A Pseudomonas bacterium with oscillatory algae-dissolving properties and its application in water bloom control

Abstract

The present invention discloses a Pseudomonas strain with oscillatory algae-dissolving properties, characterized in that: the Pseudomonas strain is named Pseudomonas sp GangLiHuanBao-Z1, deposited with the China General Microbiological Culture Collection Center in Beijing, China, with a deposit number of CGMCC No. 32315 and a deposit date of October 23, 2024. This strain can further assist in the effective control of cyanobacterial blooms. Furthermore, the strain is safe, environmentally friendly, and harmless, and has promising application prospects in the field of ecological restoration.

Description

Technical Field

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

[0002] With the development of the country and the improvement of people's quality of life, improving people's well-being and enhancing their quality of life are the current aspirations of the people. The construction of landscaped ponds, riverside parks, lakes and reservoirs, and riverside trails are key measures to improve people's quality of life. However, the influx of exogenous pollutants such as nitrogen and phosphorus has led to the proliferation of algae in the water, causing algal blooms. Algal blooms are a phenomenon in which the proliferation of cyanobacteria, green algae, and diatoms causes the water to appear blue or green. Cyanobacterial blooms are the most common in freshwater waters. When a bloom occurs in large numbers, a thick layer of green algae forms on the water surface, releasing toxins—lake indigo—that are toxic to fish. As the algae die, decay, and decompose, they consume large amounts of dissolved oxygen in the water, causing foul odors. This not only damages aquatic resources but also affects the aesthetics and recreational value of the water.

[0003] Currently, the main methods for controlling algal blooms include physical, chemical, and biological methods. Physical methods include mechanical evacuation, ultrasound, membrane filtration, adsorption, and other technologies; chemical methods utilize metals, photosensitizers, herbicides, and other chemicals to remove algal blooms; and biological methods utilize aquatic animals, plants, and microorganisms found in natural ecosystems to degrade algal cells, thereby achieving the goal of removing algal blooms. Physical and chemical methods are relatively costly and prone to secondary pollution. Microbial algae control technology is currently the most promising due to its low cost, simple operation and maintenance, environmental friendliness, and lack of secondary pollution.

[0004] A large number of studies have been conducted on cyanobacteria algae-lysing bacteria, but most of them focus on Microcystis aeruginosa. For example, patents CN113583906A, CN 105861395A, CN103484403A, CN 103937726A, CN 105502688B, and CN109749969A all studied the algae-lysing properties of Microcystis aeruginosa. Patent CN 114806942A mainly studies the significant inhibitory effect on multicellular Tetraselmis obliquus, unicellular filamentous algae, and Chlorella pyrenoidosa of the Chlorophyta, and also aims to have an inhibitory effect on Microcystis aeruginosa for cyanobacteria. Cyanobacterial blooms are primarily composed of Microcystis aeruginosa, Oscillatoria, and Aphanizomenon. Oscillatoria is a multicellular cyanobacterium with a filamentous structure, strong vitality, and year-round growth, unaffected by seasonal changes. It is the most widespread and diverse cyanobacteria. Therefore, research on the algae-dissolving properties of Oscillatoria can help further control freshwater cyanobacterial blooms. While patent CN109735472A mentions research on Oscillatoria removal, it primarily utilizes a marine Pseudomonas bacterium to convert Oscillatoria into bioelectricity, creating a microbial fuel cell to achieve algae removal. This is a direct algae-dissolving method; indirect dissolution of Oscillatoria is rarely reported. Summary of the Invention

[0005] In order to solve the above technical problems, the present invention aims to provide a Pseudomonas sp. with the ability to dissolve oscillatory algae. The second purpose is to provide a use thereof.

[0006] To achieve the above-mentioned object, the present invention provides the following technical solution: a Pseudomonas with oscillatory algae dissolving properties, 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 invention relates to an application of the Pseudomonas with the algae-dissolving property in the preparation of an algae-dissolving agent.

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

[0009] There are two ways for algae-lysing bacteria to lyse algae: direct lysis and indirect lysis. Direct lysis is when the algae-lysing bacteria directly attack the algae cells, come into contact with the algae cell surface, and invade the algae cells, causing them to lyse and die. Indirect lysis is when the algae-lysing bacteria secrete specific or non-specific extracellular substances, such as proteins, alkaloids, and peptides, during their metabolism, which destroy the algae cell structure and cause the algae to die.

