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WO2018193847A1 - Inhibiteur de croissance d'algues et procédé d'inhibition de la croissance d'algues - Google Patents

Inhibiteur de croissance d'algues et procédé d'inhibition de la croissance d'algues Download PDF

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Publication number
WO2018193847A1
WO2018193847A1 PCT/JP2018/014433 JP2018014433W WO2018193847A1 WO 2018193847 A1 WO2018193847 A1 WO 2018193847A1 JP 2018014433 W JP2018014433 W JP 2018014433W WO 2018193847 A1 WO2018193847 A1 WO 2018193847A1
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Prior art keywords
algae
nanoparticles
cyanoacrylate
microalgae
growth
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English (en)
Japanese (ja)
Inventor
飯田大介
大濱武
ドゥイヤンタリウィディヤンニンルン
小松千景
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Chikami Miltec Inc
Kochi Prefectural PUC
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Chikami Miltec Inc
Kochi Prefectural PUC
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Priority to JP2019513545A priority Critical patent/JP7134442B2/ja
Publication of WO2018193847A1 publication Critical patent/WO2018193847A1/fr
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    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N25/00—Biocides, pest repellants or attractants, or plant growth regulators, characterised by their forms, or by their non-active ingredients or by their methods of application, e.g. seed treatment or sequential application; Substances for reducing the noxious effect of the active ingredients to organisms other than pests
    • A01N25/12—Powders or granules
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N37/00—Biocides, pest repellants or attractants, or plant growth regulators containing organic compounds containing a carbon atom having three bonds to hetero atoms with at the most two bonds to halogen, e.g. carboxylic acids
    • A01N37/34—Nitriles
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N61/00—Biocides, pest repellants or attractants, or plant growth regulators containing substances of unknown or undetermined composition, e.g. substances characterised only by the mode of action

Definitions

  • the present invention relates to an algal growth inhibitor that suppresses the growth of algae and a method for suppressing the growth of algae.
  • red tide is a term that refers to the occurrence of a specific type of plankton that occurs in the sea and its surface layer accumulation phenomenon.
  • the word freshwater red tide is officially used for those that are yellow and accumulate in surface water.
  • the red tide in the sea area has great social impact, not only worsening the landscape but also causing serious damage such as the massive death of cultured fish.
  • the influence of freshwater red tide on surrounding residents is pointed out as follows.
  • Patent Document 1 describes a red tide algae using silver thiosulfate ion as a component.
  • Patent Document 2 describes an antibacterial agent containing cyanoacrylate polymer particles conjugated with an antibiotic as an active ingredient as an antibacterial agent for vancomycin-resistant gram-positive bacteria.
  • the cyanoacrylate polymer particles are obtained by anionic polymerization of a cyanoacrylate monomer that is used, for example, as an adhesive for wound closure in the surgical field. Cyanoacrylate-based polymer particles are porous, and a desired substance can be conjugated inside.
  • VRE vancomycin-resistant enterococci
  • Patent Document 3 describes that a cyanoacrylate polymer particle conjugated with an amino acid is synthesized by anionic polymerization of a cyanoacrylate monomer in the presence of an amino acid to synthesize cyanoacrylate polymer particles having an average particle diameter of less than 1000 nm.
  • the amino acid-conjugated particles of Patent Document 3 are said to be useful for cancer treatment and prevention because they can induce apoptosis-like cell death in cancer cells and damage the cancer cells.
  • cyanoacrylate polymer particles are useful for antibacterial action and cancer treatment and prevention.
  • Patent Document 1 In order to suppress the abnormal occurrence of microalgae and the surface layer accumulation phenomenon, the use of metal ions as described in Patent Document 1 may cause concern about accumulation of metals in the environment. In view of the accumulation of metal in the environment as described above, it is desirable to suppress the growth of microalgae using a metal-free component.
  • an object of the present invention is to provide an algal growth inhibitor that suppresses the growth of algae and a method for suppressing the growth of algae using metal-free nanoparticles.
  • the present invention relates to an algae growth inhibitor having an action of inhibiting the growth of algae (microalgae) constituting, for example, red tides and the like and a method for inhibiting the growth of algae.
  • the object is to prevent or reduce the above-mentioned damage by suppressing the occurrence of abnormal algae and its surface accumulation phenomenon.
  • [1] to [16] are provided.
  • An algae growth inhibitor that suppresses the growth of algae containing organic compound nanoparticles having a hydrocarbon as a main component and affinity for the cell walls of algae.
  • the algal growth inhibitor according to [3], wherein the cyanoacrylate monomer is isobutyl cyanoacrylate.
  • the microalgae are at least selected from the group of dinoflagellates belonging to the dinoflagellate, diatoms belonging to the diatomaceous plant, raffido algae belonging to the irregular planta, golden algae, or true eyed algae.
  • the algal growth inhibitor according to [5] which is a kind.
  • the algae growth inhibitor according to any one of [5] to [9] which prevents or inhibits water pollution caused by the growth of the microalgae.
  • the algae growth inhibitor according to [10] wherein the water pollution is red tide or pollution in a closed water area.
  • the algae growth inhibitor according to any one of [5] to [9] wherein the growth of the microalgae on the solid surface is prevented or suppressed.
  • the algae growth inhibitor according to [12], wherein the solid is a member used in a plant factory or an outer wall for building materials.
  • the algal growth inhibitor uses nanoparticles of organic compounds mainly composed of hydrocarbons, for example, nanoparticles composed of organic compounds such as acrylic resins. be able to. Therefore, the algal growth inhibitor does not use metal oxide nanoparticles, is metal-free, has no accumulation in the environment, and is highly safe. In addition, metal oxide nanoparticles often form large agglomerates in aqueous solutions, but organic compound nanoparticles mainly composed of hydrocarbons have little cohesiveness between the nanoparticles, and in aqueous solutions. A dispersed state can be stably maintained.
