Method for culturing Yunnan hot spring algae
Through the method of combining a closed photobioreactor and LED multi-wavelength light source with a temperature control system, the problem of unstable yield of hot spring algae in traditional culture methods is solved, and efficient and stable algae culture is achieved.
Patent Information
- Application Number
- CN202510596222.2
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-05-09
- Publication Date
- 2025-07-25
AI Technical Summary
Traditional open culture methods are greatly affected by climate, temperature and light fluctuations, resulting in unstable yields of hot spring algae, easy introduction of mixed algae and bacteria, and difficult to maintain the efficient growth of hot spring algae.
The closed photobioreactor is used to combine LED multi-wavelength light source and temperature control system to accurately control light and temperature, add sulfur-containing compounds, monitor pH and dissolved oxygen in real time, prevent contamination of miscellaneous bacteria, and achieve controllable culture.
It improves the yield and stability of hot spring algae, reduces energy consumption, prevents contamination of miscellaneous bacteria, and achieves efficient algae culture.
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Figure BDA0005394661790000081
Abstract
Description
Technical Field
[0001] This application relates to the technical field of algal bioculture, and particularly to a method for culturing Yunnan hot spring algae. Background Art
[0002] Yunnan hot spring algae are a special type of algae that can adapt to extreme high-temperature, high-acid, or high-mineral environments. They are mainly distributed in special spring water areas such as sulfur springs and silicate springs. Compared with algae in other water systems such as the ocean and lakes, the species of hot spring algae are relatively single. Hot spring algae mainly include prokaryotic cyanobacteria, as well as eukaryotic red algae, green algae, diatoms, and euglenoids. Cyanobacteria belong to prokaryotes and have a similar structural characteristic to bacteria without a cell nucleus, so they are also called blue bacteria. Hot spring algae are mainly single-celled, or in the form of single-celled clusters or filamentous aggregates. Only a few species of green algae have branched filamentous structures. Most hot spring algae, except for a few that live planktonically in water bodies, grow on the surface of sediments such as stones, sand, and mud at the bottom of hot springs, forming a biological film of varying thickness. Some filamentous cyanobacteria can form laminated algal mats together with other bacteria or diatoms and sediment particles, with a thickness of up to several centimeters. There are also some hot spring algae that often aggregate into a gelatinous mass, that is, an algal ball.
[0003] In recent years, the international research on hot spring algae has made rapid progress, bringing rapid changes to the development and utilization of hot spring algae, and deeply exploring the biological activities and functions of hot spring algae. As a very precious pharmaceutical raw material, hot spring algae have opened up new prospects and brought new hopes for biomedicine and clinical applications.
[0004] In terms of the culture of hot spring algae, traditional open culture is greatly affected by climate, temperature, and light fluctuations, resulting in unstable yields. Open culture is prone to introducing miscellaneous algae, miscellaneous bacteria, pathogens, etc., leading to culture failure. Moreover, the spectrum of natural light is broad and cannot match the efficient photosynthetic requirements of hot spring algae. Traditional culture relies on natural water temperature and it is difficult to maintain the optimal growth temperature of hot spring algae. Summary of the Invention
[0005] Aiming at the above technical defects, the purpose of this application is to provide a method for culturing Yunnan hot spring algae to stably culture hot spring algae and increase the yield.
[0006] To achieve the above purpose, this application provides a method for culturing Yunnan hot spring algae, including the following steps:
[0007] S1. Take the algal species of Yunnan hot spring algae and inoculate them into a culture medium, culture them in the dark and statically, filter, and obtain activated algal species;
[0008] S2. Inoculate the activated algal species into a culture flask, adjust the pH to 7.5 - 8.5, supplement sulfur-containing compounds, place the culture flask on a constant temperature shaker, irradiate with light, culture for 5 - 7 days and then filter to obtain primary cultured algal species;
[0009] S3. Inoculate the primary cultured algal species into a closed reactor, then adjust the pH to 7.5 - 8.5, raise the temperature programmatically, provide light, and supplement sulfur-containing compounds, and culture for 8 - 10 days.
