Haematococcus pluvialis outdoor cultivation method for improving astaxanthin yield
By adding pyrophosphate to the outdoor culture of Haematococcus pluvialis to adjust the pH and culture conditions, astaxanthin synthesis was promoted, solving the problem of unstable astaxanthin yield in large-scale outdoor culture and achieving efficient astaxanthin accumulation.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- YUNNAN BOXIN BIOTECHNOLOGY CO LTD
- Filing Date
- 2023-12-11
- Publication Date
- 2026-07-03
AI Technical Summary
Existing technologies struggle to consistently increase astaxanthin yield when cultivating Haematococcus pluvialis on a large scale outdoors. Common methods, such as altering the culture medium formula, increasing light intensity, and adding more red-transforming culture medium, are unstable and uncontrollable. Genetic modification requires high precision and has low universality.
During outdoor culture, pyrophosphate was added to adjust the pH of the algal solution to 9.0-10.5. After adding potassium pyrophosphate, stress culture was continued. This improved the efficiency of astaxanthin synthesis by promoting isopentenyl pyrophosphate synthesis and maintaining endoplasmic reticulum homeostasis.
This method increases astaxanthin yield in a shorter time, achieving an astaxanthin content of over 5.5%, shortens stress culture time, and is simple and easy to implement without affecting the food safety of algae powder.
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Abstract
Description
Technical Field
[0001] This invention belongs to the field of Haematococcus pluvialis cultivation technology, specifically, it relates to an outdoor cultivation method for Haematococcus pluvialis to improve astaxanthin yield. Background Technology
[0002] Haematococcus pluvialis is a biological source required for the large-scale commercial production of astaxanthin, and obtaining higher astaxanthin yields is the main goal of Haematococcus pluvialis aquaculture. Currently, when large-scale industrial aquaculture enters the outdoor cultivation stage, it mainly uses airlift photobioreactors (PB bags) for the initial green algae proliferation to increase the biomass of Haematococcus pluvialis green algae. Subsequently, it is transferred to outdoor tubular photobioreactors (GP pipes) or open raceway tanks for stress cultivation to accumulate astaxanthin. Common methods for outdoor stress cultivation of Haematococcus pluvialis to accumulate astaxanthin typically include changing the culture medium formula, increasing light duration and intensity, increasing the amount of reddening medium added, and genetic modification. While changing the culture medium formula can increase the accumulation of astaxanthin in Haematococcus pluvialis, the cultivation is unstable after adjustment, especially with changes in temperature and seasons, requiring constant adjustments. The increase in astaxanthin content is limited, generally between 4% and 5%. Increasing light duration and intensity is only suitable for small-scale indoor experiments or aquaculture. For large-scale outdoor aquaculture, the results are too unpredictable, especially due to the significant impact of weather and other natural factors, making it impossible to consistently improve astaxanthin yield. Increasing the amount of red-conversion medium, using a high concentration to accelerate the transformation of motile cells into sporozoites and allow for earlier astaxanthin accumulation, can also cause some cells to die and lyse due to the high osmotic pressure, leading to a decrease in cell number and final yield. Editing the gene fragment of astaxanthin synthase in Haematococcus pluvialis can increase the content of astaxanthin synthase in Haematococcus pluvialis, thereby increasing astaxanthin content. However, this method requires advanced equipment and technology, resulting in low versatility. Furthermore, most reported patents for improving astaxanthin yield involve treatment during the indoor green algae stage or small-scale outdoor aquaculture to promote astaxanthin accumulation. The applicability of these methods to large-scale outdoor production faces considerable uncertainty. Summary of the Invention
[0003] To overcome the problems existing in the background technology, the present invention provides an outdoor cultivation method for Haematococcus pluvialis to improve astaxanthin yield. By adding pyrophosphate during the outdoor cultivation process, the synthesis rate of astaxanthin is effectively increased, the stress culture time is shortened, more astaxanthin is accumulated in a shorter time, and the astaxanthin yield is improved.
