Method for screening spirulina with anti-pollution capacity
By using the contaminated bacteria Cyclonatronum proteinivorum to conduct stress evolutionary screening of spirulina, the problem of spirulina being susceptible to contamination was solved, and efficient anti-contamination algae strains were screened out, which were suitable for industrial breeding of microalgae.
Patent Information
- Application Number
- CN202510598318.2
- 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
The prior art lacks effective methods to screen out spirulina with anti-pollution ability, which leads to susceptibility to predators and pathogens during the spirulina culture, resulting in failure in culture and economic losses.
The contaminated bacteria Cyclonatronum proteinivorum and its mixed bacterial solution were used to perform stress evolution screening on Spirulina. Through light and standstill culture, spirulina strains with anti-pollution ability were screened out.
It has achieved rapid and effective screening of spirulina strains with high pollution resistance, reduced breeding costs, improved the growth rate and stress resistance of spirulina, and is suitable for industrial production of microalgae.
Smart Images
Figure CN120442748A_ABST
Abstract
Description
Technical Field
[0001] The present invention relates to the field of microalgae cultivation, and more particularly to a method for screening spirulina with anti-pollution ability. 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 biodiesel. It also has great potential in mitigating greenhouse gas emissions, wastewater treatment, and agricultural green manure.
[0003] The open culture process of Spirulina is often contaminated by unfavorable factors such as predators and pathogens. Once these unfavorable factors become dominant, it will be difficult to produce large amounts of microalgae biomass, resulting in culture failure and huge economic losses.
[0004] However, there is currently no method in the art that can effectively screen out Spirulina with anti-pollution ability. Summary of the Invention
[0005] To solve the above problems, the present invention provides a method for screening Spirulina with anti-pollution ability, comprising the step of adding a pollution system into a starting Spirulina culture environment.
[0006] In a specific embodiment, the contamination system is a mixed bacterial liquid containing the contaminating bacteria Cyclonatronum proteinivorum, and the contaminating bacteria are deposited in the General Microbiology Center of the China Culture Collection Administration of Microorganisms, with the deposit number: CGMCC No.46274 and the deposit date being December 25, 2024.
[0007] In a specific embodiment, the method comprises the following steps:
[0008] S1: inoculating the starting Spirulina into a liquid culture medium, and adding the contamination system to obtain a bacteria-algae evolution screening system;
[0009] S2: culturing the bacteria-algae evolution screening system under light until the algae cells are substantially lysed;
[0010] S3: resuscitating the bacteria-algae evolutionary screening system to obtain a resuscitated Spirulina culture;
[0011] S4: Purifying Spirulina with anti-pollution ability from the Spirulina recovery culture.
[0012] In a specific embodiment, in S2, shaking culture is performed for 2-3 days;
[0013] Static culture is performed in S3 for 1-3 months.
[0014] In one embodiment, the culture temperature in S3 is 18-38°C and the light intensity is 10-50 μmolphotons m -2 s -1 .
[0015] In a specific embodiment, the contamination system further comprises one or more combinations of the following bacteria: Halomonas, Aliidiomarina, Natronohydrobacter.
[0016] In a specific embodiment, the pollution system is prepared by the following steps:
[0017] 1) mixing the contaminating bacteria with Halomonas, Aliidiomarina, and Natronohydrobacter to obtain an initial mixed bacterial solution;
[0018] 2) inoculating the initial mixed bacterial solution into a Spirulina culture, and culturing the Spirulina cells until they die, thereby obtaining a lysate;
[0019] 3) inoculating the lysate into a Spirulina culture, culturing until the Spirulina cells die, and passage the cells for more than 4 times, to obtain the lysate as the contaminated system.
[0020] In one embodiment, the concentration of the Spirulina culture is OD 730 ≈0.01-3.
[0021] In one embodiment, the illumination intensity of the culture in steps 2 and 3 is 30-50 μmol photons m -2 s -1 ;
[0022] The culture temperature was 30°C;
[0023] The culture method is shaking culture.
[0024] In a specific embodiment, the lysis solution in step 3 is added to the Spirulina culture at a volume ratio of 0.01%-10%.
[0025] The present invention adopts a stress evolution strategy and uses a mixed bacterial solution to perform stress mutagenesis and evolutionary screening on Spirulina, thereby achieving the goal of using existing Spirulina strains to obtain Spirulina strains with higher pollution resistance, and provides a fast and effective way for Spirulina breeding for microalgae industry.
