Compound microbial agent and application thereof in prevention and control of cyanobacterial bloom

Abstract

The invention discloses a compound microbial agent and application thereof in prevention and control of cyanobacterial bloom, two algicidal bacteria are obtained through screening, the preservation number is CCTCC NO: M 2024698, and the preservation number is CCTCC NO: M 2024699, and the compound microbial agent is proved to have obvious inhibition effects on a plurality of cyanobacteria bacteria such as microcystis aeruginosa, lacustrine triphora, anabaena and cylindrocystis, and can be used for preventing and controlling cyanobacteria bloom. And the composite bacterial agent combined by the two algicidal bacteria has a remarkable algicidal effect on algal blooms in natural water areas, can be applied to the field of biological prevention and control of freshwater cyanobacterial blooms, and provides an ideal choice for treatment of cyanobacterial blooms in water bodies.

Description

Technical Field

[0001] This invention relates to the field of algal bloom control technology, specifically to compound microbial agents and their application in the prevention and control of cyanobacterial blooms. Background Technology

[0002] In the history of algal bloom control over the past few decades, various technical means have been adopted to prevent, eliminate, and mitigate the outbreaks and negative impacts of algal blooms. These technical means are mainly divided into physical, chemical, and biological methods. Physical algae control generally uses methods such as fencing, mechanical harvesting, dilution and rinsing, flocculation and sedimentation, and ultrasonic disruption to separate and break down algae. Chemical algae control mainly involves adding oxidizing or non-oxidizing algaecides such as persulfate, permanganate, ozone, and polyaluminum chloride to the algal bloom area, thereby damaging the algal cell walls and cell membranes or flocculating algal cells to accelerate their sedimentation. Both physical and chemical methods can cause damage and adverse effects on other components of the ecosystem, and the chemical algaecides used may cause secondary pollution and continuous damage to the ecological environment.

[0003] Based on the concept of green development, biological algae control is a pollution-free, low-energy-consumption, and high-efficiency method for controlling algae blooms. Among them, algicidal bacteria are microorganisms that are closely related to the outbreak and decline of algal blooms. Utilizing their ability to dissolve and inhibit algal species in algal blooms has great potential for preventing and controlling algal blooms.

[0004] Cyanobacterial blooms are characterized by strong environmental adaptability, high toxicity, and wide outbreak range. Currently, many researchers have screened alginolytic bacteria for cyanobacterial blooms, but most alginolytic bacteria only inhibit one or two types of cyanobacteria, and have no effect when faced with mixed blooms of various algae. Due to the complex and variable nature of the natural environment, single bacterial agents often suffer from reduced algae-inhibiting ability. Summary of the Invention

[0005] The purpose of this invention is to propose algicidal bacteria and their compound microbial agents for application in the prevention and control of cyanobacterial blooms, thereby overcoming the limitation of algicidal bacteria in inhibiting algae blooms and resulting in poor bloom suppression.

[0006] In view of this, the solution of the present invention is as follows: The first aspect of this invention is that it proposes an algicidal bacterium, which is deposited at the China Center for Type Culture Collection (CCTCC) with accession numbers CCTCC NO: M 2024698 or CCTCC NO: M 2024699.

[0007] The second aspect of this invention is to propose the application of the algicidal bacteria described in the first aspect in the prevention and control of cyanobacterial blooms.

[0008] Furthermore, in the above applications, the cyanobacteria are selected from at least one of Microcystis aeruginosa, Lymphaea lappa, Anabaena spp., and Cyclops spp.

[0009] A third aspect of this invention is to provide a compound microbial agent, comprising algicidal bacteria or their cultures with preservation numbers CCTCC NO: M2024698 and CCTCC NO: M 2024699.

[0010] Furthermore, the composite microbial agent is a liquid or solid preparation.

[0011] Furthermore, the composite microbial agent includes a carrier; the carrier is selected from at least one of sawdust, zeolite powder, diatomaceous earth, vermiculite, bentonite, peat moss, and weathered coal, preferably sawdust.

[0012] A fourth aspect of this invention is to propose the application of the composite microbial agent described in the third aspect in the prevention and control of cyanobacterial blooms.

[0013] Furthermore, the ratio of the two algicidal bacteria in the compound microbial agent is (1-10):(10-1); the ratio of the compound microbial agent to cyanobacteria is 10:1 or higher, preferably (10-100):1.

