KR102489018B1 - A Paucibacter sp. strain having algicidal activity against Microcystis aeruginosa and uses thereof - Google Patents

Abstract

The present invention relates to a composition for killing microalgae, comprising a Paucibacter sp. DH15 strain, a culture medium or a culture filtrate thereof as an active ingredient; It relates to a method for controlling algae, comprising the step of treating the algae composition in areas where freshwater algae are likely to occur or occur.
The composition for algae algae of the present invention inhibits the growth of microalgae that cause green algae and has the effect of decomposing and killing them, and is particularly useful for controlling algae through its selective algal activity against Microcystis aeruginosa. can be used

Description

Paucibacter genus strain having algicidal activity against Microcystis aeruginosa and its use {A Paucibacter sp. strain having algicidal activity against Microcystis aeruginosa and uses its}

The present invention relates to a composition for killing microalgae, comprising a Paucibacter sp. DH15 strain, a culture medium or a culture filtrate thereof as an active ingredient; It relates to a method for controlling algae, comprising the step of treating the algae composition in areas where freshwater algae are likely to occur or occur.

When contaminated wastewater that exceeds the natural purification capacity is introduced into a river or lake, nutrients are accumulated in the water and the bottom, resulting in eutrophication of the water system, which causes algal bloom or water bloom. Therefore, dissolved oxygen in the water decreases due to sunlight blocking on the surface and restriction of oxygen delivery, and the aquatic ecosystem may be destroyed.

In particular, in artificial lakes in Korea, microcystis aeruginosa and Anabaena Flos-aquae, which are the dominant species of blue-green algae, occur frequently as the cause of algal bloom, and toxic substances are produced in the process. This can lead to public health and hygiene problems. For example, some blue-green algae among algae produce toxic substances that can damage the liver or nervous system. For example, in Pennsylvania, USA in 1979, about 60 people after swimming in a lake where blue-green algae occurred suffered from headaches, abdominal pain, vomiting, diarrhea, Symptoms such as eye inflammation, rashes, and ear pain are known. In addition, these blue-green algae have caused direct damage to all animals or livestock including phytoplankton, zooplankton, protozoa and fish.

In addition, it may cause a decrease in the function of the process during water treatment, resulting in water use failure. For example, it causes problems such as interfering with coagulation and filtration, which are key processes in water purification, and thus increases the cost of water purification. In addition, Geosmin (recommended standard: 20 ppt), which is produced during the metabolic process of blue-green algae, causes bad odor in tap water and, in severe cases, causes economic losses by lowering the waterfront value of rivers and lakes (Y.-H. Kang, J.-D.Kim, B.-H.Kim, D.-S.Kong, M.-S.Han (2005), Isolation and characterization of a bio-agent antagonistic to diatom, Stephanodiscus hantzschii , Journal of Applied Microbiology , 98, 10301038, 2005). Above all, the stagnant water system at a high temperature facilitates the occurrence of algae due to eutrophication, and as a result, the algal bloomed water covered with algae has a negative aesthetic effect on the people, and complexes such as culture, art, tourism, and leisure It can make activation as a space impossible. Also, from an ecological point of view, the occurrence of algae has a problem in that the river itself loses its self-regulating ability to improve water quality and restore the health of the aquatic ecosystem.

Currently, various physical, chemical, and biological methods are being developed to control algae (especially blue-green algae, etc.), which are the cause of algae, but they are not effective, and practical technology that can effectively control algae while being environmentally friendly, In particular, there is a demand for development of a nature-friendly control technology for algal blooms generated in water using algal bacteria or algicidal bacteria, and research on this is being conducted. For example, Korean Registered Patent KR 10-1311837 B1 discloses a composition for controlling harmful algae using algae bacteria and has a killing activity of strains of the genus Lactobacillus or the genus Lactococcus .

