Pseudomonas with oscillatoria dissolving characteristic and application of pseudomonas in algal bloom treatment

Abstract

The pseudomonas with the oscillatoria dissolving characteristic is characterized in that the pseudomonas is named as Pseudomonas sp GangLiHuanBao-Z1, the preservation unit is China General Microbiological Culture Collection Center (CGMCC), the address is Beijing, China, the preservation number is CGMCC No.32315, and the preservation date is October 23, 2024. The invention further discloses a preparation method of the pseudomonas with the oscillatoria dissolving characteristic, and the pseudomonas with the oscillatoria dissolving characteristic is characterized in that the pseudomonas is named as Pseudomonas sp GangLiHuanBao-Z1. Further assistance can be provided for effective control of cyanobacterial bloom, and meanwhile, the bacterial strain disclosed by the invention is a safe, environment-friendly and harmless bacterial strain and has a relatively good application prospect in the field of ecological restoration.

Description

Technical Field

[0001] The invention relates to the field of ecological restoration microorganisms, and in particular to a Pseudomonas with oscillatory algae dissolving property and application thereof in water bloom control. Background Art

[0002] With the development of the country, the improvement of people's quality of life, the improvement of people's well-being and the improvement of life quality are the current pursuits of the people. The construction of landscape pools, riverside parks, lakes and reservoirs, and riverside trails are the main measures to improve people's quality of life. However, due to the discharge of exogenous N, P and other pollutants, a large number of algae are produced in the water, forming water blooms. Water bloom is a phenomenon in which the water body appears blue or green after the large-scale reproduction of cyanobacteria, green algae, diatoms, etc. Freshwater water blooms are mainly cyanobacteria. The most common is the bloom of freshwater algae. When the bloom occurs in large quantities, a thick layer of green algae is formed on the water surface, which can release toxins-lake indigo, which is toxic to fish. After the algae die, decay, and decompose, they consume a large amount of dissolved oxygen in the water, causing the water body to produce a foul odor, which not only destroys aquatic resources, but also affects the aesthetics and recreation of the water body.

[0003] At present, the main methods for controlling algal blooms are physical, chemical and biological. Physical methods include mechanical salvage, ultrasound, membrane filtration, adsorption and other technologies; chemical methods use metals, photosensitizers, herbicides and other chemicals to remove algal blooms; biological methods refer to the use of aquatic animals, aquatic plants and microorganisms in natural ecology to degrade algal cells, thereby achieving the purpose of removing algal blooms. Physical and chemical methods are costly and prone to secondary pollution. Microbial algae control technology has low cost, simple operation and maintenance, environmental protection and no secondary pollution. It is currently the most promising algae control technology.

[0004] At present, a large number of studies have been conducted on cyanobacteria algae-lysing bacteria, but most of them are based on Microcystis aeruginosa. For example, patents CN113583906A, CN 105861395A, CN103484403A, CN 103937726A, CN 105502688B, and CN109749969A all study the algae-lysing properties of Microcystis aeruginosa. Patent CN 114806942A mainly has a significant inhibitory effect on multicellular Tetraselmis obliquus, unicellular filamentous algae, and protein-nuclear Chlorella of the Chlorophyta, and is also intended to have an inhibitory effect on cyanobacteria. Cyanobacteria blooms are mainly composed of Microcystis aeruginosa, Oscillatoria, and Aphanizomenon. Oscillatoria is a multicellular cyanobacteria, filamentous, stable in structure, extremely strong in vitality, and its growth is not affected by seasonal changes. It can grow all year round. It is the most widely distributed and most diverse cyanobacteria. Therefore, the study of the algae-dissolving characteristics of Oscillatoria is helpful to further control the freshwater cyanobacteria bloom. Although patent CN109735472A mentions the removal of Oscillatoria, it mainly uses a marine Pseudomonas to convert Oscillatoria as an electron donor to obtain bioelectricity and construct a microbial fuel cell to achieve the removal of algae. It is a direct algae-dissolving method, and there are few reports on the indirect dissolution of Oscillatoria. . Summary of the invention

[0005] In order to solve the above technical problems, the present invention aims to provide a Pseudomonas with the property of lysing oscillatory algae. The second purpose is to provide the application thereof.

[0006] To achieve the above-mentioned purpose, the present invention provides the following technical solution: a Pseudomonas with oscillatory algae dissolving characteristics, characterized in that: the Pseudomonas is named Pseudomonas sp GangLiHuanBao-Z1, the preservation unit is the China General Microbiological Culture Collection Center, located in Beijing, China, the preservation number is CGMCC No.32315, and the preservation date is October 23, 2024.

