Spirulina with anti-pollution capacity and application thereof
By screening out the SR3, the Spirulina algae strain with strong anti-pollution ability, the problem of spirulina being contaminated in the open culture system was solved, stable growth and efficient production were achieved, cost reduction and product quality was improved.
Patent Information
- Application Number
- CN202510598194.8
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-05-09
- Publication Date
- 2025-08-08
AI Technical Summary
Spirulina is susceptible to external pollutants in open culture systems, resulting in unstable growth and degraded product quality. The existing prevention and control methods have problems of ecological and environmental pollution and high costs.
Through the anti-pollution evolution screening method, a spirulina algae strain SR3 was obtained. This algae strain has significant anti-pollution ability, can resist the invasion of harmful microorganisms and bacteria in an open environment, and grow stably under different water quality and temperature conditions to avoid additional pollution control measures.
The stable growth and efficient production of spirulina have been achieved, production costs have been reduced, product quality and market competitiveness have been improved, and stable industrial production in an open culture system has been achieved.
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Figure CN120442472A_ABST
Abstract
Description
Technical Field
[0001] The invention belongs to the field of biotechnology, and particularly relates to spirulina with anti-pollution ability and application thereof. Background Art
[0002] Spirulina is a prokaryotic microorganism that can perform oxygenic photosynthesis. It is also an edible filamentous cyanobacteria rich in various bioactive substances, such as protein, γ-linolenic acid, vitamins, and glycerol glucose (GG). It has anti-inflammatory, antioxidant, and immune-enhancing properties. In recent years, the industrial production of microalgae has rapidly developed. Due to its ease of large-scale cultivation and harvesting and its high economic value, spirulina is increasingly used in the production of high-value products such as functional foods, biopharmaceuticals, and cosmetics. It also has great potential in mitigating greenhouse gas emissions, wastewater treatment, and agricultural green manure.
[0003] Industrial production of spirulina primarily occurs in open culture systems, such as raceway ponds. These systems are widely adopted due to their simple process design and low construction costs. However, their major drawback is their susceptibility to external environmental influences. Intrusion by eukaryotic algae, harmful microorganisms, pathogens, and other harmful substances can lead to unstable cultivation processes and even failure. External contamination not only affects the growth efficiency of spirulina but also compromises the stability and quality of its nutrients, ultimately affecting the quality of the final product.
[0004] While there has been some exploration into spirulina pollution control within academia and industry, most research has focused on optimizing and improving cultivation conditions or using chemical reagents and pharmaceuticals. While these approaches have mitigated pollution from the spirulina aquaculture industry to some extent, they often come with potential ecological and environmental pollution, require additional control measures, and increase production costs.
[0005] Therefore, there is an urgent need for Spirulina strains that can grow stably in open culture systems and exhibit significant resistance to biofouling to ensure smooth production. Such strains would not only improve the production efficiency of the spirulina industry but also reduce production costs, enhancing the quality and market competitiveness of the final product. Developing high-yielding Spirulina strains that are resistant to contamination and stress has become a key technological need and research hotspot in the field of spirulina production. Summary of the Invention
[0006] To solve the above problems, the present invention provides a spirulina with anti-pollution ability. The spirulina was deposited in the General Microbiology Center of the China Culture Collection Administration on December 25, 2024, with the deposit number: CGMCC No.46270.
[0007] This spirulina strain exhibits significantly greater pollution resistance than wild-type spirulina, effectively resisting interference from harmful microorganisms, pathogens, and other harmful substances in open culture environments. It can grow stably across diverse water quality conditions and temperature ranges, exhibiting strong environmental adaptability and maintaining growth and metabolic activity in environments with fluctuating water quality or significant temperature changes. During the cultivation process of this spirulina strain, no additional pollution control measures are required, significantly reducing production costs and improving production efficiency.
[0008] The present invention also provides the use of the spirulina in producing target metabolites.
[0009] In a specific embodiment, the target metabolites include one or more combinations selected from glycerol glucoside, protein, γ-linolenic acid, and vitamins.
[0010] The present invention also provides a method for culturing the spirulina, comprising the step of culturing the spirulina in an open environment or an environment without additional pollution control.
[0011] In a specific embodiment, the culture temperature is 15-35°C.
[0012] In a specific embodiment, contaminating bacteria are added to the culture environment. By actively adding contaminating bacteria that are lethal to wild-type Spirulina or other Spirulina, the invasion of non-target Spirulina species can be better prevented, and the purity of the target Spirulina species can be maintained.