  • cyanoacrylate nanoparticles may be used as the organic compound nanoparticles, and the cyanoacrylate nanoparticles may be polymerized in the presence of a cyanoacrylate monomer and a surfactant.
  • the cyanoacrylate monomer is preferably isobutyl cyanoacrylate.
  • the cell wall is composed of glycoprotein, chitin, cellulose, proteoglycan and the like in algae (microalgae).
  • the above-mentioned organic compound nanoparticles mainly composed of hydrocarbons have an affinity for the cell wall composed of the above components. Therefore, the organic compound nanoparticles can adhere to and cover the cell surface due to the affinity with the cell wall.
  • Microalgae includes, for example, green algae belonging to the green algal plant gate, dinoflagellate belonging to the dinoflagellate gate, diatoms belonging to the diatom plant gate, rafido algae belonging to the irregular plant gate, golden algae or true-eye algae It is.
  • examples of the green algae belonging to the green alga plant gate include algae belonging to the green algae class or the Trevoxia algae class.
  • examples of the green algae belonging to the green alga class include Chlamydomonas, and examples of the green algae belonging to the Trevoxia algae class include Chlorella.
  • the inventors unexpectedly found that the organic compound nanoparticles are acute cell death against the eukaryotic unicellular green alga Chlamydomonas (eg Chlamydomonas reinhardtii). It was found that can be induced. Furthermore, it has been found that protoplast-like cells are generated at a high frequency by inducing the secretion of cell wall lytic enzymes in the genus Chlorella (for example, Chlorella vulgaris).
  • the suffocation effect by covering the whole cell wall with nanoparticles promoted the growth inhibition of microalgae and the secretion of cell wall lytic enzyme. If the nanoparticles are sufficiently small and can pass through damaged cell walls, it is highly likely that the nanoparticles can enter the cytoplasm and induce cell death by phagocytosis.
  • nanoparticles of organic compounds can induce cell death against a wide range of algae or induce abnormal secretion of cell wall lytic enzymes that do not directly induce cell death but are partially degraded even if the cell wall is small.
  • the cells can be easily changed to a state in which the cells are lysed by mechanical stimulation.
  • chlorophyll degradation and ROS generation it causes damage to the photosynthetic system and various metabolic abnormalities.
  • the algal growth inhibitor of the present invention prevents or suppresses water pollution caused by the growth of the above-mentioned microalgae.
  • the algae growth inhibitor can suppress the growth of microalgae, or can kill and remove microalgae that have already occurred, thereby preventing water pollution caused by the growth of microalgae or Can be suppressed.
  • By preventing or suppressing water pollution damage to fish and shellfish that live in, for example, farms and aquariums can be reduced.
  • the water pollution is red tide or pollution in a closed water area.
  • the red tide is a phenomenon in which a large amount of microalgae is generated in open waters and semi-open waters such as oceans, lakes and dam lakes, and causes various problems as described above.
  • Examples of the closed water area include artificial closed water areas such as ponds, fountains, reservoirs, moats, drains, septic tanks, water-cooled cooling towers, bathtubs, and farms in parks.
  • the algal growth inhibitor of the present invention can suppress the occurrence of abnormalities of the above-mentioned specific types of microalgae and the surface layer accumulation phenomenon, thereby preventing or suppressing contamination in the red tide or closed water area.
  • the algal growth inhibitor of the present invention prevents or suppresses the growth of microalgae on the solid surface.
  • the solid may be, for example, a member used in a plant factory or an outer wall for building materials. That is, as a member to be used in a plant factory, a microalgae that inhibits the growth of plant seedlings by applying an algae growth inhibitor to the hole for inserting the seedlings formed in the member supporting the plant seedlings. Proliferation can be prevented or suppressed.
  • an algal growth inhibitor to the outer wall for building materials, the growth of microalgae that grow on the outer wall for building materials can be prevented or suppressed.
  • [14] A method for suppressing the growth of algae using nanoparticles of an organic compound mainly composed of hydrocarbons and having affinity for the cell walls of algae. [15] The method for suppressing the growth of algae according to [14], wherein the algae are microalgae. [16] The method for inhibiting the growth of algae according to [15], wherein microalgae insensitive to the nanoparticles can be grown.
  • the organic compound nanoparticles adhere to the cell surface due to the affinity with the cell wall and cover the cell surface, so that the cell is in contact with the external environment. It becomes difficult to introduce components essential for growth, such as oxygen, carbon dioxide, and nutrients (suffocation effect), and the suffocation effect causes physiological abnormal reactions such as ROS accumulation, chlorophyll degradation, and cell wall lytic enzyme secretion. Wake up. As a result, an event occurs in which the cell becomes a protoplast or the nanoparticle enters the cytoplasm through the damaged cell wall. As a result, the homeostasis of the cells is not maintained, and it is considered that cell death is caused.
  • the suffocation effect by covering the whole cell wall with nanoparticles promoted the growth inhibition of microalgae and the secretion of cell wall lytic enzyme. If the nanoparticles are sufficiently small and can pass through damaged cell walls, it is highly likely that the nanoparticles can enter the cytoplasm and induce cell death by phagocytosis.
  • nanoparticles of organic compounds can induce cell death against a wide range of algae or induce abnormal secretion of cell wall lytic enzymes that do not directly induce cell death but are partially degraded even if the cell wall is small.
  • the cells can be easily changed to a state in which the cells are lysed by mechanical stimulation.
  • chlorophyll degradation and ROS generation it causes damage to the photosynthetic system and various metabolic abnormalities.
  • the growth of algae can be suppressed using nanoparticles of organic compounds that have an affinity for the cell wall of algae (microalgae).
  • microalgae it is useful for the oil production industry utilizing microalgae, for example, to suppress the growth of microalgae that are insensitive to nanoparticles of organic compounds described above and to suppress the growth of microalgae that are insensitive to the nanoparticles.
