Algicidal bacterium TL3 with efficient algicidal activity and application of algicidal bacterium TL3

Abstract

The invention discloses an algicidal bacterium TL3 with efficient algicidal activity and application thereof, the algicidal bacterium TL3 is named as Enterobacter cloacae TL3 and is preserved in Guangdong Microbial Culture Collection Center located at No.100, Xianlie Middle Road, Guangzhou City, Guangdong Province, the preservation number is GDMCC No: 65777, and the preservation date is January 13, 2025; the invention also provides an algicidal bacterial agent containing the algicidal bacteria TL3 for degrading or inhibiting water-blooming cyanobacteria. The algicidal bacterium TL3 is obtained by screening and separating in the algicidal period by taking the algicidal habitat of the Lake Taihu as a strain resource library of the efficient algicidal bacterium, has an efficient inhibition effect on microcystis aeruginosa, has a certain algicidal effect on chrysosporium oospermum, cuscuta larakii and anabaena pseudosciaena, is used for various types of cyanobacterial bloom water bodies, and has a good algicidal effect on the water bodies. The application prospect is wide.

Description

Technical Field

[0001] The present invention belongs to the technical field of environmental microbiology, and particularly relates to an algicidal bacterium TL3 with high algicidal activity and its application. Background Art

[0002] Cyanobacteria are ubiquitous in nature and can carry out photosynthesis. Their photosynthetic oxygen evolution contributed to the formation of the Earth's atmosphere and they have an important nitrogen fixation function. However, since the 1940s, affected by human activities, cyanobacterial blooms often occur in many freshwater ecosystems. Currently, with the increasingly serious global climate change and water eutrophication, the outbreak and expansion of cyanobacterial blooms have gradually intensified. Common bloom-forming cyanobacteria include Dolichosperum, Microcystis, Planktothrix, etc., among which Microcystis is the most common dominant species in blooms. It is reported that Microcystis has proliferated massively globally in North America, Australia, Europe, etc., except in Antarctica. Currently, large-scale Microcystis blooms frequently occur in the freshwater ecological environment of China, especially in freshwater lakes such as Taihu Lake, Chaohu Lake, Dianchi Lake, Erhai Lake, etc. and many reservoirs, among which Taihu Lake, Chaohu Lake and Dianchi Lake are the most serious.

[0003] The prevention and control of cyanobacterial blooms have always been the focus and difficulty of ecological environment restoration and protection. First, it is the complex current situation of water environmental pollution. Numerous human activities such as industry, agriculture, and medicine have led to various pollutions in the water environment. The most prominent is the significant increase in the content of nutrients such as nitrogen and phosphorus in the water. The regulation of water quality and the control of pollution sources are both major problems. Currently, the governance principle at home and abroad is to first control exogenous pollution and then reduce the endogenous nutrient load, considering the actual water body situation, so as to achieve a comprehensive pollution prevention and control effect. Second, it is the suddenness of cyanobacterial blooms, making it difficult to accurately predict the time of blooms and take preventive measures in advance. The large-scale outbreak of blooms also makes the prevention and control work difficult. It requires a large amount of time and resources and the algae need to be removed within a short time, and the secondary outbreak of blooms also needs to be prevented. Therefore, the prevention and control of cyanobacterial blooms is a difficult task. Currently, the prevention and control means are mainly divided into four types: physical method, chemical method, physical-chemical combined method, and biological ecological method. Among them, the physical algae removal method has been improved many times and the algae removal efficiency has been greatly improved. However, these new technologies cannot fundamentally solve the problems of repeated cyanobacterial blooms and secondary pollution of the water environment. Chemical algae removal will release toxins after the death of algae and cause secondary pollution. The large-scale use of chemical reagents will accumulate in animals and plants, thus harming the entire food chain and ecosystem. Biological prevention and control emphasizes the ecological balance of the water ecosystem, controls cyanobacteria through the competitive and grazing relationships between organisms, and can improve the health of the water body.

