Arthrobacter sp. having algicidal activity against harmful algae and uses thereof

Abstract

The present invention relates to a strain of the genus Arthrobactersp. having algicidal activity against harmful algae and its uses, and more specifically, to a strain of the genus Arthrobactersp. HNIBRBA5316 (accession number KCTC15442BP) having algicidal activity against harmful algae and its uses. Since the strain of the genus Arthrobactersp. HNIBRBA5316 (accession number KCTC15442BP) according to the present invention has selective algicidal activity against harmful cyanobacteria, the strain of the genus Arthrobactersp. HNIBRBA5316, a composition for controlling harmful algae containing the same, and a method for controlling harmful algae comprising treating an area where harmful algae occur or an area requiring harmful algae control with said composition can be usefully utilized as an environmentally friendly composition and / or method for removing algal blooms.

Description

Technology Field

[0001] The present invention relates to an algaecidal genus (Arthrobacter) having algicidal activity against harmful algae Arthrobacter The invention relates to a strain of the genus Arthrobacter (sp.) and its uses, and more specifically, to a strain of the genus Arthrobacter HNIBRBA5316 (accession number KCTC15442BP) having algicidal activity against harmful algae and its uses. Background Technology

[0003] When eutrophication occurs due to the accumulation of nutrients in the water and on the bottom caused by reasons such as the natural dissolution of organic matter from the soil into rivers or lakes or the unauthorized discharge of wastewater, algae present in the water, especially cyanobacteria ( Cyanobacteria ) increases explosively, causing an algal bloom or water bloom where the water turns green.

[0004] Serious problems arise in rivers where algal blooms occur. First, as the algae proliferate significantly and cover the river surface, they block sunlight underwater, severely hindering the photosynthesis of aquatic plants. Consequently, the supply of dissolved oxygen necessary for the survival of the river ecosystem decreases rapidly, leading to the collapse of the ecosystem. In addition, Microcystis aruginosa, known as the dominant cyanobacteria species in Korea ( Microcystis aeruginosa ) produces a substance called microcystin, a hepatotoxic substance with a median lethal dose of 50 μg / kg, and in addition, nodularia ( Nodularia ) genus, Anabaena( Anabaena ) genus, aphanizomenon( AphanizomenonAlgae of the genus *Cyanobacteria* are known to produce nodularin, anatoxin, and saxitoxin, respectively, which are toxins harmful to humans and animals (Hitzfeld, BC et al. "Cyanobacterial toxins: removal during drinking water treatment, and human risk assessment." Environmental health perspectives vol. 108 Suppl 1, Suppl 1 (2000): 113-22.), and in fact, damage to humans and livestock caused by these toxic substances has been reported in the United States and Japan. In particular, in temperate regions like Korea, aquatic ecosystems create an environment favorable for the growth of primary producers such as phytoplankton based on abundant nutrients, leading to the recurring occurrence of algal blooms every year.

[0005] Accordingly, various methods have been developed to suppress or prevent such algal blooms. Known technologies to date include chemical spraying, ultrasonic and ozone treatment, surface recovery and sedimentation, and loess application. However, these physicochemical methods lack specificity for the harmful algae that cause algal blooms, raising concerns about adverse effects on organisms other than the harmful algae. Furthermore, they present practical problems such as requiring significant labor and cost. Therefore, there is currently a demand for the development of methods utilizing natural enemies or algaecid bacteria with excellent algae control capabilities. Prior art literature

[0007] Republic of Korea Published Patent No. 10-2015-0117594 Republic of Korea Published Patent No. 10-2010-0134343 The problem to be solved

[0008] Accordingly, the inventors have made diligent efforts to develop a composition for controlling harmful algae capable of suppressing the generation and proliferation of harmful algae that cause algal blooms, and as a result, a newly discovered genus of Arthrobacter ( Arthrobacter It was confirmed that the strain sp.) HNIBRBA5316 possesses algicidal activity specifically against cyanobacteria, and the present invention was completed.