Under this background, the present inventors have made diligent efforts to develop a more effective composition for algae algae, and as a result, the DH15 strain of the genus Fauscibacter has the ability to kill microalgae that cause algae, especially Microcystis eruge, which is a causative species of freshwater algae. The present invention was completed by confirming that the selective algicidal activity for nosa was excellent.

One object of the present invention is to provide a composition for killing microalgae comprising a Paucibacter sp. DH15 strain, a culture medium or a culture filtrate thereof as an active ingredient.

Another object of the present invention relates to a method for controlling algae, comprising the step of treating an area where freshwater algae occurs or is likely to occur with the composition.

Another object of the present invention relates to a Paucibacter sp. DH15 strain deposited under Accession No. KCTC18831P, which has an algicidal activity against Microcystis aeruginosa.

A detailed description of this is as follows. Meanwhile, each description and embodiment disclosed in the present invention may also be applied to each other description and embodiment. That is, all combinations of the various elements disclosed herein fall within the scope of the present invention. In addition, it cannot be seen that the scope of the present invention is limited by the specific descriptions described below.

One aspect of the present invention for achieving the above object provides a composition for killing microalgae comprising a Paucibacter sp. DH15 strain, a culture medium or a culture filtrate thereof as an active ingredient.

The term "algicidal composition" as used herein refers to inhibiting or killing the growth of algae, and the algicidal composition of the present invention includes DH15 strain of the genus Fauscibacter, its culture medium or culture filtrate as an active ingredient, It may mean inhibiting the growth of microalgae or directly decomposing and killing microalgae, but is not limited thereto.

The term of the present invention, " Paucibacter sp. DH15 strain" is a new strain having an algicidal effect on microalgae, which is the cause of green algae, and is a 10-fold diluted R2A medium from environmental samples obtained near Daecheong Lake in Korea. Smeared and isolated through incubation at 25 ° C for 24-48 hours, it may mean a strain deposited with accession number KCTC18831P at the Korea Research Institute of Bioscience and Biotechnology as of July 7, 2020.

Unlike previously reported algicidal fungi, the DH15 strain of the genus Fauscibacter has an algicidal activity by secondary metabolites produced by the fungicide, the DH15 strain of the genus Faucibacter also has an excellent algicidal effect when directly treated with it. It has a high possibility of being used for algae control.

In a specific embodiment of the present invention, the algicidal activity of the microalga Microcystis aeruginosa, which is a green source, was confirmed by treating the DH15 strain of the genus Faucibacter, its culture medium and culture filtrate.

In another specific embodiment of the present invention, as a result of culturing the DH15 strain of the genus Fauscibacter with microalgae and then observing it under a microscope, it was confirmed that the DH15 strain directly attached to the microalgal cell membrane, and at the same time, the microalgal cell membrane It was confirmed that it was partially decomposed, and it was confirmed that it had the ability to inhibit the growth of microalgae and decompose them.

In addition, in another specific embodiment of the present invention, the selective algicidal activity of the DH15 strain of the genus Fausibacter against Microcystis aeruginosa was confirmed through comparison of the algicidal activity with other microalgae.

The term "microalgae" of the present invention, in a broad range, means collectively unicellular organisms capable of performing photosynthesis including photosynthetic pigments, and in a narrow range, it lives in an aquatic environment, performs photosynthesis, and as a single cell It refers to organisms that exist or can form single-celled aggregates, which are classified into freshwater algae that live in freshwater and marine algae that live in seawater, depending on the environment in which they live. For the purpose of the present invention, the microalgae may be blue-green algae or green algae. The blue-green algae are specifically Microcystis sp., Anabaena sp., Aphanizomenon sp., Oscillatoria sp., or Woronichinia sp. etc., specifically, it may be algae of the genus Microcystis.

In addition, the green algae may be microalgae of the genus Ettlia sp., the genus Chlamydomonas sp., or the genus Haematococcus sp.

