A strain of Alteromonas with algae-lysing ability and its application in algae-lysing Karenia mikimotoi
Algae-soluble agents were prepared by culturing alternating monocytogenes JY-JZ1, and the problems of high red tide treatment cost and secondary pollution in the prior art were solved, and efficient and environmentally friendly red tide control effect was achieved.
Patent Information
- Application Number
- CN202310235125.1
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- Filing Date
- 2023-03-13
- Publication Date
- 2025-09-02
- Estimated Expiration
- 2043-03-13
AI Technical Summary
The prior art is costly and prone to secondary pollution when treating red tides. The biological methods lack efficient algae-soluble bacteria, making it difficult to effectively control the growth of red tide algae.
Algae-soluble bacteria agents are prepared through culture and fermentation, and are used for red tide algae prevention and control, especially for common red tide algae such as Kellen algae, microproditinothenol and midrib strip algae.
The algae dissolved rate of Alternative Monassia JY-JZ1 on red tide algae such as Karen algae is as high as 80%, which is highly adaptable and environmentally friendly, and will not cause secondary pollution.
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Abstract
Description
Technical Field
[0001] The present invention relates to the field of environmental microorganisms, in particular to a strain of Alteromonas with algae-lysing ability, and also to the algae-lysing application of the strain of Alteromonas with algae-lysing ability on Karenia mikimotoi. Background Art
[0002] In recent decades, with the advancement of science and technology and economic progress, the impact of human activities on the environment has gradually increased. The discharge of industrial and domestic wastewater, as well as the development of agriculture and mariculture, has led to increasing eutrophication of water bodies, changes in the marine ecosystem, and frequent outbreaks of red tides worldwide, resulting in significant economic losses and environmental damage. Marine red tides are increasingly becoming an obstacle to the sustainable development of my country's marine economy.
[0003] Currently, there are physical, chemical, and biological methods for treating red tides. Physical and chemical methods are relatively expensive, and the addition of foreign substances can easily cause secondary pollution, and have direct or indirect side effects on other organisms. Compared with physical and chemical methods, biological methods are low-cost and environmentally friendly. Because microorganisms have characteristics such as short generation cycles, rapid reproduction, and rapid metabolism, the use of microorganisms to prevent and control harmful red tides occupies an important position in biological methods. In surface water environments and soils, especially in environments with algal blooms and red tide outbreaks, the growth and metabolism of algae-inhibiting bacteria usually become very active. They can inhibit the growth of red tide algae cells through direct contact or secretion of algae-inhibiting substances, and through competition for nutrients such as nitrogen and phosphorus, playing an important role in the natural reduction of red tides.
[0004] Therefore, technicians are committed to screening highly efficient algae-lytic bacteria or isolating highly efficient algae-lytic active substances, and then developing microbial algaecides as an effective means of red tide prevention and control. Summary of the Invention
[0005] In view of the above, the object of the present invention is to provide an Alteromonas JY-JZ1 having a strong algae-lyzing effect on Karenia mikimotoi and its application.
[0006] In order to achieve the above objectives, the technical solution adopted by the present invention is:
[0007] A strain of Alteromonas abrolhosensis JY-JZ1 with algae-lytic ability was deposited with the China General Microbiology Center under the China Culture Collection Administration on October 11, 2021, under the accession number CGMCC No. 23511. The 16s rRNA gene sequence of the strain is listed in GenBank as ON533324.
[0008] The method for culturing the Alteromonas comprises the following steps:
[0009] (1) Bacterial strain: the Alteromonas JY-JZ1 strain was used;
[0010] (2) Plate culture: The bacteria in step (1) were inoculated on a solid plate culture medium and cultured at 28°C for 24 hours;
[0011] (3) Primary culture: Aseptically take a plate that has been cultured and use an inoculating loop to take a loopful of culture into 20 mL of liquid culture medium. Incubate at 28°C with a shaker at 180 rpm for 24 h to obtain a primary culture solution.