[0010] Therefore, the objective of the present invention is to provide a genus of Arthrobacter ( Arthrobacter It is to provide the strain sp.) HNIBRBA5316 (accession number KCTC15442BP).

[0012] Another objective of the present invention is the genus Artrobacter ( Arthrobacter The present invention provides a composition for controlling harmful algae comprising the strain HNIBRBA5316 (accession number KCTC15442BP) or a culture medium thereof as an active ingredient.

[0014] Another objective of the present invention is to provide a method for controlling harmful algae, comprising applying the above-mentioned composition for controlling harmful algae to an area where harmful algae occur or to an area where harmful algae control is required.

[0016] Another objective of the present invention is the genus Arthrobacter ( Arthrobacter The present invention provides an algaecide comprising the strain sp.) HNIBRBA5316 (accession number KCTC15442BP) or its culture medium as an active ingredient. means of solving the problem

[0018] To achieve the above objectives, the present invention relates to a genus of Arthrobacter (characterized by having algicidal activity against harmful algae) Arthrobacter We provide the strain HNIBRBA5316 (accession number KCTC15442BP).

[0020] To achieve another objective of the present invention, the present invention relates to the genus Artrobacter ( ArthrobacterA composition for controlling harmful algae is provided, comprising the strain HNIBRBA5316 (accession number KCTC15442BP) or a culture medium thereof as an active ingredient.

[0022] To achieve another objective of the present invention, the present invention provides a method for controlling harmful algae comprising treating the above-mentioned composition for controlling harmful algae in an area where harmful algae occur or in an area where harmful algae control is required.

[0024] To achieve another objective of the present invention, the present invention relates to the genus Arthrobacter ( Arthrobacter The present invention provides an algaecide comprising the strain sp.) HNIBRBA5316 (accession number KCTC15442BP) or its culture medium as an active ingredient.

[0026] The present invention will be described in detail below.

[0028] The present invention is characterized by having algicidal activity against harmful algae, in the genus Arthrobacter ( Arthrobacter We provide the strain HNIBRBA5316 (accession number KCTC15442BP).

[0030] The inventors discovered the occurrence of an algicidal ring within a cyanobacteria culture medium isolated from a soil sample from Wando-gun, Jeollanam-do. After isolating 25 types of bacteria from the affected area and verifying their algicidal activity, one novel bacterium with excellent algicidal activity was selected and isolated. Upon confirming the 16S sequence (785F / 907R) of the isolated strain, it was found to be of the genus Arthrobacter ( Arthrobacter It was confirmed that this strain belongs to the genus sp., and the strain was named HNIBRBA5316 and deposited at the National Institute of Biotechnology and Bioengineering (NIBB) Biological Resource Center on May 10, 2023 (accession number KCTC15442BP).

[0032] According to one embodiment of the present invention, the genus Artrobacter ( ArthrobacterThe sp.) HNIBRBA5316 strain is one of two strains of the harmful cyanobacteria genus Anabaena ( Anabaena sp. HNIBRCY11, Anabaena sp. AG10663) and two strains of the genus Microcystis ( Microcystis sp. KW, Microcystis It was confirmed that it has algicidal activity against the microorganism sp. AG60752, and in particular against strains of the genus Green Algae that are not harmful cyanobacteria ( Chlorella In the case of sp. JD1-1), chlorophyll actually increased, confirming that the above HNIBRBA5316 strain exhibits selective algicidal activity against harmful cyanobacteria.

