Streptomyces sp. P-10 and application thereof

Abstract

The application discloses a marine Streptomyces sp. P-10 and application thereof, which is preserved in the China Center for Type Culture Collection on May 11, 2022, and the preservation number is CCTCC NO. M2022600. The application can directly and indirectly dissolve algae. In the indirect algal interaction mode, the application can secrete compounds for dissolving algae. In the direct algal interaction mode, the application needs to directly contact algal cells to dissolve algae, and can achieve the effect of dissolving algae by winding microalgal cells.

Description

Technical Field

[0001] This invention belongs to the field of marine biotechnology, specifically relating to a marine Streptomyces sp. P-10 and its applications. Background Technology

[0002] Eutrophication and harmful algal blooms are among the most prominent ecological and environmental problems in my country's coastal waters. The Bohai Sea, the Yangtze River estuary and its adjacent waters, and the nearshore waters of the South China Sea are three typical high-incidence areas for red tides. Large-scale red tide outbreaks have caused severe damage to mariculture and threatened marine ecological security and human health. In recent years, harmful algal blooms such as green tides and brown tides have also appeared in the southern Yellow Sea and Bohai Sea, attracting significant attention. Comprehensive analysis shows that the causes of algal blooms in my country's coastal waters are evolving towards diversification, harmfulness, and smaller scale, which brings many difficulties to the monitoring and management of algal blooms.

[0003] *Prorocentrum donghaiense* and *Karenella mikimotoi* are the most common and dominant species in the East China Sea. Since the 1990s, *Prorocentrum donghaiense* has formed continuous red tides almost every spring, with its influence lasting for a considerable period (about 30 days) and covering an area of ​​up to 105 km². 2 While marine dinoflagellates do not release biotoxins, they reduce zooplankton abundance within their growth range by forming large-scale, dense algal blooms, thereby altering the material and energy cycles of the marine ecosystem. This severely damages marine ecosystems and mariculture, causing enormous economic losses. Karenia mikimotoi is a demonstrably linked algal species to frequent neurotoxicity incidents. It releases various neurotoxins, including hemolytic toxins, ichthyotoxic toxins, cytotoxins, and reactive oxygen species. These toxins are considered the primary cause of marine mortality caused by Karenia mikimotoi, and through the food chain, they accumulate and ultimately threaten seafood safety and human health, disrupting the entire marine ecosystem.

[0004] Currently, red tide control methods mainly include physical, chemical, and biological methods. Physical methods do not cause secondary pollution, but are expensive, inefficient, and prone to recurrence, making them difficult to eradicate. Chemical methods are fast-acting and inexpensive, but can easily cause secondary pollution and other side effects on the local habitat, requiring control of drug dosage and continuous improvement. Biological methods, although slower to take effect, are easy to operate, have low technical costs, and are highly safe, providing sustained suppression of red tide disasters. Biological control methods, especially those represented by microbial and allelopathic technologies, are of great significance and have broad application prospects. Summary of the Invention

[0005] The purpose of this invention is to provide a marine Streptomyces sp. P-10.

[0006] Another object of the present invention is to provide the application of the above-mentioned marine Streptomyces sp. P-10.

[0007] The technical solution of the present invention is as follows:

[0008] A marine Streptomyces sp. P-10 was deposited at the China Center for Type Culture Collection (CCTCC) on May 11, 2022, with accession number CCTCC NO. M2022600. (Location: Wuhan University, Wuhan, Hubei Province, 430072, China)

[0009] The application of the above-mentioned marine Streptomyces sp. P-10 in the preparation of marine algae-inhibiting compositions.

[0010] In a preferred embodiment of the present invention, the marine algae-inhibiting composition further includes N-acetyltryptamine and cellulase secreted by the marine Streptomyces sp. P-10.

[0011] In a preferred embodiment of the present invention, the marine algae-inhibiting composition has an inhibitory effect on Prorocentrum donghaiense and Karenia mikimotoi.

[0012] A marine algae-inhibiting composition, the active ingredient of which includes the above-mentioned marine Streptomyces sp. P-10.

[0013] In a preferred embodiment of the present invention, the active ingredients further include N-acetyltryptamine and cellulase secreted by the marine Streptomyces sp. P-10.