[0012] (4) Fermentation: The bacterial solution prepared in step (3) was inoculated into 200 ml of liquid culture medium at an inoculum size of 1%, and cultured at 28° C. and 180 rpm for 24 hours, and the fermentation liquid was collected;
[0013] The liquid culture medium described in steps (3) and (4) is formulated as follows: 5 g / L peptone, 1 g / L yeast extract, 0.01 g / L ferric phosphate, and 1 L artificial seawater. The pH is adjusted to 7.6-8.2 and sterilized by high-temperature steam sterilization at 121°C for 20 min. Agar powder is added to the solid culture medium at a final concentration of 1.6 wt%.
[0014] The colonies of the Alteromonas strain JY-JZ1 are round, milky white, entire, slightly protruding, with a smooth, slightly moist surface, and rod-shaped bodies. Gram staining is negative. Alteromonas JY-JZ1 can utilize glucose, mannitol, sucrose, fructose, maltose, and lactose as sole carbon sources for bacterial growth and can produce amylase, gelatinase, oxidase, and catalase. The results of the methyl red test, VP test, and nitrate reduction test for Alteromonas JY-JZ1 are all negative.
[0015] The present invention also provides an algae-lytic agent for Alteromonas JY-JZ1.
[0016] The present invention also provides an algae-lytic agent prepared from the algae-lytic agent.
[0017] The present invention also provides the use of Alteromonas JY-JZ1 in the prevention and treatment of red tide algae.
[0018] The present invention discloses an application of the Alteromonas JY-JZ1 in algae lysis, wherein the algae include Karenia mikimotoi, Skeletonemacostatum, Prorocentrum minimum and the like, which are common red tide algae.
[0019] The main effects and advantages of the present invention are:
[0020] 1. The obtained Alteromonas JY-JZ1 has a good algae-lyzing effect, especially on Karenia mikimotoi. The bacterial fermentation broth has algae-lyzing activity against Karenia mikimotoi, Prorocentrum microphylum, and Skeletonema costatum, reaching more than 80% within 72 hours.
[0021] 2. The fermentation broth of Alteromonas JY-JZ1 has high adaptability to temperature (-20℃~100℃) and pH (3~13);
[0022] 3. Alteromonas JY-JZ1 originates from the marine environment, is environmentally friendly, and will not cause secondary pollution when used for red tide prevention and control, and has good application prospects. BRIEF DESCRIPTION OF THE DRAWINGS
[0023] Figure 1 The algae-lytic activity of the strain filtrates at different concentrations against Karenia mikimotoi in Example 2 is shown.
[0024] Figure 2 The changes in the cell morphology of Karenia mikimotoi observed in Example 3.
[0025] Figure 3 This is the experiment to verify the effect of the fermentation filtrate of Alteromonas JY-JZ1 on the maximum quantum yield of algal cells in Example 4.
[0026] Figure 4 This is the experiment to verify the effect of the fermentation filtrate of Alteromonas JY-JZ1 on the apparent photosynthetic efficiency of algal cells in Example 4.
[0027] Figure 5 This is an experiment to verify the effect of the fermentation filtrate of Alteromonas JY-JZ1 on the maximum relative electron transfer rate in Example 4.
[0028] Figure 6 The algae lysis rate of the three red tide algae treated with the fermentation filtrate of Alteromonas JY-JZ1 obtained in the experiment of Example 5 for 72 hours. DETAILED DESCRIPTION
[0029] In order to more clearly understand the above-mentioned objects, features and advantages of the present invention, the present invention is further described in detail below in conjunction with the accompanying drawings and specific embodiments. It should be noted that the embodiments of the present application and the features therein can be combined with each other without conflict.
[0030] Example 1: Identification of Alteromonas JY-JZ1 strain
[0031] Morphological observation, physiological and biochemical reactions, staining, and sugar fermentation experiments on the JY-JZ1 strain revealed the following biological characteristics: JY-JZ1 colonies are round, milky white, with entire, slightly protruding margins, a smooth, slightly moist surface, and rod-shaped bodies. Gram staining was negative. JY-JZ1 can utilize glucose, mannitol, sucrose, fructose, maltose, and lactose as sole carbon sources for bacterial growth and can produce amylase, gelatinase, oxidase, and catalase. JY-JZ1 tested negative in the methyl red test, VP test, and nitrate reduction test.