[0004] To achieve the above objectives, the present invention is implemented through the following technical solution:
[0005] The method for outdoor cultivation of Haematococcus pluvialis to improve astaxanthin yield includes the following steps:
[0006] (1) After Haematococcus pluvialis was cultured indoors, the algae were separated and placed in outdoor GP pipes. After three days of routine stress culture, red-converting medium was added.
[0007] (2) The next day, adjust the ratio of carbon dioxide and air to adjust the pH of the algae solution in the GP pipeline to between 9.0 and 10.5;
[0008] (3) Once the pH value reaches between 9.0 and 9.5, add pyrophosphate;
[0009] (4) After adding potassium pyrophosphate, Haematococcus pluvialis was cultured outdoors under stress for 11-22 days.
[0010] Furthermore, the algal concentration in step (1) is 6-10 × 10⁻⁶. 4 per ml.
[0011] Further, in step (2), the ratio of carbon dioxide to air is adjusted to 1:100-500V / V.
[0012] Furthermore, the pyrophosphate mentioned in step (3) is potassium pyrophosphate, and the amount added is 0.3-3 mg / L.
[0013] Furthermore, the pyrophosphate mentioned in step (3) is potassium pyrophosphate, and the amount added is 0.33-2.31 mg / L.
[0014] Furthermore, potassium pyrophosphate was added and the mixture was then cultured outdoors for 11 to 22 days.
[0015] Furthermore, the flow rate of Haematococcus pluvialis solution in the GP pipeline is 0.8-2 m / s.
[0016] The beneficial effects of this invention are:
[0017] This invention provides a method for accumulating and promoting astaxanthin during outdoor aquaculture. The method involves adjusting the pH of the algal culture solution after indoor cultivation to 9.0-10.5 when transferring it to GP pipes, creating alkaline conditions for algal cell survival. Once the pH reaches 9.0-9.5, potassium pyrophosphate is added, which hydrolyzes into pyrophosphate ions and potassium ions. The algal cells absorb pyrophosphate ions, which can be used to synthesize isopentenyl pyrophosphate (IPP). Since the synthesis pathway of natural astaxanthin in Haematococcus pluvialis cells begins with two common terpene precursors, isopentenyl pyrophosphate (IPP) and dimethylallyl pyrophosphate (DMAPP), the exogenous addition of pyrophosphate promotes astaxanthin synthesis. Furthermore, astaxanthin is synthesized and esterified within the endoplasmic reticulum (ER) of Haematococcus pluvialis cells. After exogenous addition of potassium pyrophosphate, the potassium ions hydrolyzed are absorbed by the Haematococcus pluvialis cells and enter the ER, ensuring the ER can normally release calcium ions to balance the potential difference across the ER, maintain ER homeostasis, and enable the cells to efficiently function the ER under stress, producing more astaxanthin. The method provided by this invention allows for the harvesting of algal powder with an astaxanthin content of over 5.5% after 7 days of stress culture following the addition of potassium pyrophosphate, shortening the stress culture time of Haematococcus pluvialis in GP pipelines, accumulating more astaxanthin in a shorter time, and increasing astaxanthin yield. Attached Figure Description
[0018] Figure 1 This is a graph showing the relationship between algal division ratio and astaxanthin yield in Example 1 of the present invention;
[0019] Figure 2 This is a graph showing the effect of different carbon dioxide to air ratios on astaxanthin yield in Example 2 of the present invention.
[0020] Figure 3 This is a graph showing the effect of pipeline flow rate on astaxanthin yield in Embodiment 2 of the present invention;
[0021] Figure 4 This is a graph showing the effect of potassium pyrophosphate on astaxanthin yield in Example 4 of the present invention.
[0022] Figure 5 This is a graph showing the effect of potassium pyrophosphate on astaxanthin yield under different culture environment pH values in Example 5 of the present invention;
[0023] Figure 6 This is a graph showing the effect of potassium pyrophosphate, potassium phosphate, and sodium pyrophosphate on the astaxanthin yield in Example 6 of the present invention. Detailed Implementation
[0024] To make the objectives, technical solutions, and beneficial effects of this invention clearer, the technical solutions of this invention will be described in detail below. Obviously, the described embodiments are only a part of the embodiments of this invention, and not all of them. Other embodiments obtained by those skilled in the art based on the embodiments of this invention without creative effort are all within the scope of protection of this invention.