[0033] In the present invention, the term "harmful algae" refers to algae that cause green algal blooms in rivers or lakes. The harmful algae refers to algae that cause destruction of the aquatic ecosystem through the release of harmful toxins and a decrease in dissolved oxygen levels, thereby causing adverse effects such as the death of livestock, wild animals, fish, etc., as well as visual discomfort, foul odors, and shortages of agricultural / industrial water and drinking water. The harmful algae may be cyanobacteria, diatoms, green algae, Euglenobacteria, dinoflagellates, yellow flagellates, brown flagellates, or red algae, although they are not limited thereto; more preferably, cyanobacteria ( Cyanophyceae Microcystis belonging to ) Microcystis ), Anabena( Anabaena ), Aphanijonmenon( Aphanizomenon ), Oscillatoria( Oscillatoria ) and Waronikinia( Woronichinia It may be algae of the genus ) and most preferably may be algae of the genus Microcystis or Anabaena.

[0035] genus Artrobacter according to the present invention ( ArthrobacterThe strain HNIBRBA5316 (sp.) coexists with harmful algae that cause green algae blooms and possesses selective algicidal activity against cyanobacteria; therefore, it has the advantage of being an environmentally friendly strain for controlling harmful algae that does not have an adverse effect on the aquatic ecosystem. Accordingly, the present invention relates to the genus Arthrobacter ( Arthrobacter A composition for controlling harmful algae and an algaecide are provided, comprising the strain of sp.) HNIBRBA5316 (accession number KCTC15442BP) or a culture medium thereof as an active ingredient.

[0037] All compositions and algaecides according to the present invention are of the genus Arthrobacter (according to the present invention) Arthrobacter Contains the strain sp.) HNIBRBA5316 (accession number KCTC15442BP) or its culture medium as an active ingredient, and 1×10 4 Up to 1×10 12 Inoculation concentration of cells / ml, preferably 1×10 6 Up to 1×10 10 Inoculation concentration of cells / ml, more preferably 1×10 7 Up to 1×10 9 Inoculation concentration of cells / ml, most preferably 4.6×10 8 It is preferable to include it at an inoculation concentration of cells / ml, but it is not limited thereto, and it is obvious that a person skilled in the art can select and use the appropriate inoculation concentration depending on the type of algae to be killed, the type of bacteria, and the degree of the algal bloom that has already occurred.

[0039] The composition and algaecide for controlling harmful algae according to the present invention may be prepared in various forms according to known methods and may further include pharmaceutically acceptable solid carriers, liquid carriers, liquid diluents, liquefied gaseous diluents, solid diluents, or various suitable auxiliary agents, such as surfactants, emulsifiers, dispersants, and foaming agents, to ensure stable expression of effect, enhanced attachment to target organisms, and simplified transport and processing within a range that does not impair the effect.

[0040] The composition and algaecide for controlling harmful algae according to the present invention may be formulated into emulsions, wettable powders, granules, powders, capsules, and gels, and it is preferable to provide them as contact agents for the buoyancy of the formulations. For example, the composition may be easily utilized by long-term storage or commercialization in powder form or in a high-concentration liquid form.

[0042] In addition, the present invention provides a method for controlling harmful algae, comprising treating the above-mentioned composition for controlling harmful algae in an area where harmful algae occur or in an area where harmful algae control is required.

[0044] In the present invention, the term "area where harmful algae occur or area requiring control of harmful algae" refers to an area where harmful algae have rapidly increased in rivers or lakes and algal blooms are already widespread, or an area where harmful algae are gradually increasing and algal blooms are beginning, and / or an area where algal blooms have not yet occurred but have frequently occurred in the past, or an area used for agricultural or industrial water where there is a need to prevent algal blooms, but is not limited thereto.

[0045] In the present invention, the term 'treatment' is not limited thereto, but refers to the genus Arthrobacter (according to the present invention) Arthrobactersp.) HNIBRBA5316 strain, a composition for controlling harmful algae comprising said strain, or an algaecide comprising said strain may mean spraying in an area where harmful algae occur or in an area where harmful algae control is required. The above 'spraying' preferably means spraying in an area where green algae have occurred using a vessel, but is not limited thereto, and the genus Arthrobacter according to the present invention ( Arthrobacter It may involve spraying live or dead strains of the HNIBRBA5316 strain, a composition containing said strain, and / or an algaecide, spraying an algaecidal substance derived from said strain, or spraying said strain by immobilizing it on a porous floating carrier. The phrase "spraying by immobilizing on a porous floating carrier" may mean adsorbing / immobilizing the strain according to the present invention onto various commercial carriers, including water-based floating foam sponges, and then administering it to an area where an algal bloom has occurred.