[0004] In recent years, more and more people have paid attention to the relationship between algae and microorganisms. Research has found that the factors contributing to the disappearance of cyanobacterial blooms include fungal parasitism, algicidal bacteria, zooplankton predation, phage, and some physical and chemical factors. Microorganisms such as algae-lysing fungi, algicidal bacteria, and phage have a certain control effect on harmful algal blooms. Existing research has shown that algicidal bacteria have good application prospects in regulating bloom waters, especially in algae control. Microorganisms have the advantages of being widely distributed, having diverse species and functions, and being easy to culture in natural environments. Therefore, algae-inhibiting microorganisms have become a research hotspot in environmental governance.

[0005] Algicidal bacteria are widely distributed in aquatic ecosystems such as the ocean, lakes, swamps, and soil microenvironments, and algicidal bacteria are generally screened from bloom and red tide waters. Therefore, algicidal bacteria play an important role in the natural recession process of algal blooms or red tides. Currently, some algae-inhibiting microorganisms have been discovered, and a small number of them have been applied in actual production. For example, the patent document with the publication number CN112625952A discloses the first application of a Bacillus subtilis subsp. in the control of cyanobacteria, and the patent document with the publication number CN102308852A discloses a microbial algal inhibitor composed of a mixture of Bacillus laterosporus VKPM B-10531 and expanded perlite particle carriers, which can inhibit the growth of microalgae and the formation of microalgal biofilms in water bodies, ultimately leading to the death of algae; the patent document with the publication number CN115895942A discloses an Enterobacter isolated from seawater that has a high inhibitory effect on the red tide alga Phaeocystis globosa.

[0006] Currently, the research on algicidal bacteria is still basically in the laboratory stage. The sources and biosafety of algicidal bacteria are not clear and have not been widely applied to bloom waters, especially large areas of water. In addition, the species diversity and functional diversity of microorganisms determine the diversity of algicidal bacteria species. Exploring new microbial resources is also one of the current application hotspots. Therefore, screening regional indigenous algicidal bacteria, systematically studying the algicidal characteristics and algicidal thresholds of strains, and exploring the algicidal mode can provide technical support for the biological control of cyanobacterial blooms. Summary of the Invention

[0007] Based on this, the main objective of the present invention is to provide an algicidal bacterium TL3 with high algicidal activity, using the habitat of the recession of cyanobacterial blooms in Lake Taihu, Jiangsu Province as a germplasm resource bank for highly effective algicidal bacteria, screened and isolated during the recession period of algal blooms, which has a high inhibitory effect on Microcystis aeruginosa, and at the same time has a certain algicidal effect on Chrysosphaerella ellipsoidea, Raphidiopsis raciborskii, and Pseudanabaena sp.

[0008] Another objective of the present invention is to provide the application of the algicidal bacterium TL3 with high algicidal activity in degrading or inhibiting bloom cyanobacteria.

[0009] To achieve the above object, the present invention adopts the following technical solution:

[0010] The invention provides an algalytic bacterium TL3 with high-efficiency algalytic activity, which is named Enterobactercloacae TL3, and has a taxonomic name of Enterobacter cloacae. The strain is preserved in Guangdong Microbiological Culture Collection Center located at No. 100, Xianlie Middle Road, Guangzhou City, Guangdong Province, with a preservation number of GDMCC No: 65777 and a preservation date of January 13, 2025.

[0011] Preferably, the algae-lytic bacteria TL3 is isolated from the habitat of water bloom retreat in Taihu Lake.

[0012] The present invention also provides an algae-lytic agent, which comprises the algae-lytic bacteria TL3 with high-efficiency algae-lytic activity.

[0013] The present invention also provides use of the algae-lytic bacteria TL3 or the algae-lytic agent in degrading or inhibiting water bloom cyanobacteria.

[0014] Preferably, the water bloom cyanobacteria include one or more of Microcystis aeruginosa, Chrysosporum ovalisporum, Raphidiopsis raciborskii, and Pseudanabaena cinerea.