[0014] More preferably, its active ingredients consist of the marine Streptomyces sp. P-10 and its secreted N-acetyltryptamine and cellulase.

[0015] In a preferred embodiment of the present invention, it has an inhibitory effect on Prorocentrum donghaiense and Karenia mikimotoi.

[0016] The beneficial effects of this invention are:

[0017] 1. This invention can dissolve algae through direct and indirect means. In the indirect algae-inhibiting bacterial-algae interaction mode, this invention will secrete compounds that dissolve algae. Direct algae killing requires the living cells of this invention to directly contact algal cells to dissolve algae, and can achieve the algae-dissolving effect by wrapping around microalgal cells.

[0018] 2. This invention can cover and adsorb onto the surface of algal cell shells, wrap around and entwine between the two shells at the edge of the algal cell wall, with some bacterial cells extending into the gaps between the shells. As the treatment time increases, the bacterial cells gradually destroy the algal cell wall and plasma membrane. At this point, the surface of the algal cell no longer has clear granular texture and pores, the cell shrinks, undergoes apoptosis, loses its cellular characteristics, the contents are released, and it gradually disintegrates and dies.

[0019] 3. The surface of the cells of Prorocentrum donghaiense has a hard cellulose shell. This invention can dissolve the shell by releasing a large amount of cellulase and at the same time secrete N-acetyltryptamine to destroy the cell membrane, which helps to kill algae. Attached Figure Description

[0020] Figure 1 This is a graph showing the effect of Streptomyces sp. P-10 on the cell density of Prorocentrum donghaiense in Example 1 of the present invention. The graph shows: (a) the effect of 10% fermentation supernatant on the cell density of Prorocentrum donghaiense; (b) the effect of 10% fermentation supernatant on the Fv / Fm ratio of Prorocentrum donghaiense; (c) the effect of 1% fermentation supernatant on the cell density of Prorocentrum donghaiense; (d) the effect of 1% fermentation supernatant on the Fv / Fm ratio of Prorocentrum donghaiense; (e) the effect of 10% P-10 bacterial cells on the cell density of Prorocentrum donghaiense; (f) the effect of 10% P-10 bacterial cells on the Fv / Fm ratio of Prorocentrum donghaiense; (g) the effect of 1% P-10 bacterial cells on the cell density of Prorocentrum donghaiense; and (h) the effect of 1% P-10 bacterial cells on the Fv / Fm ratio of Prorocentrum donghaiense.

[0021] Figure 2 This is a graph showing the algae-killing rate of Streptomyces sp. P-10 against Prorocentrum dinoflagellate in East China Sea in Example 1 of the present invention.

[0022] Figure 3 This is a photograph of the shake culture results of Streptomyces sp. P-10 in algal culture medium in Example 1 of the present invention.

[0023] Figure 4 This is a scanning electron microscope image of the effect of Streptomyces sp. P-10 cells on Prorocentrum donghaiense in Example 1 of the present invention. (af) Prorocentrum donghaiense cells treated with 10% Streptomyces sp. P-10 cells; (g1) Prorocentrum donghaiense cells in the blank control group.

[0024] Figure 5This is a scanning electron microscope image of the effect of Streptomyces sp. P-10 cells on Karenia mikimotoi in Example 1 of the present invention. (ae) shows Karenia mikimotoi cells treated with 10% Streptomyces sp. P-10 cells; (fj) shows Karenia mikimotoi cells in the blank control group.

[0025] Figure 6 This is the HPLC chromatogram of compound C3-1 in Example 1 of the present invention.

[0026] Figure 7 This is a molecular weight diagram of compound C3-1 in Example 1 of the present invention.

[0027] Figure 8 The figure shows the effect of N-acetyltryptamine on the growth of Prorocentrum donghaiense and Karenia mikimotoi in Example 1 of the present invention.

[0028] Figure 9 This is a photograph of a plate for detecting the cellulose activity of Streptomyces sp. P-10 in Example 1 of this invention. In the image, a is a blank control; b, c, and d are 10 μL of the strain's fermentation broth.

[0029] Figure 10 This is a diagram showing the changes in in-situ red tide algae cells treated with Streptomyces sp. P-10 in Example 1 of the present invention.