[0047] In the case of useful bacteria such as the strain developed by the inventors, they are utilized in various fields (wastewater treatment starter cultures, microbial decomposers, oil treatment agents, microbial odor removers, food waste treatment agents, etc.) as environmental clean technologies using biological control methods, and many products have been commercialized domestically and internationally. Therefore, the genus Arthrobacter ( ArthrobacterThe HNIBRBA5316 strain offers a wide range of commercial applications, including not only live bacteria but also strain-derived algaecides and carriers for live bacteria or algaecides. For instance, as a technology applicable to the restoration and conservation management of medium- and small-scale water systems (algal bloom control or prevention), it can be applied to ponds, reservoirs, lakes, and rivers within artificial fish farms, golf courses, parks, amusement parks, and recreational facilities, as well as agricultural reservoirs scattered throughout the country. By preventing algal bloom damage and controlling harmful algae, it avoids other forms of artificial environmental pollution. Furthermore, it can be used as a key component in antifouling paints for ships, dock facilities, swimming pools, and buildings, thereby replacing toxic conventional antifouling paints and preventing aesthetic damage, reduced durability, and performance degradation caused by attached algae. In addition, it can be applied as a pretreatment technology for water treatment facilities (water supply treatment, sewage treatment) to secure drinking water, agricultural water, and industrial water, thereby preventing filter clogging and excessive exposure to toxic substances, which can reduce the economic burden and ensure the safety of drinking water.

[0048] Furthermore, this technology can be applied to the improvement of water quality in relatively large-scale water systems, such as lakes (dams), water sources, and sewage systems, as well as to the development of environmental technologies. Consequently, improvements in agricultural water quality will enable the production of high-quality agricultural products, and the stabilization of drinking water will contribute to public health, thereby demonstrating tangible benefits. Additionally, through the export and technology transfer of environmental technologies to countries facing severe algal bloom problems, such as China, or those with high demand for antifouling coatings against attached algae, such as Thailand, this technology can contribute to creating high added value, enhancing the national image, and strengthening national competitiveness. Finally, depending on technological advancements, it can also be actively utilized for controlling red tides occurring in coastal areas.

[0049] As such, the bio-control technology using algaecidal bacteria as an environmentally friendly method for controlling harmful algae according to the present invention can not only be utilized for the ecological restoration and water purification of small-scale water systems, but can also serve as a core technology that can be widely utilized as a foundational technology for various forms of bio-restoration technologies (river ecological restoration, water quality restoration of water sources, wetland restoration, and coastal habitat restoration, etc.). Effects of the invention

[0051] The present invention relates to an algaecidal genus (Arthrobacter) having algicidal activity against harmful algae Arthrobacter The present invention provides a strain of sp.) HNIBRBA5316 (accession number KCTC15442BP). Since the strain according to the present invention has selective algicidal activity against cyanobacteria, it can be usefully utilized to control algal blooms by employing a harmful algae control method comprising applying a harmful algae control agent and a composition for preventing harmful algae containing said strain to an area where harmful algae occur or where harmful algae control is required. Brief explanation of the drawing

