WO2022118960A1

WO2022118960A1 - Microorganism carrier and method for producing same

Info

Publication number: WO2022118960A1
Application number: PCT/JP2021/044483
Authority: WO
Inventors: 友子荒川; 克敏堀
Original assignee: 国立大学法人東海国立大学機構
Priority date: 2020-12-04
Filing date: 2021-12-03
Publication date: 2022-06-09

Abstract

Provided are: a microorganism carrier which has good handling properties and excellent microorganism treatment performance; and a method for producing the microorganism carrier. A carrier 1 is a microorganism carrier capable of carrying a microorganism. The carrier 1 comprises an expansion-molded article comprising a thermoplastic resin 3 and an inorganic powder 4, and has asperities 2 on the surface thereof, in which the inorganic powder 4 is contained in an amount of 15 to 70% by mass relative to the total amount of the thermoplastic resin 3 and the inorganic powder 4, and the inorganic powder comprises at least one component selected from the group consisting of silicon dioxide, aluminum oxide, calcium silicate hydrate, diatomaceous earth, maifan stone, shell, bone meal, calcium carbonate, boehmite, zeolite, apatite, kaolin, mullite, spinel, olivine, sericite, bentonite, wollastonite, anorthite and mica.

Description

Microbial carrier and its manufacturing method

The present invention relates to a microbial carrier (hereinafter, also simply referred to as a carrier) and a method for producing the same, and more particularly to a microbial carrier that carries, proliferates, and desorbs microorganisms and a method for producing the same. The present invention also relates to a method for preventing diseases of fish and shellfish, and more particularly to a method for preventing diseases of fish and shellfish bred in aquaculture farms, ornamental plants, aquariums and the like.

A method using microorganisms is known as a method for purifying industrial wastewater such as factory wastewater, domestic wastewater, breeding water for fish and shellfish, breeding water for aquatic plants, and the like. For example, by putting a carrier carrying a microorganism into the water to be treated and allowing water to pass therethrough, harmful substances in the water to be treated can be decomposed by the action of the microorganism. As such microorganisms, aerobic microorganisms that require oxygen for growth and anaerobic microorganisms that do not require oxygen for growth are used.

Conventionally, a carrier made of ceramic powder, which is an inorganic powder, is known as a carrier. Since ceramics have many pores on the surface, they are suitable for the adhesion of microorganisms. For example, Patent Document 1 describes a carrier made of a ceramic molded product containing warastonite and anorthite. On the surface of this carrier, two types of voids having a large pore diameter in the range of 50 to 1000 μm and a pore diameter in the range of 0.1 to 10 μm are formed.

In addition, a technique using casting waste sand generated from the casting manufacturing process as a microbial carrier is also known. For example, Patent Document 2 describes a carrier obtained by heat-treating cast waste sand at 180 ° C. or higher and 400 ° C. or lower.

On the other hand, the demand for fish and shellfish as animal protein is increasing all over the world. In order to meet the demand, the cultivation of fish and shellfish, which is a stable supply means, has become an important position. In recent years, closed-circulation aquaculture performed on land has attracted attention. Since this aquaculture manages the environment in a closed facility, it is not easily affected by external factors such as weather and red tide, and the water temperature can be adjusted, resulting in excellent productivity. However, water quality management is particularly important due to the closed environment, and it is regarded as a problem that fish and shellfish get sick as the water quality deteriorates.

Conventionally, antibacterial agents and vaccines have been administered to prevent diseases of fish and shellfish (especially fish). For example, Patent Document 3 describes oral administration of antibacterial agents such as oxytetracycline and doxycycline to fish. Further, Patent Document 4 describes a vaccine containing an inactivated fish Streptococcus disgalactier as an antigen.

Japanese Unexamined Patent Publication No. 7-60279 Japanese Unexamined Patent Publication No. 2018-114494 Japanese Unexamined Patent Publication No. 2019-172619 Japanese Unexamined Patent Publication No. 2007-326794

Regarding the carrier on which microorganisms are carried, the carrier made of a ceramic molded body has a large surface area because it has a porous structure, and has excellent adhesion to microorganisms. However, the weight of the carrier is heavy, and it cannot be said that the handling is good. In addition, since firing is required, the cost tends to be high. The same can be said for carriers made from foundry waste sand. On the other hand, as a lightweight and relatively low-cost carrier, a carrier made of a resin molded body is also known, but the surface area is smaller than that of a carrier made of a ceramic molded body, and the carrier tends to be inferior in durability.

By the way, in order to fully exert the function of the microorganisms carried on the carrier, it is important to bring the treated water into contact with the microorganisms as much as possible. Therefore, in general, the treatment capacity is improved by flowing the water to be treated or the carrier by stirring or the like, but it is desirable that the treatment capacity of microorganisms can be improved more easily.

On the other hand, although antibacterial agents are effective against many microorganisms in the prevention of fish and shellfish diseases, there are problems such as the emergence of resistant bacteria and restrictions on use before shipment. In addition, vaccines are often administered by injection, the administration work is complicated, and the types of pathogens that can be dealt with are limited. Therefore, it is desirable to be able to continuously take preventive measures against illness by a simple method.

In addition, since the epidermis of fish is always in contact with microorganisms in water, diseases caused by transdermal infection can be a problem.

The present invention has been made to deal with such a situation, and an object of the present invention is to provide a microbial carrier having good handleability and excellent microbial processing ability and a method for producing the same. Another object of the present invention is to provide a preventive method capable of sustainably and easily preventing fish and shellfish diseases.

The microbial carrier of the present invention is a microbial carrier that carries a microbial substance, and the microbial carrier is a foamed molded product containing a thermoplastic resin and an inorganic powder, and has irregularities on the surface. , 15% by mass to 70% by mass with respect to the total amount of the thermoplastic resin and the inorganic powder. Further, the foam molded product is an extruded foam molded product, and the inorganic powder is characterized by containing 35% by mass to 70% by mass with respect to the total amount of the thermoplastic resin and the inorganic powder.

The inorganic powders include silicon dioxide, aluminum oxide, calcium silicate hydrate, diatomaceous earth, barley stone, shell, bone meal, calcium carbonate, boehmite, zeolite, apatite, kaolin, mulite, spinel, olivine, sericite, bentonite, etc. It is characterized by being at least one selected from the group consisting of wallastonite, anausite and mica.

