Method for controlling harmful algae in fresh water through combination of vegetable sponge and rose kocuria

Abstract

The invention discloses an application of Kocuria rosea (Kocuria rosea) in the control of harmful algae in fresh water. Meanwhile, the invention discloses application of the vegetable sponge combined with the rose kocuria in control of harmful algae in fresh water. The implementation of the invention provides a new technical approach for in-situ bioremediation of algae outbreak in the eutrophic freshwater body, and has important environmental significance and application prospects.

Description

Technical Field

[0001] The present invention relates to the field of controlling harmful algae in fresh water of the ecological environment, and particularly relates to a method for controlling harmful algae in fresh water by combining loofah sponge and Kocuria rosea. Background Art

[0002] The statements in this section merely provide background technical information related to the present invention and do not necessarily constitute prior art.

[0003] With the intensification of water eutrophication, harmful algal blooms (HABs) frequently occurring globally have had a significant negative impact on the ecosystem and economic society. Microcystis aeruginosa, as a representative cyanobacterium of HABs in the freshwater ecosystem, the microcystins produced by it have acute or chronic health effects on humans and other organisms. Therefore, controlling HABs has become a research hotspot in recent years. However, the application of algicidal bacteria in the prior art is mostly limited to laboratory conditions, lacking verification of effectiveness and sustainability in actual environmental water bodies, and mainly focusing on the role of a single bacterium, making the algicidal activity unstable under actual environmental conditions.

[0004] Cell immobilization is a beneficial method that can create immobilized biocatalysts, which can improve the production efficiency of cells and protect cells from environmental stress. However, there are few methods for immobilizing algicidal bacteria to inhibit harmful algal blooms at present. Summary of the Invention

[0005] The present invention aims to solve the problems that the application effect of algicidal bacteria in the prior art in actual environmental water bodies is poor and it is difficult to form a stable microecosystem, and provides a method for effectively removing harmful algae in actual environmental fresh water by jointly using loofah sponge and Kocuria rosea, further improving the control effect of harmful algae.

[0006] The present invention is achieved through the following technical solutions: The first aspect of the present invention lies in providing an application of Kocuria rosea ( Kocuria rosea ) in controlling harmful algae in fresh water.

[0007] The second aspect of the present invention lies in providing an application of a combination of loofah sponge and Kocuria rosea in controlling harmful algae in fresh water.

[0008] The third aspect of the present invention lies in providing a method for controlling harmful algae in fresh water by combining loofah sponge and Kocuria rosea, comprising the following steps: The Roseomonas gilardii was immobilized with loofah sponge. The Roseomonas gilardii bacterial solution was cultured in 1,000 mL of LB medium until OD600 = 1.5. The bacterial cells were collected by centrifugation at 18,000 g for 20 minutes, washed twice with Tris buffer at pH = 8, and then suspended in 1,000 mL of fresh Tris buffer. The loofah sponge was cut into uniform small pieces, sterilized by repeated ultraviolet irradiation, and then added to the above Tris buffer containing Roseomonas gilardii cells. The culture flask was placed in the dark and shaken at 25 °C for 6 hours (100 rpm), and then cultured for another 8 hours under the same conditions without shaking. Finally, a combined algicidal agent of Roseomonas gilardii immobilized on loofah sponge was obtained. The internal reticulated porous structure of the air-dried loofah sponge. After the sterile loofah sponge was placed alone in the Microcystis aeruginosa algal solution, it also had a certain adsorption effect on Microcystis aeruginosa, and the adsorption control rate was 11.3%. After immobilizing Roseomonas gilardii with it, its combined killing effect achieved better results.

[0009] The above-mentioned Roseomonas gilardii was made into an algicidal agent or a combined algicidal agent for controlling harmful algae.

[0010] Furthermore, the concentration of Roseomonas gilardii in the algal culture solution was 1 - 5%. More specifically, the concentration of Roseomonas gilardii in the algal culture solution was 3%.