[0015] Compared with the prior art, the present invention has the following beneficial effects:

[0016] 1. The present invention uses the water body of the Taihu Lake bloom retreat as a bacterial resource library, separates and purifies the cultivable algal microorganisms of the native Microcystis aeruginosa population by low-cost culture medium plate streaking, and screens out the highly efficient algicidal bacteria TL3 by co-cultivation with Microcystis aeruginosa. The method has the advantages of low cost, simplicity, safety, etc.

[0017] 2. The algae-lytic bacteria TL3 screened by the present invention has a certain dissolving effect on Microcystis aeruginosa, the dominant species in water bloom cyanobacteria. Under the optimal experimental conditions, the actual algae-lytic efficiency of the algae-lytic bacteria TL3 on Microcystis aeruginosa reached 100% on the 7th day, and the algae-lytic effect was very ideal.

[0018] 3. The algicidal bacterium TL3 screened by the present invention and its sterile filtrate have a high algicidal effect on the dominant species (Microcystis aeruginosa) of several cyanobacterial blooms. The photosynthetic inhibition efficiency on algal cells reaches 100% within 72 h, and the actual algicidal efficiency reaches 100% within 7 d. The algicidal effect is very ideal, and it also has a certain inhibitory effect on three common cyanobacteria, namely Chrysosporum ovalisporum, Raphidiopsis raciborskii, and Pseudanabaena cinerea. This algicidal bacterium TL3 also has no significant algicidal effect on common beneficial green algae, which makes it better play its algicidal characteristics in natural waters and is applicable to the prevention and control of various cyanobacterial blooms, with broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 (a) Colony morphology of algicidal bacterium TL3 by streak plate method; (b) Microscopic examination of Gram staining of algicidal bacterium TL3 under 100× magnification; (c) Observation of submicroscopic structure of bacterium TL3 in the examples.

[0020] Figure 2 Phylogenetic tree of algicidal bacterium TL3 in the examples.

[0021] Figure 3 Variation of algal cell density during co-culture of different inoculation amounts of algicidal bacteria and Microcystis aeruginosa in the examples, * represents p < 0.05, ** represents p < 0.01, *** represents p < 0.001.

[0022] Figure 4 Effect of different components of the culture solution of algicidal bacterium TL3 on the algal cell density of Microcystis aeruginosa in the examples, * represents p < 0.05, ** represents p < 0.01, *** represents p < 0.001.

[0023] Figure 5 Effect of the culture solution of algicidal bacterium TL3 on the algal cell density of Chrysosporum ovalisporum CCFWA01007, Raphidiopsis raciborskii FACHB - 1503, and Pseudanabaena cinerea FACHB - 1277 in the examples. DETAILED DESCRIPTION OF THE INVENTION

[0024] In order to more fully understand and demonstrate the technical solutions, objectives, and advantages of the present invention, the technical effects produced by the present invention will be further described in detail and completely below in conjunction with the accompanying drawings and specific examples. Obviously, the described examples are only a part of the examples of the present invention, not all of them. It should be noted that for those of ordinary skill in the art, other examples obtained without departing from the concept of the present invention all belong to the protection scope of the present invention.

[0025] In the following examples, an algalytic bacterium TL3 with high algalytic activity was screened and identified, named Enterobacter cloacae TL3, and its taxonomic name is Enterobacter cloacae. The strain is deposited in the Guangdong Provincial Microbiological Culture Collection Center at No. 100, Xianlie Middle Road, Guangzhou City, Guangdong Province, with a deposit number of GDMCC No: 65777, and a deposit date of January 13, 2025. It was isolated from the Taihu Lake algae bloom retreat habitat.

[0026] In some embodiments, a method for removing Microcystis aeruginosa using algicidal bacteria TL3 is provided, comprising the following steps:

[0027] Step 1, inoculate the algae-lytic bacteria TL3 into LB (Luria-Bertani) liquid culture medium, culture at 37°C and a shaking speed of 150 r / min to the logarithmic growth phase, then transfer the inoculum to fresh LB liquid culture medium at a volume fraction of 1%, and culture under the same culture conditions for 12 hours to the logarithmic phase for later use.