  • unnecessary microalgae microalgae sensitive to nanoparticles
  • useful desired microalgae microalgae insensitive to nanoparticles
  • the nanoparticles can be added to the medium for the purpose. Harmful or unnecessary growth of microalgae can be prevented.
  • FIG. 3 is a photographic diagram showing the results of culturing Chlamydomonas (wild type CC-124) sensitive to cyanoacrylate nanoparticles on a TAP medium. It is the photograph figure which showed the result of having performed the microscope observation, after adding a cyanoacrylate nanoparticle to Euglena gracilis of a logarithmic growth phase, and culturing for 12 hours.
  • ROS reactive oxygen species
  • FIG. 5 is a photographic diagram showing the results of applying a Chlamydomonas (CC-124 strain) on a sample in which a dispersion of cyanoacrylate nanoparticles is uniformly attached on the surface of an agar medium and conducting a culture test (day 11 of culture). is there.
  • CC-124 strain Chlamydomonas
  • the algae growth inhibitor that suppresses the growth of algae according to the present invention contains nanoparticles of organic compounds mainly composed of hydrocarbons and having affinity for the cell walls of algae. Moreover, the method for suppressing the growth of algae according to the present invention suppresses the growth of algae by using nanoparticles of an organic compound mainly composed of hydrocarbons and having an affinity for the cell walls of algae.
  • the algal growth inhibitor is an organic compound nanoparticle mainly composed of hydrocarbons. That is, the algal growth inhibitor may be nanoparticles composed of an organic compound such as an acrylic resin. Therefore, the algal growth inhibitor does not use metal oxide nanoparticles, is metal-free, has no accumulation in the environment, and is highly safe. In addition, metal oxide nanoparticles often form large agglomerates in aqueous solutions, but organic compound nanoparticles mainly composed of hydrocarbons have little cohesiveness between the nanoparticles, and in aqueous solutions. A dispersed state can be stably maintained.
  • the cell wall is composed of glycoprotein, chitin, cellulose, proteoglycan and the like in algae (microalgae).
  • the above-mentioned organic compound nanoparticles mainly composed of hydrocarbons have an affinity for the cell wall composed of the above components.
  • the degree of affinity is not particularly limited.
  • the affinity is such that nanoparticles of an organic compound can adhere to and cover the cell surface due to affinity with the cell wall. If there is.
  • the algae in the present specification include, for example, seaweeds (red algae, brown algae, green algae) that are multicellular organisms, and microalgae that cause red tides. This embodiment demonstrates the case where algae is used as a micro algae.
  • the microalgae includes, for example, green algae belonging to the green algal plant gate, dinoflagellate belonging to the dinoflagellate gate, diatom belonging to the diatom plant gate, rafido algae belonging to the irregular plant gate, golden algae and true-eye algae It is.
  • Examples of the green algae belonging to the green algae plant gate include algae belonging to the green algae class or the Trevoxia algae class.
  • Examples of the green algae belonging to the green alga class include Chlamydomonas
  • examples of the green algae belonging to the Trevoxia algae class include Chlorella.
  • the genus Chlamydomonas has a shape of 10 to 30 ⁇ m in a spherical shape or a smooth ellipse shape, and has two flagellums of almost the same length as the insect antennae in front of the cell body.
  • the genus Chlorella is a nearly spherical shape with a diameter of about 2 to 10 ⁇ m and has no flagella.
  • Chlamydomonas applanata Chlamydomonas assymetrica, Chlamydomonas debaryana, Chlamydomonas lam lambomona monadina), Chlamydomonas noctigama, Chlamydomonas parkeae, Chlamydomonas perpusilla, Chlamydomonas reinhardtii, but not limited to them, such as Chlamydomonas reinhardtii.
  • Chlorella ellipsoidea Chlorella saccharophila
  • Chlorella sorokiniana Chlorella ⁇ vulgaris, and the like. is not.
  • Chlamydomonas and other microalgae (Chlorophyceae) other than Chlorella genus (Astrephomene gubernaculifera), Carteria radiosa, Dismorphococcus globosus, Eudoraina elegans (Eudo, elegans) Gonium multicocum, Labochlamys culleus, Pandorina morum, Phacotuslenticularis, Tetrabaena socialis, Volbox Carteri (Volvox carti, Volvox carti) (Volvulina steiniii) and the like, but are not limited thereto.
  • microalgae other than the above-mentioned green algae include Chattonella marina (Rafido algae), Heterocapsa triketra (Heterocapsatriquetra), Thalassioneama nitzschioides (Diatomae), Karenia mikimotoi (Dinoflagellate), Keetoceros debilis (Chadiato), Calyptophaphaphaera sphaeroidea (Golden algae), Gambierdiscus sp (Dinoflagellate) , Heterosigma akashiwo (Rafido algae), Odontella longicruris (Odontella longicruris) and the like, but are not limited thereto.
  • the nanoparticles are cyanoacrylate nanoparticles.
  • the cyanoacrylate nanoparticles are preferably polymerized in the presence of a cyanoacrylate monomer and a surfactant.
  • the algal growth inhibitor in the present invention contains cyanoacrylate nanoparticles having an average particle diameter of 10 to 500 nm, preferably 25 to 350 nm, a particle dispersion, a particulate form, a granular form, etc. Any aspect may be sufficient.
  • the dispersion may take a form such as a suspension or a colloidal liquid, but is not limited thereto.
  • the algal growth inhibitor may have a cyanoacrylate nanoparticle concentration of 30 mg / L to 1 g / L, preferably 250 to 1 g / L.
  • the cyanoacrylate polymer portion is obtained by anionic polymerization of a cyanoacrylate monomer.
  • the cyanoacrylate monomer used is preferably an alkyl cyanoacrylate monomer (the alkyl group preferably has 1 to 8 carbon atoms), and is particularly used as an adhesive for sutures in the surgical field. It is preferable to use butyl cyanoacrylate represented by the formula.