[0010] In the above process, sulfur-containing compounds are supplemented because sulfur element is a component of sulfur-containing amino acids and coenzyme A, and lack of sulfur will lead to hindered chlorophyll synthesis; light is provided because algae need photosynthesis, especially having a high absorption coefficient for blue light and red light to promote photosynthetic efficiency and chlorophyll synthesis; temperature has a great impact on algae. Most of the Yunnan hot spring algae live under high-temperature conditions and have the characteristic of being heat-resistant, which can reduce the competition of other algae and promote growth.
[0011] Further, the hot spring algae is at least one of Synechococcus elongatus, Aphanothece nidulans, Chroococcus weissei, Chroococcus minutus, Chroococcus limneticus, Chroococcus minor, Gloeocapsa gelatinosa, Gloeocapsa linearis, Synechococcus oscillans, Oscillatoria chlorina, and Lyngbya gracilis.
[0012] Further, the culture medium is at least one of BG - 11 medium, MA medium, and M - 11 medium.
[0013] Further, the sulfur-containing compound is at least one of magnesium sulfate, sodium sulfide, sodium sulfate, and cysteine.
[0014] Further, the temperature of the constant temperature shaker is 35 - 50 °C.
[0015] Further, the light in step S2 is LED blue-violet light, and the intensity ratio of blue light to violet light is 3 - 5:1 - 3, and the total light intensity is 2000 - 5000 lux.
[0016] Further, the programmed temperature increase in step S3 is to increase the temperature to 45 - 70 °C.
[0017] Preferably, the programmed temperature increase in step S3 is to increase the temperature to 45 - 60 °C.
[0018] Further, the light in step S3 is LED red-blue light, and the intensity ratio of red light to blue light is 3 - 5:1 - 3, and the total light intensity is 2000 - 5000 lux.
[0019] Further, the amount of the supplemented sulfur-containing compound in step S3 is 70 - 90% of the initial concentration.
[0020] In summary, the present application has the following beneficial effects:
[0021] The light source used in the cultivation method of Yunnan hot spring algae provided by this application is an LED light source, which has higher light efficiency than traditional light sources and reduces energy consumption. By precisely matching the absorption peaks of hot spring algae through a multi-wavelength LED combination, this application accurately controls the temperature of the cultivation environment through a temperature control system, and maintains the living environment of hot spring algae by real-time monitoring of pH and dissolved oxygen. A closed photobioreactor is used to prevent contamination by miscellaneous bacteria and for controllable cultivation, stably cultivating hot spring algae and increasing the yield of hot spring algae. Detailed implementation manners
[0022] Next, in combination with the embodiments of this application, the technical solutions in the embodiments of this application will be clearly and completely described. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts belong to the protection scope of the present invention.
[0023] Unless otherwise specified, each raw material component in the following embodiments can be obtained through commercial channels, and the performance test method is a known test method in the art. In addition: the slender Synechococcus used in the following embodiments is from the Freshwater Algae Species Bank FACHB-410 of the Typical Culture Collection Committee of the Chinese Academy of Sciences; the closed reactor is a columnar photobioreactor.
[0024] Example 1
[0025] A cultivation method of Yunnan hot spring algae specifically includes the following steps:
[0026] S1. Take the algal species of slender Synechococcus and inoculate them at 1.0×10 6 cells / mL into 50 mL of sterilized BG-11 medium (the content of magnesium sulfate in the medium is 5 mg / L, and the content of sodium bicarbonate is 50 mg / L), and culture them statically in the dark at 25°C, shake 3 times a day, 5 minutes each time, and continuously culture for 3 days, then filter to obtain activated algal species.
[0027] S2. Inoculate the activated algal species into a culture flask containing 100 mL of sterilized BG-11 medium at an inoculation amount of 10%, adjust the pH to 7.5 with sodium bicarbonate, add magnesium sulfate to 8 mg / L, place the culture flask on a constant temperature shaker at 45°C (120 rpm), and irradiate it with LED blue-violet light (the intensity ratio of blue light to violet light is 3:1, and the total light intensity is 2500 lux). During this period, filter-sterilized air is introduced, aerate for 10 minutes every two hours, and the aeration volume is 0.6 L / min. After culturing for 7 days, filter to obtain the first-stage cultured algal species.