[0007] The application of the Pseudomonas with oscillatory algae dissolving property in the preparation of algae dissolving agent.

[0008] The application of the Pseudomonas with oscillatory algae dissolving property in water bloom control.

[0009] There are two ways of algae lysis by algae-lytic bacteria: direct algae lysis and indirect algae lysis. Direct algae lysis is when algae-lytic bacteria directly attack algae cells, come into direct contact with the surface of algae cells, invade into algae cells and cause algae cells to lyse and die; indirect algae lysis is when algae-lytic bacteria secrete specific or non-specific extracellular substances during metabolism, such as proteins, alkaloids, peptides and other substances, which destroy the structure of algae cells and cause algae death.

[0010] The Pseudomonas of the present invention is an indirect algae-lysing agent, and its inactivated bacterial solution can still cause an obvious yellowing effect on Oscillatoria. The Pseudomonas of the present invention also has the function of removing nitrate nitrogen and COD. It can provide further assistance for the effective control of cyanobacterial blooms. At the same time, the strain of the present invention is a safe, environmentally friendly and harmless strain, and has a good application prospect in the field of ecological restoration. BRIEF DESCRIPTION OF THE DRAWINGS

[0011] Figure 1 This is a graph showing the color change of the Oscillatoria algae liquid after 14 days.

[0012] Figure 2 This is a diagram showing the effect of suppressing cyanobacteria bloom in the reservoir.

[0013] Figure 3 This is the strain development tree. DETAILED DESCRIPTION

[0014] The present invention is further described below in conjunction with the accompanying drawings and embodiments.

[0015] Example 1

[0016] Specific screening and identification of safe and environmentally friendly Pseudomonas for indirect lysis of Oscillatoria

[0017] (1) Isolation and purification of strains: Water samples with yellow algae were collected from a water storage farm in Yongchuan, Chongqing. The water samples were broken and mixed with glass beads, and then inoculated into enrichment medium and cultured in a constant temperature shaker at 30°C and 170 rpm for 48 h. The enrichment culture solution was heated to 10 °C and then 20 °C. -1 , 10 -2 , 10 -3 , 10 -4 , 10 -5 Five gradient dilutions were performed, the diluted samples were spread on beef extract peptone solid nutrient plates for culture, and the plates were inverted and placed in a constant temperature incubator at 30°C for culture for 48 hours, and then the plaques with different morphologies were picked and cultured using enrichment culture medium; the picked strains were inoculated into 1 / 8 beef extract peptone culture medium and cultured in a constant temperature shaker at 30°C and 170rpm for 48 hours; the enrichment culture medium was: 30 g / L sucrose, 1 g / L dipotassium hydrogen phosphate, 0.5 g / L potassium chloride, 0.5 g / L magnesium sulfate heptahydrate, 0.01 g / L ferrous sulfate, and 2 g / L sodium nitrate.

[0018] (2) Screening: The cultured strains were inoculated into the algae solutions of Oscillatoria, Chromococcus, Microcystis, Anabaena, and Aphanizomenon, respectively, and cultured together. The algae culture medium was BG11 medium, and the culture conditions were 12 h of light culture, the light intensity was 2500-3500 lux, and 12 h of dark culture. The absorbance of the solution at OD720 was detected every 7 days to observe the degree of algae yellowing. The yellowing inhibition effect is shown in Table 1 below:

[0019] Table 1 Effect of Pseudomonas on the yellowing inhibition of different algae (after 14 days)

[0020]

[0021]

[0022] The calculation formula of yellowing inhibition rate is:

[0023] H=(BA)÷B×100%

[0024] Where: H—yellowing inhibition rate, %;

[0025] A—absorbance of algae suspension 14 days after adding bacterial agent;

[0026] B—Absorbance measured in the control experiment (blank algae solution).

[0027] By measuring the absorbance of each algae at OD720, the strain GangLiHuanBao-Z1 had an inhibition rate of more than 95% on the chlorosis of Oscillatoria odorifera, 70% on Chromococcus chlorella, and 74% on Microcystis chlorella within 14 days, and had no obvious chlorosis inhibition effect on the other algae.

[0028] The GangLiHuanBao-Z1 Pseudomonas solution was inactivated and then added to the Oscillatoria solution. After 14 days, the yellowing inhibition rate of Oscillatoria reached 82%, indicating that its algae lysis method was indirect algae lysis.