[0014] Furthermore, the ratio of the two algicidal bacteria in the compound microbial agent is 1:1.

[0015] The fifth aspect of the present invention is to provide a method for preventing or controlling cyanobacteria in water, comprising the step of adding the composite microbial agent described in the third aspect to water containing one or more types of cyanobacteria and co-culturing them.

[0016] Compared with the prior art, the beneficial effects of the present invention are as follows: The present invention obtained two algicidal bacteria through screening and biological preservation. It has been verified that they have significant inhibitory effects on many cyanobacteria such as Microcystis aeruginosa, Lycopodium clavatum, Anabaena spp., and Cyclops spp., and can be used to prevent and control cyanobacterial blooms in water bodies. This invention contains a compound bacterial agent containing two algicides, which has a significant algae control effect on algal blooms in natural waters and can be applied to the field of biological control of freshwater cyanobacterial blooms, providing an ideal choice for the treatment of cyanobacterial blooms in water bodies. Attached Figure Description

[0017] Figure 1 This is a schematic diagram of the phylogenetic tree obtained by analyzing the 16S rRNA gene sequence of Algophilus 1 in Example 1 of the present invention.

[0018] Figure 2 In Example 2 of this invention, the algicidal bacteria MERYL1-35 and MERXLGS1 respectively inhibited... C. raciborskii FACHB-1503 Limnothrix_g2 filamentous algae of the genus Anabaena sp.PCC 7120 M. aeruginosa A schematic diagram of FACHB-524.

[0019] Figure 3 This is a schematic diagram illustrating the inhibitory effects of alginolytic bacteria MERYL1-35 and MERXLGS1 on four cyanobacteria in Example 2 of this invention.

[0020] Figure 4 Microscopic images showing the composition of artificial cyanobacterial blooms and the inhibitory effect of the compound microbial agent in Example 3 of the present invention.

[0021] Figure 5 This is a comparison of the effects of two algicidal bacteria and their combination on artificial cyanobacterial blooms in Example 3 of the present invention.

[0022] Figure 6 This illustrates the control effect of the compound microbial agent in Example 4 of the present invention on a complex artificial algal bloom in a 15-liter system.

[0023] Figure 7 This invention demonstrates the effect of the compound microbial agent in Example 5 on the control of algal blooms in lakes under natural conditions.

[0024] Figure 8 This is the result of controlling the algal blooms in lakes under natural conditions by adjusting the bacteria-algae ratio and the proportion of algicidal bacteria in Example 5 of the present invention. Detailed Implementation

[0025] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0026] Example 1: Screening and Identification of Alginolytic Bacteria

[0027] 1. Isolation and screening of viable strains

[0028] Water samples were collected from lakes such as Yilong Lake and Xiaolvgu, where algal blooms occurred. Single colonies of all culturable bacteria were obtained by dilution and plating using 2 / TY and R2A media. The media composition is as follows: 2 / TY medium composition: Tryptone 2.5g / L, YEAST EXTRACT 1.5g / L, agar powder 15g / L.

[0029] R2A medium composition: Tryptone 0.5 g / L, Yellow Extract 0.5 g / L, Glucose 0.5 g / L, Solublestarch 0.5 g / L, KH2PO4 0.3 g / L, MgSO4 7H2O 0.05g / L, C3H3NaO3 0.3g / L, agar powder 15g / L.

[0030] Single bacterial colonies were picked up with toothpicks and cultured on the same plates. After preliminary screening and elimination based on the size, shape, edge, surface characteristics, elevation, color, transparency, and hardness of the bacterial colonies, more than 200 bacterial strains were obtained.

[0031] 2. Screening of algicidal bacteria and identification of 16S rRNA gene sequence

[0032] The bacterial 16S rRNA gene was amplified by PCR using universal primers 27F (5'-AGAGTTTGATCCTGGCTCAG-3') and 1492R (5'-GGTTACCTTGTTACGA CTT-3'), and then sequenced. The sequence was compared with the EzBioCloud database to find the closest bacteria and its 16S rRNA gene sequence.