[0025] The method for outdoor cultivation of Haematococcus pluvialis to improve astaxanthin yield includes the following steps:
[0026] (1) After being cultured indoors, Haematococcus pluvialis was transferred to outdoor GP pipelines at a ratio (initial inoculation concentration) of 6-10 × 10⁻⁶. 4 per ml.
[0027] (2) After three days of stress culture, add red-turning medium. The next day, adjust the carbon dioxide and air ratio to 1:100-500 V / V and adjust the pH of the algal solution in the GP pipeline to between 9.0 and 10.5.
[0028] (3) When the pH value reaches between 9.0 and 9.5, add potassium pyrophosphate. The concentration of potassium pyrophosphate in the algal solution is 0.3-3 mg / L.
[0029] (4) Control the flow rate of Haematococcus pluvialis liquid in the GP pipeline to 0.8-2 m / s.
[0030] Under stress conditions, Haematococcus pluvialis accumulates astaxanthin in its immobile spore cells to counteract unfavorable growth conditions. By adding red-converting medium to the GP pipeline to provide salt stress, and then adjusting the pH to between 9.0 and 10.5, an alkaline stress condition was created for the algal cells. Increasing the flow rate of the algal solution also generated significant fluid shear stress. These combined measures provided the algal cells with multiple stress factors. Furthermore, the addition of potassium pyrophosphate further promoted astaxanthin accumulation in Haematococcus pluvialis cells by both supplying raw materials for astaxanthin synthesis and maintaining endoplasmic reticulum homeostasis at the astaxanthin synthesis site.
[0031] (5) After adding potassium pyrophosphate, continue outdoor cultivation for 11 to 22 days.
[0032] The method provided by this invention enables the harvesting of algal powder with an astaxanthin content of over 5.5% within 11 days of stress cultivation after the addition of potassium pyrophosphate, thus shortening the stress and cultivation time and increasing the astaxanthin yield. The algal concentration, pH value, aeration rate, and pump speed that need to be controlled in this invention are all simple and easy to implement. The added potassium pyrophosphate is a food additive that meets national standards; it is inexpensive, structurally stable, and can be added to food. Therefore, the food properties of the algal powder harvested after adding potassium pyrophosphate are not adversely affected.
[0033] To illustrate the present invention more clearly, the following embodiments will be described in detail. Example 1
[0034] An outdoor cultivation method for Haematococcus pluvialis to improve astaxanthin yield includes the following steps: (1) increasing the number of motile cells cultured in indoor PB bags to 20 × 10⁻⁶. 4 The number of spores / ml and the number of dead cells were less than 1×10⁻⁶. 4 Algal solutions of 6 × 10⁶ cells / ml were respectively used at 6 × 10⁻� 4 8 x 10 cells / ml 4 cells / ml, 10×10 4 (1) The algae were cultured in a GP tube at a concentration of 100 cells / ml under normal stress for three days, and then red-converted medium was added. (2) The next day, the carbon dioxide and air ratio was adjusted to 1:500 V / V, and the pH of the algal solution in the GP tube was adjusted to between 9.0 and 10.5. (3) The pH was measured every hour. When the pH reached between 9.0 and 9.5, potassium pyrophosphate was added to the algal solution at a concentration of 1.65 mg / L. (4) The flow rate of the algal solution in the GP tube was 1.6 m / s. (5) Samples were taken daily to test the astaxanthin content.
