WO2014017233A1 - Microbe activity modulator and method of modulating microbe activity - Google Patents

Abstract

Provided is a microbe activity modulator which contains a microbial activity modulating substance and a carrying medium and which is present in a reaction tank in a biological wastewater treatment facility.

Description

Microbial activity regulator and method for regulating microbial activity

The present invention relates to a microorganism activity regulator and a method for regulating microorganism activity.

It is known that microorganisms transmit information through intercellular signal transmitters (hereinafter referred to as “signal substances”) and control the secretion of pathogenic substances and biofilm production depending on the density of the microorganisms. It has been. Such an information transmission mechanism is called quorum sensing (see, for example, Non-Patent Document 1).

However, many microbial activity regulating substances including signal substances are insoluble in water. In order to dissolve such a microbial activity-regulating substance in water, it may be necessary to use an organic solvent or the like, and the operability may be poor. Accordingly, an object of the present invention is to provide a microbial activity regulator with improved operability. The present invention also aims to provide a method for regulating the activity of microorganisms.

The present invention provides a microbial activity regulator present in a reaction tank of a biological wastewater treatment facility, which comprises a microbial activity regulator and a transport medium. The present invention also provides the use of a composition comprising a microbial activity regulator and a carrier medium for regulating the activity of microorganisms present in a reaction vessel of a biological wastewater treatment facility.

According to the microbial activity regulator or use of the present invention, regardless of whether the microbial activity regulator is hydrophilic or not, the microbial activity regulator can be easily added to an aqueous system and added to the environment in which the microorganism is present. It can be diffused and delivered to microorganisms, and the activity of microorganisms can be efficiently regulated. For this reason, the operability of the microbial activity regulating substance can be improved, and it is suitable for regulating the activity of microorganisms present in the reaction tank of the biological wastewater treatment facility, particularly microorganisms involved in wastewater treatment.

The microbial activity-regulating substance may be one or more substances selected from the group consisting of a signal substance, a nucleic acid that affects prokaryotic activity, and a protein that affects prokaryotic activity.

By delivering such a microbial activity regulating substance to a microorganism, the activity of the microorganism can be regulated.

The microbial activity-regulating substance may be N-acyl-L-homoserine lactone. If the microbial activity regulator is N-acyl-L-homoserine lactone, the microbial activity of many types of gram-negative bacteria can be regulated.

The transport medium may be one or more substances selected from the group consisting of vesicles, liposomes, micelles, emulsions, peptides, proteins, metal nanoparticles, carbon nanotubes, fullerenes and first polymers. Also, the first polymer as the carrier medium may be in the form of a hydrogel, nanosphere, microsphere, or dendrimer.

The microbial activity regulator or use of the present invention can further improve the operability of the microbial activity regulator by including such a transport medium. Moreover, decomposition | disassembly of a microbial activity regulator can be suppressed, it can spread | diffuse easily with the whole reaction tank of a biological wastewater treatment facility, and microbial activity can be regulated more reliably.

The above transport medium may be modified with a sugar chain, an antibody, or a second polymer. The second polymer that modifies the carrier medium may be polyethylene glycol, polyethyleneimine, polyamidoamine.

The carrier medium is modified with these substances, so that the above-mentioned activity regulator for microorganisms can be selectively delivered to microorganisms having a specific antigen, or the survivability (remaining time) of the activity regulator for microorganisms can be adjusted. It becomes possible to do.

The transport medium may be a liposome. The liposome preferably contains egg yolk lecithin or hydrogenated soybean phosphatidylcholine. The liposome may further contain cholesterol.

When the liposome contains cholesterol, the fluidity of the liposome can be adjusted, and the liposome membrane can be more stably present.

The liposome may further contain stearic acid.

When the liposome contains stearic acid, the liposome is negatively charged, the stability of the membrane is lowered, and the microorganism activity regulator tends to be released more easily.

The microbial activity is preferably nitrification activity.

If the microbial activity is nitrification activity, the removal efficiency of ammonia in the wastewater is improved.

The present invention is also a method for regulating the activity of microorganisms present in a reaction tank of a biological wastewater treatment facility, wherein the above-mentioned microorganism activity regulator is added to the reaction tank of the biological wastewater treatment facility. A method comprising the steps is provided.

According to the method of the present invention, the activity of microorganisms present in the reaction tank of the biological wastewater treatment facility can be adjusted easily and efficiently. For this reason, the waste water treatment performed in the reaction tank can be promoted or suppressed as necessary.

According to the present invention, a microbial activity regulator with improved operability can be provided. Moreover, the method of adjusting the activity of microorganisms can be provided.

FIG. 1 is a graph showing the results of promoting the nitrification reaction. FIG. 2 is a graph showing the results of promoting the nitrification reaction. FIG. 3 is a molecular phylogenetic tree showing the results of simple molecular phylogenetic analysis of Paracoccus AS6 strain. FIG. 4 is a graph showing the action of 3oxoC12-HSL when liposomes are used. FIG. 5 is a graph showing the action of 3oxoC12-HSL when the liposome composition is changed. FIG. 6 is a schematic diagram showing a state in which the microbial activity-regulating substance is conveyed by vesicles. FIG. 7 is a schematic diagram showing a state in which a microorganism activity regulator is transported by micelles. FIG. 8 is a schematic diagram showing a state in which the activity regulating substance for microorganisms is carried by a protein. FIG. 9 is a schematic diagram showing a state in which the activity regulating substance for microorganisms is transported by metal nanoparticles. FIG. 10 is a schematic diagram showing a state in which a microorganism activity regulator is transported by carbon nanotubes. FIG. 11 is a schematic diagram showing a state in which a microorganism activity regulator is transported by fullerene. FIG. 12 is a schematic diagram showing a state in which the microorganism activity regulator is transported by the first polymer. FIG. 13 is a schematic diagram showing a state in which a microorganism activity-regulating substance is transported by a hydrogel. FIG. 14 is a schematic diagram showing a state in which a microorganism activity regulator is transported by nanospheres. FIG. 15 is a schematic diagram showing a state in which a microorganism activity regulator is transported by a dendrimer. FIG. 16 is a schematic diagram of a transport medium according to an embodiment of the present invention. FIG. 17 is a schematic view of a transport medium according to another embodiment of the present invention. FIG. 18 is a schematic view of a transport medium according to still another embodiment of the present invention. FIG. 19 is a schematic configuration diagram illustrating a biological wastewater treatment apparatus according to an embodiment of the present invention. FIG. 20 is a schematic configuration diagram illustrating a biological wastewater treatment apparatus according to another embodiment of the present invention. FIG. 21 is a schematic configuration diagram illustrating a biological wastewater treatment apparatus according to still another embodiment of the present invention. FIG. 22 is a schematic configuration diagram illustrating a biological wastewater treatment apparatus according to still another embodiment of the present invention.

(Microbial activity regulator)
The microbial activity regulator of the present invention includes a microbial activity regulator and a transport medium. Examples of the microbial activity regulating substance include an intercellular signal transduction substance (signal substance), a nucleic acid that affects the prokaryotic activity, a protein that affects the prokaryotic activity, and the like. These can be used alone or in combination of two or more. The microbial activity regulating substance may be a substance that improves the activity of the microorganism or a substance that decreases the activity of the microorganism.