The term of the present invention, " Microcystis aeruginosa " is a representative species of the genus Microcystis, which is a blue-green algae, and lives in fresh water such as rivers, lakes, and ponds, and the occurrence of green algae or cyanobacteria due to eutrophication ( It is the most representative photosynthetic bacterium that causes cyanobacterial bloom.

Microcystis aeruginosa produces microcystin, which is known as a toxin that causes liver disease, and hundreds to thousands of cells gather to form colonies and grow while floating on the water surface. Microcystis causes an unpleasant odor, and microcystin, a toxic substance, is leached into water and accumulated in aquatic organisms, causing serious ecological and environmental problems.

As used herein, the term "culture medium" refers to a medium in which the newly isolated DH15 strain of the genus Fauscibacter is cultured, and preferably is a culture medium containing the strain, and may be a concept including a concentrate or dried product thereof. there is.

In the present invention, the culture medium means a medium containing nutrients necessary for growing animal cells, plant cells, or bacteria, and the culture medium means a culture medium inoculated with a strain.

The concentrated solution of the culture medium refers to concentrated the culture medium, and the dry matter of the culture medium means that the culture medium is dried. Drying methods include air drying, natural drying, spray drying and freeze drying, but are not limited thereto.

As used herein, the term “culture filtrate” may refer to a culture medium in which the newly isolated DH15 strain of the genus Fauscibacter is inoculated into a culture medium and then eradicated, that is, the strain is removed.

In a specific embodiment of the present invention, after culturing the DH15 strain of the genus Fauscibacter in a culture medium, the culture medium was centrifuged, and the supernatant was separated using a filter to separate only the filtrate for use.

In a specific embodiment of the present invention, the algicidal activity of the microalgae Microcystis aeruginosa, which is a green source, was confirmed by treating not only the novel strain Pausibacter genus DH15 strain, but also its culture medium and culture filtrate.

The term "cultivation" of the present invention refers to a series of activities for growing microorganisms under appropriately artificially controlled environmental conditions. In the present invention, the method of culturing the DH15 strain of the genus Fausibacter can be performed using a method widely known in the art.

The medium used for culture must meet the requirements of a specific strain in an appropriate manner while controlling temperature, pH, etc. under aerobic conditions in a conventional medium containing appropriate carbon sources, nitrogen sources, amino acids, vitamins, etc. As a carbon source that can be used, a mixed sugar of glucose and xylose is used as the main carbon source, and in addition to sugars and carbohydrates such as sucrose, lactose, fructose, maltose, starch, and cellulose, soybean oil, sunflower oil, castor oil, and coconut Oils and fats such as oil, fatty acids such as palmitic acid, stearic acid and linoleic acid, alcohols such as glycerol and ethanol, and organic acids such as acetic acid. These materials may be used individually or as a mixture. Nitrogen sources that can be used include inorganic nitrogen sources such as ammonia, ammonium sulfate, ammonium chloride, ammonium acetate, ammonium phosphate, ammonium carbonate, and ammonium nitrate; Amino acids such as glutamic acid, methionine, and glutamine, and organic nitrogen sources such as peptone, NZ-amine, meat extract, yeast extract, malt extract, corn steep liquor, casein hydrolysate, fish or decomposition products thereof, defatted soybean cake or decomposition products thereof, may be used. can These nitrogen sources may be used alone or in combination. The medium may contain monopotassium phosphate, dipotassium phosphate and corresponding sodium-containing salts as phosphorus. Persons that may be used include potassium dihydrogen phosphate or dipotassium hydrogen phosphate or the corresponding sodium-containing salts. In addition, as the inorganic compound, sodium chloride, calcium chloride, iron chloride, magnesium sulfate, iron sulfate, manganese sulfate, and calcium carbonate may be used. Finally, essential growth substances such as amino acids and vitamins may be used in addition to the above substances.