[0029] (3) Rescreening: The bacteria with yellowing effect are stored at low temperature and subjected to the acute Daphnia test in accordance with HJ / T 415-2008 Environmental Safety Assessment of Environmentally Friendly Microbial Agents to determine safety;

[0030] The GangLiHuanBao-Z1 bacteria with yellowing effect was stored at low temperature, and the acute activity inhibition of Daphnia was verified in accordance with the "Environmental Safety Evaluation of Environmental Microbial Agents for Environmental Protection" 40 Daphnia of uniform size were selected, and 10 were randomly selected from each group and divided into 4 groups, one of which was used as the control. Except for the control, the remaining groups were added with the corresponding 10%, 20%, and 30% bacterial solution for cultivation, and the activity of Daphnia was observed every 24 hours. The experimental results are as follows:

[0031] Table 2 Results of the acute activity inhibition test of Daphnia

[0032] Components concentration 24h inhibition 48h inhibition rate (%) Comparison 0 0 0 GangLiHuanBao-Z1 10％ 0 0 GangLiHuanBao-Z1 20％ 0 0 GangLiHuanBao-Z1 30％ 0 0

[0033] The experimental results showed that when the dosage of GangLiHuanBao-Z1 was 10-30%, the inhibition rate of acute activity of Daphnia was 0, indicating that it is a safe and environmentally friendly strain.

[0034] (4) Identification of Pseudomonas

[0035]

[0036] The sequencing results were compared with NCBI Blast to determine the species of the rescreened strains. After comparison with the NCBI database, the similarity between the strain GangLiHuanBao-Z1 of the present invention and the model bacteria Pseudomonas sp ADM2-2 was as high as 99%; based on the morphological characteristics and physiological and biochemical characteristics of the strain GangLiHuanBao-Z1, the strain GangLiHuanBao-Z1 was identified as Pseudomonas sp and named Pseudomonas sp GangLiHuanBao-Z1. At the same time, a developmental tree was constructed for the strain, and the developmental tree was as follows: Figure 3 .

[0037] Example 2

[0038] Application of blue algae bloom control in a reservoir

[0039] The reservoir often breaks out blue algae in summer. It is found that the blue algae in the reservoir are mainly composed of Oscillatoria. The algae liquid in the reservoir is taken back and evenly divided into 2 groups. One group is used as the control, and the other group is inoculated with the activated strain Pseudomonas sp GangLiHuanBao-Z1 at a volume ratio of 1%. The activated strain Pseudomonas sp GangLiHuanBao-Z1 is cultured under the conditions of 12h of light, 3300lux of light intensity, and 12h of darkness. The absorbance at OD720 of the solution is detected, and the algae yellowing rate is determined as shown in Table 3. The water quality changes are shown in Table 4:

[0040] Table 3 Algae yellowing inhibition rate

[0041] Components 7d yellowing rate 14d yellowing inhibition Comparison 0 0 GangLiHuanBao-Z1 52％ 92％

[0042] Table 4 Water quality changes

[0043] Components 7dCOD 7d Nitrate 14dC 14d Nitrate Comparison 32 4.3 34 4.2 GangLiHuanBao-Z1 25 3.1 18 1.8

[0044] It can be seen that the Pseudomonas sp GangLiHuanBao-Z1 strain still has a 92% cyanobacteria yellowing inhibition rate in actual cyanobacteria control applications, indicating that it has a good yellowing inhibition effect on Oscillatoria, and can provide an effective measure for the inhibition of cyanobacteria blooms. At the same time, the strain also has a certain removal effect on COD and nitrate nitrogen, and is a safe and environmentally friendly strain. It can be seen that it has a good application prospect in the field of freshwater bloom restoration.

[0045] Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions and variations may be made to the embodiments without departing from the principles and spirit of the present invention, and that the scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A Pseudomonas with oscillatory algae dissolving properties, characterized in that: The Pseudomonas is named Pseudomonassp GangLiHuanBao-Z1, the preservation unit is the China General Microbiological Culture Collection Center, located in Beijing, China, the preservation number is CGMCC No.32315, and the preservation date is October 23, 2024.

2. Use of the Pseudomonas with oscillatory algae-dissolving property according to claim 1 in the preparation of an algae-dissolving agent.

3. Use of the Pseudomonas with oscillatory algae dissolving property as claimed in claim 1 in water bloom control.

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