[0030] Figure 11 This is a scanning electron microscope image of Streptomyces sp. P-10 fermentation broth used in Example 1 of the present invention to treat in-situ red tide microalgae in Pingtan. Detailed Implementation

[0031] The technical solution of the present invention will be further explained and described below with reference to specific embodiments and accompanying drawings.

[0032] Example 1

[0033] I. Materials and Methods

[0034] 1. Strain screening

[0035] The sponge was rinsed 5 times in sterile seawater, then ground into a homogenate in a sterile mortar with 10 mL of sterile seawater. The homogenate was filtered through a 10 µm filter membrane to obtain the bacterial solution. The solution was centrifuged at 5000 rpm for 10 min and concentrated to 1 mL, which was the original bacterial solution. The original bacterial solution was then diluted 10 mL... -1 10 -2 10 -3Store at 4℃ until use. Before loading, activate the bacterial culture in a 28℃ water bath for 10 min. Droplet formation during loading was performed using the following method:

[0036] (1) Set up a microfluidic platform: Use a 500 µL microsyringe to draw 500 µL of dispersed phase and 500 µL of continuous phase respectively. Set up the microfluidic platform according to 2.1.2.1, turn on the microsyringe pump that controls the dispersed phase and continuous phase, and observe under an inverted microscope until droplets of stable size and flow rate are generated.

[0037] (2) Determine the bacterial concentration: Use the original bacterial solution and the solution diluted 10% respectively. -1 10 -2 10 -3 The diluted solution was used as the dispersed phase. The droplets generated by the chip were collected using a sterilized glass slide and observed under an inverted microscope. The appropriate bacterial concentration was determined by the presence of one bacterial cell in each droplet.

[0038] (3) Droplet collection: After determining the appropriate bacterial concentration, the small droplets containing single bacterial cells generated by the microfluidic chip are aspirated into a 6-well plate pre-filled with 1 mL of continuous phase for dilution. The single-cell droplets are then collected in a 96-well plate pre-filled with 200 µL of the corresponding culture medium under backlight conditions using a 2.5 µL pipette. 1 to 5 small droplets are collected from each well for subsequent culture.

[0039] Preservation method of sporulating Streptomyces: Single colonies were picked and cultured in seawater Gao's No. 1 liquid medium for 120 h, followed by spreading on solid Gao's No. 1 medium and culturing for 120-192 h until spores grew. The spores were then collected, centrifuged and concentrated to 900 µL, 300 µL of 80% glycerol was added, mixed well, pre-frozen and stored at -80℃. Three tubes of each sample were preserved. Marine Streptomyces sp. P-10 was obtained from the samples (deposited at the China Center for Type Culture Collection on May 11, 2022, accession number: CCTCC NO. M2022600).

[0040] 2. Experimental algal strains

[0041] Two typical red tide algae were selected: *Prorocentrum donghaiense*, belonging to the class Diplodocidae, order Prorocentrales, family Prorocentrales, and genus *Prorocentrum*, is one of the algae species that frequently cause large-scale red tides along the coast of my country; and *K. mikimotoi*, belonging to the phylum Dinophyta, class Dinophyta, order Gynodinales, family Gynodinaceae, and genus *K. mikimotoi*, is poisonous and can produce paralytic shellfish poisoning, hemolytic toxins, and neurotoxins, causing poisoning to organisms. All algal strains used in the experiment were obtained from the Algal Strain Center of the College of Ocean and Earth Sciences, Xiamen University.

[0042] 3. Activation conditions and growth curves of experimental microalgae

[0043] Both experimental microalgae were cultured in natural membrane-filtered seawater. After two activation cultures to reach the logarithmic growth phase, the algal strains were inoculated into a new L1 medium at a ratio of 1:5. The culture was carried out in a light incubator at 22°C, light intensity of 100 µmol / (m2·s), and light-dark ratio of 14h:10h. 30 mL of algal solution in the exponential growth phase was added to a 50 mL cell culture flask. After acclimatization for 24 h, the culture was ready for experiments.

[0044] 4. Experimental Design for Co-culture of Bacteria and Algae

[0045] To investigate whether Streptomyces sp. P-10 has the ability to kill experimental algae species and to explore its algicidal mechanism—specifically, whether Streptomyces sp. P-10 inhibits algae growth indirectly by suppressing cell death in red tide microalgae through mycelial entanglement or by secreting substances that kill algal cells or inhibit cell proliferation—microalgal cell density (cells / L), algicidal rate (%), and chlorophyll fluorescence (Fv / Fm) were used as indicators to determine algal cell activity and growth status.