The specific surface area of the microbial carrier is 1 m ² / g to 5 m ² / g.

The production method for producing the microbial carrier of the present invention is characterized in that at least the above-mentioned thermoplastic resin, the above-mentioned inorganic powder and the foaming agent are kneaded and foam-molded.

The manufacturing method is characterized in that raw materials other than the foaming agent are pelletized in advance, the obtained pellets and the foaming agent are kneaded, and extrusion foam molding is performed.

The preventive method of the present invention is a preventive method for preventing a disease of fish and shellfish, and is a step of adding a microorganism having an inhibitory action against the pathogenic microorganism of the disease to a microbial carrier having a desorbing action of the microorganism. It is characterized by having a step of putting the above-mentioned microbial carrier into water in which the above-mentioned fish and shellfish grow and fixing the above-mentioned microorganism on the epidermis of the above-mentioned fish and shellfish, and the above-mentioned microbial carrier is the microbial carrier of the present invention. do. Here, the "inhibitory action against pathogenic microorganisms" includes an action of reducing the growth and proliferation of pathogenic microorganisms and an action of killing pathogenic microorganisms. Further, the "desorption action of microorganisms" refers to the action of releasing the microorganisms once carried on the carrier into water.

The preventive method of the present invention is a preventive method for preventing a disease of fish and shellfish, and is a step of adding a microorganism having an inhibitory action against the pathogenic microorganism of the disease to a microbial carrier having a desorbing action of the microorganism. It is characterized by having a step of putting the above-mentioned microbial carrier into water in which the above-mentioned fish and shellfish grow, and fixing the above-mentioned microorganism on the epidermis of the above-mentioned fish and shellfish.

The above-mentioned fish and shellfish are characterized as being farmed fish and shellfish, fish and shellfish bred in an aquarium, ornamental fish, and tropical fish. For example, fish and shellfish cultivated include eel, ayu, yellowtail, trout, Thai, carp, amberjack, tuna, salmon, horse mackerel, flounder, tirapia, blowfish, hamachi, hata, mackerel, yellowtail, catfish, butterfly shark, crab, etc. Examples include shrimp, octopus, amberjack, and catfish. Examples of ornamental fish and tropical fish include goldfish, killifish, tetra, betta, and osteoglossidae.

The microorganism added to the microorganism carrier is characterized by being a microorganism derived from the epidermis of the fish and shellfish.

The above-mentioned microorganism is a microorganism of the genus Pseudomonas.

The microbial carrier of the present invention is a foam molded product containing a thermoplastic resin and an inorganic powder, and has irregularities on the surface, and the inorganic powder is 15 with respect to the total amount of the thermoplastic resin and the inorganic powder. Since it is contained in an amount of% to 70% by mass, the amount of microorganisms attached to the carrier is increased, and the processing capacity of the microorganisms can be improved. Further, by combining the thermoplastic resin and the inorganic powder, it becomes a microbial carrier having the features of a lightweight resin and a durable inorganic powder having a large specific surface area.

Further, surprisingly, the above-mentioned microbial carrier has an action of releasing the microbial carrier once carried on the carrier into water (desorption action) in addition to the action of supporting and propagating the microbial carrier. As shown in Examples described later, the microbial carrier of the present invention has an excellent desorption action because the potential of the carrier and the potential of the microorganism have an appropriate relationship by combining the thermoplastic resin and the inorganic powder. It is thought that it will be. As described above, according to the microbial carrier of the present invention, the water to be treated can be treated even by the microorganism desorbed from the carrier, and the treatment capacity of the microorganism can be easily improved.

In the method for producing a microbial carrier of the present invention, at least a thermoplastic resin, an inorganic powder and a foaming agent are kneaded and foam-molded, so that unlike a carrier made of a ceramic molded body, firing is not required and the cost is relatively low. Can produce carriers.

In the above manufacturing method, raw materials other than the foaming agent are pelletized in advance, and the obtained pellets and the foaming agent are kneaded and extruded and foamed. Therefore, even if the content of the inorganic powder is increased, the inorganic powder in the carrier It is easy to secure the dispersibility of the body. In addition, it becomes easy to disperse the foaming agent in the entire composition, and it becomes easy to prevent uneven distribution of pores and the like.

The preventive method of the present invention includes a step of adding a microorganism having an inhibitory effect on pathogenic microorganisms (useful microorganism) to a microbial carrier having a desorbing action of the microorganism, and putting the microbial carrier into water in which fish and shellfish grow. Since the microorganism carrier of the present invention is used as the microbial carrier, it has an excellent desorption action of useful microorganisms into water and has a preventive effect on microbial diseases. Can be improved.

The preventive method of the present invention includes a step of adding a microorganism having an inhibitory effect on pathogenic microorganisms (useful microorganism) to a microbial carrier having a desorbing action of the microorganism, and putting the microbial carrier into water in which fish and shellfish grow. Since it has a step of fixing the microorganisms on the epidermis of fish and shellfish, the useful microorganisms are continuously grown on the epidermis of fish and shellfish by proliferating the useful microorganisms on the carrier and further desorbing the proliferated useful microorganisms. Can be supplied. That is, by using useful microorganisms as probiotics of the epidermis of fish and shellfish, diseases of fish and shellfish can be prevented sustainably and easily. This preventive method can replace traditional fish and shellfish disease control such as antibacterial and vaccination.

The above-mentioned fish and shellfish are the fish and shellfish listed above, and by applying the preventive method of the present invention to aquaculture, ornamental use, breeding in an aquarium, etc., the diseases of those fish and shellfish can be effectively prevented. For example, in aquaculture, fish and shellfish are often bred under high density, so the epidermis is easily damaged by contact between fish and shellfish, and the damage may cause infectious diseases, especially fish and shellfish. This method using probiotics for the epidermis of aquaculture is particularly useful as a countermeasure against diseases of fish and shellfish in aquaculture and the like.

Since the microorganism added to the microbial carrier is a microorganism derived from the epidermis of fish and shellfish, it has excellent colonization to the epidermis and supplements the useful microorganisms originally possessed by the fish and shellfish. Can protect the epidermis of.

In particular, since the above-mentioned microorganism is a microorganism belonging to the genus Pseudomonas, it has an excellent inhibitory effect on pathogenic microorganisms.

