A composite microbial agent for controlling cyanobacteria blooms

Abstract

The present invention relates to the field of microbial engineering technology, and specifically discloses a composite microbial agent for controlling cyanobacterial blooms. The Bacillus velezinoff provided by the present invention destroys algal cells and inhibits the activity of related biological enzymes by secreting heterocyclic algaecidal active substances, while causing the leakage of cell contents (such as algal toxins, polysaccharides, and lipids); the extracellular enzyme system (such as alkaline phosphatase and β-glucosidase) of Sphingomonas decomposes free algal toxins into low-toxic linear peptide segments, while hydrolyzing the β-1,4-glycosidic bonds in EPS, converting algal organic fragments into small-molecule soluble substances, and providing substrates for subsequent metabolism of Rhodococcus; the lipase and esterase secreted by Rhodococcus can hydrolyze triglycerides and wax components, and convert fatty acids into energy and biosurfactants through a β-oxidation pathway, thereby enhancing the hydrophilicity of algal fragments and promoting overall degradation efficiency.

Description

Technical Field

[0001] The present invention relates to the technical field of microbial engineering, and in particular to a composite microbial agent for controlling cyanobacteria blooms. Background Art

[0002] Eutrophication is a common environmental pollution problem characterized by excessive concentrations of nutrients such as nitrogen and phosphorus in water, which triggers the proliferation of algae. The harm caused by algae is mainly manifested in several aspects: large-scale coverage of algae will form a thick green or brown "algae blanket" on the water surface, which will hinder the contact between air and water, causing water quality corruption, resulting in a sharp drop in dissolved oxygen content in the water, and causing fish and other aquatic organisms to suffocate and die from lack of oxygen. Many blue-green algae can produce algal toxins, which are highly toxic and can be transmitted through the food chain, ultimately posing a serious threat to human health. In addition, excessive algae growth can also affect key environmental indicators such as water transparency and dissolved oxygen, leading to a continuous deterioration of water quality, which in turn affects the rational use of water resources and brings many inconveniences and safety hazards to agricultural irrigation, industrial water use, and human water use.

[0003] Currently, the commonly used algae removal methods are physical, chemical and biological. The physical method has relatively limited processing capacity for large-scale algal blooms, and may cause certain disturbances to the water body during operation, affecting the ecological balance of the water body; the chemical algae removal method is quick to take effect, but chemical agents are prone to secondary pollution, which has an adverse impact on the ecosystem.

[0004] Microbial algae control technology is an emerging ecological restoration technology with the advantages of high efficiency, environmental safety, and pollution-free, and has broad application prospects. Summary of the Invention

[0005] In view of the shortcomings of the prior art, the object of the present invention is to provide a composite microbial agent for controlling cyanobacteria blooms.

[0006] In order to achieve the above object, the present invention adopts the following technical solutions:

[0007] A composite microbial agent for controlling cyanobacteria blooms, characterized in that the composite microbial agent consists of a Bacillus Velez-like agent, a Sphingomonas agent, and a Rhodococcus agent.

[0008] In the technical solution disclosed in the present invention, the classification name of Bacillus velezensis HY16 is Bacillus velezensis, and the deposit number is: CGMCC No. 31645; the deposit unit is: General Microbiology Center of China Culture Collection Administration (CGMCC); the address is: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences; the deposit date is: August 9, 2024.

[0009] In the technical solution disclosed in the present invention, the volume ratio of the Bacillus Velez-Neanderthalensis agent, the Sphingomonas agent and the Rhodococcus agent is 10-20:10-15:10-15.

[0010] In the technical solution disclosed in the present invention, the number of viable bacteria in the Bacillus Velezii inoculum is not less than 2.0×10 10 cfu / mL.

[0011] In the technical solution disclosed in the present invention, the number of viable bacteria in the Sphingomonas inoculum is not less than 1.0×10 9 cfu / mL.

[0012] In the technical solution disclosed in the present invention, the number of viable bacteria in the Rhodococcus inoculum is not less than 1.0×10 9 cfu / mL.

[0013] In the technical solution disclosed in the present invention, the composite microbial agent also includes a carrier.

[0014] In the technical solution disclosed in the present invention, the carrier includes diatomaceous earth, bentonite, zeolite powder or hydrotalcite.

[0015] The present invention also provides application of the composite microbial agent in controlling cyanobacteria blooms.

[0016] In the technical solution disclosed in the present invention, a composite microbial agent is added to the cyanobacteria water body at a volume ratio of 1-5% of the bacteria-algae liquid.