The method of culturing the DH15 strain of the genus Faucibacter is not particularly limited, but may be performed by a known batch culture method, continuous culture method, fed-batch culture method, or the like. At this time, the culture conditions are not particularly limited thereto, but a suitable pH (eg, pH 5 to 9, specifically, using a basic compound (eg, sodium hydroxide, potassium hydroxide, or ammonia) or an acidic compound (eg, phosphoric acid or sulfuric acid) can adjust pH 6 to 8, most specifically pH 6.8).

In addition, antifoaming agents such as fatty acid polyglycol esters may be used to suppress foam formation. Oxygen or an oxygen-containing gas (eg, air) may be injected into the culture to maintain aerobic conditions.

In addition, the culture temperature may be 20 ° C to 37 ° C, preferably 24 ° C to 26 ° C, more preferably 25 ° C, and the culture time may be 10 hours to 60 hours, preferably 24 hours to 48 hours. It may be time, the culture pH may be between 6 and 8, preferably 7, but is not particularly limited thereto.

The composition for killing microalgae of the present invention, as an active ingredient, is a DH15 strain of the genus Fauscibacter, stable expression of the effect within the range that does not impair the effect of its culture medium or culture filtrate, enhancement of attachment to the organism to be applied, transport and treatment For the sake of convenience, a pharmaceutically acceptable solid carrier, liquid carrier, liquid diluent, liquefied gas diluent, solid diluent, or other suitable auxiliary agent, for example, surfactant such as an emulsifier, dispersant or foaming agent may be further included.

In addition, the algicidal composition may be preferably formulated into emulsions, wettable powders, granules, powders, capsules, and gel formulations, and may be provided as a contact agent through a donut-type formulation for buoyancy of the formulation, It is not limited to this.

Another aspect of the present invention for achieving the above object provides a method for controlling algae, comprising the step of treating an area where freshwater algae occurs or is concerned about the occurrence of algae with the composition for algae.

The term "algae control" as used herein refers to suppressing the growth of microalgae causing algae, killing them, or suppressing the motility of microalgae to prevent the growth of algae and to eliminate the phenomenon that causes algae. In addition, it may also include the meaning of killing or suppressing the growth of algae that cause algae more broadly. In the present invention, it may be used in the sense of killing or suppressing the growth of Microcystis aeruginosa, which is a causative algae of green algae, but is not limited thereto.

The method for controlling algae is to treat areas where freshwater algae are likely to occur or occur by methods such as spraying an algicidal composition containing the DH15 strain of the genus Fauscibacter, its culture medium or culture filtrate, or spraying after immobilizing the composition on a porous floating carrier. However, it is not limited thereto and may be variously made by methods used in the art.

In addition, the method for controlling algae is a DH15 strain of the genus Fauscibacter that is suitable for the control of microalgae, especially Microcystis aeruginosa, which is the cause of green algae, and its culture solution or culture filtrate is used for the type of microalgae, the type of bacteria, etc. Accordingly, those skilled in the art may be appropriately selected and used at an inoculation concentration with excellent control effect, but preferably, the optimal treatment concentration is 1% or more, specifically 1 to 20%, 1 to 1% when the optical density of the culture medium is 0.3. Inoculation at a concentration of 10, 1 to 5% may exhibit a high algicidal effect, but is not limited thereto.

Another aspect of the present invention for achieving the above object provides a Paucibacter sp. DH15 strain deposited with accession number KCTC18831P, which has a salicidal activity against Microcystis aeruginosa.

The term of the present invention, Microcystis aeruginosa, Fauscibacter genus DH15 strain is as described above.