[0046] 5. Grouping of bacterial-algae co-culture

[0047] In this embodiment, the experiment was designed to study the inhibitory effect of bacterial fermentation broth, bacterial cells, and bacterial secretions on the test algae species as individual variables. In order to separate various substances in the Streptomyces culture medium, two bacterial agents, namely fermentation supernatant and bacterial cell resuspension, were prepared and tested separately.

[0048] Preparation of fermentation supernatant: Take 50 mL of the bacterial strain fermentation broth, centrifuge at 8000 rpm for 5 min, collect the supernatant, and then filter it through a 0.22 μm filter membrane to obtain a cell-free fermentation supernatant. This supernatant contains Gao's No. 1 culture medium and P-10 secretions. Using the Gao's No. 1 experimental group as a control, if algal cells in this group treated with the bacterial agent die, it indicates that the algal lysis is caused by the secretions of bacterial cells. Preparation of bacterial cell washing resuspension: Take 50 mL of the bacterial strain fermentation broth in the exponential phase, centrifuge at 5000 rpm for 10 min. After discarding the supernatant, the cells were washed three times with sterile L1 medium and resuspended in an equal volume of L1 algal medium. The prepared bacterial cell resuspension, after three washes, removed residual Gao's No. 1 medium and extracellular secretions, leaving only the mycelial cells of Streptomyces P-10 and the L1 algal medium used for resuspension. If the number of algal cells or their activity decreased under this treatment, the L1 algal medium treatment group could be used as a control to investigate whether Streptomyces P-10 has an algicidal effect. By preparing these two agents, the two components in the Streptomyces fermentation broth were separated, and the main active components were explored by co-culturing the two agents with algal cells, thereby investigating their algicidal mechanism.

[0049] 6. Time-series scanning electron microscopy observation of cell state during co-culture of Streptomyces sp. P-10 mycelium and red tide dinoflagellates.

[0050] To investigate the inhibitory pattern of mycelium on algal cells, namely whether Streptomyces mycelium has direct contact with algal cells and penetrates the algal cell wall, and to visually obtain the intercellular interaction relationship between mycelium and algal cells, scanning electron microscopy was used to observe the experimental group under mycelium treatment during co-culture.

[0051] Sampling and slide preparation for scanning electron microscopy (SEM): In the bacterial cell resuspension treatment group, 2 mL of algal solution from co-cultured for 24 h, 48 h, 72 h, 96 h, and 120 h, and 2 mL of algal solution from the blank control group at the same time point, were taken, fixed, and the morphology of normally independently growing bacterial and algal cells was observed under a scanning electron microscope. This was done in a co-culture system treated with 10% volume of bacterial cell resuspension.

[0052] II. Results and Analysis

[0053] 1. Response of *Prorocentrum donghaiense* to different bacterial agents

[0054] The supernatant of the fermentation broth of Streptomyces sp. P-10 was added to the algal culture of Prorocentrum donghaiense in the exponential growth phase at different concentration gradients, and the physiological data of the algae during the co-culture period were monitored.