  • butyl cyanoacrylate such as isobutyl cyanoacrylate, n-butyl-2-cyanoacrylate, sec-butyl cyanoacrylate, tert-butyl cyanoacrylate, etc. can be used, and methyl cyanoacrylate, ethyl cyanoacrylate ( Other alkyl cyanoacrylates such as adhesive for false eyelashes) and propyl cyanoacrylate may be selected.
  • isobutyl cyanoacrylate, n-butyl-2-cyanoacrylate, and ethyl cyanoacrylate are excellent in safety.
  • a surfactant is used to stabilize the polymerization.
  • a nonionic surfactant or an ionic surfactant can be used, but the surfactant is not limited thereto.
  • the ionic surfactant is preferably an anionic surfactant, but is not limited thereto.
  • nonionic surfactants for example, polysorbates (Tween 20, 40, 60, 80, etc.) can be used, and as anionic surfactants, for example, alkylbenzenesulfonic acid or a salt thereof, sodium lauryl sulfate, sodium laureth sulfate, sodium dodecylbenzenesulfonate. , Sodium 1-pentanesulfonate, sodium 1-decanesulfonate and the like can be used, but are not limited thereto.
  • the nonionic surfactant and the anionic surfactant may be used at the same time.
  • a nonionic surfactant and an anionic surfactant in combination, the cyanoacrylate polymer particles hardly aggregate over time.
  • the above-mentioned surfactant can be used in combination with those having a function of stabilizing the polymerization of anionic polymerization such as polyethylene glycol and saccharide.
  • the saccharide is not particularly limited and may be any of a monosaccharide having a hydroxyl group, a disaccharide having a hydroxyl group, and a polysaccharide having a hydroxyl group, and is particularly preferably a polysaccharide.
  • monosaccharides include glucose, mannose, ribose and fructose.
  • disaccharide include maltose, trehalose, lactose and sucrose.
  • the polysaccharide dextran, mannan, or the like used for the polymerization of conventionally known cyanoacrylate polymer particles can be used.
  • the cyanoacrylate polymer particles are porous, and a desired substance can be conjugated inside.
  • the desired substance may be conjugated to the inside of the cyanoacrylate polymer particles by immersing the cyanoacrylate polymer particles in an aqueous solution of the desired substance or adding the desired substance.
  • the desired substance may be conjugated to the produced particles by performing the above-described anionic polymerization in the presence of the desired substance.
  • amino acids such as glycine and aspartic acid can be conjugated to cyanoacrylate polymer particles. Conjugation refers to a state in which a foreign substance is held in, for example, a hydrophilic molecule.
  • Water can be used as a solvent for the polymerization reaction.
  • Purified water, ion-exchanged water, distilled water, pure water, tap water, ground water, etc. may be appropriately selected as the water depending on the product application having different required purity.
  • the algal growth inhibitor of the present invention can be produced by performing anion polymerization of a cyanoacrylate monomer in the presence of a cyanoacrylate monomer and a surfactant.
  • the polymerization reaction can be performed, for example, by dissolving a surfactant as a polymerization stabilizer in water as a solvent, adding a cyanoacrylate monomer with stirring, and continuing stirring.
  • reaction temperature is not specifically limited, It is good to carry out at room temperature.
  • the reaction time is not particularly limited because the reaction rate varies depending on the pH of the reaction solution, the type of solvent and the concentration of the polymerization stabilizer, and may be appropriately selected depending on these factors, but is usually about 1 to 6 hours. It is.
  • the concentration of the cyanoacrylate monomer in the polymerization reaction solution at the start of the reaction is not particularly limited, but is usually about 0.01 to 5%, preferably about 0.1 to 3%.
  • the concentration of the surfactant in the polymerization reaction solution at the start of the reaction is not particularly limited, but is usually about 0.01 to 5%, preferably about 0.1 to 3%.
  • the concentration of the saccharide in the polymerization reaction solution at the start of the reaction is not particularly limited, but is usually about 0.01 to 5%, preferably about 0.1 to 3%.
  • the cyanoacrylate monomer is anionically polymerized to synthesize cyanoacrylate nanoparticles, whereby the algal growth inhibitor of the present invention can be produced.
  • the synthesized cyanoacrylate nanoparticles can be used as an algal growth inhibitor in the state of a particle dispersion dispersed in a solvent.
  • the obtained particle dispersion hardly changes over time in the particle size distribution during storage, and the particles do not aggregate or settle even when stored at rest, and is excellent in dispersion stability.
  • the synthesized cyanoacrylate nanoparticles can be collected by conventional filtration such as centrifugal ultrafiltration and used as an algal growth inhibitor in a particulate or granular state. Furthermore, the cyanoacrylate nanoparticles recovered by filtration can be used as an algal growth inhibitor in the state of a particle dispersion in which the particles are dispersed in a solvent such as water.
  • the particle size of the synthesized cyanoacrylate nanoparticles can be adjusted by adjusting the concentration of cyanoacrylate monomer in the reaction solution and the reaction time.
  • a surfactant is used as the polymerization stabilizer
  • the particle size can be adjusted by changing the concentration and type of the polymerization stabilizer.
  • the pH of the reaction solution affects the polymerization rate.
  • the pH of the reaction solution is high, the hydroxyl ion concentration is high, so that the polymerization is fast, and when the pH is low, the polymerization is slow. Therefore, the pH is preferably about 1 to 4.
  • the algal growth inhibitor of the present invention prevents or suppresses water pollution caused by the growth of the above-mentioned microalgae. That is, by spreading an appropriate amount of the algal growth inhibitor containing the above-mentioned cyanoacrylate nanoparticles to the ocean, lakes, or water tanks, it is possible to suppress the growth of microalgae, or to kill already generated microalgae. Therefore, it is possible to prevent or suppress water pollution caused by the growth of microalgae. By preventing or suppressing water pollution, damage to fish and shellfish that live in, for example, farms and aquariums can be reduced. In addition, by preventing or suppressing water pollution, it is possible to suppress odors and improve the landscape.