[0028] S3. Inoculate the primary cultured algal seeds into a closed reactor containing 30 L of sterilized BG-11 medium at an inoculation amount of 5%, then adjust the pH to 7.5 with sodium bicarbonate. At 50 °C, raise the temperature by 2 °C per day from the 1st to the 3rd day, and keep the temperature constant from the 4th to the 10th day. Under the irradiation of LED red and blue light (the intensity ratio of red light to blue light is 3:1, the total light intensity is 3000 lux, 14 h of light, 10 h of darkness), stir magnetically (200 rpm). During the cultivation, filter-sterilized air is introduced, with microporous aeration, and the aeration rate is 0.8 L / min. Supplement magnesium sulfate to 4 mg / L every 48 hours. The cultivation cycle is 10 days.
[0029] Example 2
[0030] A cultivation method for Yunnan hot spring algae specifically includes the following steps:
[0031] S1. Take the algal seeds of Synechococcus elongatus and inoculate them into 50 mL of sterilized BG-11 medium (the magnesium sulfate content in the medium is 5 mg / L, and the sodium bicarbonate content is 50 mg / L) at an inoculation density of 1.0×10 6 cells / mL. Cultivate statically in the dark at 25 °C, shake 3 times a day, 5 min each time, and continue to cultivate for 3 days. Then filter to obtain activated algal seeds.
[0032] S2. Inoculate the activated algal seeds into a culture flask containing 100 mL of sterilized BG-11 medium at an inoculation amount of 10%. Adjust the pH to 8.0 with sodium bicarbonate, and supplement magnesium sulfate to 8 mg / L. Place the culture flask on a constant temperature shaker at 40 °C (120 rpm). Under the irradiation of LED blue and violet light (the intensity ratio of blue light to violet light is 3:1, the total light intensity is 2500 lux), filter-sterilized air is introduced during the process, aerate for 10 min every two hours, and the aeration rate is 0.6 L / min. After cultivating for 7 days, filter to obtain the primary cultured algal seeds.
[0033] S3. Inoculate the primary cultured algal seeds into a closed reactor containing 30 L of sterilized BG-11 medium at an inoculation amount of 5%, then adjust the pH to 8.0 with sodium bicarbonate. At 45 °C, raise the temperature by 2 °C per day from the 1st to the 3rd day, and keep the temperature constant from the 4th to the 10th day. Under the irradiation of LED red and blue light (the intensity ratio of red light to blue light is 3:1, the total light intensity is 3000 lux, 14 h of light, 10 h of darkness), stir magnetically (200 rpm). During the process, filter-sterilized air is introduced, with microporous aeration, and the aeration rate is 0.8 L / min. Supplement magnesium sulfate to 4 mg / L every 48 hours. The cultivation cycle is 10 days.
[0034] Example 3
[0035] A cultivation method for Yunnan hot spring algae specifically includes the following steps:
[0036] S1. Take the algal seeds of Synechococcus elongatus and inoculate them into6 Inoculate at 1.0×10 cells / mL into 50 mL of sterilized BG-11 medium (the content of magnesium sulfate in the medium is 5 mg / L and the content of sodium bicarbonate is 50 mg / L), and culture statically in the dark at 25 °C, shake 3 times a day for 5 min each time, continuously culture for 3 days, and filter to obtain the activated algal species.
[0037] S2: Inoculate the activated algal species into a culture flask containing 100 mL of sterilized BG-11 medium at an inoculation amount of 10%, adjust the pH to 8.5 with sodium bicarbonate, add magnesium sulfate to 8 mg / L, place the culture flask on a constant temperature shaker at 40 °C (120 rpm), and irradiate with LED blue-violet light (the intensity ratio of blue light to violet light is 3:1, and the total light intensity is 4000 lux). During this period, filter-sterilized air is introduced, aerate for 10 min every two hours, and the aeration volume is 0.6 L / min. After culturing for 7 days, filter to obtain the first-stage cultured algal species.
[0038] S3: Inoculate the first-stage cultured algal species into a closed reactor containing 30 L of sterilized BG-11 medium at an inoculation amount of 5%, then adjust the pH to 8.5 with sodium bicarbonate. At 45 °C, from the 1st to the 3rd day, raise the temperature by 2 °C every day, and keep a constant temperature from the 4th to the 10th day. Under the irradiation of LED red-blue light (the intensity ratio of red light to blue light is 3:1, the total light intensity is 5000 lux, 14 h of light, 10 h of darkness), stir magnetically (200 rpm). During this period, filter-sterilized air is introduced, micro-aeration is carried out, and the aeration volume is 0.8 L / min. Supplement magnesium sulfate to 4 mg / L every 48 hours, and the culture period is 10 days.