It is a figure which shows an example of the microbial carrier of this invention. It is a figure which shows an example of the manufacturing method of the microbial carrier of this invention. It is a schematic diagram which used the microbial carrier of this invention for the probiotic of the fish epidermis. It is a schematic diagram which used the microbial carrier of this invention for a biological deodorization system. It is a figure which shows the schematic diagram of the prevention method of the disease of the fish and shellfish of this invention. It is a figure which shows an example of the screening of useful microorganisms. It is a schematic diagram of a closed circulation type aquaculture system. It is a photograph of the appearance of the carrier of Test Examples 1 to 3. It is a micrograph and an SEM photograph of the cross section of the carrier of Test Example 1. It is a micrograph and an SEM photograph of the cross section of the carrier of Test Example 2. 3 is a photomicrograph and an SEM photograph of a cross section of the carrier of Test Example 3. It is a figure which shows the result of the growth inhibition test against the pathogenic microorganism of KH-ZF1. It is a figure which shows the property of the carrier used for the growth / desorption test of a microorganism. It is a schematic diagram of the growth / desorption test of microorganisms. It is a photograph showing the result of the growth / desorption test of microorganisms. It is a photograph which shows the result of the colonization test to the epidermis of a fish. It is a figure which shows the result of the infection control test.

First, an example of the microbial carrier of the present invention will be described with reference to FIG. FIG. 1A shows a perspective view of the carrier, and FIG. 1B shows a schematic cross-sectional view thereof. This carrier carries a microorganism, and is used, for example, in water, and the action of the microorganism causes decomposition, inactivation, removal, growth inhibition, killing, and the like of a specific substance in the water to be treated. Specific substances include substances that adversely affect the human body and fish and shellfish (for example, harmful substances such as ammonia and nitrite and pathogenic microorganisms), and unnecessary substances (sludge, suspended solids, odors) and the like.

As shown in FIG. 1 (a), the carrier 1 is an extruded foam molded product containing a thermoplastic resin and an inorganic powder, and has a large number of irregularities 2 on the surface. The unevenness 2 is an unevenness formed on the surface of the molded body when the carrier is extruded and foamed, and is also called a melt fracture. Specifically, the unevenness 2 is formed by partially raising the resin portion by foaming, and is coarser than the fine unevenness (several micro-order) formed by a roughening treatment such as general etching. be. The unevenness 2 is formed over substantially the entire outer surface 1a of the cylinder and the inner surface 1b of the cylinder. Although the extruded foam molded product is shown in FIG. 1, an injection foam molded product may be used as the foam molded product. In this case, unevenness is formed on the surface by the foamed resin portion and the inorganic powder exposed on the surface.

The size of the carrier 1 is not particularly limited, but for example, the outer diameter φ is 10 mm to 50 mm, and the axial length L is 20 mm to 100 mm. The axial length L of the carrier 1 is in the same direction as the direction of extrusion from the extruder, and is adjusted by the cut length of the molded body extruded from the extruder. The shape of the carrier 1 is not limited to a cylinder, and a cylinder, a sphere, a cube, a rectangular parallelepiped, a plate, or the like can be adopted.

As shown in FIG. 1 (b), in the carrier 1, the inorganic powder 4 is dispersed in the thermoplastic resin 3. The carrier 1 is formed with communication holes 6 and closed pores 7 as pores 5 generated by foam molding. The communication hole 6 is a hole that communicates from the cylindrical surfaces 1a and 1b, and the air-closing hole 7 is a hole that is surrounded by a wall surface. As shown in FIG. 1 (b), a part of the inorganic powder 4 is exposed inside the pores 5 and on the cylindrical surfaces 1a and 1b, and is configured to come into contact with the water to be treated.

As shown in FIG. 1 (b), the carrier 1 has a porous structure, and its specific surface area is preferably 0.1 m ² / g or more from the viewpoint of microbial adhesion. As a specific range, the specific surface area is preferably 0.1 m ² / g to 10 m ² / g, more preferably 1 m ² / g to 5 m ² / g. The specific surface area is a value measured by the nitrogen gas adsorption method. The nitrogen gas adsorption method is performed by measuring the amount of nitrogen gas adsorbed and desorbed per unit mass.

The bulk density of the carrier 1 is preferably 0.9 g / cm ³ to 2 g / cm ³ . Bulk density is measured using an apparent density measuring device compliant with JIS K6911. By setting the bulk density within the above range, the carrier can be easily flowed in water, and the processing capacity by microorganisms can be improved. The bulk density is more preferably 0.9 g / cm ³ to 1.5 g / cm ³ , and even more preferably 0.9 g / cm ³ to 1.2 g / cm ³ .

The composition of the composition constituting the microbial carrier of the present invention will be described below.

The thermoplastic resin is not particularly limited, and is a polyolefin resin such as polyethylene (PE) resin or polypropylene (PP) resin, a polystyrene resin, an ABS (acrylonitrile butadiene styrene) resin, a vinyl chloride resin, a polyethylene terephthalate or a polyethylene na. Examples thereof include polyester-based resins such as phthalate and polycarbonate-based resins. These may be used alone or in combination of two or more. Among the above-mentioned thermoplastic resins, polyolefin-based resins are preferable because they are excellent in lightness and moldability.

The thermoplastic resin is preferably contained in an amount of 30% by mass to 85% by mass, more preferably 30% by mass to 65% by mass, based on the total amount of the thermoplastic resin and the inorganic powder. When the content is less than 30% by mass, it tends to be difficult to secure the fluidity at the time of molding, and the retention of the inorganic powder may decrease. Further, the content of the thermoplastic resin may be lower than that of the inorganic powder in terms of mass ratio.

In the microbial carrier, the inorganic powder is preferably contained in an amount of 15% by mass to 70% by mass and preferably 35% by mass to 70% by mass with respect to the total amount of the thermoplastic resin and the inorganic powder. The inorganic powder is not particularly limited, and minerals, fine ceramics, natural products such as shells, bone powder and the like can be used. Examples of minerals include diatomaceous earth, bakuhan stone, boehmite, zeolite, apatite, kaolin, mullite, spinel, olivine, sericite, bentonite, wallastonite, anausite, and mica. In addition, these minerals may be natural minerals or artificially synthesized minerals (for example, synthetic zeolites). Examples of fine ceramics include aluminum nitride, silicon carbide, silicon nitride, boron nitride, and alumina. Examples of the shell include oysters and Akoya pearl oyster shells. Further, as the inorganic powder, silicon dioxide, which is a component contained in minerals and shells, aluminum oxide, calcium silicate hydrate, calcium carbonate and the like can also be used. The inorganic powder may be used alone or in combination of two or more.