[0032] The 16s rRNA of JY-JZ1 strain was amplified, and the gene sequence was cloned and sequenced. The results showed that it was similar to that of Alteromonas abrolhosensis PEL67E. T The homology was 99.3%, so it was identified as Alteromonas abrolhosensis and named Alteromonas JY-JZ1.
[0033] The strain was deposited at the General Microbiology Center of China Culture Collection Administration on October 11, 2021, with the deposit number: CGMCC No.23511.
[0034] The accession number of the 16srRNA gene sequence of the strain in GenBank is ON533324.
[0035] Table 1 Physiological and biochemical reaction results of JY-JZ1 strain
[0036]
[0037] Example 2: Algal-lyzing effect of different concentrations of Alteromonas JY-JZ1 fermentation filtrate on Karenia mikimotoi
[0038] (1) Karenia mikimotoi was cultured in F / 2 medium (see Table 2). Stock solution D was sterilized by filtration through a 0.22 μm PVDF membrane. 1 mL of each of stock solutions ABC was added to 1 L of sterile seawater and sterilized at 121°C for 20 min. Before use, 1 mL of stock solution D was added.
[0039] Table 2F / 2 culture medium formula
[0040]
[0041] (2) Alteromonas JY-JZ1 was cultured in 2216E medium. The formula is shown in Table 3. The medium was sterilized at 121°C for 20 min and then used.
[0042] Table 32216E culture medium formula
[0043]
[0044] (3) The strain was inoculated into liquid 2216E medium for activation. The activated bacterial solution was inoculated into liquid 2216E medium at 1% (v / v) for fermentation and culture for 24 hours. The fermentation filtrate was added to the Karenia mikimotoi algae solution in the exponential growth phase at 1%, 2.5%, 5%, 7.5%, and 10% (v / v) for co-cultivation. A blank control was set up, and the control group was algae solution of the same period with 1%, 2.5%, 5%, 7.5%, and 10% volume of sterile 2216E medium added. Three replicates were set up for both the experimental group and the control group. Samples were taken at 0h, 12h, 24h, 36h, and 48h, the algae cell concentration was counted, and the algae lysis rate was further calculated.
[0045] As shown in the figure, the changes in algae lytic activity in different concentration groups indicate that filtrate treatment inhibits the growth of algal cells. There is a dose effect on algae lytic activity. When the filtrate is added at a low concentration (1%, 2.5%), it can be reflected that the growth rate of algal cells is hindered or stagnant. High concentrations (5%, 7.5%, 10%) have obvious algae lytic effects. The algae lytic rate of the 10% group is close to 100% within 12 hours.
[0046] Example 3: Microscopic observation of the algae lysis process of Karenia mikimotoi using the fermentation filtrate of Alteromonas JY-JZ1
[0047] Alteromonas JY-JZ1 fermentation filtrate was added to the Karenia mikimotoi algae solution at a rate of 5% (v / v). Algal cells were observed every 24 hours using a Leica microscope, and differential interference imaging was used to record morphological changes in Karenia mikimotoi.
[0048] Microscopic examination revealed that over time, the algal cells swelled and deformed, becoming spherical. Organelles moved closer to the nucleus. After 24 hours, the cells began to stratify, with increased blanking. Cytochromes shrank into clusters, and structures resembling nuclei were visible. Filtrate stress likely weakened the cell membrane, and after 48 hours, the contents of the algal cells were generally precipitated.
[0049] Example 4: Effect of Alteromonas JY-JZ1 Fermentation Filtrate on the Photosynthetic Activity of Karenia mikimotoi. The photosynthetic fluorescence parameters of Karenia mikimotoi were measured using a Phyto-PAM Phytoplankton Analyzer (HeinzWalz GmbH, Effeltrich, Germany). Strain filtrate or 2216E medium was added to the Karenia mikimotoi algae at a 5% (v / v) concentration. Samples were collected every 12 hours for 48 hours. Before measurement, each sample was acclimated in the dark for 15 minutes to ensure that all reaction centers were open.