As the signal substance, N-acyl-L-homoserine lactone (AI1: autoinducer 1); 4,5-dihydroxy-2,3-pentanedione (AI2: autoinducer 2); HHQ (2-alkyl-4 -Quinolones), quinolones and quinolines such as PQS (2-alkyl-3-hydroxy-4-quinolones); indoles; peptides; cyclic dipeptides; diketopiperazines and the like. Examples of N-acyl-L-homoserine lactone include N-butanoyl-L-homoserine lactone, N-3-oxobutanoyl-L-homoserine lactone, N-3-hydroxybutanoyl-L-homoserine lactone, N- Pentanoyl-L-homoserine lactone, N-3-oxopentanoyl-L-homoserine lactone, N-3-hydroxypentanoyl-L-homoserine lactone, N-hexanoyl-L-homoserine lactone, N-3-oxohexanoyl- L-homoserine lactone, N-3-hydroxyhexanoyl-L-homoserine lactone, N-heptanoyl-L-homoserine lactone, N-3-oxoheptanoyl-L-homoserine lactone, N-3-hydroxyheptanoyl-L- Homoserine lactone, N-octanoyl-L Homoserine lactone, N-3-oxooctanoyl-L-homoserine lactone, N-3-hydroxyoctanoyl-L-homoserine lactone, N-nonanoyl-L-homoserine lactone, N-3-oxononanoyl-L-homoserine lactone, N -3-hydroxynonanoyl-L-homoserine lactone, N-decanoyl-L-homoserine lactone, N-3-oxodecanoyl-L-homoserine lactone, N-3-hydroxydecanoyl-L-homoserine lactone, N-undecanoyl-L -Homoserine lactone, N-3-oxoundecanoyl-L-homoserine lactone, N-3-hydroxyundecanoyl-L-homoserine lactone, N-dodecanoyl-L-homoserine lactone, N-3-oxododecanoyl-L -Homoserine lactone N-3-hydroxydodecanoyl-L-homoserine lactone, N-tridecanoyl-L-homoserine lactone, N-3-oxotridecanoyl-L-homoserine lactone, N-3-hydroxytridecanoyl-L-homoserine lactone, N-tetradecanoyl-L-homoserine lactone, N-3-oxotetradecanoyl-L-homoserine lactone, N-3-hydroxytetradecanoyl-L-homoserine lactone, N-pentadecanoyl-L-homoserine lactone, N-3-oxopentadecanoyl-L-homoserine lactone, N-3-hydroxypentadecanoyl-L-homoserine lactone, N-hexadecanoyl-L-homoserine lactone, N-3-oxohexadecanoyl-L- Homoserine lactone, N-3-hydroxyhexadeca Examples thereof include, but are not limited to, N-acyl-L-homoserine lactone having 4 to 16 carbon atoms in the acyl group, such as noyl-L-homoserine lactone.

The signal substance may be prepared by chemical synthesis, or may be prepared by purifying the signal substance secreted into the medium by the signal substance-producing bacteria. Moreover, you may use the culture of a signal substance producing microbe as a signal substance, without refine | purifying. Examples of the signal substance-producing bacteria include bacteria such as Paracoccus, Burkholderia, Pseudomonas, Vibrio, Aeromonas, Bacillus, Streptomyces, Streptococcus, and Lactobacillus.

Examples of nucleic acids that affect the activity of prokaryotes include enzymes involved in protein synthesis and synthesis inhibition, and DNA and RNA encoding ATP synthase expression. These DNAs are preferably linked downstream of the promoter so that they can be expressed.

Nucleic acids that affect the activity of prokaryotes can be obtained by PCR or the like using genomic DNA of prokaryotes such as Escherichia coli and Bacillus as a template. Nucleic acids that affect the activity of prokaryotes are cloned into vectors such as pGEM (Promega), pBluescript (Stratagene), pUC (Takara Bio Inc.), and introduced into Escherichia coli, for example. Can be maintained. Moreover, it can prepare in large quantities from the said E. coli if necessary.

Proteins that affect prokaryotic activity include polymerases, galactosidases, proteases, and the like.

Proteins that affect the activity of prokaryotes may be prepared by purification from microorganisms that produce the proteins, for example. Alternatively, an expression vector incorporating a DNA encoding a protein that affects the prokaryotic activity may be expressed in a large amount in a body such as Escherichia coli and purified for preparation.

(Transport medium)
The microorganism activity regulator includes a microorganism activity regulator and a transport medium. The carrier medium may contain a microbial activity-regulating substance, or may form a complex with the microbial activity-regulating substance by a covalent bond or a non-covalent bond. The transport medium is not particularly limited as long as it can form a complex with the microbial activity regulator, and specific examples include vesicles, liposomes, micelles, emulsions, peptides, proteins, metal nanoparticles, carbon nanotubes, fullerenes, 1 polymer and the like. These can be used alone or in combination of two or more. Further, cyclodextrin, cyclophane, calixarene and the like generally used in the field of host-guest chemistry may be used.

A vesicle is generally a vesicle formed of a lipid bilayer membrane, the inside and outside of the vesicle exhibit hydrophilicity, and the inside of the lipid bilayer membrane exhibits hydrophobicity. In particular, a vesicle composed of a lipid bilayer mainly composed of phospholipid is called a liposome. As the phospholipid, phosphatidylcholine, sphingomyelin, phosphatidylethanolamine, phosphatidylserine, egg yolk lecithin, hydrogenated soybean phosphatidylcholine and the like can be used. Liposomes incorporating a hydrophobic substance can be easily added to water and allowed to diffuse. The vesicles may be secreted by microorganisms or chemically synthesized.

When liposomes are used as the transporting medium, liposomes having no charge may be used, or those having a charge may be used. Examples of the charged liposome include a cationic liposome and an anionic liposome.

The cationic liposome is a vesicle composed of a lipid bilayer mainly composed of phospholipid and having a positive surface on the surface in contact with the outside world. As the phospholipid, phosphatidylcholine, sphingomyelin, phosphatidylethanolamine, phosphatidylserine and the like can be used. As other components constituting the lipid bilayer membrane, lipids other than phospholipids such as cholesterol, sugars, proteins and the like can be used.

Cationic liposomes are preferred in that the liposome membrane is positively charged and is easily taken up by electrostatic interaction with the negatively charged microbial cell membrane. Cationic liposomes attached to the sludge attract the microorganisms in the sludge and form aggregates. The formation of aggregates brings the physical distance between the cationic liposome and the microorganism closer, increases the probability that the cationic liposome will be fused or biodegraded with the cell membrane of the microorganism, and the microorganism activity regulator is delivered to the microorganism. Efficiency.

As the cationic liposome, for example, those used as gene transfer vectors can be used. Cationic liposomes can be obtained by including cationic lipids, cationic polysaccharides and the like in the liposome as constituents of the lipid bilayer.

The cationic liposome can contain, for example, a lipid to which a primary amine, secondary amine, tertiary amine or quaternary ammonium cation is bound; a cationic polysaccharide. Specific examples of such lipids include N, N-dioleyl-N, N-dimethylammonium chloride (DODAC), N- [1- (2,3-dioleyloxy) propyl] -N, N, N-trimethyl. Ammonium chloride (DOTMA), dimethyldioctadecyl ammonium bromide (DDAB), N- [1- (2,3-dioleyloxy) propyl] -N, N, N-trimethylammonium methyl sulfate (DOTAP), 3β- [N -(N ', N'-dimethylaminoethane) -carbamoyl] cholesterol (DC-Chol), (1,2-dimyristyloxypropyl-3-dimethylhydroxyethylammonium bromide (DMRIE), dioctadecylamide glycylspermine ( DOGS), N, N-dimethyl- (2,3-dioleyl) Alkoxy) propylamine (DODMA), 1,2-dioleoyl-3-dimethylammonium propane (DODAP), specific examples of. Cationic polysaccharides stearylamine and the like and chitosan.

Anionic liposomes (hereinafter also referred to as negatively charged liposomes) are vesicles composed of a lipid bilayer mainly composed of phospholipids, and have a negative surface on the surface in contact with the outside world. As the phospholipid, phosphatidylcholine, sphingomyelin, phosphatidylethanolamine, phosphatidylserine and the like can be used. As other components constituting the lipid bilayer membrane, lipids other than phospholipids such as cholesterol, sugars, proteins and the like can be used.

Anionic liposomes can contain, for example, anionic lipids or anionic polysaccharides. Examples of the anionic lipid include fatty acids such as stearic acid, oleic acid, and palmitic acid; and alkyl sulfuric acids such as dodecyl sulfate and hexadecyl sulfate. Examples of the anionic polysaccharide include carrageenan and dextran sulfate.