The average particle size of the inorganic powder is not particularly limited, but is preferably 1 μm to 100 μm. If the average particle size of the inorganic powder exceeds 100 μm, the dispersibility in the resin may decrease, and the amount of the inorganic powder exposed on the surface of the carrier or the pores of the carrier may decrease. On the other hand, if the average particle size of the inorganic powder is less than 1 μm, aggregation between the particles is likely to occur, and the dispersibility may be impaired. The average particle size is obtained by measuring by a laser diffraction / scattering method.

Inorganic powders include silicon dioxide, aluminum oxide, calcium silicate hydrate, diatomaceous earth, barley stone, shells, bone meal, calcium carbonate, boehmite, zeolite, apatite, kaolin, mulite, spinel, olivine, sericite, bentonite, and wa. It is preferable to use at least one selected from the group consisting of lastnite, anausite and mica, and it is more preferable to use at least one of diatomaceous earth and zeolite. Diatomaceous earth is a porous rock whose main component is silicon dioxide, which is composed of fossils of diatom, which is a kind of algae. Diatomaceous earth contains a small amount of alumina, iron oxide, and other components. Further, by using diatomaceous earth having excellent water absorption and hygroscopicity as the inorganic powder, water absorption can be imparted to the carrier.

Zeolites are referred to as crystalline aluminosilicates and contain silicon, aluminum and oxygen atoms. Many skeleton structures of zeolite are known, and LTA type zeolite, FER type zeolite, MWW type zeolite, MFI type zeolite, MOR type zeolite, LTL type zeolite, FAU type zeolite and the like can be used. Zeolites are also known to have a function of adsorbing harmful ammonia emitted from fish droppings and food residues to release sodium, and are therefore suitable for, for example, fish breeding water.

In the configuration containing diatomaceous earth as the inorganic powder, diatomaceous earth is contained, for example, 15% by mass to 55% by mass, preferably 20% by mass to 45% by mass, based on the total amount of the thermoplastic resin and the inorganic powder. Further, in the configuration containing zeolite as the inorganic powder, zeolite is contained, for example, 15% by mass to 55% by mass, preferably 20% by mass to 45% by mass, based on the total amount of the thermoplastic resin and the inorganic powder. Is done. Further, even in a configuration containing diatomaceous earth and zeolite, each of the above numerical ranges can be adopted. Further, in this case, it is preferable that the content of diatomaceous earth is higher than the content of zeolite.

The carrier of the present invention is a foam molded product and can be obtained by foam molding using a foaming agent. This carrier may contain a component derived from a foaming agent. As the foaming agent, a physical foaming agent, a chemical foaming agent, or the like is used. Physical foaming agents include hydrocarbons such as propane, butane and pentane, hydrocarbons such as dichloroethane and dichloromethane, fluorinated hydrocarbons such as trichloromonofluoromethane and dichlorodifluoromethane, carbon dioxide and nitrogen. Examples include gas and water. Examples of the chemical foaming agent include azo compounds such as sodium hydrogen carbonate and barium azodicarboxylate, and nitroso compounds such as N, N-dinitrosopentamethylenetetramine.

A lubricant may be added to the carrier in order to improve fluidity during molding. Examples of the lubricant include aliphatic hydrocarbons such as synthetic paraffin, higher fatty acids such as stearic acid, higher fatty alcohols, metal soaps such as sodium stearate, and higher fatty acid esters. The blending amount of the lubricant is, for example, 0.5% by mass to 10% by mass with respect to the total amount of the thermoplastic resin and the inorganic powder. In addition, other well-known additives may be added to the microbial carrier of the present invention.

The water absorption rate of the carrier is preferably 5% or more. When the water absorption rate is 5% or more, the carrier quickly adapts to water, so that it is easy to fill when immersed in a filter tank. In addition, since the contact with the water to be treated becomes frequent, the grown microorganisms are easily released. The water absorption rate of the carrier is more preferably 5% to 15%. The water absorption rate is measured by the method described in Examples described later.
Further, the pH of the solution leached from the carrier is preferably near neutral. Specifically, the pH is preferably 6.0 to 8.5, more preferably 6.5 to 8.0.

Next, an example of the method for producing the carrier of the present invention will be described with reference to FIG. Although FIG. 2 shows a manufacturing method by extrusion foam molding, the carrier may be manufactured by injection foam molding. In the method shown in FIG. 2, raw materials other than the foaming agent are obtained in advance as pellets 18. The pellet 18 is obtained, for example, by kneading a thermoplastic resin, an inorganic powder, and a lubricant, extruding the kneaded product with a pelletizer, and then cutting the pellet into a predetermined length. The lubricant is added as needed.

As shown in FIG. 2, by charging the pellet 18 and the foaming agent 19 into the hopper 12 of the extruder 11, these are introduced into the cylinder 13. In the cylinder 13, the pellet 18 and the foaming agent 19 are heated and melted by the heater 14, kneaded by the screw 15, and transferred from the cylinder nozzle 16 to the die 17. Then, bubbles are formed by the pressure drop when the molten kneaded product flows through the die 17, and an extruded foam molded product is obtained.

The blending amount of the foaming agent and the foaming ratio in foam molding are adjusted as appropriate. The blending amount of the foaming agent is not particularly limited, but is, for example, 1% by mass to 60% by mass, preferably 20% by mass to 60% by mass, more preferably, with respect to the total amount of the thermoplastic resin and the inorganic powder. Is blended in an amount of 40% by mass to 60% by mass.

In the method shown in FIG. 2, a chemical foaming agent is used as the foaming agent 19, but a physical foaming agent may be used instead. For example, when the foaming agent is a gas such as carbon dioxide, the pressure-controlled gas is added to the cylinder 13 so as to have a predetermined ratio with respect to the molten pellets 18 and kneaded.

Further, as shown in FIG. 2, each raw material may be directly charged into the hopper 12 of the extruder 11 without obtaining the pellet 18 in advance. In that case, the thermoplastic resin, the inorganic powder, the lubricant, and the foaming agent are put into the hopper 12, respectively, kneaded, and extruded and foamed as described above. However, since the amount of the inorganic powder to be blended can be increased and bubbles can be formed uniformly and well over the entire carrier, a method of pre-kneading to obtain pellets 18 in advance is preferable.