The composition for algae algae of the present invention inhibits the growth of microalgae that cause green algae and has the effect of decomposing and killing them, and is particularly useful for controlling algae through its selective algal activity against Microcystis aeruginosa. can be used

Figure 1 is a result of comparing the algicidal effect on Microcystis aeruginosa for 48 hours after the addition of the culture solution of the DH15 strain of the genus Fauscibacter, (A) relates to the algicidal activity by concentration and treatment time of the strain, (B) is a comparison photograph by concentration of the treated strain after 48 hours of treatment.
Figure 2 relates to the change in photosynthetic efficiency of Microcystis aeruginosa for 48 hours after the addition of the culture solution of the DH15 strain of the genus Fausibacter, (A) relates to active chlorophyll a, (B) relates to Fv / Fm.
Figure 3 is an electron microscope result of Microcystis aeruginosa, (A) relates to a control group, (B) relates to a treatment group.
Figure 4 is a result of comparing the algicidal effects of the DH15 strain of the genus Fauscibacter, its culture medium and culture filtrate. (A) is a comparative photograph of the algicidal effect on Microcystis aeruginosa, and (B) is a comparison of the algicidal activity against Microcystis aeruginosa for each treatment method.
Figure 5 is a result of comparing the algicidal activity of each microalgae of the DH15 strain of the genus Fausibacter.

Hereinafter, the present invention will be described in more detail through examples. However, these examples are intended to illustrate the present invention by way of example, and the scope of the present invention is not limited to these examples.

Example 1. Isolation and identification of fungicidal strains

In order to isolate various strains in the vicinity of Daecheong Lake in Korea, they were plated on 10-fold diluted R2A medium and cultured at 25 ° C for 24-48 hours. In order to isolate pure microorganisms, passages were performed three or four times. Microorganisms with excellent killing activity were finally selected from the pure separated microorganisms, and the selected strain was named “ Paucibacter sp. It was deposited with the Biological Resource Center (Korean Collection for Type Culture, hereinafter abbreviated as "KCTC") under accession number KCTC18831P.

Example 2. Confirmation of the killing activity of the isolated strain

2-1. Microcystis aeruginosa ( Microcystis aeruginosa )

In order to examine the killing activity of the isolated DH15 strain of the genus Fauscibacter of the present invention against Microcystis aeruginosa, the concentration of the strain pre-cultured in R2A medium was adjusted to OD 600 nm 0.3. Diluted culture medium was inoculated with 0.1, 1, 2, 5% (v / v) microalgae culture medium. At this time, the cell number of the Microcystis aeruginosa culture was used at a concentration of 2Х10 ⁶ cells / ml, and the algicidal effect was confirmed at 12-hour intervals for 48 hours (Fig. 1).

As a result of confirming the above results using a microscopic counting method, when 1% or more of the culture medium was treated after 24 hours, an algicidal effect of 45% or more was shown. After 36 hours, all treatments showed a high algicidal effect of 50% or more, and 1% or more culture medium treatment showed a very high algicidal effect of 95% or more (FIG. 1A).

In addition, as a result of examining the culture flask after 48 hours, it was confirmed that most of the Microcystis aeruginosa cells died in the culture solution treatment group of 1% or more, and the color of the culture solution all changed to yellow (FIG. 1B).

In addition, as a result of confirming the algicidal effect by measuring the photosynthetic efficiency under each treatment condition with a photosynthetic measuring device PHYTO-PAM, the active chlorophyll a concentration and Fv/Fm (indicator of photosynthetic activity) values rapidly decreased after 12 hours of cultivation. It was confirmed that it was, and after 48 hours, it was confirmed that there was a high algicidal effect of 80% or more (FIG. 2).

2-2. In the Fauscibacter ( Paucibacter sp. ) Microscopic results of Microcystis aeruginosa degraded by strain DH15

In order to confirm the direct algicidal effect of the DH15 strain of the genus Fauscibacter on Microcystis aeruginosa, the DH15 strain of the genus Fauscibacter was pre-cultured in R2A medium, then diluted to an O.D. value of 0.3 and inoculated with 1%, After 36 hours, the culture medium was examined using an electron microscope.

As a result, in the case of the control group, the surface of Microcystis aeruginosa was well maintained (Fig. 3A), but in contrast, in the treatment group, the DH15 strain, an algicidal microorganism, was directly attached to the surface of Microcystis aeruginosa. It was confirmed that , it was confirmed that the cell membrane surface was peeled off in the middle as if dissolved (Fig. 3B).