[0028] Step 2: The algae-lysing bacteria TL3 cultured to the logarithmic phase were inoculated at an initial algae density of 2×10 6 cells / mL of fresh Microcystis aeruginosa algae liquid, co-cultured in a light incubator at 25°C and a light intensity of 1500 lux, the Microcystis aeruginosa can be removed, and the light cycle is set to 12h:12h (Day:Light). The composition of the LB liquid medium is peptone 10g, yeast powder 5g, NaCl 10g, distilled water 1L, pH adjusted to about 7.0, and solid culture medium needs to add 2% agar.

[0029] Step 3: Inoculate fresh algae at a volume ratio of bacteria to algae greater than 1:30 to a density of 2×10 6 The bacteria and algae were co-cultured in an algae solution of Microcystis aeruginosa with a concentration of 1 cells / mL in a light incubator at 25°C, a light intensity of 1500 lux, and a photoperiod of 12h:12h (Day:Light) for 7 days.

[0030] Example 1

[0031] 1. Isolation and purification of algae bacteria:

[0032] Preparation of LB liquid culture medium: Accurately weigh 10 g of peptone, 5 g of yeast powder and 10 g of NaCl into a beaker, and fully dissolve them with a small amount of distilled water. Transfer them to a 1 L volumetric flask and make up to 1 L with distilled water, and adjust the pH to about 7.0. Sterilize in a high-pressure steam autoclave at 121 ° C for 20 min. Solid culture medium needs to add 2% agar.

[0033] Microbial source sample collection: Collect 200 mL of water samples containing Microcystis colonies from the area where the Taihu Lake bloom is about to subside, and collect them three times respectively. Store the collected samples at 4 °C and transport them to the laboratory immediately. Immediately isolate and purify the epiphytic bacteria in the laboratory.

[0034] (1) Method for isolating and purifying epiphytic bacteria:

[0035] Pick a single Microcystis aeruginosa colony from the collected bloom water samples. In a laminar flow hood, wash the single Microcystis aeruginosa colony 6 times with sterile water in a sterile petri dish, and take 0.2 mL of the water from the 3rd to 6th washings and spread it on an LB solid medium. And spread the washed Microcystis aeruginosa colony on the LB solid medium. Incubate the spread solid plates inverted at 37 °C for 12 - 24 h, and observe the growth status of the bacteria at 12 h and 24 h and record. If there are single colonies with good growth status, record the colony morphological characteristics in time, and pick them to the LB liquid medium with an inoculation loop in time (do three parallels for each colony). After culturing for 24 h, perform dilution coating to detect the purity of the strain. Culture the purified bacterial liquid to the logarithmic growth phase, and store the bacterial liquid and 30% glycerol in equal volume in a storage tube and store it at -20 °C to obtain multiple pure epiphytic bacteria.

[0036] (2) Screening of highly efficient algicidal bacteria:

[0037] Preparation of cyanobacteria culture solution: Weigh 1.7 g of BG-11 into a beaker, dissolve it thoroughly with a small amount of distilled water, and transfer it to a 1 L volumetric flask, accurately make up the volume to 1 L, and sterilize it at 121 °C for 20 min in a high-pressure steam sterilizer.

[0038] Use LB liquid medium to uniformly activate and expand the multiple pure epiphytic bacteria screened out at 37 °C and 150 r / min. Measure the OD value of the bacterial liquid with an ultraviolet spectrophotometer to about 1.0, and use LB liquid medium as the solution to adjust the concentration of each bacterial liquid to the same level.

[0039] Take 50 mL of logarithmically growing Microcystis aeruginosa (FACHB 905, purchased from the Freshwater Algae Culture Collection of the Chinese Academy of Sciences) cultured in the laboratory into 400 mL of BG-11 culture solution, and then take 50 mL of the bacterial culture solution into the co-culture system. The algal cell concentration of Microcystis aeruginosa in the whole culture system is 1.0×10 6 cells / mL. Set up control experiments by adding LB liquid medium and BG-11 medium with the same volume as the bacteria respectively. The experimental culture conditions are 25 °C, the light cycle is 12 h:12 h (Day:Light), and the light intensity is 1500 lx. Screen out a strain of bacteria with high algicidal ability by comparing the chlorophyll a content among the experimental groups and with the control group.