[0050] The initial value of the maximum quantum yield Fv / Fm of algae cells was 0.523. As the stress time increased, the Fv / Fm value of the treatment group gradually decreased, showing a significant difference from the control group. Compared with the control group, the Fv / Fm value at 24 hours decreased by 65.08%, indicating that the photosynthetic activity of algae cells was inhibited. The maximum relative electron transfer rate rETR of the treatment group was 0.523. max The value gradually decreases over time after 6 hours, and the rETR after 24 hours max The filtrate stress significantly reduced the efficiency of Karenia mikimotoi's light energy utilization. After 24 hours, the apparent photosynthetic efficiency (α) of the control group was 0.165, while that of the treated group was only 0.064. The growth of the algal cells in the control group was significantly better than that in the treated group. The filtrate significantly interfered with the normal photosynthesis of the algal cells.
[0051] Example 5: Killing effect of Alteromonas JY-JZ1 on three common red tide algae
[0052] The fermentation product of algae-lytic bacteria JY-JZ1 was inoculated into Karenia mikimotoi, Skeletonemacostatum, and Prorocentrum minimum in the logarithmic growth phase at a volume concentration of 15% (v / v). Three replicates were set for each algae species. The culture medium was collected at 72 h, the algae cell concentration was counted, and the algae lysis rate was further calculated (the results are shown in Table 4 below). Table 4 Algae lysis rate of JY-JZ1 for three common red tide algae
[0053]
[0054] The algae-lyzing rate of the fermentation filtrate of Alteromonas JY-JZ1 on the three red tide algae was above 80%.
[0055] Obviously, those skilled in the art may make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if such changes and modifications fall within the scope of the claims and their equivalents, the present invention is intended to include such changes and modifications.
Claims
1. A strain of Alteromonas with algae-lysing ability, Alteromonas ( Alteromonas abrolhosensis )JY-JZ1 was deposited in the General Microbiology Center of China Culture Collection Administration on October 11, 2021, with the deposit number: CGMCC No.23511.
2. The method for culturing the algae-lytic Alteromonas according to claim 1, wherein: The following steps are involved: (1) Bacterial strain: the Alteromonas JY-JZ1 described in claim 1; (2) Plate culture: inoculate the bacteria in step (1) onto a solid plate culture medium and culture at 28°C for 24 hours; (3) Primary culture: Take the cultured plate under sterile conditions, take a loop with an inoculating loop and put it into 20 mL of liquid culture medium, culture it at 28°C and shake it at 180 rpm for 24 hours to obtain the primary culture solution; (4) Fermentation culture: The bacterial solution prepared in step (3) was inoculated into 200 ml of liquid culture medium at an inoculum size of 1%, cultured at 28°C and 180 rpm for 24 hours, and the fermentation liquid was collected; The liquid culture medium described in steps (3) and (4) is formulated as follows: 5 g / L peptone, 1 g / L yeast extract, 0.01 g / L ferric phosphate, and 1 L artificial seawater. The pH is adjusted to 7.6-8.2 and sterilized by high-temperature steam sterilization at 121°C for 20 min. Agar powder is added to the solid culture medium at a final concentration of 1.6 wt%.
3. An algae-lytic agent comprising the Alteromonas JY-JZ1 according to claim 1.
4. An algae-lytic agent prepared from the algae-lytic agent according to claim 3.
5. Use of the Alteromonas JY-JZ1 according to claim 1 in the prevention and treatment of red tide algae.
6. The algae-lysing application of the Alteromonas JY-JZ1 according to claim 1, characterized in that: The algae include Karenia mikimotoi ( Karenia mikimotoi )、Skeletonea costatum( Skeletonema costatum )、Prorocentrum microphyllum( Prorocentrum minimum ).