[0055] like Figure 1As shown in figures a to d, the fermentation supernatant had a sustained inhibitory effect on the density of *Prorocentrum donghaiense*, and the inhibitory effect of the high-concentration (10%) treatment group was stronger than that of the low-concentration group. Under the 10% supernatant treatment, the algal cell density continued to decrease. After deducting the influence of Gao's No. 1, the algal dissolution rate of the 10% sterile supernatant treatment group reached 65% after 72 hours of co-cultivation. Figure 2 After 120 hours of co-cultivation, the concentration differed from the initial concentration by an order of magnitude, and after 144 hours of co-cultivation, the inhibition rate against *Prorocentrum donghaiense* cells reached 94%. Figure 2 The 1% concentration treatment group consistently showed a highly significant difference from the blank control. The number of *Prorocentrum donghaiense* cells in the 1% concentration treatment group maintained a low-density, fluctuating growth trend, but consistently showed a significant difference from Gao's No. 1 medium and the blank control group. Figure 1 The comparison of e and g shows that both concentrations of bacterial cells can significantly inhibit algal cell density, with the high concentration showing a greater inhibitory effect than the low concentration. Furthermore, the high concentration group achieved a maximum algicidal rate of 71% at 72 hours. Figure 2 Afterwards, the algae-killing rate fluctuated between 35% and 50%, still exhibiting good algae-killing effects; the cell density did not continuously decrease but remained as... Figure 1 After 72 hours of co-culturing with e and g, both the 10% and 1% concentration groups showed a rebound in cell density; Figure 1 It can be seen that the 10% concentration of bacterial cell resuspension consistently had a significant inhibitory effect on the algal cell density of the East China Sea. The algal cell density reached its lowest point after 72 hours of co-cultivation, at which point the algal inhibition rate reached 71%. Figure 2 Although a highly significant difference remained between this control and the blank control, a rebound in algal cell density occurred. Figure 1 It can be seen that the inhibitory effect of the 1% bacterial cell treatment group also reached its peak after 72 hours of co-culture. After that, the algal cells began to proliferate in large quantities, almost returning to a level with no significant difference from the blank control group. The algae killing rate reached its highest value of 34% at 72 hours. Figure 2 ). Reference Figure 1 The f and h graphs show that the Fv / Fm ratio at 72h was significantly lower than that of the previous two days, which also proves that the cell activity of Prorocentrum donghaiense was significantly inhibited at this time point.

[0056] During the co-culture period of bacterial cells and algal solution, the bacterial cell density was maximally inhibited at 72 hours, followed by a rapid recovery in algal cell density. This is because the bacterial cells lack a stable carbon source in the algal cell culture medium, leading to a gradual decrease in their activity. To verify whether the algal culture medium can sustain bacterial cell growth and proliferation, a colony activated on a plate was transferred to the algal culture medium and cultured in a shake flask at 28°C and 200 rpm. After three days, no mycelial growth was observed (e.g., ...). Figure 3As shown in the figure, algal culture medium is not conducive to bacterial growth and cannot maintain its activity for a long time in algal culture medium environment; therefore, the reason for the sudden increase in algal cell density in the bacterial cell treatment group after 72 hours may be that the activity of Streptomyces in the algal solution decreased and could not continuously inhibit the reproduction of algae.

[0057] 2. Exploring the Patterns of Bacterial-Algal Interactions under Scanning Electron Microscopy

[0058] like Figure 4 As shown, the cells of *Prorocentrum donghaiense* in the control group exhibited an asymmetrical elliptical shape, slightly pointed at the top, widest in the middle, and rounded at the bottom. The cell structure was intact and plump, and no other bacteria or debris were observed in the field of view. In the sample treated with the bacterial agent, a large number of bacteria gathered around the algal cells. These bacterial cells covered and adhered to the surface of the algal cell shell, wrapping and entwining between the two shell plates at the edge of the algal cell wall. Some bacterial cells extended into the gaps between the shell plates. As the treatment time increased, the bacterial cells gradually destroyed the algal cell wall and plasma membrane. At this point, the surface of the algal cells no longer had clear granular texture and stomata. The cells shrank, underwent apoptosis, lost their cellular characteristics, and their contents were released. They gradually disintegrated and died, and finally only algal cell fragments and the bacterial cells that had been in contact with them could be observed. These phenomena indicate that P-10 bacterial cells may have a direct contact algal-killing mode, and its specific mechanism still needs further investigation.

[0059] like Figure 5 As shown, normally growing Karenia mikimotoi cells are oval, approximately 20-30 μm in size, with transverse and longitudinal grooves. They belong to the order Gymnodinales, lack an external cellulose shell structure, and have plump, structurally intact cells. After washing and processing the bacterial cells, a large number of bacterial cells in direct contact with the cells can be observed in the field of view. From 24 hours of co-culture, the mycelium can attack and entangle the algal cells, and many algal cells can be observed crossing and contacting the mycelium in the field of view. At 48 hours, some algal cells can be seen to have dissolved. In the algal culture medium with a low carbon source, these decomposed algal cell remnants will become raw materials for the expansion of a large number of bacterial cells. At 72 hours, dead algal cell fragments can be seen directly interacting with the aggregated mycelium, but structurally intact cells are still present in the field of view. In the subsequent co-culture system, almost no intact cells can be seen. The remaining fragments of lysed algal cells are scattered in the field of view, consistent with the light microscopy results. The electron micrograph shows that P-10 cells are in contact with and entangled with Karenia mikimotoi. During co-culture, the morphology and structure of Karenia mikimotoi were significantly affected compared with the control group. This can be considered as one of the supporting evidences that P-10 has a direct algicidal effect.