[0026] Microbial Deposits
[0027] The contaminating bacteria used in the present invention are deposited in the General Microbiology Center of the China Culture Collection Administration of Microorganisms with the deposit number of CGMCC No. 46274 and the deposit date of December 25, 2024. The deposit address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing. The scientific name is Cyclonatronum proteinivorum. BRIEF DESCRIPTION OF THE DRAWINGS
[0028] Figure 1 This is a laser confocal three-dimensional reconstruction image of the contaminating strain attached to the surface of Spirulina cells.
[0029] Figure 2 Flow chart for screening antibacterial algae strains.
[0030] Figure 3 The growth of wild algae and the antibacterial algae strain SR3 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 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. DETAILED DESCRIPTION
[0031] 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.
[0032] 1. Isolation of contaminating bacteria
[0033] During our research on Spirulina cultivation, we isolated a strain that can contaminate Spirulina. Its morphology is shown in Figure 1, and the sequence of its 16S rRNA gene is shown in SEQ ID NO: 1.
[0034] The strain was deposited in the General Microbiology Center of China Culture Collection Administration with the deposit number: CGMCC No.46274, the deposit date is December 25, 2024, the deposit address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, and the scientific name is Cyclonatronum proteinivorum.
[0035] 2. Preparation of the Pollution System
[0036] The preparation method of the pollution system is as follows:
[0037] Prepare a mixed bacterial solution containing Cyclonatronum proteinivorum, Halomonas, Aliidiomarina, and Natronohydrobacter, and inoculate the mixed bacterial solution into fresh pure Spirulina cultured with Zarrouk medium at a rate of 1% (v / v) for several times. The initial OD of Spirulina is 730 The light intensity was 30-50 μmol photons m -2 s -1 The shaking speed is 150 rpm, the culture temperature is 30°C, and after 2 days, the subculture is continued as described above. After 4-5 subcultures, the content of each bacteria in the obtained lysate is stable, and the contaminated system is obtained. The contaminated system is stored in a -80°C refrigerator with a glycerol concentration of 20%-30% for seed preservation.
[0038] Details on the preparation of the contaminated system
[0039] 1) Concentration of Spirulina: Spirulina can be infected in any range of initial inoculation concentration, such as OD 730 The range is 0.01-3, but the best effect is in the range of OD 730 0.2-0.5, usually choose OD 730 ≈0.3.
[0040] 2) Proportion of infection solution addition: The initial mixed bacterial solution is added to the Spirulina culture solution according to a volume ratio. The Spirulina solution can be infected by adding any proportion, such as 0.01%-10% (v / v). Adding too low an amount will result in a longer infection time (for example, when adding 1% contamination system, the infection time takes 48 hours; when adding 0.1% contamination system, the infection time takes 96 hours). Adding too high an amount will introduce too many contaminating bacteria. In order to ensure the infection rate and not introduce too many contaminating bacteria, a ratio of 1% (v / v) is usually selected for the best effect.
[0041] 3) Culture conditions: After the infection solution is added to the Spirulina culture medium, the algal cells can be infected regardless of static culture, shaking culture or ventilation culture, but the efficiency varies. The light conditions are not limited. In order to ensure the highest activity of the infection solution, the light condition of 50 μmol m -2 s -1 Cultured in a shaking incubator.
[0042] 3. Screening of anti-pollution spirulina
[0043] The method of screening resistant algae strains adopts the evolutionary rescue strategy, such as Figure 2As shown, the following steps are included:
[0044] S1: Spirulina was heated to 0D 730 ≈0.3 concentration was inoculated into the culture medium, and the above-mentioned contamination system was added to obtain the bacteria-algae evolution screening system;
[0045] S2: The bacteria-algae evolution screening system was cultured in a shaker under light for 2-3 days (30°C, 30-50 μmol m -2 s -1 , 150 rpm) until the algal cells are basically lysed;
[0046] S3: The bacteria-algae evolution screening system was transferred to a static culture for 1-3 months at a temperature of 18-38°C and a light intensity of 10-50 μmol m -2 s -1 , Spirulina recovery occurred in some lysates;
[0047] S4: Isolate the revived Spirulina into single filaments and use them for subsequent resistance verification.