[0017] Compared with the prior art, the present invention has the following beneficial effects:

[0018] The Bacillus Velez subtilis, Sphingomonas subtilis and Rhodococcus subtilis provided by the present invention exert a synergistic algae control effect, achieving efficient cyanobacteria degradation through multi-level metabolic complementarity and functional coupling. The possible mechanism of action is that Bacillus Velez subtilis secretes heterocyclic algaecidal active substances, destroys algal cells and inhibits the activity of related biological enzymes, while causing the leakage of cell contents (such as algae toxins, polysaccharides, and lipids); the extracellular enzyme system of Sphingomonas (such as alkaline phosphatase and β-glucosidase) decomposes free algae toxins into low-toxic linear peptide segments. , while hydrolyzing the β-1,4-glycosidic bonds in EPS, converting algal organic fragments into small soluble molecules, providing substrates for the subsequent metabolism of Rhodococcus; the lipase and esterase secreted by Rhodococcus can hydrolyze triglycerides and wax components, and convert fatty acids into energy and biosurfactants through the β-oxidation pathway, enhancing the hydrophilicity of algal fragments to promote overall degradation efficiency; Sphingomonas further converts the oligosaccharides produced by Bacillus Velezii into organic acids, and Rhodococcus uses organic acids for deep oxidation to form a complete closed loop. In addition, the applicant unexpectedly discovered during the experiment that compared with Microcystis aeruginosa, the composite microbial agent provided by the present invention has a better algae-dissolving effect on Oscillatoria. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 The diagram shows the algae-lyzing effects of different groups on Microcystis aeruginosa.

[0020] Figure 2 These are physical pictures of the algae-lysing effect of the composite microbial agent provided in Example 1 of the present invention, wherein A is the control group, B is the physical picture of the algae-lysing effect after 3 days, and C is the physical picture of the algae-lysing effect after 5 days.

[0021] Figure 3 This is a comparison chart of the algae-lyzing effect of the composite microbial agent provided in Example 1 on Microcystis aeruginosa and Oscillatoria. DETAILED DESCRIPTION

[0022] The present invention is further described in detail below through specific preferred embodiments, but the present invention is not limited to the following embodiments.

[0023] It should be noted that, unless otherwise specified, all chemical reagents involved in the present invention were purchased through commercial channels.

[0024] The type of Bacillus Velez subtilis used in the examples of the present invention is Bacillus Velez subtilis HY16, with a deposit number of CGMCC No. 31645.

[0025] The model of Sphingomonas was SHMCC D72199, which was purchased from Pusrui (Shanghai) Biopharmaceutical Co., Ltd.;

[0026] The model of Rhodococcus was SHMCC D10827, which was purchased from Pusrui (Shanghai) Biopharmaceutical Co., Ltd.;

[0027] The number of viable bacteria in the Bacillus Velezii inoculum used in the examples and comparative examples of the present invention was 5.0×10 10 cfu / mL, the number of viable bacteria in the Sphingomonas inoculum was 2.0×10 9 cfu / mL, the number of viable bacteria in the Rhodococcus inoculum is 2.0×10 9 cfu / mL.

[0028] Example 1

[0029] A composite microbial agent for controlling cyanobacterial blooms, wherein the composite microbial agent consists of a Bacillus velez agent, a Sphingomonas agent, and a Rhodococcus agent, and the volume ratio of the Bacillus velez agent, the Sphingomonas agent, and the Rhodococcus agent is 15:10:10.

[0030] Example 2

[0031] A composite microbial agent for controlling cyanobacteria blooms, wherein the composite microbial agent consists of a Bacillus velez agent, a Sphingomonas agent, and a Rhodococcus agent, and the volume ratio of the Bacillus velez agent, the Sphingomonas agent, and the Rhodococcus agent is 20:15:15.

[0032] Example 3

[0033] A composite microbial agent for controlling cyanobacterial blooms, wherein the composite microbial agent consists of a Bacillus velez agent, a Sphingomonas agent, and a Rhodococcus agent, and the volume ratio of the Bacillus velez agent, the Sphingomonas agent, and the Rhodococcus agent is 10:15:10.

[0034] Example 4

[0035] A composite microbial agent for controlling cyanobacterial blooms, wherein the composite microbial agent consists of a Bacillus velez agent, a Sphingomonas agent, and a Rhodococcus agent, and the volume ratio of the Bacillus velez agent, the Sphingomonas agent, and the Rhodococcus agent is 10:10:10.

[0036] Comparative Example 1

[0037] A composite microbial agent for controlling cyanobacterial blooms, wherein the composite microbial agent is composed of a Sphingomonas belcheri agent and a Rhodococcus agent, and the volume ratio of the Sphingomonas agent to the Rhodococcus agent is 10:10.

[0038] Compared with Example 1, Comparative Example 1 did not add Bacillus Velez subtilis.

[0039] Comparative Example 2

[0040] A composite microbial agent for controlling cyanobacteria blooms, wherein the composite microbial agent consists of a Bacillus Velez subtilis agent and a Rhodococcus agent, and the volume ratio of the Bacillus Velez subtilis agent to the Rhodococcus agent is 15:10.

[0041] Compared with Example 1, Comparative Example 2 did not add Sphingomonas.

[0042] Comparative Example 3

[0043] A composite microbial agent for controlling cyanobacterial blooms, wherein the composite microbial agent consists of a Bacillus subtilis agent, a Sphingomonas agent, and a Rhodococcus agent, and the volume ratio of the Bacillus subtilis agent, the Sphingomonas agent, and the Rhodococcus agent is 15:10:10.