[0053] Figure 1 shows the occurrence of a cyanobacteria ring in a culture medium isolated from a soil sample. Figure 2 shows the genus Anabaena ( Anabaena genus Artrobacter according to the present invention for sp.) strains ( Arthrobacter This is a photograph showing the results of the algaecidic activity test of the sp.) HNIBRBA5316 strain. Figure 3 shows the results of confirming the phylogenetic tree based on the 16s sequence of the selected strain. Fig. 4 shows two species of the Anabaena genus ( Anabaena sp. HNIBRCY11 and AG10663) strains, 2 species of the genus Microcystis ( Microcystis sp. KW and AG60752) strains and 1 species of green algae ( ChlorellaThis shows the results of confirming the selective algicidal ability against harmful cyanobacteria through changes in chlorophyll after treating sp. JD1-1) with the strain according to the present invention. Figure 5 shows the Anabaena genus ( Anabaena This shows the results of the change in cell number after 5 days of culture following treatment with the strain according to the present invention on the sp. HNIBRCY23 strain. Figure 6 shows the Anabaena genus ( Anabaena This shows the results of changes in cell number after 5 days of culture following treatment with the strain according to the present invention on the sp. HNIBRCY23 strain, compared with optical microscope images. Figure 7 shows the Anabaena genus ( Anabaena This shows the results of chlorophyll changes after 5 days of culture following treatment with the strain according to the present invention on the sp. HNIBRCY23 strain. Fig. 8 shows the Anabaena genus ( Anabaena This shows the results of chlorophyll changes after 5 days of culture following treatment with the strain according to the present invention on the sp. HNIBRCY23 strain, compared with fluorescence microscope images. Specific details for implementing the invention

[0054] The present invention will be described in detail below. However, the following examples are merely illustrative of the present invention, and the scope of the present invention is not limited to the following examples.

[0056] Example 1. Isolation and Identification of Algaecidal Strains

[0057] 1-1. Isolation of strains with algicidal activity against cyanobacteria

[0058] In order to develop a composition for controlling harmful algae that can suppress the generation and proliferation of harmful algae that cause algal blooms, a novel strain having algalicidal ability was isolated from a soil sample from Wando-gun, Jeollanam-do.

[0059] It was confirmed that an algal bloom occurred in the cyanobacteria culture medium isolated from a soil sample (see Fig. 1), and 25 strains were isolated from the affected area.

[0060] As a result of inoculating each isolated strain into cyanobacteria and verifying, the corresponding strain with excellent algaecidic ability was selected, as can be seen in Figure 2.

[0062] 1-2. Identification of Isolated Strains

[0063] Based on the 16S sequence (785F / 907R) of the selected strain, the phylogenetic tree was confirmed to be of the genus Arthrobacter ( Arthrobacter It was confirmed that it was a sp. (see Fig. 3). Subsequently, the isolated strain was named HNIBRBA5316 and deposited with a depositary (accession number KCTC15442BP).

[0065] Example 2. Confirmation of the selective algicidal activity of the HNIBRBA5316 strain against cyanobacteria

[0066] The strain isolated in Example 1 above was cultured in NA medium under medium temperature (25℃) conditions for 48 hours, and then four types of harmful cyanobacteria ( Anabaena sp. HNIBRCY11, Anabaena sp. AG10663, Microcystis sp. AG60752 and Microcystis sp. KW) and 1 species of green algae ( Chlorella The amount of chlorophyll was measured after inoculation into sp. JD1-1.

[0067] As a result, as can be seen in Fig. 4, it was confirmed that the chlorophyll of four types of harmful cyanobacteria decreased by 300 to 1400 μg / L, while the chlorophyll of green algae actually increased by 170 μg / L. From the above results, the genus Arthrobacter according to the present invention ( Arthrobacter It was confirmed that the HNIBRBA5316 strain possesses selective algicidal activity against harmful cyanobacteria.

[0069] Example 3. Confirmation of optimal conditions for algicidal action against harmful cyanobacteria

[0070] The strain isolated in Example 1 above was cultured in NA medium under medium temperature (25°C) conditions for 48 hours, and then 4.6×10 8 harmful cyanobacteria at an inoculation concentration of cells / ml Anabaena The cell number and changes in chlorophyll were measured after inoculation into sp. HNIBRCY23.