In addition, in order to adjust fluidity, the liposome may contain lipids other than phospholipids such as cholesterol. By containing lipids other than phospholipids, the fluidity of the lipid bilayer membrane of the liposome can be adjusted. The content of lipids other than phospholipids is preferably 50% by mass or less, more preferably 30% by mass or less, based on the mass of the whole liposome, from the viewpoint of adjusting the stability of the liposome membrane. The content is preferably 5 to 20% by mass.

Liposomes used as a transport medium may have a plurality of layer structures. A transport medium 1600 shown in FIG. 16 is formed of two layers, an outermost layer 1601 and an inner layer liposome 1602. Inner layer liposome 1602 contains microbial activity regulating substance 1603. The carrier medium 1600 may have a layer structure of three or more layers. In that case, for example, a liposome layer enclosing a microbial activity regulating substance may be formed in the inner layer liposome 1602.

Examples of the microbial activity regulating substance 1603 include intercellular information transmission substances (signal substances), nucleic acids that affect the activity of prokaryotes, proteins that affect the activity of prokaryotes, and the like. These can be used alone or in combination of two or more. The microbial activity regulating substance may be a substance that improves the activity of the microorganism or a substance that decreases the activity of the microorganism.

Since the outermost layer 1601 is formed of liposomes, the carrier medium 1600 is easily diffused and delivered to microorganisms even in an aqueous environment. Even if the outermost layer 1601 of the transport medium 1600 is broken and the contents are released, the microbial activity regulating substance 1603 is encapsulated in the inner layer liposome 1602. Therefore, it can be avoided that the microbial activity regulating substance is directly subjected to the microbial degradation action. As a result, the delivery efficiency of the microbial activity regulator is improved.

FIG. 17 shows a schematic diagram of a transport medium 1700 according to another embodiment of the present invention. The carrying medium 1700 shown in FIG. 17 is the same as the carrying medium shown in FIG. 16 except that the outermost layer 1701 contains the microbial activity regulating substance 1704. The microbial activity regulating substance 1703 and the microbial activity regulating substance 1704 may be the same substance or different substances.

When the outermost layer 1701 encloses the microbial activity regulating substance 1704, the release timing of the microbial activity regulating substance 1703 encapsulated in the inner layer liposome 1702 can be shifted. When the outermost layer 1701 is broken or fused with the cell membrane of the microorganism, the microbial activity regulating substance 1704 and the inner layer liposome 1702 are released. Thereafter, the inner layer liposome 1702 is broken or fused with the cell membrane of the microorganism, whereby the microbial activity regulating substance 1703 is released. Since the outermost layer 1701 and the inner layer liposome 1702 are broken at different timings, the release timings of the microbial activity regulating substance 1704 and the microbial activity regulating substance 1703 contained therein are also different.

By making the microbial activity regulating substance 1703 and the microbial activity regulating substance 1704 the same substance, the same substance can be allowed to act on the microorganism for a long time. In addition, the amount of the microbial activity modulator per carrier medium can be increased, and a larger amount of the microbial activity modulator can be delivered to the microorganism. By making the microbial activity regulating substance 1703 and the microbial activity regulating substance 1704 different, it is possible to cause different substances to act on different parts of the microorganism.

FIG. 18 shows a schematic diagram of a transport medium 1800 according to still another embodiment of the present invention. A transport medium 1800 shown in FIG. 18 encloses a microbial activity-regulating substance 1804 inside the lipid bilayer of the liposome which is the outermost layer 1801, and encloses a microbial activity-regulating substance 1803 inside the lipid bilayer of the inner-layer liposome 1802. The microbial activity regulating substance 1803 and the microbial activity regulating substance 1804 may be the same substance or different substances.

When the water solubility of the microbial activity regulators 1803 and 1804 is high, they are taken into liposome vesicles as shown in FIGS. 16 and 17, and the microbial activity regulators 1803 and 1804 are low in water solubility (highly hydrophobic). In some cases, it is incorporated into the lipid bilayer of the liposome as shown in FIG. As described above, the transport medium of the present invention can be efficiently transported to microorganisms regardless of whether it is a water-soluble microbial activity regulator or a hydrophobic microbial activity regulator.

The inner-layer liposome 1802 enclosing the microbial activity-regulating substance 1803 is the same as the known method for producing liposomes, and for example, the Bangham method can be used. Specifically, liposome constituents such as phospholipids and microbial activity regulator 1803 are dissolved in an organic solvent such as chloroform, and the organic solvent is distilled off to obtain a lipid thin film. Water is added thereto, and hydrated and dispersed using ultrasonic treatment or a vortex mixer, whereby inner-layer liposomes 1802 enclosing the microbial activity-regulating substance 1803 are obtained. Other methods for producing liposomes, such as reverse phase evaporation, ethanol injection, and ether evaporation, may be used.

The transport medium 1800 can be produced by incorporating the inner-layer liposome 1802 thus obtained into the liposome or biodegradable polymer that is the outermost layer 1801 by a known method.

In the case where the outermost layer 1801 is a liposome, it is possible to manufacture a transport medium 1800 that is a liposome including the inner layer liposome 1802 enclosing the microbial activity regulating substance 1803 by controlling the manufacturing conditions of the liposome.

Micelle is a collection of amphiphilic compounds in a spherical shape. In micelles, the lipophilic part of the amphiphilic compound is gathered toward the center of the sphere and the hydrophilic part is gathered towards the outside of the sphere. For this reason, the micelle can take in a hydrophobic substance and dissolve it in water. Examples of the amphiphilic compound include a copolymer of a hydrophilic polymer (such as polyethylene glycol) and a hydrophobic polymer (such as polyamino acid).

Emulsion is one in which two liquids that do not mix together become fine droplets and are dispersed in the other phase. By emulsifying an oil phase in which a hydrophobic substance is dissolved, it can be easily diffused into water as fine droplets. Examples of the oil phase include cyclohexane, dodecane, and ethyl acetate.

Examples of peptides and proteins that can be used as a transport medium include albumin, α1-acid glycoprotein, and elastin. Proteins and peptides can be used as transport media because they have high affinity for specific sites and are easily taken up by living organisms.

Examples of metal nanoparticles that can be used as a transport medium include gold, silver, platinum, copper, nickel, and iron oxide. For example, since various compounds such as polyethylene glycol can be bonded to gold nanoparticles through a thiol group, various substances can be attached and used as a transport medium. Commercially available metal nanoparticles can be used. In addition, crown ether, azacrown ether (for example, cyclen), porphyrin, or the like that is chemically modified with polyethylene glycol or the like on an alkali metal or alkaline earth metal such as sodium, potassium, or calcium may be used.

Examples of carbon nanotubes that can be used as a transport medium include single-walled carbon nanotubes and multi-walled carbon nanotubes. By utilizing the hydrophobic properties of carbon nanotubes and the space inside, it is possible to attach a substance and use it as a transport medium. A commercially available carbon nanotube can be used.

Fullerene is a spherical substance composed of many carbon atoms. Examples of fullerene that can be used as a transport medium include C ₆₀ fullerene having ₆₀ carbon atoms and higher-order fullerene having _70, 74 carbon atoms and the like. Various substances can be attached to fullerenes by covalent bonds or the like and used as a transport medium.

Examples of the first polymer that can be used as a transport medium include polyethyleneimine, polyamidoamine, and polyethylene glycol. These polymers can be used as a transport medium because they can stably hold a substance inside by interaction with the substance. It is also characterized by being able to be stably maintained in an environment where microorganisms are present.

The first polymer may be in the form of a hydrogel, nanosphere, microsphere, or dendrimer. Since these polymers form a three-dimensional structure, they are suitable as a carrier medium.

A biodegradable polymer can be used as the first polymer. In the case of a bilayer structure similar to the example of the liposome shown in FIG. 16, the biodegradable polymer is used as the outermost layer of the transport medium of the transport medium, thereby diffusing the microbial activity regulator in the water environment where the microbes are present. Can be made. Since the outermost layer of the carrier medium is formed of a biodegradable polymer, it can be easily diffused and delivered to microorganisms even in an aqueous environment. Even if the outermost layer of the transport medium is broken and the contents are released, the microbial activity regulating substance is encapsulated in the inner layer liposome. Therefore, it can be avoided that the microbial activity regulating substance is directly subjected to the microbial degradation action. As a result, the delivery efficiency of the microbial activity regulator is improved.