[0033] After activating and culturing the obtained bacteria for 48 hours, bacterial plates were scraped off, and the bacterial cells were washed three times with culture medium BG11. The bacteria were then inoculated onto cyanobacteria at a bacterial-to-algae ratio of 100:1. Microcystis aeruginosa In FACHB 524, it was placed in a light-controlled incubator and cultured under full light (25℃, 120 μmol photons). 2 1 (Light intensity). After 4 days of cultivation, the algae were observed to see if they lost their green color, and the changes in chlorophyll content were measured. Several algicidal strains with strong algicidal abilities were ultimately obtained, among which MERYL1-35 and MERXLGS1 showed superior bacterial activity and algicidal effect, with an algicidal rate exceeding 90%. The phylogenetic tree obtained after 16S rRNA gene sequence analysis of the algicidal bacteria is shown below. Figure 1 As shown. The bacterium most homologous to the algicidal bacterium MERYL1-35 is Chromobacterium rhizoryzae Lame1188 (KT951843) was identified as... Chromobacterium The genus; the one most closely homologous to the alginolytic bacterium MERXLGS1 is Paucibacter aquatile CR182 (POSP01000003), identified as Paucibacter Therefore, the algicidal bacteria MERYL1-35 and MERXLGS1 are classified and named as follows: Chromobacterium rhizoryzae and Paucibacter aquatile It was deposited at the China Center for Type Culture Collection (Wuhan University) on April 16, 2024, with accession number CCTCC M 2024699 for MERYL1-35 and CCTCC M2024698 for MERXLGS1.

[0034] Example 2 Alginate inhibitory ability of algaecides alone

[0035] MERYL1-35 and MERXLGS1 respectively with C. raciborskii FACHB-1503, filamentous algae, Anabaena sp. PCC 7120 and M. aeruginosa Co-culturing with FACHB-524 revealed that either ingredient alone could inhibit these four algae species. Figure 2 As shown, the algae MERYL1-35 and MERXLGS1 can respectively inhibit C. raciborskii FACHB-1503 Limnothrix_g2 filamentous algae of the genus Anabaena sp. PCC 7120 M. aeruginosa FACHB-524. The bar chart showing the ability of alginolytic bacteria MERYL1-35 and MERXLGS1 to inhibit the growth of the four algae species by extracting and calculating chlorophyll content in various algae is shown below. Figure 3 As shown.

[0036] Example 3: Screening of Combination Microbial Agents

[0037] Twenty-eight algicidal bacteria strains capable of dissolving prokaryotic algae were screened from the obtained algicidal bacteria library and paired together. Using artificially constructed simulated algal blooms as materials, the algicidal bacteria were co-cultured with them to screen out composite microbial agents that can inhibit artificial algal blooms.

[0038] The steps for preparing artificial cyanobacterial blooms are as follows: (1) A plant obtained by separating water samples from Houhu Lake (Wuhan City, Hubei Province). Limnothrix_g2 Filamentous algae of the genus, after being cultured to the stable phase, were compared with those cultured to the stable phase. M. aeruginosa FACHB-524 Anabaena sp PCC7120 and C. raciborskii FACHB-1503 was mixed at a cell density ratio of 1:1:1:1. Microscopic images of the four algae are shown below. Figure 4 As shown in AD Figure 4 A is a filamentous algae. Figure 4 B is M. aeruginosa FACHB-524 Figure 4 C is Anabaena sp. PCC7120 and Figure 4 D is C . raciborskii FACHB-1503.

[0039] (2) Referring to the N and P content of water bodies during algal blooms in natural water bodies, a simulated micro-natural water body was prepared using lake water samples, distilled water, and BG11 culture medium (BG11 culture medium was about 1 / 40 of the system, lake water about 30%, and distilled water about 70%). Four types of algae were mixed in a 1:1:1:1 cell density ratio and placed in the mixture. The mixture was then statically cultured at 25°C for about one month until the community stabilized, resulting in a planktonic algal system with multiple components. A significant amount of *Microcystis aeruginosa*, filamentous cyanobacteria, *Anabaena*, and *Strombocytosporium* were clearly observed coexisting. Figure 4 E), the cell density ratio obtained by optical microscopy was approximately, Microcystis aeruginosa: 10 7 , Lake Thread Algae: 10 4 10g of Anabaena 4 Cylindrica pseudocytophyta: 10 3 .

[0040] By adding microbial agents and controlling the bacteria-to-algae ratio at 50:1 (CFU / ml), the algicidal efficiency was tested to screen combined algicidal bacteria. It was found that the combination of MERYL1-35 and MERXLGS1 significantly inhibited the formation of artificially simulated complex algal systems. Figure 4 F, Figure 5 This indicates that it may have some application in simple algal bloom waters.