[0071] After 5 days of culture Anabaena The cell number of sp. HNIBRCY23 is 2.4×10 5 cells / ml (see Fig. 5), while the chlorophyll concentration was found to be 2617 μg / L (see Fig. 7), the genus Arthrobacter ( Arthrobacter Under conditions inoculated with the sp.) HNIBRBA5316 strain Anabaena No normal cells of the sp. HNIBRCY23 strain were observed (see Fig. 6), and the chlorophyll concentration was found to be 346 µg / L, a decrease of 87%. Through fluorescence microscopy Anabaena It was confirmed that most of the sp. HNIBRCY23 strain cells were destroyed and the chlorophyll was also eliminated (see Fig. 8). Industrial applicability

[0073] As examined above, the genus Artrobacter according to the present invention ( Arthrobacter The strain sp.) HNIBRBA5316 (accession number KCTC15442BP) possesses selective algicidal activity against harmful cyanobacteria. Therefore, the above-mentioned genus Arthrobacter ( Arthrobacter The HNIBRBA5316 strain, a composition for controlling harmful algae containing the same, and a method for controlling harmful algae comprising treating an area where harmful algae occur or an area where harmful algae control is required can be usefully utilized as an environmentally friendly composition and / or method for removing algal blooms.

[0074] No content

[0075] Depository Name: Korea Research Institute of Biotechnology and Bioengineering Biological Resource Center (KCTC) Trustee Number: KCTC15442BP Date of Deposit: 2023-05-10

Claims

Claim 1 The genus Arthrobacter deposited under accession number KCTC15442BP for the control of harmful algal blooms ( Arthrobacter As the sp.) HNIBRBA5316 strain, the above harmful algae is Microcystis ( Microcystis ) or Anabena( Anabaena It is a cyanobacteria of the genus ), and the strain has algicidal activity against the cyanobacteria, and Chlorella ( Chlorella genus Artrobacter deposited under accession number KCTC15442BP that increases chlorophyll in green algae of the ) genus ( Arthrobacter sp.) HNIBRBA5316 strain. Claim 2 In claim 1, the strain is characterized by the fact that the harmful algae cause an algal bloom. Claim 3 delete Claim 4 delete Claim 5 The genus Arthrobacter deposited under deposit number KCTC15442BP ( Arthrobacter A composition for controlling harmful algae comprising the sp.) HNIBRBA5316 strain or a culture solution thereof as an active ingredient, wherein the harmful algae is Microcystis ( Microcystis ) or Anabena( Anabaena It is a cyanobacteria of the genus ), and the above strain has algicidal activity against the above cyanobacteria, and Chlorella ( Chlorella A composition for controlling harmful algae that increases chlorophyll in green algae of the genus ) Claim 6 A composition for controlling harmful algae according to claim 5, characterized in that the harmful algae cause an algal bloom. Claim 7 delete Claim 8 delete Claim 9 A method for controlling harmful algae comprising treating a harmful algae occurrence area or an area requiring harmful algae control with the composition of claim 5 or 6, wherein the harmful algae is Microcystis ( Microcystis ) or Anabena( Anabaena Method for controlling harmful algae, specifically cyanobacteria. Claim 10 A method for controlling harmful algae according to claim 9, characterized in that the harmful algae cause an algal bloom. Claim 11 A method for controlling harmful algae according to claim 9, characterized in that the treatment is carried out in the form of spraying using a vessel. Claim 12 The genus Arthrobacter deposited under deposit number KCTC15442BP ( Arthrobacter An algaecide for controlling harmful algae comprising the sp.) HNIBRBA5316 strain or a culture solution thereof as an active ingredient, wherein the harmful algae is Microcystis ( Microcystis ) or Anabena( Anabaena It is a cyanobacteria of the genus ), and the above strain has algicidal activity against the above cyanobacteria, and Chlorella ( Chlorella An algaecide for controlling harmful algae that increases chlorophyll in green algae of the genus )

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