The carrier of the present invention is used, for example, under water and is used for improving the water quality of the water to be treated. The microorganism carried on this carrier may be any microorganism that decomposes a specific substance in the water to be treated, and either an aerobic microorganism or an anaerobic microorganism can be used. For example, the genus Pseudomonas, the genus Rhodococcus, the genus Alcaligenes, the genus Burkholderia, the genus Bacillus, the genus Corynebacterium, the genus Corynebacterium. Genus Rhodobacter, Genus Ralstonia, Genus Acidovorax, Genus Serratia, Genus Flavobacterium, Genus Nitrosomonas, Nitrobacter-Nit And other microorganisms can be used. The microorganism to be used is appropriately selected depending on the conditions of use of the carrier and the like. For example, when it is used for breeding water for fish and shellfish, it is appropriately selected depending on the type of fish and shellfish, the type of fish disease, and the like.

The carrier of the present invention can carry (fix) the above-mentioned microorganisms, proliferate, and desorb (sustained release). Conventionally, the performance of a general carrier (especially water treatment) is judged by how to fix and increase microorganisms, and there is no concept of "sustained release". In a carrier that has only the action of adhering and growing microorganisms, it is the contact surface that mainly exerts the action, so it is important how the carrier is brought into contact with the water to be treated. On the other hand, since the carrier of the present invention having the three actions of fixation, proliferation and sustained release releases microorganisms slowly, it is possible to reduce the measures for contacting the carrier with the water to be treated. In addition, the microorganisms desorbed (sustainedly released) from the carrier can further adhere to other carriers in the water to be treated, structures, surfaces of living organisms such as fish and shellfish, and exert microbial activity there. Further, the desorbed microorganisms may aggregate to form flocs or granules. As described above, by using the carrier of the present invention, excellent water treatment capacity can be exhibited, and various wastewater, aquaculture farms, aquariums, and the like can be efficiently improved in water quality.

Examples of the use of the carrier of the present invention include use in aquaculture farms, ornamental breeding, and aquariums. In recent years, closed-circulation aquaculture performed on land (see FIG. 7 described later) has attracted attention. However, in this aquaculture, water quality management is particularly important because it is a closed environment, and it is regarded as a problem that fish and shellfish get sick as the water quality deteriorates. For example, in aquaculture, fish are often bred under high density, so that the epidermis of fish is easily damaged by contact between fish, and there is a risk of developing an infectious disease due to the damage. Under these circumstances, it is conceivable to use probiotics targeting the epidermis of fish.

FIG. 3 shows a schematic diagram of using the microorganism supported on the carrier of the present invention as a probiotic of fish epidermis. First, microorganisms useful as probiotics are added to the carrier, and the carrier is put into the water in which cultured fish and ornamental fish are bred. The microorganisms carried on the carrier proliferate under a normal breeding environment (for example, about 20 to 30 ° C., pH 6.0 to 8.5) to form a biofilm. After that, the grown microorganisms are constantly desorbed from the carrier and settled on the epidermis of fish. By the sustained release of microorganisms from the carrier, the microorganisms can be continuously supplied to fish. Then, the action of the microorganisms settled on the epidermis can prevent infection by pathogenic microorganisms. The details will be described in the section of the preventive method of the present invention.

Further, as another example of use of the carrier of the present invention, there is a use in a biological deodorization system. FIG. 4 shows a schematic diagram of an example of a biological deodorizing system. The biological deodorization system is mainly used as an odor treatment device in sewage treatment plants and organic waste treatment such as livestock manure. As shown in FIG. 4, the biological deodorizing system 21 mainly includes a biological deodorizing tower 22 that deodorizes odorous gas using a microbial carrier 23, and an activated carbon adsorption tower 28 that absorbs and desorbs odorous gas using activated carbon. Have. In FIG. 4, the eliminator 26 and the deodorizing fan 27 are installed in the path connecting the biological deodorizing tower 22 and the activated carbon adsorption tower 28.

First, the odorous gas introduced from the lower part of the biological deodorizing tower 22 passes through the microbial carrier 23 and is discharged from the upper exhaust port. A sprinkler pipe 24 is installed in the upper part of the biological deodorizing tower 22, and the water supply water containing nutrients and the like is sprayed on the microbial carrier 23 by the sprinkler pipe 24 to supply a nutrient source to the microorganisms. The water collected at the bottom of the biological deodorizing tower 22 is drained through a process such as pH measurement. The odorous gas discharged from the biological deodorizing tower 22 is introduced into the activated carbon adsorption tower 28 via the eliminator 26 and the deodorizing fan 27. Then, it is introduced into the lower part of the activated carbon adsorption tower 28, and as it rises, it is deodorized by passing through the activated carbon filled in the activated carbon filling portion 29. The gas discharged from the activated carbon adsorption tower 28 is released to the atmosphere or the like.

Conventionally, in order to fix a microbial carrier having a deodorizing effect to a microbial carrier used in such a biological deodorizing system, the carrier (string-like, pumice-like, etc.) is tightly packed and water is run for 1 to 2 months for training. I had to do it. In addition, the carrier is densely packed in order to increase the contact area, but in reality, the microorganisms did not spread over the entire carrier, and in many cases, the microorganisms adhered and proliferated only partially. As a result, there is a problem that the device becomes large in order to increase the effectiveness and the initial cost increases.

By applying the carrier of the present invention to the microbial carrier 23 in the biological deodorizing tower 22, it is considered that the training time can be significantly shortened because the microorganisms grown on the carrier are effectively released. In addition, the sustained release action facilitates the distribution of microorganisms throughout the carrier, leading to miniaturization of the device. Further, even during use, the water sprinkled on the sprinkler pipe 24 releases microorganisms, which is expected to have a further deodorizing effect.

The carrier of the present invention is considered to be effective for other soil treatment and sewage treatment. At present, in soil treatment, the method of waiting for the diffusion of useful microorganisms or the treatment by forced stirring is adopted, but by using the carrier of the present invention, the microorganisms are effectively released and the treatment ability by the microorganisms is enhanced. Can be improved.