[0039] Control Example 1
[0040] The difference between this control example and Example 2 is that the light used in this control example is full-spectrum light.
[0041] Control Example 2
[0042] A cultivation method for Yunnan hot spring algae specifically includes the following steps:
[0043] S1: Take the algal species of Synechococcus elongatus and inoculate at 1.0×10 6 Inoculate at 1.0×10 cells / mL into 50 mL of sterilized BG-11 medium (the content of magnesium sulfate in the medium is 5 mg / L and the content of sodium bicarbonate is 50 mg / L), and culture statically in the dark at 25 °C, shake 3 times a day for 5 min each time, continuously culture for 3 days, and filter to obtain the activated algal species.
[0044] S2. Inoculate the activated algal species into a culture flask containing 100 mL of sterilized BG-11 medium at an inoculation amount of 10%, adjust the pH to 6.0 with sodium bicarbonate, supplement magnesium sulfate to 8 mg / L, place the culture flask on a constant temperature shaker at 30 °C (120 rpm), irradiate it with LED blue-violet light (the intensity ratio of blue light to violet light is 3:1, and the total light intensity is 2500 lux), during which filter-sterilized air is introduced, ventilate for 10 min every two hours, the ventilation volume is 0.6 L / min, filter after culturing for 7 days to obtain the primary cultured algal species.
[0045] S3. Inoculate the primary cultured algal species into a closed reactor containing 30 L of sterilized BG-11 medium at an inoculation amount of 5%, then adjust the pH to 6.0 with sodium bicarbonate, at 30 °C, from the 1st to the 3rd day, increase the temperature by 2 °C every day, from the 4th to the 10th day, keep the temperature constant, under the irradiation of LED red-blue light (the intensity ratio of red light to blue light is 3:1, the total light intensity is 3000 lux, 14 h of light, 10 h of darkness), magnetic stirring (200 rpm), during which filter-sterilized air is introduced, microporous aeration, the ventilation volume is 0.8 L / min, supplement magnesium sulfate to 4 mg / L every 48 hours, and the culture period is 10 days.
[0046] Control Example 3
[0047] A cultivation method of Yunnan hot spring algae specifically includes the following steps:
[0048] S1. Inoculate the algal species of Synechococcus elongatus at 1.0×10 6 cells / mL into 50 mL of sterilized BG-11 medium (the content of magnesium sulfate in the medium is 5 mg / L, and the content of sodium bicarbonate is 50 mg / L), statically culture in the dark at 25 °C, shake 3 times a day, 5 min each time, continuously culture for 3 days, filter to obtain the activated algal species.
[0049] S2. Inoculate the activated algal species into a culture flask containing 100 mL of sterilized BG-11 medium at an inoculation amount of 10%, adjust the pH to 8.0 with sodium bicarbonate, supplement magnesium sulfate to 8 mg / L, place the culture flask on a constant temperature shaker at 40 °C (120 rpm), irradiate it with LED blue-violet light (the intensity ratio of blue light to violet light is 3:1, and the total light intensity is 6000 lux), during which filter-sterilized air is introduced, ventilate for 10 min every two hours, the ventilation volume is 0.6 L / min, filter after culturing for 7 days to obtain the primary cultured algal species.
[0050] S3. Inoculate the first-stage cultured algal seeds into a closed reactor containing 30 L of sterilized BG-11 medium at an inoculation amount of 5%. Then adjust the pH to 8.0 with sodium bicarbonate. At 45 °C, from the 1st to the 3rd day, increase the temperature by 2 °C daily. From the 4th to the 10th day, maintain a constant temperature. Under the irradiation of LED red and blue light (the intensity ratio of red light to blue light is 3:1, the total light intensity is 6500 lux, 14 h of light, 10 h of darkness), stir magnetically (200 rpm). During this period, filter and sterilize the air and aerate through micropores with an aeration rate of 0.8 L / min. Supplement magnesium sulfate to 4 mg / L every 48 hours. The culture period is 10 days.
[0051] Performance Test
[0052] Perform content detection and detection of contamination by miscellaneous bacteria on the Synechococcus elongatus cultured by the culture methods described in Examples 1-3 and Comparative Examples 1-3. Measure the content of chlorophyll a in mg / g FW and the biomass in g / L respectively, and detect the pH change of the culture solution to determine whether there is contamination by miscellaneous bacteria.