As the biodegradable polymer, starch, alginic acid, cellulose, chitin, chitosan, gelatin, collagen, etc. can be used. The biodegradable polymer can be used as it is, and can also be used in the form of microspheres or gels using a curing agent. Further, a plurality of layer structures as shown in FIGS. 16 to 18 may be formed using a biodegradable polymer. By making the outermost layer a biodegradable polymer, the release timing of the microbial activity regulator tends to be easily adjusted.

Hydrogel is a polymer that is cross-linked to form a three-dimensional network structure, and a large amount of water is taken inside. Therefore, the substance can be included in the internal water. Specific examples of the hydrogel include gelatin, carboxymethylcellulose, collagen, alginic acid, acrylamide, and polyvinyl alcohol.

A spherical substance composed of a polymer having a diameter of 1 μm or less is called a nanosphere, and a substance having a diameter of several μm is called a microsphere. Substances can be encapsulated in nanospheres and microspheres by the interaction between the polymer and the substance. Specific examples of the nanosphere / microsphere include polyglycolic acid, polylactic acid, and lactic acid glycolic acid copolymer.

Dendrimer is a spherical polymer having a regularly branched structure and a particle size of about several nanometers. Substances can be held and transported in the internal space by hydrophobic interaction or ionic interaction. Since the structure can be controlled at the molecular level and many functional groups exist on the surface, it is advantageous for functionalization. Specific examples of the dendrimer include a polyamidoamine dendrimer, a polypropyleneimine dendrimer, a polylysine dendrimer, and a polyphenyl ether dendrimer.

(Method for preparing microorganism activity regulator)
The microorganism activity regulator includes a microorganism activity regulator and a transport medium. When liposomes are used as the carrier medium, the activity regulator for microorganisms can be prepared, for example, as follows. First, a phospholipid dissolved in an organic solvent is mixed with a microbial activity-regulating substance solution. Subsequently, after removing the organic solvent, purified water is added and heated to a temperature equal to or higher than the phase transition temperature of the phospholipid. Thereby, a liposome can be produced. At this time, when the microbial activity-regulating substance is a fat-soluble substance, it is taken into the inside of the lipid bilayer by hydrophobic interaction as shown in FIG. Thus, it is taken into the inner aqueous phase formed inside the lipid bilayer membrane.

When vesicles are used as the transport medium, the activity regulator for microorganisms can be prepared using, for example, microorganisms. When a microorganism that secretes the target microbial activity regulating substance in the form of a vesicle is cultured in the medium, the vesicle encapsulating the microbial activity regulating substance in the culture is secreted. This vesicle can be used as a microorganism activity regulator. The vesicle encapsulating the microbial activity regulator may be used after being purified from the above culture, or the above culture may be used as a microbial activity regulator without purification. Examples of microorganisms that secrete microbial activity-regulating substances in the form of vesicles include Paracoccus and Pseudomonas.

The method for producing a cationic liposome encapsulating a microbial activity-regulating substance is the same as the method for producing a known liposome, and for example, the Bangham method can be used. Specifically, constituent components of cationic liposomes such as phospholipids are dissolved in an organic solvent such as chloroform, and the organic solvent is distilled off to obtain a lipid thin film. A microbial activity regulator and water are added thereto and hydrated and dispersed using an ultrasonic treatment or a vortex mixer to obtain a cationic liposome encapsulating a microbial activity regulator. Other methods for producing liposomes, such as reverse phase evaporation, ethanol injection, and ether evaporation, may be used.

When micelles are used as the transport medium, for example, an activity regulator for microorganisms can be prepared by forming micelles by hydrophobic interaction or electrostatic interaction between an amphiphilic compound and a microorganism activity regulator. At this time, as shown in FIG. 7, the micelle forms a spherical micelle with the microbial activity regulating substance 703 that is a hydrophobic substance as the center, the hydrophobic group 702 facing inside, and the hydrophilic group 701 facing outside. Can be dissolved in water.

When an emulsion is used as the transport medium, it can be prepared, for example, by adding a surfactant to an oil phase in which a microbial activity regulator is dissolved and dispersing it in water.

When a peptide or protein is used as a transport medium, for example, a microorganism activity regulator can be prepared by binding a peptide or protein and a microorganism activity regulator by hydrophobic interaction or ionic interaction. It is preferable to use albumin when the microbial activity-regulating substance is an acidic or neutral substance, and α1-acid glycoprotein when it is a basic substance. In addition, the microbial activity regulating substance is incorporated into a protein and transported as shown in FIG. The mode in which the microbial activity-regulating substance 802 is incorporated into the peptide or protein may be a mode in which the three-dimensional structure 801 of the protein is enclosed in a plurality of helix structures or loop structures, or a mode in which the microbial activity regulating substance 802 is attached to the surface of the three-dimensional structure, particularly a recess. .

When metal nanoparticles are used as the transport medium, for example, as shown in FIG. 9, physical adsorption of the metal nanoparticles 901 bonded to the metal particles 901 via the thiol group of the modification group 902 and the microbial activity regulator 903 is performed. Thus, an activity regulator for microorganisms can be prepared.

When carbon nanotubes are used as the transport medium, for example, as shown in FIG. 10A, a microbial activity regulating substance 1002 is adhered to the surface or inside of the carbon nanotubes 1001 by hydrophobic interaction, adsorption action, or the like. A microorganism activity regulator can be prepared. When the microbial activity regulating substance 1002 is a hydrophilic substance, a chain-like modifying group 1003 such as polyethylene glycol is covalently bonded to the surface of the carbon nanotube 1001 outward as shown in FIG. Carbon nanotubes can be used.

When fullerene is used as the transport medium, if the microbial activity-regulating substance 1102 is a hydrophobic substance, it can be attached to the side surface of the fullerene 1101 by hydrophobic interaction or the like as shown in FIG. It can also be included. Further, if the microorganism regulating substance 1102 is a hydrophilic substance, as shown in FIG. 11B, a chain-like modifying group 1102 such as polyethylene glycol is covalently bonded from the surface of the fullerene three-dimensional structure to the outside. The fullerene 1101 can be prepared.

When the first polymer is used as the transport medium, as shown in FIG. 12, for example, the first polymer 1201 and the microbial activity regulator 1202 are bonded by a hydrophobic interaction or the like, thereby microbial activity regulator. Can be prepared.

When the first polymer is a hydrogel, the hydrogel is swollen in a solvent in which a microbial activity regulator is dissolved or dispersed, so that the first polymer 1301 has a network structure as shown in FIG. The microorganism 130 may be held in the solvent 1303 and the microorganism 130 may be dissolved in the solvent 1303 to prepare a microorganism activity regulator.

When the first polymer is nanosphere / microsphere, the activity regulator for microorganisms can be prepared as follows. By mixing the microbial activity regulator and the nanosphere / microsphere in a solvent, as shown in FIG. 14, the microbial activity regulator is converted into the nanosphere / microsphere by the interaction between the nanosphere / microsphere 1401 and the microbial activity regulator 1402. Can be included in the sphere.

When the first polymer is a dendrimer, as shown in FIG. 15, by mixing the microbial activity regulator 1502 and the dendrimer 1501 in a solvent, the hydrophobic interaction between the dendrimer and the microbial activity regulator, ions A microbial activity regulating substance can be retained in the internal space of the dendrimer by the interaction. Further, the microbial activity regulating substance may be chemically bonded to a dendrimer. The microbial activity regulator chemically bonded to the dendrimer is released as the dendrimer is decomposed.

(Modification of transport medium)
The carrier medium may be modified with a sugar chain, an antibody, or a second polymer. By modifying the carrier medium with these substances, it is possible to selectively deliver the above-mentioned microbial activity regulator to microorganisms having a specific antigen, or to control the persistence of the microbial activity regulator. become. Examples of the sugar chain that modifies the transport medium include oligosaccharide, galactose, and fucose. Examples of the antibody that modifies the transport medium include those that use lipopolysaccharide, flagella, capsule, pili and the like as antigens. In addition, examples of the second polymer that modifies the transport medium include polyethylene glycol, polyethyleneimine, and polyamidoamine. The second polymer that modifies the delivery medium may be in the form of a hydrogel, nanosphere, microsphere, or dendrimer.