Next, the outline of the preventive method of the present invention will be described with reference to FIG. FIG. 5 shows an example of applying this method to aquaculture farms such as closed circulation land farms. In this case, the target fish and shellfish are, for example, eel, ayu, yellowtail, trout, tie, carp, campachi, tuna, salmon, horse mackerel, flatfish, tirapia, blowfish, hamachi, hata, mackerel, sama, catfish, butterfly shark, etc. Fish, crabs, shellfish such as shrimp, head and foot such as octopus, shellfish such as abalone, and catfish.

As shown in FIG. 5, this method includes a step A of adding useful microorganisms to a carrier and a step B of putting the carrier into water in which fish and shellfish are bred to fix the useful microorganisms on the epidermis of fish and shellfish. Has. As used herein, the term "useful microorganism" refers to a microorganism having an inhibitory effect on pathogenic microorganisms of fish and shellfish diseases. By finally colonizing the epidermis of fish and shellfish with useful microorganisms, it is possible to prevent infection from the epidermis and wounds even in an aquatic environment exposed to pathogenic microorganisms. The epidermis of fish and shellfish includes the surface of the body of fish, eyes, scales, fins, gills, mouth and the like, and the surface of crustaceans such as crabs and shrimp.

Diseases of fish and shellfish to be targeted are preferably diseases caused by transcutaneous infection, for example, vibrio disease, asthma disease, atypical Eromonas salmonicida infection, Eromonas hydrophila infection, Edowagellosis, red spot disease. , Ayu's Pseudomonas disease, Redmouth disease, Bacterial gill disease, Columnnaris disease (selected, tailed, torn, squeezed), cold water disease, gliding bacillosis, bacterial kidney disease, mycobacteriosis, nocardiosis, Examples include Lenza bacillosis.

Below, each process will be described in detail.

[Step A]
In this step, useful microorganisms are added to the carrier. As shown in FIG. 5, the useful microorganism used here is preferably a microorganism derived from the epidermis of the target farmed fish or the like. For example, when the farmed fish is trout, it is preferable to use microorganisms derived from the epidermis of trout. By using microorganisms originally present in the epidermis of fish and shellfish, the fish and shellfish can be safely protected.

As useful microorganisms used in step A, both aerobic microorganisms and anaerobic microorganisms can be used. For example, the genus Pseudomonas, the genus Rhodococcus, the genus Alcaligenes, the genus Burkholderia, the genus Bacillus, the genus Corynebacterium, the genus Corynebacterium. Genus Rhodobacter, Genus Ralstonia, Genus Acidovorax, Genus Serratia, Genus Flavobacterium, Genus Nitrosomonas, Nitrobacter-Nit And other microorganisms can be used. The useful microorganism to be used is appropriately selected depending on the type of farmed fish and the type of fish disease. Moreover, as a useful microorganism, various commercially available microorganisms can be used.

Further, as one form of step A, useful microorganisms may be obtained by screening after collecting candidate microorganisms from the epidermis of fish and shellfish. Candidate microorganisms can be collected by rubbing the epidermis of fish and shellfish with a cotton swab or the like. Then, the collected candidate microorganisms are cultured on the medium, and useful microorganisms are selected from the microorganisms derived from each formed colony.

In the screening, a growth inhibition test and a competitive test as shown in FIG. 6 are performed. In the growth inhibition test shown in FIG. 6 (a), first, the candidate microorganisms are linearly drawn on the Nutrient Broth agar medium (NB agar medium) and cultured at 28 ° C. overnight. Subsequently, the pathogenic microorganism is drawn vertically from the candidate microorganism so as not to touch the candidate microorganism, and the cells are cultured at 28 ° C. overnight. After this culture, it is judged from the image line of the candidate microorganism that there is a growth inhibitory effect when a clear zone exists over a range of, for example, 10 mm or more. The clear zone can be determined visually.

The method for the growth inhibition test is not limited to the method shown in FIG. 6A, and the culture conditions and the like can be appropriately changed. Further, as shown in FIG. 12 described later, the candidate microorganism may be placed in the center of the medium in the petri dish, and the pathogenic microorganism may be radially drawn around the candidate microorganism to determine the presence or absence of the growth inhibitory action.

In the competitive test shown in FIG. 6 (b), first, the candidate microorganisms are linearly drawn on the NB agar medium and cultured at 28 ° C. overnight. Subsequently, the pathogenic microorganism is drawn in a direction perpendicular to the image so as to intersect the image of the candidate microorganism, the candidate microorganism and the pathogenic microorganism are mixed, and then cultured at 28 ° C. overnight. After this culture, it is judged that there is a competitive advantage when the candidate microorganism grows preferentially in the portion after crossing the image of the candidate microorganism on the image of the pathogenic microorganism. When there is a competitive advantage, it is possible to prevent pathogenic microorganisms from adhering to the epidermis of fish and shellfish.

The useful microorganism used in the preventive method of the present invention is preferably an aerobic microorganism because it is fixed on the epidermis of fish and shellfish to prevent diseases. Specifically, microorganisms of the genus Pseudomonas are more preferable, and Pseudomonas mossellii, Pseudomonas marginalis, Pseudomonas colenesis (Pseudomonas korensis) Pseudomonas korensis (Pseudomonas korensis) Pseudomonas parafulva) is more preferred.

As a microbial carrier to which useful microorganisms are added, a carrier having a desorbing action of useful microorganisms is used. The shape and material of the carrier are not particularly limited as long as they have an eliminating action of microorganisms. As the shape of the carrier, for example, a cylinder, a cylinder, a sphere, a cube, a rectangular parallelepiped, a plate, or the like can be adopted. Further, as the material of the carrier, a resin such as a polyolefin resin, an inorganic powder, a metal, glass, carrageenan, dextrin, gelatin, charcoal or the like can be used, and two or more kinds may be used.

As the microbial carrier used in the preventive method of the present invention, it is preferable to use the carrier of the present invention described above, and for example, the carrier 1 of FIG. 1 can be used.

[Step B]
In this step, first, a carrier to which useful microorganisms are added is put into water in which cultured fish and the like are bred (see FIG. 5). The useful microorganisms carried on the carrier grow in a normal breeding environment (for example, about 20 to 30 ° C., pH 6.0 to 8.5) to form a biofilm. After that, the proliferated useful microorganisms are constantly desorbed from the carrier and settled on the epidermis of cultured fish and the like. By slowly releasing the useful microorganisms from the carrier, the useful microorganisms can be continuously supplied to farmed fish and the like. Then, by the action of useful microorganisms established on the epidermis, infection by pathogenic microorganisms can be prevented.