[0010] The Pseudomonas strains of the present invention are indirect algae-lysing agents, and their inactivated bacterial solution can still cause significant yellowing of Oscillatoria algae. The Pseudomonas strains of the present invention also remove nitrate nitrogen and COD, further aiding the effective control of cyanobacterial blooms. Furthermore, the strains of the present invention are safe, environmentally friendly, and harmless, and have promising application prospects in ecological restoration. BRIEF DESCRIPTION OF THE DRAWINGS

[0011] Figure 1 This is a diagram 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 will be further described below with reference to the accompanying drawings and embodiments.

[0015] Example 1

[0016] Screening and identification of safe and environmentally friendly Pseudomonas bacteria 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. After the water samples were collected, they were broken and mixed with glass beads, and then inoculated into enrichment culture medium and cultured in a constant temperature shaker at 30°C and 170 rpm for 48 h. The enrichment culture solution was then heated to 10 -1 , 10 -2 , 10 -3 , 10 -4 , 10 -5 Five gradient dilutions were performed, and the diluted samples were spread on beef extract peptone solid nutrient plates for culture. The plates were inverted and cultured in a 30°C constant temperature incubator for 48 hours. 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 co-cultured. The algae culture medium was BG11 medium, and the culture conditions were 12 h of light culture with a light intensity of 2500-3500 lux and 12 h of dark culture. The absorbance at OD720 of the solution was measured 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 for 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 the inoculant;

[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 yellowing of Oscillatoria, 70% on the yellowing of Chromococcus, and 74% on the yellowing of Microcystis within 14 days, and had no obvious yellowing inhibition effect on the other algae.

[0028] After the GangLiHuanBao-Z1 Pseudomonas solution was inactivated, it was 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 acute Daphnia test in accordance with HJ / T 415-2008 Environmental Safety Evaluation of Microbial Agents for Environmental Protection to confirm safety;

[0030] The GangLiHuanBao-Z1 bacteria, which has a yellowing effect, was cryopreserved and tested for its ability to inhibit the acute activity of Daphnia according to the "HJ / T415-2008 Environmental Safety Assessment of Environmentally Friendly Microbial Agents." Forty daphnia of uniform size were selected, 10 of which were randomly selected from each group and divided into four groups. One group served as a control. The remaining groups, excluding the control, were incubated with 10%, 20%, and 30% of the corresponding bacterial solution. The activity of the 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, as shown in the following figure: Figure 3 .

[0037] Example 2

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

[0039] The reservoir often breaks out in the summer. After testing, it was found that the cyanobacteria in the reservoir were mainly composed of Oscillatoria. The algae liquid in the reservoir was taken back and evenly divided into two groups. One group was used as a control, and the other group was inoculated with the activated strain Pseudomonas sp GangLiHuanBao-Z1 at a volume ratio of 1%. The cells were cultured under the conditions of 12 hours of light, 3300 lux of light intensity, and 12 hours of darkness. The absorbance of the solution at OD720 was tested, and the algae yellowing rate was determined as shown in Table 3. The changes in water quality 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 nitrogen 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 cyanobacterial 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 good application prospects in the field of freshwater bloom remediation.

[0045] While 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 these embodiments without departing from the principles and spirit of the invention, and that the scope of the invention is defined by the appended claims and their equivalents.

Claims

1. A Pseudomonas sp. having the indirect dissolving property of Oscillatoria, characterized in that: The Pseudomonas sp. is named Pseudomonas sp. GangLiHuanBao-Z1, and the depository is China General Microorganism Culture Collection Center, located in Beijing, China. The deposit number is CGMCC No. 32315, and the deposit date is October 23, 2024.

2. Use of the Pseudomonas sp. having the indirect lysis property of Oscillatoria as claimed in claim 1 in the preparation of an algae-dissolving agent.

3. Use of the Pseudomonas sp. having the indirect dissolving property of Oscillatoria as claimed in claim 1 in water bloom control.

Images