[0040] To accurately describe the algicidal efficiency of the reagent of the algicidal bacterium, the algal cell density was used as a reference index to calculate the actual algicidal efficiency:

[0041] R(%) = [(N0 - N) / N0] × 100%; where N0 and N represent the algal cell densities of the control group and the treatment group, respectively.

[0042] (3) Identification of highly efficient algicidal bacteria:

[0043] Physiological and biochemical tests and 16S rDNA identification were carried out on the obtained pure strains of highly efficient algicidal bacteria.

[0044] Analysis of the homology of the 16srDNA sequence of the strain: By comparing the 16S rDNA sequence of the strain with the database in the National Center for Biotechnology Information (NCBI) Genebank in the United States, this strain belongs to the genus Enterobacter and is most closely related to Enterbacter cloacae, with a similarity of 98% ( Figure 2 ), and the serial number was uploaded to the NCBI database, and the accession number is PQ849188.

[0045] Morphology and physiological and biochemical properties of the strain: When cultured for 24 h, the colonies of this bacterium were slightly smaller, the surface was dry, and there was a slightly raised shape, and the colony color was milky white ( Figure 1 a), and it could reach the logarithmic growth phase when cultured for 12 h. The results of Gram staining showed that this bacterium was a Gram-negative bacterium ( Figure 1 b), and the morphology of the bacteria was rod-shaped under the microscope ( Figure 1 c). The physiological characteristics of the strain were positive for β-galactosidase, arginine dihydrolase, lysine decarboxylase, ornithine decarboxylase, citrate utilization, acetoin (VP test), glucose fermentation, mannitol fermentation, inositol fermentation, sorbitol fermentation, amygdalin fermentation, and arabinose fermentation; negative for H2S production, urease, tryptophan deaminase, and indole test; the optimal growth temperature, salinity, and pH were 37 °C, 1%, and pH 7, the growth range was 25 - 40 °C, 0% - 5% salinity, and the pH range was about 6.0 - 9.0.

[0046] Combined with molecular and biological identification, it is one of Enterbacter cloacae and is named Enterbacter cloacae TL3.

[0047] The following examples provide the effects of the above strain Enterbacter cloacae TL3 on Microcystis aeruginosa, Chrysocapsa oocystis, Rhaphidiopsis raciborskii, and Pseudanabaena sp.

[0048] Example 2

[0049] Inoculate the mother liquor of Microcystis aeruginosa cultured to the logarithmic phase into BG-11 medium, and set the algal cell concentration in the unified culture system to 2.0×10 6 cells / mL. At the same time, dilute the TL3 bacterial solution pre-activated to the logarithmic growth phase with LB medium to OD 600 = 1.0 ≈ 1×10 8 CFU / mL. Set the volume ratio of bacteria to algae (V_bacteria / V_algae) in the experiment to 0:300, 1:300, 5:300, 10:300, 20:300, 30:300. Set 3 parallel groups for each experimental group, and the experimental period is 8 days. Determine the algicidal effect of bacterial solutions with different inoculation amounts by measuring the algal cell density and chlorophyll a. As Figure 3 shown, the algicidal effect is the best at the inoculation volume ratio of 30:300. The actual algicidal efficiency at 48 h of the experiment is 57%, the number of algal cells drops to more than half, and the algicidal rate reaches 100% on the 7th day, and all algal cells disappear.

[0050] Example 3

[0051] Activate and culture the strain to the logarithmic growth phase, adjust the OD of the culture solution to about 1.0, and prepare three different components: strain cells, cell-free culture supernatant, and cell lysate.