(Transportation medium modification method)
As a method for modifying the transport medium with a sugar chain, an antibody or a second polymer, for example, a method of binding a sugar chain, an antibody or a second polymer to a molecule constituting the transport medium using a chemical crosslinking agent. Can be mentioned. Chemical crosslinking agents include 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride (EDC), N-hydroxysuccinimide (NHS), succinimidyl-4- (N-maleimidomethyl) cyclohexane-1-carboxylate (SMCC).

(Method of regulating the activity of microorganisms)
By administering the above-mentioned microorganism activity regulator to a microorganism, the activity of the microorganism can be regulated. Here, the microbial activity refers to an activity in which a microorganism decomposes a specific substance (pollutant substance). Improvement of microbial activity means that the ability of a unit amount of microorganisms to decompose a specific pollutant is improved. In addition, a decrease in microbial activity means a decrease in the ability of a unit amount of microorganisms to decompose a specific pollutant. Examples of the pollutant include sugars such as glucose and maltose; alcohols such as methanol; aldehydes such as formaldehyde; organic solids such as straw; starch, protein, ammonia, nitrate, dimethyl sulfoxide (DMSO) and the like. In particular, microbial activity that decomposes ammonia into nitrate is called nitrification activity, and microbial activity that decomposes nitrate into nitrogen is called denitrification activity.

Examples of microorganisms to which the above activity regulator for microorganisms acts effectively include Vibrio, Aeromonas, Streptomyces, Streptococcus, Lactobacillus, Alcaligenes, Ralstonia, Achromobacter, and Halomonas ( Halomonas, Burkholderia, Pseudomonas, Rhodobacter, Paracoccus, Sphingobacterum, Flavobacterium, Flavobacterium Bacillus), Aerobacter, Brevibacterium, Corynebacterium, Comomanas, Micrococcus, Spirillum, Zogrelo e (Clostridium), Dehalococcoides, Aminomonas, Geobacter, Desulfuromonas, Desulfovibrio, Syntrobacter s. Sta hylococcus), Methanobacterium genus (Methanobacterium), Mesanosupiriramu genus (Methanospirillum), Mesanosarushina genus (Methanosarcina), Metanorinea genus (Methanolinea) meth knob Levi genus (Methanobrevibacter), include methanosaeta (Methanosaeta) or the like.

The above-described method for regulating the activity of microorganisms can be applied to various objects such as marine structures and pipelines that are affected by biofilm formation. For example, in a microbial battery, it can be applied to a technique for forming a dense biofilm on an anode and a cathode. In addition, it can be applied to a technology for forming a biofilm to prevent the attachment of large organisms to offshore structures.

(Method of adjusting the activity of microorganisms present in the reaction tank of biological wastewater treatment equipment)
In one embodiment, the above-mentioned activity regulator for microorganisms is for microorganisms present in a reaction vessel of a biological wastewater treatment facility. By adding the above-described activity regulator for microorganisms to the reaction tank of the biological wastewater treatment facility, the activity of microorganisms existing in the reaction tank can be improved or decreased. Thereby, the waste water treatment performed in the reaction tank can be promoted or suppressed as necessary.

Hereinafter, the biological wastewater treatment facility will be described in detail with reference to FIGS. In the description of the drawings, the same elements are denoted by the same reference numerals, and redundant description is omitted.

(Biological wastewater treatment facility 1)
FIG. 19 is a schematic configuration diagram showing an activated sludge treatment apparatus according to the biological wastewater treatment facility 1. As shown in FIG. 19, the activated sludge treatment apparatus 1900 includes an adjustment tank 1901, an aeration tank 1902, and solid-liquid separation means 1903.

The organic wastewater is first introduced into the adjustment tank 1901 via the organic wastewater introduction line L1, and after removing suspended matters and the like, is introduced into the aeration tank 1902 via the line L2. Organic waste water is introduced into the aeration tank 1902 and aerated to decompose organic matter by activated sludge (aerobic microorganisms), thereby generating stable substances such as water, carbon dioxide, sulfate, nitrate, and the like, and the sludge is propagated. Treated water containing sludge after being aerated in the aeration tank 1902 is introduced into the solid-liquid separation means 1903 via the line L3. In the solid-liquid separation means 1903, sludge and treated water are separated into solid and liquid, the separated treated water is discharged out of the system from the treated water discharge line L4, and the separated sludge is returned to the aeration tank 1902 as returned sludge. A part of the separated sludge is returned via the line L5, and is discharged to the outside from the sludge discharge line L6 as surplus sludge. Here, the solid-liquid separation means 1903 is a settling tank, and the separation treated water is the supernatant and the separation sludge is the sedimentation sludge. However, if solid-liquid separation is possible, for example, a membrane separation device or a centrifugal separation device may be used. good.

Here, the activated sludge treatment apparatus 1900 according to the biological wastewater treatment facility 1 includes a signal substance addition means 1904 (addition means) for introducing an intercellular information transmission substance (signal substance) into the aeration tank 1902.

The signal substance added by the signal substance addition means 1904 may be, for example, in a state of being encapsulated by a transport medium or in a state of being combined with the transport medium to form a complex. Examples of the transport medium for transporting the signal substance include vesicles, liposomes, micelles, emulsions, peptides, proteins, metal nanoparticles, carbon nanotubes, and polymers. These can be used alone or in combination of two or more.

In the activated sludge treatment apparatus 1900 according to the biological wastewater treatment facility 1, when the signal substance is added to the aeration tank 1902 by the signal substance addition means 1904, at least the operation of the aeration tank 1902 in the activated sludge treatment apparatus 1900 is performed. The signal substance may be added to the activated sludge in a state where the activated sludge in the aeration tank 1902 has settled in the lower part of the tank.

The activated sludge that has settled in the lower part of the aeration tank 1902 has a higher concentration than the activated sludge concentration in the aeration tank 1902 during the aeration treatment operation. Thus, with a configuration in which the signal substance is added to the activated sludge in a state where the concentration is higher than the concentration of the activated sludge in the aeration tank 1902 during normal operation, the activated sludge is more concentrated. Signaling substances can be added. Accordingly, since the signal substance can be effectively reached with respect to the microorganisms in the activated sludge, the activity of the microorganisms by the signal substance can be regulated more efficiently.

In this regard, for example, a case where a signal substance is added to an aeration tank 1902 having a volume of 1000 m ³ will be considered. Here, when a signal substance is added in the operating aeration tank 1902, it is assumed that a signal substance having a molecular weight of 500 MW needs to be administered at 10 μmol / L in order to suitably adjust the activity of the microorganism. In this case, 5 kg of the signal substance needs to be added to the aeration tank 1902. On the other hand, it is assumed that the operation of the aeration tank 1902 is stopped and the activated sludge is naturally settled, so that the volume of the activated sludge is reduced to 30% compared to that during operation. In this case, assuming that the signal substance is administered at a concentration of 1 μmol / L with respect to the activated sludge that has been concentrated by sedimentation, 1.5 kg of the signal substance is sufficient, and the less signal substance activates in the aeration tank 1902. It is possible to regulate the activity of microorganisms.

In the biological wastewater treatment facility 1, the configuration in which the aeration process in the aeration tank 1902 is stopped and the signal substance is added has been described. However, for example, by reducing the driving power without completely stopping the aeration process. It can also be set as the state where activated sludge is stored in the tank downward compared with the time of normal operation, and a signal substance is added with respect to the stored activated sludge. Even in this case, it is possible to obtain an effect of regulating the activity by causing the signal substance to act on the microorganism more effectively.

In addition, although it is characterized by providing the addition process which adds a signal substance to an aeration tank in this invention, it is not limited to the aspect of the said biological waste water treatment equipment 1 about how an addition process is performed. Hereinafter, other aspects of the addition process will be described using the biological wastewater treatment facilities 2 to 4.