[0013] The present invention screened out a Spirulina strain with pollution resistance through an anti-pollution evolution screening method. The Spirulina strain can effectively resist the interference of external contaminating bacteria in open culture. The Spirulina strain has a significantly higher pollution resistance than the wild-type Spirulina and can be cultured in an open culture. It is an excellent algae species suitable for Spirulina cultivation and related industrial production, which can effectively reduce production costs and realize stable industrial production.
[0014] Microbial Deposit
[0015] The Spirulina described in the present invention is deposited in the General Microbiology Center of the China Culture Collection Administration of Microorganisms with the deposit number of CGMCC No. 46270 and the deposit date of December 25, 2024. The address of the depository is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing. The scientific name is Spirulina platensis. BRIEF DESCRIPTION OF THE DRAWINGS
[0016] Figure 1 This is an optical micrograph of the algae strain SR3.
[0017] Figure 2Statistical graphs of filament length and cell width of wild-type algae strain and antibacterial algae strain SR3.
[0018] Figure 3 The growth of wild and antibacterial algae strains simulates actual production. The left figure shows the growth curve, and the right figure shows photos of culture samples on days 3, 4, 5, 7, 11, and 14 of culture. ZKLZ-CK represents the wild-type strain without contamination, ZKLZ-CG represents the wild-type strain with contamination, SR3-CK represents the SR3 strain without contamination, and SR3-CG represents the SR3 strain with contamination.
[0019] Figure 4 Statistical charts of GG (glycerol glucoside) content at different time points during simulated production of wild-type and antibacterial algae strains. ZKLZ-CK represents the wild-type group without contaminating bacteria, ZKLZ-CG represents the wild-type group with contaminating bacteria, SR3-CK represents the SR3 group without contaminating bacteria, and SR3-CG represents the SR3 group with contaminating bacteria.
[0020] Figure 5 Flow chart for the culture of antibacterial algae strain expansion system.
[0021] Figure 6 To test the antibacterial properties of antibacterial algae strains cultured outdoors.
[0022] Figure 7 Statistics of GG production in April, May, November and December of different years (wild-type algae strains were used for production and cultivation in 2021-2023, and SR3 was used for production and cultivation in 2024). DETAILED DESCRIPTION
[0023] The principles and features of the present invention are described below with reference to examples. The examples are only used to explain the present invention and are not used to limit the scope of the present invention.
[0024] 1. Obtaining Spirulina strains resistant to biofouling based on evolutionary rescue strategies
[0025] Spirulina plate was cultured in Zarrouk medium. 730 ≈0.1 was inoculated into fresh sterile Zarrouk medium and cultured in a 50 mL small shake flask with a culture volume of 20 mL. The culture was continued on a shaker for 5-7 days. The conditions for the shaker culture were: light intensity of 30-50 μmol m -2 s -1 , the shaking speed was 150 rpm, the culture temperature was 30°C, and the culture was continued until the logarithmic phase.
[0026] Contaminating bacteria were added to a spirulina culture system and cultured on a shaker for five days, causing nearly all of the spirulina in the culture system to die. The culture system was then placed in a static incubation at room temperature under natural light, allowing the spirulina in the culture system to recover and develop resistance to the contaminating bacteria. The resuscitated spirulina was isolated and purified, then expanded and passaged. The expanded spirulina was then infected with the contaminating bacteria and cultured on a shaker for five to seven days to verify its antibacterial properties, thereby identifying antibacterial spirulina strains with contamination resistance.
[0027] 2. Identification and preservation of antibacterial Spirulina strains with anti-pollution capabilities
[0028] Through the above method, an antibacterial Spirulina strain SR3 was screened, and its morphology is as follows Figure 1 As shown in the figure, 100 algal filaments were selected from each of the wild-type algae strain and the antibacterial algae strain for statistical analysis. It was found that the antibacterial algae strain had significant differences in morphology (filament length, cell width, etc.) compared with the wild-type strain, as shown in the figure. Figure 2 In addition, whole genome sequencing revealed mutations in the genome.
[0029] Spirulina is deposited in the General Microbiology Center of China Culture Collection Administration of Microorganisms with the deposit number of CGMCC No. 46270 and the deposit date of December 25, 2024. The address of the depository is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing. The scientific name is Spirulina platensis.
[0030] 3. Growth characteristics of Spirulina SR3
[0031] Spirulina SR3 and wild-type algae were cultured in column culture. In order to simulate the actual culture conditions of outdoor production, the algae were cultured at OD 730 ≈0.1 inoculation, 1% (v / v) contaminating bacteria were added to the ZKLZ-CG group and the SR3-CG group after 1 day of culture. On the 3rd day of culture, NaCl was added to all groups to make the final concentration of NaCl in the culture medium reach 0.8M / L. On the 7th day of culture, the microalgae in the column reactor were harvested until the algal cell concentration in each column returned to OD 730 ≈0.1, continue to cultivate for another cycle.
