensive evaluation of the parameters of each algal strain (Table 4) revealed that LT4012 was the optimal strain in terms of both dry weight and astaxanthin production. After 21 days of optimized greenhouse cultivation, the algal cell dry weight reached 1.48 g / L and the astaxanthin production reached 3.4 mg / L / day. Although LT2126 and LT2112 exhibited slower cell proliferation and transformation rates, these improvements were significantly achieved after optimized cultivation, demonstrating their strong adaptability to the external environment. With increased culture time, the astaxanthin content in both strains exceeded 5% after 26 days, and the astaxanthin production reached over 2.5 mg / L / day.

[0075] Finally, to evaluate the astaxanthin production capacity of the three well-acclimated strains during outdoor cultivation, growth and astaxanthin accumulation experiments were conducted in 1L outdoor column reactors. The cultivation process was identical to the optimized greenhouse cultivation process, and the cell dry weight and astaxanthin yield were compared with those obtained from the greenhouse column reactor (Table 5). LT2126 was found to be highly adaptable to the outdoor environment. After 26 days of cultivation, the astaxanthin content reached 5.35%, and the astaxanthin yield reached 2.6 mg / L / day, comparable to those obtained from the optimized greenhouse cultivation. The other two strains showed some decreases in astaxanthin yield, with LT4012 experiencing the largest decrease of 15%. Despite this, due to the rapid cell transformation process of LT4012, the algae could be harvested after 21 days. Its outdoor astaxanthin yield reached 2.9 mg / L / day, making it a high-quality strain with great potential for outdoor production.

[0076] Table 5: Evaluation of growth and astaxanthin accumulation of excellent algae strains in greenhouse and outdoor column reactors

[0077]

[0078]

[0079] The drought-combined three-cycle step-by-step weak-light-induced acclimation method of the present invention has strong directionality. The acclimated and cultivated Haematococcus pluvialis can sensitively sense changes in light intensity, achieve stable growth under weak light, and achieve stable conversion of algal cells and accelerated astaxanthin accumulation after changes in light intensity. The astaxanthin content and yield in the light-induced culture system are significantly higher than those of the original strain.

[0080] Compared with the traditional shake flask induction and domestication method, the 96-well plate-based large-scale induction and domestication method adopted in the present invention can simultaneously set different environmental factors and gradient conditions, and complete high-throughput screening of excellent domesticated algae strains suitable for various combinations of gradient light intensities and different drought resistance abilities, providing an effective method for industrial directed breeding.

[0081] Although the present invention has been described in detail above using general descriptions and specific embodiments, it will be apparent to those skilled in the art that modifications and improvements may be made thereto. Therefore, such modifications and improvements, without departing from the spirit of the present invention, are intended to be within the scope of protection claimed herein.

Claims

1. Haematococcus pluvialis LT4012, whose deposit number in Guangdong Provincial Microbial Culture Collection Center is GDMCC No.65798.

2. Any of the following uses of Haematococcus pluvialis LT4012 according to claim 1: (A1) for preparing a product for producing astaxanthin; (A2) is used to produce astaxanthin under weak light; the weak light has an intensity of photosynthetically active radiation of 150 μmolphotons / (m 2 ·s).

3. An algae culture, wherein the active ingredient is the Haematococcus pluvialis LT4012 according to claim 1.

4. The algae culture according to claim 3, wherein The culture has the following uses: for producing astaxanthin under weak light; the weak light is a photosynthetically active radiation intensity of 150 μmol photons / (m 2 ·s).

5. Any of the following uses of the algae culture according to claim 3: (A1) for preparing a product for producing astaxanthin; (A2) is used to produce astaxanthin under weak light; the weak light has an intensity of photosynthetically active radiation of 150 μmolphotons / (m 2 ·s).

6. A method for preparing astaxanthin, comprising: Astaxanthin is prepared by culturing the Haematococcus pluvialis LT4012 according to claim 1.

7. The method according to claim 6, comprising inoculating the Haematococcus pluvialis LT4012 according to claim 1 into BBM medium for cultivation, wherein the BBM medium is: NaNO3 0.25g / L, CaCl2·2H2O 0.025g / L, MgSO4·7H2O 0.075g / L, K2HPO4 0.075g / L, KH2PO40.175 g / L, NaCl 0.025g / L, trace elements PIV 6ml / L, vitamin B11.2 mg / L, vitamin B 12 10μg / L.

8. The method according to claim 6, wherein The trace element PIV is EDTA-Na20.75 g / L, FeCl3·6H2O 0.097 g / L, MnCl2·4H2O 0.041 g / L, ZnCl2·7H2O 0.005 g / L, CoCl2·6H2O 0.002 g / L, and Na2MoO4·2H2O 0.004 g / L.

9. A product for preparing astaxanthin, comprising the Haematococcus pluvialis LT4012 according to claim 1 and the BBM culture medium according to claim 7.

10. Use of the product according to claim 9 in the preparation of astaxanthin.

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