[0053] Table 1
[0054]
[0055] As can be seen from the data in Table 1, when comparing Comparative Example 1 with Example 2, the light source was changed. The light source was changed to full spectrum, and the results showed a large gap in biomass and chlorophyll A content compared with Example 2, and it was inferior to Example 2. When comparing Comparative Example 2 with Example 1, the culture temperature and pH were reduced. Compared with Example 1, both the biomass and chlorophyll A content decreased; when comparing Comparative Example 3 with Example 3, the light intensity was increased, and compared with Example 3, the biomass and chlorophyll A content decreased significantly, far inferior to Example 3; compared with other examples, the biomass of Synechococcus elongatus in Examples 1-3 of the present application was higher and the content of chlorophyll a was more. Further, the biomass and chlorophyll a content of Example 2 of the present application were the highest compared with other example schemes. The scheme of Example 2 of the present application effectively improved the yield of hot spring algae and the content of chlorophyll a; as can be seen from the table, for the detection of contamination by miscellaneous bacteria in Examples 1-3 and Comparative Examples 1-3, there was only a slight change in the pH of the culture solution, effectively preventing contamination by miscellaneous bacteria. In summary, the best implementation mode of the present application is Example 2.
[0056] The above content is only an example and explanation of the concept of the present application. Those skilled in the art of the present technology can make various modifications or supplements to the described specific embodiments or use similar methods for substitution, as long as they do not deviate from the concept of the invention or exceed the scope defined by the claims of the present application, they should fall within the protection scope of the present application.
Claims
1. A cultivation method of Yunnan hot spring algae, characterized in that, It includes the following steps: S1. Take the algal species of Yunnan hot spring algae and inoculate them into a culture medium, statically culture them in the dark, and filter to obtain activated algal species; S2. Inoculate the activated algal species into a culture flask, adjust the pH to 7.5 - 8.5, supplement with sulfur-containing compounds, place the culture flask on a constant temperature shaker, irradiate with light, and after culturing for 5 - 7 days, filter to obtain the first-stage cultured algal species; S3. Inoculate the first-stage cultured algal species into a closed reactor, then adjust the pH to 7.5 - 8.5, raise the temperature programmatically, irradiate with light, supplement with sulfur-containing compounds, and culture for 8 - 10 days.
2. The culture method of a Yunnan hot spring alga according to claim 1, characterized in that The hot spring algae is at least one of Synechococcus elongatus, Aphanothece nidulans, Chroococcus weissei, Chroococcus minutus, Chroococcus limneticus, Chroococcus minor, Gloeocapsa gelatinosa, Gloeocapsa linearis, Synechococcus oscillans, Oscillatoria chlorina, and Lyngbya tenerrima.
3. The culturing method of a Yunnan hot spring alga according to claim 1, characterized in that The culture medium is at least one of BG-11 medium, MA medium, and M-11 medium.
4. The culturing method of a Yunnan hot spring alga according to claim 1, characterized in that The sulfur-containing compound is at least one of magnesium sulfate, sodium sulfide, sodium sulfate, and cysteine.
5. The culturing method of a Yunnan hot spring alga according to claim 1, characterized in that, The temperature of the constant temperature shaker is 35 - 50°C.
6. The culture method of a Yunnan hot spring alga according to claim 1, characterized in that, The light in step S2 is LED blue-violet light, and the intensity ratio of blue light to violet light is 3 - 5:1 - 3, and the total light intensity is 2000 - 5000 lux.
7. The culture method of a Yunnan hot spring alga according to claim 1, characterized in that, The programmed temperature increase in step S3 is to increase the temperature to 45 - 70°C.
8. The culturing method of a Yunnan hot spring alga according to claim 7, characterized in that, The programmed temperature increase in step S3 is to increase the temperature to 45 - 60°C.
9. The culture method of a kind of Yunnan hot spring alga according to claim 8, characterized in that, The amount of the supplemented sulfur-containing compound in step S3 is 70 - 90% of the initial concentration.
10. A cultivation method of Yunnan hot spring algae according to claim 1, characterized in that, The light in step S3 is LED red-blue light, and the intensity ratio of red light to blue light is 3 - 5:1 - 3, and the total light intensity is 2000 - 5000 lux.