[0035] Experimental results: Algal concentration was 8 × 10⁻⁶ 4 On day 11 after potassium pyrophosphate stress culture, the astaxanthin content in the algal solution of the experimental group (cells / ml) reached 5.7%; the algal fraction concentration was 6×10⁻⁶. 4 The astaxanthin content in the algal solution of the experimental group reached 5.1% on day 19 of culture; the algal concentration was 10 × 10⁻⁶. 4 The astaxanthin content in the algal culture of the experimental group (cells / ml) reached 5.0% on day 16 of culture. The relationship between the algal fraction ratio and astaxanthin content is shown in the appendix. Figure 1 . Example 2
[0036] An outdoor cultivation method for Haematococcus pluvialis to improve astaxanthin yield includes the following steps: (1) increasing the number of motile cells cultured in indoor PB bags to 20 × 10⁻⁶. 4 The number of spores / ml and the number of dead cells were less than 1×10⁻⁶. 4 Algal solution of 8 × 10⁶ cells / ml 4(1) The algae were cultured in the GP pipeline at a concentration of 1 / ml under normal stress for three days, and then the algae were cultured in red medium. (2) The next day, the carbon dioxide and air ratio (V / V) were adjusted to 1:500, 1:200 and 1:100 respectively, and the pH of the algae in the GP pipeline was adjusted to between 9.0 and 10.5. (3) After inoculation, the pH was measured every hour. When the pH reached between 9.0 and 9.5, potassium pyrophosphate was added to the algae at a concentration of 1.65 mg / L. (4) The flow rate of the algae in the GP pipeline was 1.6 m / s. (5) Samples were taken daily to detect the astaxanthin content.
[0037] Experimental results: In the group with a carbon dioxide to air ratio of 1:500, the pH value reached 10 after 4 hours, and the astaxanthin content in the algal solution reached 5.7% after 11 days of continued stress culture following the addition of potassium pyrophosphate; in the group with a carbon dioxide to air ratio of 1:200, the pH value reached 9 after 6 hours, and the astaxanthin content in the algal solution reached 5.3% after 14 days of continued stress culture following the addition of potassium pyrophosphate; in the group with a carbon dioxide to air ratio of 1:100, the pH value reached 8.5 after 8 hours, and the astaxanthin content in the algal solution reached 5.0% after 18 days of continued stress culture following the addition of potassium pyrophosphate. The effect of different carbon dioxide to air ratios on astaxanthin yield is shown in the appendix. Figure 2 . Example 3
[0038] An outdoor cultivation method for Haematococcus pluvialis to improve astaxanthin yield includes the following steps: (1) increasing the number of motile cells cultured in indoor PB bags to 20 × 10⁻⁶. 4 The number of spores / ml and the number of dead cells were less than 1×10⁻⁶. 4 Algal solution of 8 × 10⁶ cells / ml 4 (1) The algae were cultured in a GP tube at a concentration of 1 / ml under normal stress for three days, and then red-converted medium was added. (2) The next day, the carbon dioxide and air ratio was adjusted to 1:500 V / V, and the pH of the algae solution in the GP tube was adjusted to between 9.0 and 10.5. (3) The pH was measured every hour. When the pH reached between 9.0 and 9.5, potassium pyrophosphate was added to the algae solution at a concentration of 1.65 mg / L. (4) The flow rate of the algae solution in the GP tube was adjusted to 0.8 m / s, 1.6 m / s, and 2 m / s respectively. (5) Samples were taken daily to test the astaxanthin content.