[0011] Beneficial effects By jointly using loofah sponge and Roseomonas gilardii, the present invention effectively solves the problem of removing harmful algae in the actual environmental water body. This method has high efficiency, low ecological risk, and shows good sustainability and algicidal effect in practical applications. The implementation of the present invention provides a new technical approach for the in-situ bioremediation of algal blooms in freshwater eutrophic water bodies, and has important environmental significance and application prospects. Description of the drawings

[0012] Figure 1 It is the colony morphology of Roseomonas gilardii on the plate; Figure 2 It is the killing effect of 3% concentration Roseomonas gilardii culture solution on Microcystis aeruginosa; Figure 3 It is the loofah sponge after sterilization treatment (repeated ultraviolet irradiation); Figure 4 It is the killing effect of loofah sponge immobilizing 3% concentration Roseomonas gilardii on Microcystis aeruginosa; Figure 5 It is the intact (without adding bacteria) Microcystis aeruginosa algal solution; Figure 6 It is the killing effect of loofah sponge immobilizing Roseomonas gilardii after repeatedly adding Microcystis aeruginosa algal solution; Figure 7 Changes in photosynthetic pigments of algal cells treated with sponge-fixed Roseateles depolymerans Figure 8 Changes in the maximum photosynthetic efficiency Fv / Fm of algal cells treated with sponge-fixed Roseateles depolymerans Figure 9 Normal Microcystis aeruginosa cells photographed by field emission scanning electron microscope Figure 10 Algal cells photographed by field emission scanning electron microscope after 24 hours of treatment with sponge-fixed Roseateles depolymerans Figure 11 Algal cells photographed by field emission scanning electron microscope after 96 hours of treatment with the culture solution of sponge-fixed Roseateles depolymerans Detailed implementation mode

[0013] The technical solutions of the present invention will be further explained and illustrated below through specific examples. All experimental supplies used in the present invention are commercially available.

[0014] Example 1 Cultivation of algae and bacteria: Roseateles depolymerans was purchased from Beina Biotechnology Co., Ltd. and cultured by shaking at 30 °C at a speed of 150 rpm; Microcystis aeruginosa was obtained from the Freshwater Algae Culture Collection and cultured in BG-11 medium under the conditions of a 25 °C incubator with a light intensity of 50 μmol m Kocuria rosea s Microcystis aeruginosa and a light-dark cycle of 14:10 h. The population density of Microcystis aeruginosa cells was determined by counting with a hemocytometer under a microscope. The colony morphology of Roseateles depolymerans is shown in −2 s −1 and 14:10 h light-dark cycle. The population density of Microcystis aeruginosa cells was determined by counting with a hemocytometer under a microscope. The colony morphology of Roseateles depolymerans is shown in Figure 1 .

[0015] Water body configuration: The test water body was prepared according to the nutrient salt concentration of Class V water body in the "Surface Water Environment Quality Standard" of China, containing nutrients such as COD, TN, and TP to simulate the actual environmental water body conditions.

[0016] Algal killing by strains: Microcystis aeruginosa was set at level V (≥1.0×10^8 cells / L, severe algal bloom level), and a specific proportion of the culture solution of Roseateles depolymerans (1%, 2%, 3%, 4%, 5%) was added to its culture solution to test its algicidal effect.

[0017] Optimal concentration: By setting different doses of *Kocuria rosea* to verify the killing effect on *Microcystis aeruginosa*, the test results show that the killing rates of 1%, 2%, 4%, and 5% *Kocuria rosea* solutions on *Microcystis aeruginosa* after 120 hours are 10.1%, 21.5%, 42.1%, and 29.8% respectively, and the killing effect is the best at 3% dose, which is 50%.

[0018] Single-strain algae killing effect: Compared with the control group, under the action of *Kocuria rosea* culture solution at an inoculation concentration of 3%, it has a certain killing effect on *Microcystis aeruginosa*, and can remove 50% of the algal cells within 120 hours. See the specific killing effect in Figure 2 .

[0019] Example 2 Carrier fixation: Loofah sponge was purchased from Alibaba and irradiated with ultraviolet rays before use to kill bacteria. The completely air-dried loofah sponge ( Figure 3 ) was used as the carrier for bacterial immobilization, and *Kocuria rosea* cells were fixed on the loofah sponge to form an immobilized cell sponge, which was compared with free suspended bacterial cells.