[0052] TL3 bacterial cells: Aliquot 150 mL of the bacterial solution into 15 mL centrifuge tubes, centrifuge at 8000 rpm for 5 min, remove the supernatant, and then centrifuge and wash 3 times with sterile BG-11 culture solution under the same conditions. Resuspend the bacterial pellet in the same volume of sterilized BG-11 culture solution.

[0053] TL3 cell-free filtrate: Aliquot 150 mL of the bacterial solution into 15 mL centrifuge tubes, centrifuge at 8000 rpm for 5 min, and use a 0.22 μm sterile syringe filter with a PES sterile needle filter membrane to remove residual bacterial cells to obtain a cell-free filtrate.

[0054] TL3 bacterial cell lysate: Aliquot 150 mL of the bacterial solution into 15 mL centrifuge tubes, centrifuge at 8000 rpm for 5 min, remove the supernatant, and then centrifuge and wash 3 times with sterile BG-11 culture solution under the same conditions. Resuspend the bacterial pellet in the same volume of sterilized BG-11 culture solution. Ultrasonically disrupt the resuspended bacterial cells in an ice bath for 1 h (ultrasonic conditions: power 150 W, 3 s / 3 s, 4.5 min each time, repeat 3 times). Centrifuge the disrupted liquid at 8000 rpm for 5 min at 4°C, take the supernatant, and use a 0.22 μm sterile syringe filter with a PES sterile needle filter membrane to remove residual cell debris to prepare a cell lysate.

[0055] The best algal-bacterial volume ratio of 1:10 was selected for co-culture experiments. The algal cell density of Microcystis aeruginosa and the actual algicidal efficiency were monitored daily. The results are as Figure 4 shown. In the experimental group with the addition of cell-free filtrate, the number of algal cells decreased by more than half at 48 h of the experiment, and the algicidal rate reached 100% on the 7th day, and all algal cells disappeared. The strain exerted its algicidal effect through its extracellular metabolites.

[0056] Example 4

[0057] Chrysosporum ovalisporum CCFWA01007, Raphidiopsis raciborskii FACHB-1503, and Pseudanabaena cinerea FACHB-1277 were cultured in BG11 culture medium until the logarithmic growth phase.

[0058] The algicidal bacteria were cultured until the logarithmic growth phase, and the OD of the culture solution was adjusted to about 1.0. Then they were co-cultured with the algal solutions of Chrysosporum ovalisporum, Raphidiopsis raciborskii, and Pseudanabaena cinerea respectively. The algal-bacterial volume ratio was set to 1:10, that is, 10 mL of the bacterial solution was inoculated into 100 mL of the algal solution. Good algicidal effects were achieved within 7 days, and the algicidal efficiencies reached 88.32%, 95.18%, and 100% respectively ( Figure 5 ).

[0059] The above are only the preferred embodiments of the present invention and are not used to limit the present invention. For those skilled in the art, various modifications and changes can be made to the present invention. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims

1. An algalytic bacterium TL3 with high algalytic activity, named Enterobacter cloacae TL3, with a taxonomic name of Enterobacter cloacae. The strain is deposited in the Guangdong Microbiological Culture Collection Center at No. 100, Xianlie Middle Road, Guangzhou City, Guangdong Province, with a deposit number of GDMCC No: 65777 and a deposit date of January 13, 2025.

2. The algae-lytic bacteria TL3 according to claim 1, characterized in that: The algae-lytic bacteria TL3 was isolated from the habitat of water bloom retreat in Taihu Lake.

3. An algae-lysing agent, characterized in that: It comprises the algae-lytic bacteria TL3 with high-efficiency algae-lytic activity as claimed in claim 1 or 2.

4. Use of the algae-lytic bacteria TL3 according to claim 1 or 2, or the algae-lytic agent according to claim 3, in degrading or inhibiting blue algae blooms.

5. The use according to claim 4, characterized in that: The bloom-like cyanobacteria include one or more of Microcystis aeruginosa, Chrysocytia oosporon, Acrocephalum rabatii and Pseudo-Anabaena.

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