[0044] Comparative Example 3 is compared with Example 1. In the case of replacing Bacillus Velezii with Bacillus subtilis, the number of viable bacteria in the Bacillus subtilis inoculum is 5.0×10 10 cfu / mL.

[0045] Comparative Example 4

[0046] A composite microbial agent for controlling cyanobacteria blooms, wherein the composite microbial agent consists of a Bacillus velez agent, a Sphingomonas agent and a lactic acid bacteria agent, and the volume ratio of the Bacillus velez agent, the Sphingomonas agent and the lactic acid bacteria agent is 15:10:10.

[0047] Comparative Example 4 is compared with Example 1. In this example, lactic acid bacteria is used to replace Rhodococcus. The number of viable bacteria in the lactic acid bacteria agent is 2.0×10 9 cfu / mL.

[0048] Comparative Example 5

[0049] A microbial agent for controlling blue algae blooms, wherein the microbial agent is a Bacillus Velezii agent.

[0050] Comparative Example 5 is compared with Example 1, in which a single bacterial agent, Bacillus velezensis, is used for the test.

[0051] The microbial agents prepared in Example 1 and Comparative Examples 1-5 were tested, and the specific steps were as follows:

[0052] The Microcystis aeruginosa and Oscillatoria used were purchased from the Wuhan Institute of Hydrobiology, Chinese Academy of Sciences. After activation, the algae were cultured at 25°C, with a light intensity of 2000 lx and a light-dark ratio of 12h:12h.

[0053] Take 100mL of Microcystis aeruginosa algae solution (OD 680The microbial agents prepared in Example 1 and Comparative Examples 1-5 were inoculated into the Microcystis aeruginosa algae solution at a volume ratio of 3% of the algae solution, and LB medium was added as a control group. The system was cultured at a temperature of 35° C., a light intensity of 2000 lux, and a light-dark cycle of 12 h:12 h. The algae lysis rate was calculated based on the chlorophyll a content on the 3rd and 5th days.

[0054] The test results are as follows Figure 1 As shown, from Figure 1 It can be seen from the data that the composite microbial agent provided by the present invention has a good algae-lyzing effect, the algae-lyzing rate can reach more than 85% in 3 days, and the algae-lyzing rate can reach more than 90% in 5 days.

[0055] Figure 2 These are physical pictures of the algae-lysing effect of the composite microbial agent provided in Example 1 of the present invention, wherein A is the control group, B is the physical picture of the algae-lysing effect after 3 days, and C is the physical picture of the algae-lysing effect after 5 days.

[0056] Take 100mL of Oscillatoria algae solution (OD 680 The composite microbial agent prepared in Example 1 was then inoculated into the oscillatoria algae liquid at a volume ratio of 3% to the algae liquid, and LB medium was added as a control group. The system was cultured at a temperature of 35°C, a light intensity of 2000 lux, and a light-dark cycle of 12h:12h. The algae lysis rate was calculated based on the chlorophyll a content on the 3rd and 5th days.

[0057] The test results are as follows Figure 3 As shown, from Figure 3 It can be seen that compared with Microcystis aeruginosa, the composite microbial agent provided in Example 1 of the present invention has a better algae-lysing effect on Oscillatoria.

[0058] Finally, it should be noted that the above embodiments do not limit the present invention in any form. Those skilled in the art will appreciate that modifications and improvements can be made based on the present invention. Therefore, any modifications or improvements made without departing from the spirit of the present invention are intended to fall within the scope of protection claimed in the present invention.

Claims

1. A composite microbial agent for controlling cyanobacteria blooms, characterized in that: The composite microbial agent is composed of Bacillus Velezii agent, Sphingomonas agent and Rhodococcus agent; Among them, the type of Bacillus Velez is Bacillus Velez HY16, and the deposit number is: CGMCC No.31645; The model of Sphingomonas is SHMCC D72199; The model of Rhodococcus is SHMCC D10827; The volume ratio of the Bacillus Velez bacteria agent, the Sphingomonas bacteria agent and the Rhodococcus bacteria agent is 10-20:10-15:10-15; The number of viable cells in the Bacillus Velez inoculum was 5.0×10 10 cfu / mL, the number of viable bacteria in the Sphingomonas inoculum was 2.0×10 9 cfu / mL, the number of viable bacteria in the Rhodococcus inoculum is 2.0×10 9 cfu / mL.

2. The composite microbial agent according to claim 1, characterized in that The composite microbial agent also includes a carrier.

3. The composite microbial agent according to claim 2, characterized in that The carrier includes diatomaceous earth, bentonite, zeolite powder or hydrotalcite.

4. Use of the composite microbial agent according to any one of claims 1 to 3 in controlling cyanobacterial blooms.

5. The use according to claim 4, characterized in that Add the composite microbial agent to the cyanobacteria water body at a volume ratio of 1-5% of the bacteria-algae liquid.

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