In the configuration of FIG. 5, the preventive method of the present invention can be said to be a method of breeding and cultivating fish and shellfish, and this method desorbs useful microorganisms having an inhibitory effect on pathogenic microorganisms of fish and shellfish diseases. It is characterized by having a step of adding it to an actionable microbial carrier and a step of putting the microbial carrier into water in which fish and shellfish grow to fix the microorganism on the epidermis of the fish and shellfish.

FIG. 7 shows a schematic diagram of a closed circulation type land-based aquaculture system as an example. In this system, the breeding water in the breeding aquarium circulates. Suspended solids and the like are removed from the water discharged from the breeding aquarium in the settling tank. Further, in the biological filtration tank, for example, ammonia in water is nitrified by the action of aerobic microorganisms, and nitrate produced by nitrification is reduced by the action of anaerobic microorganisms. The water purified in this way is returned to the breeding aquarium by a pump. As shown in FIG. 7, the microbial carrier is charged into the breeding water and is arranged along the bottom surface and the side surface of the breeding aquarium. The water flow in the tank causes the microorganisms to be released slowly from the microbial carrier and settles on the epidermis of farmed fish. By putting the microbial carrier together with the farmed fish in the breeding water in this way, it is possible to prevent diseases such as the farmed fish. Further, the installation of the microbial carrier is not limited to this, and may be installed so as to be suspended in a tank, for example. Further, if necessary, the breeding water may be agitated by a stirring blade or the like.

In the explanation of FIG. 5 above, an example in which the preventive method of the present invention is applied to aquaculture is shown, but for example, ornamental fish and shellfish can be targeted as fish and shellfish, and the preventive method of the present invention can be applied to aquarium tanks and homes. It can also be applied to water tanks.

Using the raw materials shown below, microbial carriers were prepared by each molding method.
Thermoplastic resin: Polypropylene resin Inorganic powder: Diatomaceous earth and zeolite (mass ratio 3: 2)
Lubricant: Stearic acid and paraffin foaming agent: Inorganic foaming agent Cellmic 417 (manufactured by Sankyo Kasei Co., Ltd.)

Test Example 1
40.0% by mass of the thermoplastic resin and 60.0% by mass of the inorganic powder were used with respect to the total amount of the thermoplastic resin and the inorganic powder. Further, the foaming agent was used in an amount of 50.0% by mass based on the total amount of the thermoplastic resin and the inorganic powder. After pelletizing the thermoplastic resin and the inorganic powder in advance according to the method of FIG. 2, the pellet and the foaming agent were simultaneously put into the hopper of the extruder and kneaded for extrusion foam molding. The rod-shaped molded product discharged from the base was cooled in a water bath and then cut to a length of 10 mm. An external photograph of the obtained extruded foam molded product is shown in FIG. 8 (a). Further, FIG. 9A shows a micrograph of a cross section of the extruded foam molded body cut along the radial direction, and FIG. 9B shows a micrograph of a cross section cut along the axial direction. 9 (c) and (d) show SEM photographs of each magnification.

Test Example 2
57.1% by mass of the thermoplastic resin, 42.9% by mass of the inorganic powder, and 0.9% by mass of the lubricant were used with respect to the total amount of the thermoplastic resin and the inorganic powder. Further, the foaming agent was used in an amount of 1.4% by mass based on the total amount of the thermoplastic resin and the inorganic powder. The thermoplastic resin, inorganic powder, lubricant, and foaming agent were directly charged into an extrusion molding machine without pre-kneading and extruded and foamed. An external photograph of the obtained extruded foam molded product is shown in FIG. 8 (b). Further, FIG. 10 shows a photograph similar to that in FIG.

Test Example 3
76.7% by mass of thermoplastic resin, 23.3% by mass of inorganic powder, 3.8% by mass of lubricant (2.2% by mass of stearic acid) with respect to the total amount of thermoplastic resin and inorganic powder. , Paraffin 1.6% by mass) was used. Further, the foaming agent was used in an amount of 5.4% by mass based on the total amount of the thermoplastic resin and the inorganic powder. The thermoplastic resin, the inorganic powder, the lubricant, and the foaming agent were directly injected into an injection molding machine without pre-kneading to perform injection foam molding. An external photograph of the obtained injection foam molded product is shown in FIG. 8 (c). Further, FIG. 11 shows a photograph similar to that of FIG. In FIGS. 9 to 11, the black portion in the carrier indicates pores, the white portion indicates inorganic powder, and the remaining portion indicates resin.

As shown in FIGS. 8 to 11, the carriers of Test Examples 1 to 3 have irregularities formed on the surface by foam molding. In addition, the carrier of the extruded foam molded product had a larger degree of unevenness on the cylindrical surface than the carrier of the injection foam molded product, and many pores were observed. In particular, the carrier of Test Example 1 had irregularities formed over the entire surface, and had higher shape uniformity than the carrier of Test Example 2. Further, in the carrier of Test Example 1, the inorganic powder and pores are evenly dispersed in the carrier (see FIG. 9), whereas in the carrier of Test Example 2, the inorganic powder and pores tend to be unevenly distributed. It was seen (see FIG. 10).

Subsequently, the physical characteristics of each obtained molded product were measured by the following method. The results are shown in Table 1.

The water absorption rate was calculated from the following formula (1). In the following formula (1), W1 indicates the weight measured after drying a 10 mm × 10 mm × 10 mm test piece collected from each molded body at 105 ° C. for one day, and W2 indicates the weight after drying. The weight W2 measured after immersing each test piece in 50 mL of distilled water for 24 hours and then removing the water adhering to the surface of the test piece is shown.
Water absorption rate (%) = (W2-W1) / W1 × 100 ... (1)

Five test pieces with a length of about 10 mm × φ10 to 15 mm collected from each molded product were immersed in 50 mL of distilled water for 30 minutes for pre-cleaning. The excess water of the test piece after washing was wiped off, immersed in a container containing 200 mL of distilled water, the lid of the container was closed, and the mixture was allowed to stand in a room temperature environment. The lid was opened regularly (1 day, 3 days, 1 week) and the pH was measured.

The specific surface area was measured by the nitrogen gas adsorption method using a specific surface area meter (manufactured by Nippon Bell Co., Ltd.).