[0048] Currently, we use a 10-μm irradiance at 18°C and 50 μmol m -2 s -1 The proportion of recovered algae strains screened under these conditions was 11 / 100, the proportion of resistant algae strains was 1 / 100, and the screening period was 3 months; at 30°C and 50 μmol m -2 s -1 Under these conditions, the proportion of recovered algae strains screened was 75 / 100, the proportion of resistant algae strains was 1 / 100, and the screening period was 1 month; at 38°C and 50 μmol / m -2 s -1 The proportion of recovered algae strains screened under the following conditions was 32 / 100, and the proportion of resistant algae strains was 3 / 100, with a screening period of 2 months; the proportion of recovered algae strains screened under room temperature and natural light was 19 / 100, and the proportion of resistant algae strains was 2 / 100, with a screening period of 3 months; the proportion of recovered algae strains screened under 35°C and 100 μmol m -2 s -1 Under the conditions of shaking culture at 220 rpm, the proportion of revived algae strains screened was 1 / 20, the proportion of resistant algae strains was 1 / 20, and the screening period was 20 days.
[0049] Taking one of the resistant algae strains screened as an example, the resistant strain SR3 and the wild-type algae strain were cultured in a column. In order to simulate the culture conditions of actual outdoor production, the algae strains were measured 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. On the 7th day of culture, the biomass in the column was harvested to make the algae concentration in each column return to OD 730 ≈0.1, continue to cultivate for another cycle.
[0050] Monitor the culture status of each group, and the results are as follows Figure 3 As shown, three days after the addition of the contaminant, the wild-type algae strain, affected by the increased salt concentration, experienced a decrease in biomass and a slowing of growth, indicating poor growth. After one round of harvesting, the algae ceased growth, leaving almost no biomass in the culture system. However, the contamination-resistant strain SR3 maintained a similar growth rate to the ZKLZ control during salt stress, regardless of whether the contaminant was added or not, both during culture and harvest.
[0051] This method can specifically screen for algal strains that are resistant to contaminating bacteria, while also simultaneously identifying strains with other desirable traits (such as high growth rate, high yield of compatible substances, and high / low temperature resistance). Compared to traditional microalgae mutagenesis breeding (physical and chemical induction), this method offers lower breeding costs, simpler procedures, higher mutation rates, stronger genetic stability, and the absence of chemical residues, allowing for rapid acquisition of target algal strains.
[0052] 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 method for screening Spirulina with anti-pollution ability, characterized in that: The method includes the steps of adding a pollution system into the initial spirulina cultivation environment.
2. The method according to claim 1, characterized in that The contamination system is a mixed bacterial liquid containing the contaminating bacteria Cyclonatronum proteinivorum. The contaminating bacteria are deposited in the General Microbiology Center of the China Microorganism Culture Collection Administration, with the deposit number: CGMCC No.46274 and the deposit date being December 25, 2024.
3. The method according to claim 2, characterized in that The following steps are involved: S1: inoculating the starting Spirulina into a liquid culture medium, and adding the contamination system to obtain a bacteria-algae evolution screening system; S2: culturing the bacteria-algae evolution screening system under light until the algae cells are substantially lysed; S3: resuscitating the bacteria-algae evolutionary screening system to obtain a resuscitated Spirulina culture; S4: Purifying Spirulina with anti-pollution ability from the Spirulina recovery culture.
4. The method according to claim 3, characterized in that In S2, shaking culture is performed for 2-3 days; Static culture is performed in S3 for 1-3 months.
5. The method according to claim 4, characterized in that The culture temperature in S3 was 18–38 °C and the light intensity was 10–50 μmol photons m -2 s -1 .
6. The method according to claim 2, characterized in that The contamination system further comprises one or more combinations of the following bacteria: Halomonas, Aliidiomarina, and Natronohydrobacter.
7. The method according to claim 6, characterized in that Prepared by the following steps: 1) mixing the contaminating bacteria with Halomonas, Aliidiomarina, and Natronohydrobacter to obtain an initial mixed bacterial solution; 2) inoculating the initial mixed bacterial solution into a Spirulina culture, and culturing the Spirulina cells until they die, thereby obtaining a lysate; 3) inoculating the lysate into a Spirulina culture, culturing until the Spirulina cells die, and passage the cells for more than 4 times, to obtain the lysate as the contaminated system.
8. The pollution system according to claim 7, characterized in that The concentration of the Spirulina culture was OD 730 =0.01-3.
9. The pollution system according to claim 7, characterized in that The light intensity in steps 2 and 3 was 30-50 μmol photons m -2 s -1 ; The culture temperature was 30°C; The culture method is shaking culture.
10. The pollution system according to claim 7, characterized in that In step 3, the lysis solution is added to the Spirulina culture at a volume ratio of 0.01%-10%.