  • the algae growth inhibitor is not only sprayed directly on the ocean, lakes, or aquariums, but also solids containing the algae growth inhibitors may be introduced into the oceans, lakes, or aquariums.
  • the solid is solidified (filmed) by mixing cyanoacrylate nanoparticles with polyethylene glycol or gelled by mixing with polyethylene oxide, the mode is particularly limited. It is not a thing.
  • the water pollution is red tide or pollution in a closed water area.
  • the red tide is water pollution caused by the generation of a large amount of microalgae in open and semi-open water systems such as the ocean and lake water.
  • examples of the closed water area include artificial closed water areas such as ponds, fountains, reservoirs, moats, drains, septic tanks, water-cooled cooling towers, bathtubs, and farms in parks, but are not limited thereto. Absent. In these closed water areas, the production of a large amount of microalgae causes water pollution.
  • the above-mentioned algal growth inhibitor containing cyanoacrylate nanoparticles may be added to or sprayed on the surface of a ship where the red tide is generated or expected from the air, or a solid substance containing an algal growth inhibitor may be added. You may throw into the said area
  • the above-described algal growth inhibitor containing cyanoacrylate nanoparticles may be added to or sprayed on the water surface, and solid matter containing the algal growth inhibitor may be added to a pond, fountain, reservoir, moat in a park. , It may be put into artificial closed water areas such as drains, septic tanks, water-cooled cooling towers, bathtubs, and farms.
  • the algal growth inhibitor of the present invention prevents or inhibits the growth of microalgae on a solid surface.
  • the solid may be, for example, a member used in a plant factory or an outer wall for building materials. That is, as a member to be used in a plant factory, a microalgae that inhibits the growth of plant seedlings by applying an algal growth inhibitor to a hole for inserting the seedlings formed in a member that supports plant seedlings in advance. Can be prevented or suppressed.
  • an algae growth inhibitor to building material outer walls (for example, shaded areas with less sunlight), it is possible to prevent or suppress the growth of microalgae that grow on the outer wall of building materials in a wet state. Can do.
  • algae for example, it is possible to prevent or inhibit the growth of seaweeds, which are multicellular organisms, on solids such as ship bottoms by the algal growth inhibitor of the present invention.
  • an algal growth inhibitor to the ship bottom or the like in advance, it is possible to prevent or suppress the growth of seaweed on the ship bottom or the like.
  • Fig. 1 Chlamydomonas reinhardtii wild type
  • Fig. 2 Chlorella vulgaris
  • ROS reactive oxygen species
  • FIG. 9 an event occurs in which the cells become protoplasts or the nanoparticles pass through the damaged cell wall and enter the cytoplasm
  • the homeostasis of the cells is not maintained, and it is considered that cell death is caused.
  • the suffocation effect by covering the whole cell wall with nanoparticles promoted the growth inhibition of microalgae and the secretion of cell wall lytic enzyme.
  • the nanoparticles need only have an affinity to stably adhere to the outermost layer of cells (so-called cell walls), and high specificity such as binding to specific receptor proteins is necessary. There is no.
  • nanoparticle which has affinity with the outermost layer of a target cell, possibility that a metabolic disorder will be caused not only with a cyanoacrylate nanoparticle but with various species by the choking effect is high. If the nanoparticles are sufficiently small and can pass through damaged cell walls, it is highly likely that the nanoparticles can enter the cytoplasm and induce cell death by phagocytosis.
  • Cyanoacrylate nanoparticles have the ability to induce cell death against a wide range of algae, or induce abnormal secretion of cell wall lytic enzymes that do not directly induce cell death but are partially degraded even if the cell wall is at least small.
  • the cells can be easily changed to a state in which the cells are lysed by mechanical stimulation.
  • chlorophyll degradation and ROS generation it causes damage to the photosynthetic system and various metabolic abnormalities.
  • microalgae that are insensitive to nanoparticles can be grown. According to this method, for example, in the case of culturing in an open culture tank in the field, by adding nanoparticles to the medium, the growth of unnecessary microalgae (microalgae sensitive to nanoparticles) is eliminated.
  • the useful desired microalgae microalgae insensitive to nanoparticles can be selectively grown.
  • Examples of useful microalgae that are insensitive to nanoparticles include Euglena gracilis (Euglena algae), Haematococcus lacustris (Hematococcus algae), etc., but are not limited thereto. Absent.
  • Cyanoacrylate nanoparticles (isobutyl cyanoacrylate nanoparticles), which are nanoparticles of an organic compound contained in the algal growth inhibitor of the present invention, were prepared by the following method.
  • Example 2 The effect of cyanoacrylate nanoparticle exposure on Chlamydomonas reinhardtii (wild type CC-124) was investigated.
  • Wild-type CC-124 Chlamydomonas used in this example was provided by the Chlamydomonas Resource Center at the University of Minnesota (USA). Chloramonas of wild-type CC-124 is mixed nutritionally under constant fluorescence (84 mmol photon m ⁇ 2 s ⁇ 1 ) while gently shaking in Tris-Acetate-Phosphate (TAP) medium (pH 7.0). Cells that had been cultured and reached the middle logarithmic growth phase (OD750 ca. 0.8) were used for the assay.
  • TAP Tris-Acetate-Phosphate
  • the cyanoacrylate nanoparticles used various sizes (25 nm, 180 nm, 350 nm), and assayed at concentrations of 250 mg / L, 500 mg / L, and 1 g / L at each size.
  • the co-incubation of the Chlamydomonas and cyanoacrylate nanoparticles was performed with very gentle rotation (10 rpm).