(Biological wastewater treatment facility 2)
FIG. 20 is a schematic configuration diagram showing an activated sludge treatment apparatus according to the biological wastewater treatment facility 2. The activated sludge treatment apparatus 2000 is different from the activated sludge treatment apparatus 1900 according to the biological wastewater treatment facility 1 in the following points. That is, in place of the signal substance addition means 1904, signal substance addition means 2004 and 2014 are provided.

The signal substance addition means 2004 shows a configuration in which a signal substance is added to the activated sludge after the solid-liquid separation by the solid-liquid separation means 1903. The activated sludge after solid-liquid separation is concentrated as compared with the activated sludge in the tank during the aeration operation. Since the concentrated activated sludge after such solid-liquid separation is returned to the aeration tank 1902 via the return line L5, a signal substance is added to the activated sludge separated in the solid-liquid separation means 1903. Compared with the configuration in which the signal substance is added to the aeration tank 1902 during the aeration treatment operation, the activity of the microorganisms by the signal substance can be adjusted more efficiently.

In addition, the signal substance adding unit 2014 is configured to add the signal substance to the activated sludge moving in the return line L5 for returning the activated sludge separated into the solid-liquid separation unit 1903 to the aeration tank 1902. Show. The activated sludge moving in the return line L5 is concentrated compared to the activated sludge in the tank during the aeration operation. By adding a signal substance to the activated sludge returned to the aeration tank 1902 via the return line L5, the signal substance is added to the configuration in which the signal substance is added to the aeration tank 1902 during the aeration treatment operation. The activity of the microorganism can be regulated more efficiently.

In addition, although the activated sludge treatment apparatus 2000 shows a configuration in which two signal substance addition units 2004 and 2014 are provided, only one of the signal substance addition units 2004 and 2014 may be provided. And by setting it as the aspect provided with the process of adding a signal substance by a signal substance addition means in an activated sludge processing method, the regulation of the activity of microorganisms by a signal substance can be performed more efficiently.

(Biological wastewater treatment facility 3)
FIG. 21 is a schematic configuration diagram showing an activated sludge treatment apparatus according to the biological wastewater treatment facility 3. The activated sludge treatment apparatus 2100 is different from the activated sludge treatment apparatus 1900 according to the biological wastewater treatment facility 1 in the following points. That is, instead of the signal substance adding means 1904, a plurality of signal substance adding means 2104 to 2124 are provided at different positions along the return line L5.

Further, the signal substance addition means 2104 to 2124 are provided at different positions along the return line L5, and a return line for returning the activated sludge separated by the solid-liquid separation means 1903 to the aeration tank 1902. It has a function of adding a signal substance to the activated sludge moving in L5. Further, the addition amount of the signal substance by the signal substance addition means 2104 to 2124 is different from each other. In FIG. 3, as an example, the configuration in which the addition amount increases in the order of the signal substance addition means 2104, 2114, 2124 is indicated by the size of the arrow.

As described above, by providing a plurality of signal substance addition means 2104 to 2124 at different positions along the return line L5, an activity having a higher concentration than the concentration during operation in the aeration tank 1902 moving in the return line L5. In addition to improving the regulation effect of microbial activity by the signal substance by adding the signal substance to the sludge, the signal substance can be added more uniformly to the activated sludge. Can be adjusted more efficiently.

Further, the amount of the signal substance added in the plurality of signal substance addition means 2104 to 2124 is made different from each other, for example, depending on the state of the activated sludge such as how the activated sludge flows in the return line L5. The signal substance can be added more uniformly to the activated sludge, and the activity of the microorganisms by the signal substance can be adjusted more efficiently.

Note that the types of signal substances added from the plurality of signal substance addition means 2104 to 2124 may be different from each other.

(Biological wastewater treatment facility 4)
FIG. 22 is a schematic configuration diagram showing an activated sludge treatment apparatus according to the biological wastewater treatment facility 4. The activated sludge treatment apparatus 2200 is different from the activated sludge treatment apparatus 1900 according to the biological wastewater treatment facility 1 in the following points. That is, instead of the signal substance addition means 1904, a sludge amount detection means 2205 is provided in the return line L5, and the signal substance addition means 2204 adds the signal substance based on the result detected by the sludge amount detection means 2205. It is a point that has been.

The sludge amount detection means 2205 is a means for measuring the amount of return sludge (activated sludge) returned to the aeration tank 1902 via the return line L5, for example, a flow meter for measuring the flow rate of sludge flowing through the return line L5. Alternatively, a densitometer that detects the concentration of sludge can be applied. Further, both a flow meter and a concentration meter may be used.

The signal substance addition means 2204 is provided downstream of the sludge amount detection means 2205 in the return line L5, and the signal substance addition means 2204 is based on the amount of sludge (flow rate and / or concentration) detected by the sludge amount detection means 2205. It has the function of adding by adjusting the amount of substance added.

In the activated sludge treatment method using the activated sludge treatment apparatus 2200, the signal substance is added from the signal substance addition means 2204 based on the flow rate and / or concentration of the activated sludge detected by the sludge amount detection means 2205 in the signal substance addition step. Added. In this way, by adopting a configuration in which the addition amount of the signal substance is changed based on the flow rate and / or concentration of the activated sludge, the addition amount of the signal substance can be appropriately controlled according to the return amount of the activated sludge. Thus, the activity of the microorganism by the signal substance can be regulated more efficiently.

The biological wastewater treatment facility has been described above, but the present invention is not necessarily limited to the biological wastewater treatment facility described above, and various modifications can be made without changing the gist thereof.

For example, in the biological wastewater treatment facility, the return sludge is returned to the line L2 upstream from the aeration tank 1902, but may be returned directly to the aeration tank 1902.

(MLSS)
In the following experiments, the amount of activated sludge was measured based on MLSS (Mixed liquor suspended solids). The measurement of MLSS was performed by the following method. First, the activated sludge sample was placed in a centrifuge tube, centrifuged at 3000 rpm for 10 minutes, and then the supernatant was discarded. Next, water was added to the resulting precipitate and mixed well, followed by centrifugation in the same manner as above to discard the supernatant. The obtained precipitate was put in a pre-weighed evaporating dish and dried in a dryer at 105 to 110 ° C. for half a day. Subsequently, the mixture was allowed to cool in a desiccator and weighed. The mass obtained by subtracting the mass of the empty evaporating dish from the measured mass was taken as MLSS.

(Preparation of N-hexadecanoyl-L-homoserine lactone encapsulated in vesicles)
A Paracoccus AS6 strain that secretes N-hexadecanoyl-L-homoserine lactone (hereinafter sometimes referred to as “C16-HSL”) in the form of a vesicle (membrane vesicle) is cultured in a medium. Used as C16-HSL encapsulated in vesicles. The Paracoccus AS6 strain will be described later. The concentration of C16-HSL was measured by mass spectrometry.

(Examination of promotion of nitrification reaction)
An inorganic salt medium prepared so that ammonia nitrogen (NH ₄ ⁺ ) was 50 mM was placed in a test tube. Subsequently, activated sludge was added to the test tube so that MLSS was 5000 mg / L. Further, C16-HSL encapsulated in vesicles was added to the test tube to a final concentration of 10 μM. C16-HSL encapsulated in vesicles could be easily diffused into the medium even though C16-HSL was a hydrophobic substance. As a negative control, the above-mentioned inorganic salt medium added with only activated sludge was used. At the start of the experiment, after 24 hours of culture and 48 hours of culture, the ammonia nitrogen concentration in the medium was measured by the ion electrode method. The results are shown in FIG. In the system in which C16-HSL encapsulated in vesicles was added, the rate of decrease of ammonia nitrogen was increased compared to the negative control, indicating that the nitrification reaction was promoted.