[0039] Experimental results: In the experimental group with an algal solution flow rate of 0.8 m / s in the GP pipe, the astaxanthin content in the algal solution reached 5.0% after 18 days of continued culture following the addition of potassium pyrophosphate. In the experimental group with an algal solution flow rate of 1.6 m / s in the GP pipe, the astaxanthin content in the algal solution reached 5.7% after 11 days of continued culture following the addition of potassium pyrophosphate. In the experimental group with an algal solution flow rate of 2 m / s in the GP pipe, the astaxanthin content in the algal solution reached 5.2% after 15 days of continued culture following the addition of potassium pyrophosphate. Furthermore, the experimental results also showed that the astaxanthin content was not directly proportional to the flow rate of the algal solution in the GP pipe, because excessively high flow rates may damage the cell structure of algal cells, ultimately leading to cell death and astaxanthin loss. Example 4
[0040] An outdoor cultivation method for Haematococcus pluvialis to improve astaxanthin yield includes the following steps: (1) increasing the number of motile cells cultured in indoor PB bags to 20 × 10⁻⁶. 4 The number of spores / ml and the number of dead cells were less than 1×10⁻⁶. 4 Algal solution of 8 × 10⁶ cells / ml 4 (1) The algae were cultured in a GP tube at a concentration of 1 / ml under normal stress for three days, and then red-converted medium was added. (2) The next day, the carbon dioxide and air ratio was adjusted to 1:500 V / V, and the pH of the algal solution in the GP tube was adjusted to between 9.0 and 10.5. (3) The pH was measured every hour. When the pH reached between 9.0 and 9.5, potassium pyrophosphate was added to the algal solution at concentrations of 0.33 mg / L, 0.99 mg / L, 1.65 mg / L, and 2.31 mg / L, respectively. At the same time, no potassium pyrophosphate was added as a blank control. (4) The flow rate of the algal solution in the GP tube was 1.6 m / s. (5) Samples were taken daily to detect the astaxanthin content.
[0041] Experimental results: The astaxanthin content in the algal broth reached 5.2% on day 18 after further culture, achieved in the experimental group with a potassium pyrophosphate concentration of 0.99 mg / L; 5.3% on day 15; 5.7% on day 11; and 5.8% on day 11 after culture, in the blank control group without potassium pyrophosphate. The astaxanthin content in the algal broth reached 4.3% after 22 days of culture under red-conversion medium stress. The effect of potassium pyrophosphate concentration on astaxanthin yield will be discussed in the appendix. Figure 4 . Example 5
[0042] Since Haematococcus pluvialis thrives best in neutral environments, the pH for outdoor cultivation of Haematococcus pluvialis is typically controlled between 7.0 and 8.5. However, through extensive experiments, the inventors discovered that using the method of this invention, the astaxanthin content of Haematococcus pluvialis is reduced when cultivated under conventional neutral conditions.
[0043] A method for outdoor cultivation of Haematococcus pluvialis includes the following steps: (1) increasing the number of motile cells cultured in indoor PB bags to 20 × 10⁻⁶. 4 The number of spores / ml and the number of dead cells were less than 6 × 10⁶. 4 Algal solution of 8 × 10⁶ cells / ml 4 (1) The algae were cultured in GP pipes at a concentration of 1 / ml under normal stress for three days, and then red-converted medium was added. (2) The next day, the carbon dioxide and air ratio was adjusted to 1:100 V / V, and the pH of the algae solution in the GP pipes was adjusted to 7.0-8.5 as the neutral condition culture control group; at the same time, the carbon dioxide and air ratio was adjusted to 1:500, and the pH of the algae solution in the GP pipes was adjusted to 9.0-10.5 as the experimental group. (3) The pH was measured every hour. When the pH of the algae solution in the neutral condition culture control group reached 8.0-8.5, potassium pyrophosphate was added to the algae solution at a concentration of 1.65 mg / L; when the pH of the algae solution in the experimental group reached 9.0-9.5, potassium pyrophosphate was added to the algae solution at a concentration of 1.65 mg / L. (4) The flow rate of the algae solution in the GP pipes was 1.6 m / s. (5) Samples were taken daily to detect the astaxanthin content.
[0044] Experimental results: The astaxanthin content of the algal liquid cultured in the experimental group of Haematococcus pluvialis cultured at pH 9.0-10.5 reached 5.8% after 11 days of stress culture with the addition of potassium pyrophosphate; while the astaxanthin content of the algal liquid cultured in the neutral environment was only 4.1% after 11 days of stress culture with the addition of potassium pyrophosphate. Example 6
[0045] An outdoor cultivation method for Haematococcus pluvialis includes the following steps: (1) increasing the number of motile cells cultured in indoor PB bags to 20 × 10⁻⁶. 4 The number of spores / ml and the number of dead cells were less than 6 × 10⁶. 4 Algal solution of 8 × 10⁶ cells / ml 4(1) The algae were cultured in a GP tube at a concentration of 1 / ml under normal stress for three days, and then red-converted medium was added. (2) The next day, the carbon dioxide and air ratio was adjusted to 1:500 V / V, and the pH of the algae solution in the GP tube was adjusted to between 9.0 and 10.5. (3) The pH was measured every hour. When the pH reached between 9.0 and 9.5, potassium pyrophosphate was added to the algae solution at a concentration of 1.65 mg / L. At the same time, potassium phosphate or sodium pyrophosphate was added to the algae solution at a concentration of 1.65 mg / L for comparison. (4) The flow rate of the algae solution in the GP tube was 1.6 m / s. (5) Samples were taken daily to test the astaxanthin content.