[0060] 3. Anti-algae activity of N-acetyltryptamine

[0061] Compound C3-1, secreted by *Streptomyces* sp. P-10, is a pale yellow powder, readily soluble in organic reagents such as dichloromethane and methanol. It is colored under 254 nm UV light. Thin-layer chromatography using dichloromethane:methanol (10:1) as the developing solvent yielded an Rf value of 0.68 and a molecular weight of [M+H]+ 203.11784. Using deuterated methanol as the solvent, compound C3-1 was matched to N-acetyltryptamine in the microspectral data from Xiamen University, with the following structural formula: The HPLC chromatogram and molecular weight chromatogram are as follows: Figure 6 and Figure 7 As shown. The effects of different concentrations of N-acetyltryptamine on the growth of two red tide dinoflagellates after 96 h of culture were analyzed. All data are average values. Figure 8 Table 1 shows the IC50 values ​​of N-acetyltryptamine against two microalgae after 96 h. The toxicity grading standard for the algal growth inhibition experiment is as follows: IC50 96h 100 mg / L, low toxicity. Therefore, N-acetyltryptamine has a very strong inhibitory effect on the growth of *Prorocentrum donghaiense* and a strong inhibitory effect on the growth of *Karenella mikimotoi*.

[0062] Table 1. Algal inhibitory activity of N-acetyltryptamine against two types of algae.

[0063]

[0064] 4. Cellulase activity of Streptomyces sp. P-10

[0065] This embodiment uses the plate method to detect the cellulase activity of extracellular products of Streptomyces sp. P-10, such as... Figure 9 As shown, the extracellular product of Streptomyces sp. P-10 can form a clear, transparent 5 cm zone on cellulose screening plates.

[0066] 5. Inhibition of in-situ red tide algae by Streptomyces sp. P-10

[0067] To verify whether Streptomyces sp. P-10 has an inhibitory effect on actual red tide algal blooms in the ocean, in-situ red tide algae blooms in the Pingtan sea area of ​​Fujian Province were collected in May 2023. The algae were identified as mainly *Prorocentrum donghaiense*. The addition of a certain concentration (10%, v / v) of Streptomyces sp. P-10 significantly reduced the algal cell density, while the untreated group maintained a high algal cell density. Figure 10 Meanwhile, electron microscopy observations showed that the algal cells were destroyed. Figure 11 This study confirms that Streptomyces sp. P-10 has potential application value in the prevention and control of red tide outbreaks.

[0068] The above description is merely a preferred embodiment of the present invention, and therefore should not be construed as limiting the scope of the present invention. All equivalent changes and modifications made in accordance with the scope of the patent and the contents of the specification should still fall within the scope of the present invention.

Claims

1. A marine Streptomyces sp. P-10, characterized by: It was deposited at the China Center for Type Culture Collection on May 11, 2022, with accession number CCTCC NO. M2022600.

2. The application of the marine Streptomyces P-10 according to claim 1 in the preparation of a marine algae-inhibiting composition, characterized in that: The marine algae-inhibiting composition has an inhibitory effect on *Prorocentrum donghaiense* and *K. mikimotoi*.

3. Use according to claim 2, wherein: The marine algae-inhibiting composition also includes N-acetyltryptamine and cellulase secreted by the marine Streptomyces P-10.

4. A marine algal growth inhibiting composition characterized in that: Its active ingredient includes the marine streptomyces P-10 described in claim 1, which has an inhibitory effect on *Prorocentrum donghaiense* and *Karenella mikimotoi*.

5. A marine algal growth inhibiting composition according to claim 4, wherein: Its active ingredients also include N-acetyltryptamine and cellulase secreted by the marine streptomyces P-10.

6. A marine algal growth inhibiting composition according to claim 5, wherein: Its active ingredients consist of the marine streptomyces P-10 and its secreted N-acetyltryptamine and cellulase.

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