[0020] Combined action algae killing effect: Under the combined action of *Kocuria rosea* immobilized with loofah sponge, the killing effect on *Microcystis aeruginosa* was greatly improved, and almost all algal cells could be removed within 120 hours ( Figure 4 ).

[0021] Example 3 Sustainability test: By repeatedly adding *Microcystis aeruginosa* culture solution ( Figure 5 ), the algicidal sustainability of the combined action of loofah sponge and bacteria was tested. After adding the algal solution repeatedly 3 times, the algicidal performance of bacteria immobilized on the loofah sponge was still very high ( Figure 6 ).

[0022] Assessment of the impact on the photosynthetic system: Samples were collected regularly to determine the impact of the combined action of loofah sponge and bacteria on the photosynthetic system of *Microcystis aeruginosa*, including the content of chlorophyll a ( Figure 7 ) and the change in the maximum photosynthetic efficiency Fv / Fm ( Figure 8 ). Under the combined action of loofah sponge and bacteria, the chlorophyll of cells decreased sharply, the photosynthetic efficiency decreased severely, and the photosynthetic system of algal cells was severely damaged.

[0023] Chlorophyll degradation kinetics: The chlorophyll degradation process under the combined action conforms to the first-order kinetic model and can accurately predict the algicidal effect.

[0024] Assessment of environmental impact: The additional introduced nutrients have limited impact on the water body nutrient concentration.

[0025] Algae-killing sustainability: The combination after immobilizing Kocuria rosea with loofah sponge can still maintain a high algae-killing effect after repeated use.

[0026] Algae-killing mechanism: It destroys the photosynthetic system of Microcystis aeruginosa, the cell structure is damaged, the cell membrane is damaged and cell lysis occurs, ultimately achieving the killing of algae. As can be seen from Figure 9 , the cell structure of normal Microcystis aeruginosa is complete and the morphology is intact. Figure 10 In , the algal cells after being treated with the culture solution of Kocuria rosea immobilized by loofah for 24 hours are severely deformed and the structural integrity is lost. Figure 11 In , the algal cells after being treated with the culture solution of Kocuria rosea immobilized by loofah for 96 hours are completely decomposed into fragments.

[0027] The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. For those skilled in the art, the present invention can have various modifications and changes. 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. Use of *Kocuria rhizophila* Kocuria rosea in controlling harmful algae in fresh water.

2. Application of Luffa cylindrica sponge combined with Kocuria rosea in controlling harmful algae in fresh water.

3. An algicidal bactericide, characterized in that, It contains Kocuria rosea.

4. The algicidal microbial agent according to claim 3, wherein Fix Kocuria rosea with Luffa cylindrica sponge. Culture the Kocuria rosea bacterial solution in 1,000 ml of LB medium until OD600 = 1.5, collect the bacterial cells by centrifugation at 18,000 g for 20 minutes, wash twice with Tris buffer at pH = 8, and then suspend in 1,000 ml of fresh Tris buffer; cut the Luffa cylindrica sponge into uniform small pieces, add them to the above Tris buffer containing Kocuria rosea cells after repeated ultraviolet sterilization; place the culture flask in the dark, culture on a shaker at 100 rpm at 25 °C for 6 hours, then continue to culture for 8 hours under the same conditions, and let it stand to finally obtain the combined algicidal agent of Luffa cylindrica sponge immobilized Kocuria rosea.

5. A method for controlling harmful algae in fresh water by using sponge gourd pulp in combination with Kocuria rhizophila, which is characterized in that, It includes the following steps: adding the Kocuria rosea described in claim 1 or the algicidal agent described in claim 3 into the algae culture solution to kill Microcystis aeruginosa.

6. A method for controlling harmful algae in fresh water by using loofah sponge combined with Roseateles depolymerans according to claim 5, characterized in that, The concentration of Kocuria rosea in the algae culture solution is 1 - 5%.

7. A method for controlling harmful algae in fresh water by using loofah sponge combined with Roseateles depolymerans according to claim 6, characterized in that, The concentration of Kocuria rosea in the algae culture solution is 3%.

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