[0046] Experimental results: The astaxanthin content in the algal solution of the experimental group with added potassium pyrophosphate at a concentration of 1.65 mg / L reached 5.8% after 11 days of continued stress culture following the addition of potassium pyrophosphate; the astaxanthin content in the algal solution of the experimental group with added potassium phosphate at a concentration of 1.65 mg / L reached 3.8% after 11 days of continued stress culture following the addition of potassium phosphate; and the astaxanthin content in the algal solution of the experimental group with added sodium pyrophosphate at a concentration of 1.65 mg / L reached 4.4% after 11 days of continued stress culture following the addition of sodium pyrophosphate.
[0047] Comparative experiments show that the reason why adding pyrophosphate to algal solutions significantly increases astaxanthin yield is not because the hydrolysis of pyrophosphate produces more phosphorus-containing nutrients, but because the absorption of pyrophosphate ions by algal cells promotes astaxanthin synthesis. Furthermore, although pyrophosphate ions significantly promote astaxanthin synthesis, the addition of potassium pyrophosphate provides additional potassium ions, which helps maintain the homeostasis of the endoplasmic reticulum, the site of astaxanthin synthesis. Therefore, its effect on promoting astaxanthin synthesis is superior to that of sodium pyrophosphate.
[0048] Finally, it should be noted that the above preferred embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described in detail through the above preferred embodiments, those skilled in the art should understand that various changes can be made to it in form and detail without departing from the scope defined by the claims of the present invention.
Claims
1. A method for outdoor cultivation of Haematococcus pluvialis to improve astaxanthin yield, characterized in that, Includes the following steps: (1) After Haematococcus pluvialis was cultured indoors, the algae were separated and placed in outdoor GP pipes. After three days of routine stress culture, red-converting medium was added. (2) The next day, adjust the ratio of carbon dioxide and air to adjust the pH of the algae solution in the GP pipeline to between 9.0 and 10.5; (3) When the pH value reaches between 9.0 and 9.5, add potassium pyrophosphate; (4) After adding potassium pyrophosphate, Haematococcus pluvialis was cultured outdoors under stress for 11-22 days.
2. The method for outdoor cultivation of Haematococcus pluvialis to improve astaxanthin yield according to claim 1, characterized in that, The algal concentration mentioned in step (1) is 6-10×10 4 per ml.
3. The method for outdoor cultivation of Haematococcus pluvialis to improve astaxanthin yield according to claim 1, characterized in that, Step (2) Adjust the carbon dioxide to air ratio to 1:100-500V / V.
4. The method for outdoor cultivation of Haematococcus pluvialis to improve astaxanthin yield according to claim 1, characterized in that, The amount of potassium pyrophosphate added in step (3) is 0.3-3 mg / L.
5. The method for outdoor cultivation of Haematococcus pluvialis to improve astaxanthin yield according to claim 4, characterized in that, The amount of potassium pyrophosphate added in step (3) is 0.33-2.31 mg / L.
6. The method for outdoor cultivation of Haematococcus pluvialis to improve astaxanthin yield according to claim 1, characterized in that, After adding potassium pyrophosphate, culture in outdoor GP pipes for 11 to 22 days.
7. The method for outdoor cultivation of Haematococcus pluvialis to improve astaxanthin yield according to claim 1, characterized in that, The flow rate of Haematococcus pluvialis solution in the GP pipeline is 0.8-2 m / s.