  • As a negative control a culture solution containing only cells and a dispersing agent and subjected to simultaneous incubation was used.
  • Chlamydomonas By co-incubating with cyanoacrylate nanoparticles of various sizes, Chlamydomonas immediately showed an abnormal migration pattern with rapid and frequent orbital changes and eventually stopped migration. In addition, due to this co-incubation, the cyanoacrylate nanoparticles adhere to and cover the cell surface due to the affinity with the cell wall (FIG. 1), and the original elliptical shape of Chlamydomonas gradually changes to a spherical shape. Observed. Most of the cells that stopped migration swelled spherically, and some of them disintegrated, releasing the cytoplasmic contents. This was thought to mean that such swollen globular cells are protoplasts or have very thin cell walls (spheroplasts).
  • the dead cell rate when co-incubated with cyanoacrylate nanoparticles of 25 nm size (250 mg / L) for 2 hours was about 30% (FIG. 3), but cyanoacrylate nanoparticles of 25 nm size (500 mg / L)
  • the dead cell rate when co-incubated with 2 hours was about 65% (FIG. 4), and the dead cell rate when co-incubated with 25 nm size cyanoacrylate nanoparticles (1 g / L) for 2 hours was 100%. (FIG. 5). From this, it was recognized that many dead cells could be induced by increasing the concentration of cyanoacrylate nanoparticles even in the same incubation period.
  • the order in which cell death can be rapidly induced at a low concentration was confirmed to be 25 nm, 180 nm, and 350 nm in descending order of their action. It was also suggested that the cells stained with trypan blue were dead cells with severe damage to the plasma membrane.
  • cyanoacrylate nanoparticles having a particle size of 25 nm and 180 nm were not observed to aggregate even after 8 hours co-incubation with Chlamydomonas.
  • Cyanoacrylate nanoparticles with a particle size of 350 nm produced aggregates consisting of 20-30 nanoparticles with co-incubation for more than 4 hours. From this, it was recognized that the organic compound nanoparticles (cyanoacrylate nanoparticles) contained in the algal growth inhibitor of the present invention have almost no cohesiveness between the nanoparticles, and can stably maintain a dispersed state in an aqueous solution. .
  • the concentration at which dead cells could be induced against Chlamydomonas wild-type CC-124 was 30 mg / L (results not shown).
  • cyanoacrylate nanoparticles of different sizes if the same molar concentration (number of particles) is used and co-incubation with Chlamydomonas, cyanoacrylate nanoparticles with a larger particle size (180 nm, 350 nm) can be obtained (death)
  • the ratio at which cells can be induced tended to be large (FIG. 6).
  • Example 3 In Chlamydomonas reinhardtii, the dead cell rate in wild type CC-124, very thin cell wall mutant CC-503 and cell wall deletion mutant CC-400 was examined. The two variants were also provided by the Chlamydomonas Resource Center at the University of Minnesota (USA). As described in Example 2, the dead cell rate was determined by co-incubating with cyanoacrylate nanoparticles (250 mg / L) having a size of 25 nm and performing a trypan blue staining assay. The results are shown in FIG.
  • cell wall deletion mutant CC-400, thin cell wall mutant CC-503, and wild type CC-124 are in descending order of sensitivity to cyanoacrylate nanoparticles. This order of sensitivity suggested that the cell wall acts as a kind of mechanical barrier that inhibits dead cell induction by cyanoacrylate nanoparticles.
  • Example 4 Cellular ultrastructure of Chlamydomonas (wild-type CC-124) after co-incubation with cyanoacrylate nanoparticles (65 mg / L) of 25 nm size for 20 minutes was observed by TEM. The results are shown in FIG.
  • Example 5 The effect of cyanoacrylate nanoparticle exposure on Chlorella vulgaris was investigated. Chlorella vulgaris used in this example was provided by the National Institute for Environmental Studies (NIES).
  • the cyanoacrylate nanoparticles used various sizes (25 nm, 180 nm, 350 nm), and assayed at a concentration of 1 g / L at each size. Due to this co-incubation, the cyanoacrylate nanoparticles adhere to and cover the surface of the cell due to the affinity with the cell wall (FIG. 2). As a result, chlorella cells are not induced into dead cells, and protoplasts or spherospheres are not induced. Changed to plast (protoplast / spheroplast).
  • Chlorella vulgaris protoplasts were prepared using a commercially available lytic enzyme mixture.
  • Commercially available lytic enzyme mixtures are 0.5% cellulidine (produced by Calbio Chem), 2% maceroteam R-10 (produced by Yakult Pharmaceutical Co., Ltd.) and 1% chitosanase derived from Bacillus R-4 (Kei Kasei Co., Ltd.).
  • the color of the non-enzyme-treated cells was pinkish red as a result of the fusion of the pure red autofluorescence of chlorophyll and the blue fluorescence of fluorescent brightener 28 and was used as a reference standard for non-protoplasts.
  • Example 6 It was investigated whether cell wall lytic enzyme was secreted by exposure to cyanoacrylate nanoparticles against Chlorella vulgaris.
  • control in FIG. 11 is obtained by co-incubation with a culture solution containing only cells and a dispersing agent, and “NP (350 nm)” is produced by protoplast / spheroplast by co-incubation with cyanoacrylate nanoparticles. Indicates what was induced.
  • Example 7 The cellular ultrastructure of chlorella after co-incubation with 25 nm sized cyanoacrylate nanoparticles (1 g / L) for 3 hours was observed by TEM. The results are shown in FIG.
  • cyanoacrylate nanoparticles were not detected inside the cell wall and between the cell wall and the plasma membrane (periplasmic space).
  • Example 8 In the above-mentioned Examples, the effect of cyanoacrylate nanoparticle exposure was examined in Chlamydomonas reinhardtii belonging to the genus Chlamydomonas. In this example, the effects of exposure to cyanoacrylate nanoparticles (25 nm) were examined for other Chlamydomonas species and Green algae. The experimental conditions were the same as in the above-described example. For reference, the results are also shown for the four Chlamydomonas reinhardtii species used in the above-described Examples (Table 1-1, Table 1-2).