[0032] The status of each group of algae strains was tested, and the results were as follows Figure 3 As shown, three days after the addition of the contaminating bacteria, the wild-type algae strain exhibited poor growth and slowed growth, resulting in a significant decrease in biomass, under the dual stresses of contamination and high salt levels. After one round of harvesting, the algae ceased growth, and no significant accumulation of microalgal biomass was observed in the culture system. In contrast, the antibacterial algae strain SR3 maintained a similar growth rate to the ZKLZ control during both salt stress culture and harvesting, regardless of whether the contaminating bacteria were added.
[0033] In terms of GG yield evaluation, systematic testing was conducted on samples taken on the 5th, 7th and 14th days. The results were as follows: Figure 4 As shown, the GG production of the wild-type algae strain was severely affected after the addition of contaminating bacteria. After one round of harvest, the biomass decreased sharply and almost no GG production was achieved. However, the GG production of the antibacterial algae strain SR3 was not affected by the contaminating bacteria.
[0034] In a column culture system simulating actual production, the GG synthesis capacity of SR3 algae strain under the interference of contaminating bacteria was tested ( Figure 4 ) and found that the GG production of the SR3-CG group (under contamination conditions) was not significantly different from that of the SR3-CK group (under non-contamination conditions), and both were significantly higher than that of the wild-type ZKLZ-CG group. Specifically, on the 7th day of culture, the GG production of the SR3-CG group was 163 mg / L / OD 730 , while the ZKLZ-CG group was only 76 mg / L / OD 730 .
[0035] 4. Gradual expansion of algae cultivation and production verification of anti-pollution ability
[0036] After verification in a small system, the antibacterial Spirulina was expanded step by step:
[0037] First, aerate the 1 L system using sterile Zarrouk medium at 25–30°C and 50–100 μmol m -2 s -1 After 5-7 days of cultivation, the algae growth rate is the same as that of the wild type. Then it is expanded to a 10L plate reactor, and the culture conditions are the same as above. After 5-7 days, it is expanded to a 100L plate reactor. After another 5-7 days of cultivation, it is further expanded to a 100L raceway tank, and finally to a ton-level raceway tank for actual production ( Figure 5 ), outdoor cultivation, culture medium is not sterilized, algae strains can adapt to water temperature of 15-35 ℃. After the algae strains are put into production, samples from the runway pool are collected regularly every month for anti-pollution ability testing. The results are as follows Figure 6 As shown, the SR3 strain cultured outdoors was genetically stable and maintained its antimicrobial capacity, while the wild-type control was infected by contaminating bacteria. A year of practical production validation demonstrated that the resistant strain remained free of biofouling throughout the entire cultivation cycle.
[0038] 5. Production and verification of bioactive substances under large-scale production and cultivation
[0039] In actual production in 2024 ( Figure 7 ), the GG production of SR3 strain remained stable in different months of the year (April: 145 mg / L / OD 730 December: 129 mg / L / OD 730), unaffected by seasonal temperature fluctuations and pollution, demonstrating its anti-pollution capabilities and stable GG synthesis. Furthermore, the cultivation of pollution-resistant algae strains surpassed the traditional cultivation time window, achieving for the first time the stable production of multiple batches of GG under low-temperature conditions in December.
[0040] The above experiments show that the antibacterial Spirulina strain SR3 of the present invention has good anti-pollution ability, can effectively resist the interference of external contaminating bacteria in open culture, and realize stable industrial production.
[0041] Although GG is listed as an example of its industrial production for illustrative purposes in the specific embodiments of this application, the use of the algae strain of the present invention should not be limited to this. The use of the algae strain of the present invention or its engineered algae strain to produce GG or other biologically active substances (such as protein, γ-linolenic acid, one or more combinations of vitamins, etc.) should be included in the scope of protection of the present invention.
[0042] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.
Claims
1. A Spirulina with anti-pollution ability, characterized in that: It was deposited in the General Microbiology Center of China Culture Collection Administration on December 25, 2024, with the accession number: CGMCC No.46270.
2. Use of the Spirulina according to claim 1 in producing target metabolites.
3. The use according to claim 2, characterized in that The target metabolites include one or more combinations selected from glycerol glucoside, protein, γ-linolenic acid, and vitamins.
4. A method for culturing the Spirulina according to claim 1, characterized in that: The method comprises the steps of culturing the spirulina in an open environment or an environment without additional pollution control.
5. The method according to claim 4, characterized in that The culture temperature is 15-35℃.
6. The method according to claim 4, characterized in that Contaminating bacteria were added to the culture environment.