[0041] Example 4: Control of complex artificial algal blooms in a 15-liter system by compound microbial inoculants

[0042] Based on the nitrogen and phosphorus levels observed during summer algal blooms in natural water bodies, the four aforementioned prokaryotic algae were added to a mixture of Houhu water, BG11 culture medium, and distilled water to prepare a 15-liter artificial algal bloom system. This system was placed in natural light and an open environment for one week to stabilize the community, which remained stable during subculturing. Cell density ratios obtained under optical microscopy were approximately: *Microcystis aeruginosa* : 10-1 7 , Lake Thread Algae: 10 4 10g of Anabaena 4 Cylindrica pseudocytophyta: 10 3 .

[0043] Subsequently, either MERYL1-35 or MERXLGS1 strains were added separately, or a compound microbial agent formed by the combination of the two strains (MERYL1-35:MERXLGS1=1:1) was added, with a strain-to-algae ratio of 50-100:1. The mixture was then left to stand for another week before chlorophyll content was extracted and calculated.

[0044] like Figure 6As shown in Figure A, the combined bacterial agent of MERYL1-35 and MERXLGS1 can significantly inhibit 15 liters of complex algae in a simulated open natural environment. Figure 6 B. Chlorophyll content indicates that the algae inhibition rate can also reach about 60%. Figure 6 This indicates that it has a good control ability over algal bloom waters with complex composition.

[0045] Example 5: Control of Algal Blooms in Lakes under Natural Environment by Compound Microbial Agents

[0046] Natural algal bloom samples were taken from a lake in Jiangsu Province during an algal bloom outbreak. Microscopic examination confirmed that the dominant algal species was Microcystis aeruginosa. Figure 7 A) Add the above-mentioned MERXLGS1 and MERYL1-35 compound microbial preparation at a 1:1 ratio, controlling the bacteria-to-algae ratio to be 50-100:1. Figure 7 B consists of three groups from left to right: 60:1, 70:1, and 90:1. The algae suppression efficiency was tested after one week. Figure 7 Results B showed that the combined bacterial agent of MERYL1-35 and MERXLGS1 (+Bacteria group) could significantly inhibit algal blooms in lakes under natural conditions, indicating that it has a good control ability on actual algal bloom areas.

[0047] By controlling the bacteria-to-algae ratio at 100:1, and adjusting the ratios of MERXLGS1 (bacterium A) and MERYL1-35 (bacterium B) respectively, good combined algae-suppressing efficiencies were observed at ratios of 5:1, 10:1, 1:5, and 1:10. Figure 8 The ratio of MERXLGS1 (bacterium A) to MERYL1-35 is between 10:1 and 1:10, and the combination of the two algicidal bacteria has an algicidal effect when the bacteria-algae ratio is higher than 50:1.

[0048] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

Claims

1. Algophilic bacteria, characterized in that, It is deposited at the China Center for Type Culture Collection, with accession numbers CCTCC NO: M2024698 or CCTCC NO: M 2024699.

2. The application of the algicidal bacteria described in claim 1 in the prevention and control of cyanobacterial blooms.

3. The application according to claim 2, characterized in that, The cyanobacteria are selected from at least one of Microcystis aeruginosa, Lycopodium clavatum, Anabaena spp., and Cyclops spp.

4. A compound microbial inoculant, characterized in that, This includes algicidal bacteria or their cultures with accession numbers CCTCC NO: M 2024698 and CCTCC NO: M2024699.

5. The compound microbial agent according to claim 4, characterized in that, The compound microbial agent is a liquid or solid preparation.

6. The compound microbial agent according to claim 4, characterized in that, The composite microbial agent includes a carrier; the carrier is selected from at least one of sawdust, zeolite powder, diatomaceous earth, vermiculite, bentonite, peat moss, and weathered coal.

7. The application of the compound microbial agent according to any one of claims 4-6 in the prevention and control of cyanobacterial blooms.

8. The application according to claim 7, characterized in that, The ratio of the two algicidal bacteria in the compound microbial agent is (1-10):(10-1); the ratio of the compound microbial agent to cyanobacteria is 10:1 or higher.

9. The application according to claim 7, characterized in that, The ratio of the two algicides in the compound microbial agent is 1:

1.

10. A method for preventing or controlling cyanobacteria in aquatic bodies, characterized in that, The method includes the step of adding the compound microbial agent described in any one of 4-6 to a water body containing one or more cyanobacteria and co-culturing them.

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