  • Chlamydomonas globosa Chlamydomonas applanata
  • Chlamydomonas assymetrica Chlamydomonas debaryana
  • Chlamydomonas clamosa Dead cell induction was observed in Chlamydomonas monadina, Chlamydomonas noctigama, Chlamydomonas parkeae, and Chlamydomonas perpusilla.
  • Example 9 The time course of reactive oxygen species (ROS) production by exposure to cyanoacrylate nanoparticles was examined in Chlamydomonas reinhardtii wild-type CC-124.
  • ROS reactive oxygen species
  • H2DCFDA 2 ', 7'-dichlorodihydrofluorescein diacetate (D668, manufactured by Sigma-Aldrich) was used.
  • Non-fluorescent H2DCFDA is converted to highly fluorescent 2 ', 7'-dichlorofluorescein (DCF) by reactive oxygen species in the cytoplasm.
  • a culture medium containing only cells and a dispersing agent was used, and as a comparative control, two types of metal oxide nanoparticles, that is, ZnO containing less than 100 nm (544906, Sigma-Aldrich). And TiO 2 (anatase form) (205-01715, manufactured by Wako Pure Chemical Industries, Ltd.) were used.
  • Example 10 In this example, the effects of exposure to cyanoacrylate nanoparticles (25 nm) were examined for other microalgae other than the green alga class described above.
  • the experimental conditions were the same as in the above-described example.
  • the microalgae used are dinoflagellates belonging to the dinoflagellate, diatoms belonging to the diatomaceous plant, rafidoalgae or golden algae belonging to the irregular planta, and specifically, Shutnera marina ( Chattonella marina (NIES-1), Heterocapsatriquetra (NIES-7), Thalassioneama nitzschioides (NIES-534), Karenia mikimotoi (NIES-2411), rosdece NIES-3710), Calyptrosphaera sphaeroidea (NIES-1308), Gambierdiscus sp (NIES-2766), Heterosigma akashiwo (NIES-5), Odontera Longicurlis (Odontella longicruris: NIES-590).
  • Shutnera marina Chattonella marina (NIES-1), Heterocapsatriquetra (NIES-7), Thalassioneama nitzschioides (NIES-534), Karenia mikimoto
  • the use of the algal growth inhibitor of the present invention can prevent or suppress water pollution (red tide or pollution in a closed water area) caused by the growth of the above-mentioned microalgae.
  • Example 11 Regarding microalgae insensitive to cyanoacrylate nanoparticles and microalgae sensitive to cyanoacrylate nanoparticles, whether or not each microalgae grew when the concentration of cyanoacrylate nanoparticles was changed in various ways was investigated.
  • Cyanoacrylate nanoparticles having a particle size of 180 nm are used, and microalgae insensitive to cyanoacrylate nanoparticles is Euglena gracilis (NIES-49), which is sensitive to cyanoacrylate nanoparticles.
  • NIES-49 Euglena gracilis
  • Chlamydomonas reinhardy wild type CC-124 was used.
  • 0.03%, 0.01%, 0.003%, and 0.001% cyanoacrylate nanoparticles were respectively placed at different positions on a TAP medium (pH 7.0) containing 1.5% agar. 20 ⁇ L was dropped and adsorbed on the TAP medium. Next, the culture solution of Chlamydomonas (CC-124 strain) that reached the logarithmic growth phase was adsorbed onto a cotton swab, spread on a TAP medium, and cultured for 10 days.
  • cyanoacrylate nanoparticles were added to Chlamydomonas (CC-124 strain) in the logarithmic growth phase to a final concentration of 0.01% (100 mg / L) and cultured in a liquid medium for 12 hours. However, few cells were observed to swim (results not shown). Similar results were obtained when the final concentration of cyanoacrylate nanoparticles was 0.03% (results not shown).
  • cyanoacrylate nanoparticles are added to the culture medium (for example, final concentration is 0.01 to 0.03%), so that Eliminate the growth of microalgae sensitive to particles (eg Chlamydomonas reinhardi) and selectively select microalgae insensitive to cyanoacrylate nanoparticles (eg useful microalgae such as Euglena gracilis, Haematococcus laxtris) It was recognized that it could be grown.
  • microalgae sensitive to particles eg Chlamydomonas reinhardi
  • microalgae insensitive to cyanoacrylate nanoparticles eg useful microalgae such as Euglena gracilis, Haematococcus laxtris
  • Example 12 An experiment was conducted to determine the minimum number of cyanoacrylate nanoparticles necessary for an effect as an algae growth inhibitor on a solid surface and the area density (mass per square meter).
  • Chlamydomonas (CC-124 strain) that reached the logarithmic growth phase was adsorbed onto a cotton swab, spread on a TAP medium, and cultured for 10 days.
  • Chlamydomonas cells were observed to be growing at the dropping position of 10, 30, 100 ppm (level 1 to 3) of cyanoacrylate nanoparticles, but at the dropping position of 300 ppm (level 4) cyanoacrylate nanoparticles. was found not to grow Chlamydomonas cells (results not shown).
  • Level 4 cyanoacrylate nanoparticles had a mass per square meter of 0.076 g / m 2 .
  • Example 13 Chlamydomonas (CC-124 strain) was applied to a sample in which a dispersion of cyanoacrylate nanoparticles (particle size 30 nm) was uniformly attached on the surface of an agar medium, and a culture test was performed. Table 3 shows the results of determining the addition amount of the cyanoacrylate nanoparticle liquid added to each sample A to D, the mass of the cyanoacrylate nanoparticles, and the number of cyanoacrylate nanoparticles. Sample E was a control to which no cyanoacrylate nanoparticles were added.