(Preparation of an activity regulator for microorganisms containing N-3-oxododecanoyl-L-homoserine lactone)
N-3-oxododecanoyl-L-homoserine lactone (hereinafter sometimes referred to as “3oxoC12-HSL”) was encapsulated in liposomes to prepare an activity regulator for microorganisms. The composition of the liposome was hydrogenated soybean phosphatidylcholine (HSPC) 29 g / L and cholesterol 13 g / L. Liposomes were made by the following procedure known as the Bangham method.

First, HSPC, cholesterol and 3oxoC12-HSL (1 mM) were dissolved in 10 mL of chloroform. Subsequently, chloroform was removed using an evaporator, and a lipid film was formed on the bottom of the eggplant-shaped flask. Subsequently, a phosphate buffer was added to the eggplant-shaped flask on which the lipid film was formed, and hydrated and dispersed at 60 ° C. while stirring using a vortex mixer. By the above operation, a microorganism activity regulator containing 3oxoC12-HSL was obtained.

(Examination of promotion of nitrification reaction)
An inorganic salt medium prepared so that ammonia nitrogen (NH ₄ ⁺ ) was 14 mM was placed in a test tube. Subsequently, activated sludge was added to the test tube so that MLSS was 5000 mg / L. Furthermore, a microorganism activity regulator (liposome solution containing 3oxoC12-HSL) was added to the test tube in the same volume as the medium. The final concentration was 10 μM. The above-mentioned activity regulator for microorganisms could be easily diffused into the medium despite the fact that 3oxoC12-HSL is a hydrophobic substance. As a negative control, a phosphate buffer hydrated with liposomes was prepared in the same manner as the above-mentioned microbial activity regulator except that activated sludge was added to the above inorganic salt medium and no 3oxoC12-HSL was contained. The same volume as the medium was added. At the start of the experiment, after 24 hours, 48 hours and 72 hours of culture, the ammonia nitrogen concentration in the medium was analyzed by ion chromatography. The results are shown in FIG. In the system to which the activity regulator for microorganisms containing 3oxoC12-HSL was added, it was shown that the decrease rate of ammonia nitrogen was faster and the nitrification reaction was promoted as compared with the negative control.

(Paracoccus AS6 strain)
(Screening for high homoserine lactone-producing bacteria)
When homoserine lactone is present in the culture medium, a high homoserine lactone-producing bacterium was screened from activated sludge using a reporter strain that produces violetsein, a violet pigment. More specifically, the strain Chromobacterium violaceum CV026 responding to N-acyl-L-homoserine lactone (C4 to C8-HSL) having 4 to 8 carbon atoms in the acyl group and 10 to 10 carbon atoms in the acyl group. Chromobacterium violaceum strain VIR07 responding to 16 N-acyl-L-homoserine lactones (C10-C16-HSL) was used. Here, as N-acyl-L-homoserine lactone to which CV026 strain responds, N-butanoyl-L-homoserine lactone, N-3-oxobutanoyl-L-homoserine lactone, N-3-hydroxybutanoyl-L -Homoserine lactone, N-pentanoyl-L-homoserine lactone, N-3-oxopentanoyl-L-homoserine lactone, N-3-hydroxypentanoyl-L-homoserine lactone, N-hexanoyl-L-homoserine lactone, N- 3-oxohexanoyl-L-homoserine lactone, N-3-hydroxyhexanoyl-L-homoserine lactone, N-heptanoyl-L-homoserine lactone, N-3-oxoheptanoyl-L-homoserine lactone, N-3- Hydroxyheptanoyl-L-homoserine lactone N- octanoyl -L- homoserine lactone, N-3- oxo-octanoyl -L- homoserine lactone, N-3- hydroxy octanoyl -L- homoserine lactone, and the like. The homoserine lactone to which the VIR07 strain responds includes N-decanoyl-L-homoserine lactone, N-3-oxodecanoyl-L-homoserine lactone, N-3-hydroxydecanoyl-L-homoserine lactone, N-undecanoyl-L. -Homoserine lactone, N-3-oxoundecanoyl-L-homoserine lactone, N-3-hydroxyundecanoyl-L-homoserine lactone, N-dodecanoyl-L-homoserine lactone, N-3-oxododecanoyl-L -Homoserine lactone, N-3-hydroxydodecanoyl-L-homoserine lactone, N-tridecanoyl-L-homoserine lactone, N-3-oxotridecanoyl-L-homoserine lactone, N-3-hydroxytridecanoyl-L -Homoserine lactone, N- Tradecanoyl-L-homoserine lactone, N-3-oxotetradecanoyl-L-homoserine lactone, N-3-hydroxytetradecanoyl-L-homoserine lactone, N-pentadecanoyl-L-homoserine lactone, N-3- Oxopentadecanoyl-L-homoserine lactone, N-3-hydroxypentadecanoyl-L-homoserine lactone, N-hexadecanoyl-L-homoserine lactone, N-3-oxohexadecanoyl-L-homoserine lactone, N -3-Hydroxyhexadecanoyl-L-homoserine lactone and the like.

The activated sludge was diluted and applied to an agar medium to obtain an isolated strain. An isolated strain and a reporter strain obtained from activated sludge were applied to adjacent positions on the agar medium. After culturing for several days, the isolated strain was judged to be a homoserine lactone-producing bacterium for the reporter strain that secreted a purple pigment (violacein). The results are shown in Table 1. In the table, the greater the number of “+”, the greater the amount of violacein produced. By this method, the AS6 strain, which is a high homoserine lactone-producing bacterium, was obtained. The AS6 strain was deposited on May 18, 2012 at the Patent Microorganism Depositary, National Institute of Technology and Evaluation, under the accession number NITE P-1363.

(Analysis of signal substance produced by AS6 strain)
The AS6 strain was cultured in LB medium, and the culture solution was centrifuged at 6,000 × g for 15 minutes. Subsequently, the centrifuged supernatant was filtered with a cellulose acetate filter having a pore size of 0.2 μm, and then ultracentrifuged at 1500,000 × g for 3 hours at 4 ° C. After discarding the supernatant, the pellet was washed with HEPES-NaCl solution (10 mM HEPES-0.85 (w / v)% NaCl). Next, 40%, 35, 30, 25, 20, 15, 10 (v / v)% of Optiprep (trade name, Axis Shield) mixed in HEPES-NaCl solution, Then, a gradient layer was formed by overlaying in a centrifuge tube, and a pellet dissolved in a 40 (v / v)% Optiprep-HEPES-NaCl solution was added on top. Subsequently, this gradient was ultracentrifuged at 1000000 × g for 3 hours. After ultracentrifugation, each layer was collected, protein concentration was determined by Bradford method (Bradford, M. 1976. Anal. Biochem. 72, 248-254.), And Stewart method (Stewart, J. 1980. Anal. Biochem. 104, 10-14.) And the phospholipid concentration was analyzed. A layer having a high protein concentration and a high phospholipid concentration was defined as a layer from which vesicles were extracted.

(Vesicle confirmation)
The sample of the layer from which the vesicles were extracted was observed by transmission electron microscope (TEM) observation using negative staining. As a result, it was confirmed that vesicles exist.

(Identification of signal substance)
The sample of the layer from which the vesicles were extracted was analyzed by mass spectrometry, and the substance was identified and the concentration was measured by comparison with a standard signal substance. As a result, it was revealed that the AS6 strain secreted about 1.0 μM of C16-HSL in the culture solution, and that at least half of the AS6 strain was contained in the vesicle. Hydrophobic substances such as C16-HSL are believed to be likely to remain on the cell membrane. For this reason, it was considered that the AS6 strain secretes a large amount of C16-HSL by enclosing C16-HSL in vesicles and discharging it outside the cell.

From the above results, the inventors found that C16-HSL produced by the AS6 strain is secreted in the form of vesicles. When a hydrophobic signal substance is chemically synthesized, the operability may be poor, for example, it is necessary to use an organic solvent to dissolve in water. In contrast, C16-HSL produced by the AS6 strain was hydrophilic because it was secreted in the form of vesicles, and it was revealed that it can be used as it is added to the aqueous system.

(Identification of AS6 strain)
The attribution taxon of the AS6 strain was estimated by 16S rDNA (16S rRNA gene) base sequence analysis, morphology observation and physiological / biochemical tests.