As a result, it was confirmed that the DH15 strain of the genus Pausibacter of the present invention exerts an algicidal effect by directly attaching to and degrading the cell membrane of microalgae.

Example 3. Comparison of algicidal effects of the DH15 strain of the genus Fauscibacter and its culture filtrate

In order to confirm the direct or indirect algicidal effect of the DH15 strain of the genus Fauscibacter isolated in Example 1, only the culture medium, the culture filtrate, and the DH15 strain of the genus Fausibacter were treated and the respective algicidal effects were compared (FIG. 4).

After culturing the DH15 strain of the genus Fauscibacter in R2A medium for 24 hours, the supernatant and the pellet were separated from the culture medium through a centrifuge. For the treatment of the supernatant, only the filtrate was separated using a 0.22 μm filter. The obtained pellet was used after washing three times using sterilized 0.85% NaCl. After adding 1% (v / v) of the pre-treated culture medium to the culture medium of Microcystis aeruginosa, the algicidal effect was confirmed after 36 hours.

As a result, it was confirmed that all of the culture filtrate, the culture medium, and the DH15 strain of the genus Fausibacter had an algicidal effect against Microcystis aeruginosa.

In addition, when the culture filtrate was treated, the algicidal activity was about 30%, whereas the culture medium of the strain was 89%, and the case where only the DH15 strain of the genus Fauscibacter was treated was 78%. Higher algicidal activity was confirmed (FIG. 4B).

Example 4. Confirmation of selective algicidal activity against Microcystis aeruginosa of DH15 strain of the genus Faucibacter

In order to confirm the selective algicidal activity of the DH15 strain of the genus Faucibacter against Microcystis aeruginosa, the algicidal activity against other microalgal species was confirmed and compared.

Microalgae Etlia sp. YC001, Chlamydomonas reinhardtii CC-124, Haematococcus lacustris LIMS-PS-1464, Anabaena sp. .) AG10818, Anabaena sp. U13 was used for comparative experiments.

The DH15 strain of the genus Pausibacter pre-cultured in R2A medium was mixed with the microalgae culture medium and cultured for 36 hours. Microcystis aeruginosa showed high algicidal activity of more than 90%, but most green algae and cyanobacteria showed 15 % or less showed very low algicidal activity (FIG. 5).

That is, it was confirmed that the DH15 strain of the genus Pausibacter had a very high selective algicidal activity against Microcystis aeruginosa, a major generating species of green algae.

From the above description, those skilled in the art to which the present invention pertains will be able to understand that the present invention may be embodied in other specific forms without changing its technical spirit or essential features. In this regard, it should be understood that the embodiments described above are illustrative in all respects and not limiting. The scope of the present invention should be construed as including all changes or modifications derived from the meaning and scope of the claims to be described later and equivalent concepts rather than the detailed description above are included in the scope of the present invention.

Korea Research Institute of Bioscience and Biotechnology Biological Resources Center KCTC18831P 20200707

Claims (8)

A composition for killing microalgae, comprising Paucibacter sp. DH15 strain deposited under accession number KCTC18831P, its culture medium or culture filtrate as an active ingredient,
The microalgae is Microcystis aeruginosa ( Microcystis aeruginosa ), a composition for killing microalgae.
delete delete The composition for killing microalgae according to claim 1, wherein the composition has selective algicidal activity against Microcystis aeruginosa.
The composition for killing microalgae according to claim 1, wherein the composition has an ability to inhibit or decompose the growth of microalgae.
An algae control method comprising the step of treating an area where freshwater algae is generated or concerned about occurrence of the algae composition according to any one of claims 1 and 4 to 5,
The algae control method, wherein the algal bloom is caused by Microcystis aeruginosa.
delete Paucibacter sp. DH15 strain deposited with accession number KCTC18831P, which has algicidal activity against Microcystis aeruginosa.

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