  • the cyanoacrylate nanoparticle liquid (concentration 1%) of each sample A to D was separately added to a TAP medium (surface area 64 cm 2 ) containing 1.5% agar, and the petri dish was placed in a thermostat set at 50 ° C. The cyanoacrylate nanoparticle liquid was dried.
  • a culture solution of Chlamydomonas (CC-124 strain) that reached the logarithmic growth phase was adsorbed onto a cotton swab, spread on a TAP medium, and cultured at room temperature (25 ⁇ 2 ° C.) for 2 weeks. The results are shown in FIG. 19 (culture day 0), FIG. 20 (culture day 7), and FIG. 21 (culture day 11).
  • cyanoacrylate nanoparticles square meter per mass is equal 0.076 g / m 2, or 0.108 g / m 2 or more, preventing or inhibiting the growth of Chlamydomonas with solid surface It was recognized that it was possible. Similar results were obtained for microalgae other than Chlamydomonas (results not shown).
  • the outer wall of the building materials square meter per mass be previously coated with cyanoacrylate nanoparticles solution so that 0.076 g / m 2, or 0.108 g / m 2 or more, the outer wall of the building materials It can be expected to prevent or suppress the growth of microalgae on the surface.
  • the volume of 0.1 g of the cyanoacrylate nanoparticle dispersion is 10 cm 3 when the dispersion concentration is 1%.
  • the dispersion concentration is 1%.
  • a method in which the dispersion is sprayed and dried can be used.
  • the drying time it is advantageous that the amount of liquid to be diluted is small.
  • Example 14 In outdoor ponds, cyanoacrylate nanoparticles were added to investigate the effect on the growth of microalgae.
  • the isobutyl cyanoacrylate nanoparticles having a diameter of 25 nm were introduced into one of the 42-ton water ponds arranged in parallel on the Kochi University of Technology campus in a final concentration of 100 ppm. Isobutyl cyanoacrylate nanoparticles were not added to the other pond, which was the target experimental group. After the lapse of 24 days, 500 mL of water was collected from each of the pond to which the cyanoacrylate nanoparticles were added and the pond of the target experimental group, and filtered through a mesh having a mesh size of 1 ⁇ m to collect organisms such as microalgae.
  • the cells were disrupted by repeating freezing and thawing three times for the organism on the filter.
  • Total DNA extraction from the disrupted cells was performed using QIAamp DNA Mini Kit (Qiagen).
  • Qiagen QIAamp DNA Mini Kit
  • an amplicon amplified by the PCR method using the following primer set was analyzed for end-pair sequences under the conditions of 2 ⁇ 300 bp using a next-generation sequencer MiSeq (manufactured by Illumina).
  • a quality check was performed on the sequence data obtained using the DNA of the organism obtained from the pond into which the cyanoacrylate nanoparticles were introduced, and 49,376 reads (bp) were finally obtained. Moreover, the quality of the sequence data obtained using the DNA of the organism obtained from the pond of the target experimental section was checked, and finally 67,268 reads (bp) were obtained.
  • the present invention can be used for an algal growth inhibitor that suppresses the growth of algae and a method for suppressing the growth of algae.

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Abstract

La présente invention concerne : un inhibiteur de croissance d'algues qui inhibe la croissance des algues et qui contient des nanoparticules d'un composé organique qui fait preuve d'affinité pour une paroi de cellule d'algue et qui possède un hydrocarbure comme constituant majeur ; et un procédé d'inhibition de la croissance d'algues utilisant les nanoparticules d'un composé organique qui fait preuve d'affinité pour une paroi de cellule d'algue et qui possède un hydrocarbure comme constituant majeur.
PCT/JP2018/014433 2017-04-18 2018-04-04 Inhibiteur de croissance d'algues et procédé d'inhibition de la croissance d'algues Ceased WO2018193847A1 (fr)

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JP2021116265A (ja) * 2020-01-27 2021-08-10 三菱鉛筆株式会社 防蟻粒子水分散体
CN113533786A (zh) * 2021-01-24 2021-10-22 大理大学 一种基于电子显微成像技术探讨双吲哚生物碱对新月菱形藻的抗污损作用机制研究方法
JP2023000739A (ja) * 2021-06-18 2023-01-04 三菱鉛筆株式会社 抗菌性粒子分散体

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WO2008126846A1 (fr) * 2007-04-09 2008-10-23 Yokohama City University Agent antibactérien actif contre les bactéries à gram positif résistantes à la vancomycine
WO2010101178A1 (fr) * 2009-03-03 2010-09-10 公立大学法人横浜市立大学 Particules de polymère de cyanoacrylate conjuguées à un acide aminé
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JP2005060273A (ja) * 2003-08-08 2005-03-10 Daiichi Seimou Co Ltd 有機酸製剤
JP2007332039A (ja) * 2006-06-12 2007-12-27 Sumiya Yoji 赤潮の除藻剤および赤潮の除藻方法
WO2008126846A1 (fr) * 2007-04-09 2008-10-23 Yokohama City University Agent antibactérien actif contre les bactéries à gram positif résistantes à la vancomycine
WO2010101178A1 (fr) * 2009-03-03 2010-09-10 公立大学法人横浜市立大学 Particules de polymère de cyanoacrylate conjuguées à un acide aminé
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JP2021116265A (ja) * 2020-01-27 2021-08-10 三菱鉛筆株式会社 防蟻粒子水分散体
JP7454387B2 (ja) 2020-01-27 2024-03-22 三菱鉛筆株式会社 防蟻粒子水分散体
CN113533786A (zh) * 2021-01-24 2021-10-22 大理大学 一种基于电子显微成像技术探讨双吲哚生物碱对新月菱形藻的抗污损作用机制研究方法
JP2023000739A (ja) * 2021-06-18 2023-01-04 三菱鉛筆株式会社 抗菌性粒子分散体

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