(Base sequence analysis of 16S rDNA)
The AS6 strain was cultured on an agar medium at 30 ° C. for 24 hours to extract DNA. For example, “Yoshiyoshi Nakagawa, Hiroko Kawasaki, Gene analysis method, 16S rRNA gene base sequencing method, edited by the Japanese Society for Actinomycetes,“ Classification and Identification of Actinomycetes ”, P. 88-117, Japan Society for the Science and Technology (2001) The base sequence of 16S rDNA was determined based on the general method described in the above, and homology search with a database and simple molecular phylogenetic analysis were performed. Simplified molecular phylogenetic analysis was performed using the Apollon DB-BA database Ver. 7.0 (trade name, Techno Suruga Laboratories, March 2011 edition, search date May 2, 2012).

Fig. 3 shows the results of simple molecular phylogenetic analysis. In FIG. 3, the lower left line represents the scale bar, and the number located at the branch of the system branch represents the bootstrap value. The T at the end of the stock name indicates that type of reference stock. As a result of homology search, the base sequence of 16S rDNA of AS6 strain showed high homology to the base sequence of rDNA belonging to the genus Paracoccus. In addition, as a result of simple molecular phylogenetic analysis, the AS6 strain is contained in a cluster formed by species of the genus Paracoccus, and is closely related to P. versustus and P. bengalerensis. It has been shown. However, there was a possibility that the AS6 strain was attributed to Paracoccus verustus or Paracoccus bengalensis and a different species from these, and the species name was not identified.

(Morphological observation and physiological / biochemical test)
Morphological observation with an optical microscope and, for example, “BARROW and FELTHAM, Cowan and Steel's Manual for the Identification of Medical Bacteria. 3rd edition. 1993, Cambridge University, based on the method described in Cambridge Presity. Tests were performed for reaction, aoxidase reaction, acid / gas evolution from glucose, and glucose oxidation / fermentation. Also, biochemistry of nitrate reduction, indole production, glucose acidification, arginine dihydrolase, urease, esculin hydrolysis, gelatin hydrolysis, β-galactosidase and cytochrome oxidase using API20NE kit (trade name, manufactured by bioMerieux) Test, as well as glucose, L-arabinose, D-mannose, D-mannitol, N-acetyl-D-glucosamine, maltose, potassium gluconate, n-capric acid, adipic acid, dl-malic acid, sodium citrate, acetic acid An assimilation test on phenyl was performed. Furthermore, the test for the presence or absence of growth under anaerobic conditions and the presence or absence of growth at 20 ° C., and the assimilation test for glycerol, saccharose, D-fructose, L-alanine, L-sodium aspartate and sodium lactate Went.

Results are shown in Tables 2-4. The AS6 strain was a gram-negative bacillus having no motility, did not oxidize glucose, and both the catalase reaction and oxidase reaction were positive. As a result of the test using the API20NE kit, the AS6 strain did not reduce nitrate, assimilated glucose, L-arabinose and D-mannose, and did not assimilate n-capric acid and sodium citrate. These properties were similar to those of Paracoccus verustus and Paracoccus bengalensis but were not completely consistent. In particular, it differs from the properties of Paracoccus bengalensis in assimilating D-mannose, and it differs from the properties of Paracoccus verustus in the assimilation of lactose, and it does not reduce nitrates in both Paracoccus verustus and Paracoccus bengalensis. It was different.

+: Positive,-: Negative

From the above results, it was estimated that the AS6 strain is a bacterium belonging to the genus Paracoccus which is closely related to Paracoccus verustus and Paracoccus bengalensis.

(Reporter assay)
Evaluation was performed using a reporter strain Pseudomonas aeruginosa PAO1 δlasI pME PlasI GFP that fluoresces in response to 3oxoC12-HSL. In the above reporter strain, a GFP (green fluorescent protein, aequorin) expression gene is inserted into a plasmid, and a lasI promoter induced by 3oxoC12-HSL is inserted upstream of the GFP expression gene. It is incorporated into the strain Pseudomonas aeruginosa PAO1. Although the reporter strain has its own ability to produce 3oxoC12-HSL, it expresses GFP in response to 3oxoC12-HSL in the environment and exhibits fluorescence. Liposomes were produced by the Bangham method.

Experimental Method 1) A plurality of systems in which a reporter strain was introduced into an LB liquid medium was prepared, and 3oxoC12-HSL (50 nM) was dissolved in DMSO or added in the form of being encapsulated in liposomes having the compositions shown in Tables 5 and 6.
2) The culture was stationary at 37 ° C. for 12 hours.
3) Using a fluorometer, the culture solution was irradiated with ultraviolet rays to analyze the degree of fluorescence development of GFP.

As shown in FIG. 4, when the 3oxoC12-HSL-containing liposome was used, the fluorescence intensity was enhanced to about 15 times compared to the case where only 3oxo-C12-HSL was added. In addition, uncharged liposomes and negatively charged liposomes showed almost the same fluorescence intensity.

As shown in FIG. 5, in liposomes A, B, and C in which cholesterol was added to the liposome to adjust the fluidity of the lipid bilayer, liposome B had the highest fluorescence intensity.

600,700,800,900,1000,1100,1200,1300,1400,1500 ... microbial activity regulator, 601,701 ... hydrophilic group, 602,702 ... hydrophobic group, 801,901,1001,1101, 1201, 1301, 1401, 1501 ... transport medium, 603, 703, 802, 903, 1002, 1102, 1202, 1302, 1402, 1502 ... microbial activity regulator, 902, 1003, 1103 ... modifying group, 1303 ... solvent, 1600 , 1700, 1800 ... transport medium, 1601, 1701, 1801 ... outermost layer, 1602, 1702, 1802 ... inner liposome, 1603, 1703, 1704, 1803, 1804 ... microbial activity regulator, 1901 ... adjustment tank, 1902 ... aeration tank 1903 ... Solid- liquid separation tank 1904,2004,2014,2104,2114,2124,2204 ... signal substances adding means, 2205 ... sludge quantity detecting means, 1900,2000,2100,2200 ... activated sludge treatment apparatus.

Claims (13)

1. An activity regulator for microorganisms present in a reaction tank of a biological wastewater treatment facility containing a microorganism activity regulator and a transport medium.
2. The microbial activity-regulating substance is one or more substances selected from the group consisting of an intercellular signal transduction substance, a nucleic acid that affects prokaryotic activity, and a protein that affects prokaryotic activity. The activity regulator for microorganisms described in 1.
3. The microorganism activity regulator according to claim 1 or 2, wherein the microorganism activity regulator is N-acyl-L-homoserine lactone.
4. The carrier medium is one or more substances selected from the group consisting of vesicles, liposomes, micelles, emulsions, peptides, proteins, metal nanoparticles, carbon nanotubes, fullerenes and first polymers. The activity regulator for microorganisms as described in any one of these.
5. The microbial activity regulator according to claim 4, wherein the first polymer is a hydrogel, nanosphere, microsphere, or dendrimer.
6. The microorganism activity regulator according to any one of claims 1 to 5, wherein the transport medium is modified with a sugar chain, an antibody, or a second polymer.
7. The microorganism activity regulator according to claim 6, wherein the second polymer is polyethylene glycol, polyethyleneimine, or polyamidoamine.
8. The microbial activity regulator according to any one of claims 1 to 7, wherein the transport medium is a liposome.
9. The microbial activity regulator according to claim 8, wherein the liposome comprises egg yolk lecithin or hydrogenated soybean phosphatidylcholine.
10. The microbial activity regulator according to claim 9, wherein the liposome further comprises cholesterol.
11. The activity regulator for microorganisms according to claim 9 or 10, wherein the liposome further comprises stearic acid.
12. The microbial activity regulator according to any one of claims 1 to 11, wherein the microbial activity of the microbial activity regulating substance is nitrification activity.
13. A method for adjusting the activity of microorganisms present in a reaction tank of a biological wastewater treatment facility, wherein the microorganism is used in the reaction tank of a biological wastewater treatment facility. Adding the activity regulator.

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