The bulk density was obtained from the following formula (2) using an apparent density measuring instrument conforming to JIS K6911.
Bulk density = [(Weight of graduated cylinder containing sample (g))-(Weight of graduated cylinder (g))] / (Capacity of graduated cylinder (cm ³ )) ... (2)

As shown in Table 1, the carriers of Test Example 1 and Test Example 2 had a specific surface area of 1 m ² / g or more, which was larger than that of the carrier of Test Example 3. It is considered that this result is largely due to the difference in the surface shape of each carrier. Also, regarding the water absorption rate, the carriers of Test Example 1 and Test Example 2 were higher than the carriers of Test Example 3. In Test Example 1 and Test Example 2, a large amount of inorganic powder was exposed on the surface of the cylinder and inside the pores, and it is considered that the exposed inorganic powder exerted a water absorption effect. The pH of the liquids leached from the carriers of Test Example 1 and Test Example 2 was almost neutral.

Next, microorganisms were added to a plurality of carriers, and the growth and desorption of the microorganisms in water were observed.

<Acquisition of useful microorganisms>
First, candidate microorganisms were collected from the epidermis of fish, and then useful microorganisms were obtained by screening. The specific procedure is shown below.

Adult zebrafish (Dario rerio) was used as the fish. During the experiment, the zebrafish were acclimatized in the breeding water tank for several days. First, the mucous membrane on the body surface of the zebrafish was rubbed with a cotton swab, and the cotton swab was immersed in 1 mL of ultrapure water and stirred to obtain a suspension. 200 μL each of this suspension and diluted solution (a solution obtained by diluting the suspension with ultrapure water 5 times) was dropped onto NB agar medium, Enriched Cytophaga agar medium, and modified Zobel 2216E agar medium and sprayed. .. Then, each agar medium was cultured at 28 ° C. for 2 days. After culturing, the obtained colonies (124) were individually screened by the CrossStrak method.

In the screening, the growth inhibition test shown in FIG. 6A was carried out, and it was determined that there was a growth inhibition effect when a clear zone was present over a range of 10 mm or more from the image line of the candidate microorganism. For pathogenic microorganisms, Aeromonas hydrophila (ATCC 700183), Aeromonas hydrophila (JCM 1027), Aeromonas caviae (JCM 1043), Flavobacterium colorYer35eri (JCM2), Yersinia (JCM2) 13 (JCM2) As a result of this test, among various candidate microorganisms, KH-ZF1 (NITE BP-20967) showed an excellent growth inhibitory effect. This KH-ZF1 was identified as Pseudomonas mossellii by molecular phylogenetic analysis using a 16s ribosomal RNA gene sequence.

FIG. 12 shows the results of a growth inhibition test against a pathogenic microorganism of KH-ZF1.この試験では、病原微生物として、Ａｅｒｏｍｏｎａｓｃａｖｉａｅ、Ａｅｒｏｍｏｎａｓｈｙｄｒｏｐｈｉｌａ（ＡＴＣＣ７００１８３）、Ａｅｒｏｍｏｎａｓｈｙｄｒｏｐｈｉｌａ（ＪＣＭ１０２７）、Ｙｅｒｓｉｎｉａｒｕｃｋｅｒｉ（ＮＶＨ３５７８）、Ｙｅｒｓｉｎｉａｒｕｃｋｅｒｉ（ＤＳＭＺ１８５０６）、Ｅｄｗａｒｄｓｉｅｌｌａｔａｒｄａ（ＮＲＩＡ４４）、Ｅｄｗａｒｄｓｉｅｌｌａｔａｒｄａ（ＮＲＩＡ 51), Vibrio angillalum, Vibrio ordalii, Streptococcus iniae were used. In addition, two types of non-pathogenic microorganisms were used as negative controls. Cell suspension of KH-ZF1 (after culturing KH-ZF1 on NB agar medium, centrifuge at 5000 × g for 10 minutes to collect the precipitated cells, and OD ₆₀₀ is 1.0 in sterile water. (Resuspended so as to be) was adjusted. This suspension was soaked in a paper disc placed in the center of the NB agar medium in a petri dish and allowed to stand at 20 ° C. for 2 days. Then, the above-mentioned test microorganisms were radially drawn from the addition site of KH-ZF1 so as not to touch KH-ZF1, and cultured at 20 ° C. for 2 days. As shown in FIG. 12, selective suppression was confirmed in pathogenic microorganisms as compared with non-pathogenic microorganisms.

<Preparation of microbial carrier>
Next, the carriers of Test Examples 4 to 8 shown in FIG. 13 were prepared respectively. The carrier of Test Example 8 was prepared in the same manner as the carrier of Test Example 1 described above.

The carrier of Test Example 4 was a carrier mainly composed of foundry waste sand, and BTS (biotope sand, manufactured by Esaki Sangyo Co., Ltd.) black clay was used. The main constituents of this carrier are silicon dioxide (about 49%), carbon (about 15%) and Fe ₂ O ₃ (about 11%).

The carrier of Test Example 5 was a carrier containing casting waste sand as a main component, and the carrier of Test Example 4 was calcined at 700 to 800 ° C. was used.

The carrier of Test Example 6 was a carrier made of a ceramic molded product, and was produced as follows. With respect to 60 parts by mass of diatomaceous earth, 20 parts by mass of kaolin-based clay and 20 parts by mass of frog-like clay, which is an aluminum-containing clay mineral, and 20 parts by mass of rice bran were mixed. An appropriate amount of water was added to this mixture and kneaded, and coating granulation was performed to obtain a spherical molded product. This molded product was calcined at 900 to 950 ° C. to obtain a carrier. The main constituents of this carrier are silicon dioxide (about 78%) and Al ₂ O ₃ (about 15%).

The carrier of Test Example 7 was a carrier made of a ceramic molded product, and was produced as follows. A mixture of 65 parts by mass of crushed material of lightweight cellular concrete (ALC) containing tovamorite, which is a kind of calcium silicate hydrate, with 30 parts by mass of frogme clay and 5 parts by mass of inorganic reinforcing fiber (sepiolite). Then, 20 parts by mass of a powdery and granular organic void forming material was further mixed with 100 parts by mass of the obtained mixture to obtain a raw material for molding. An appropriate amount of water was added to this molding raw material and kneaded, and the obtained kneaded product was supplied to an extrusion molding machine to obtain a molded product. This molded product was calcined at 1000 to 1100 ° C. to obtain a carrier. The main constituents of this carrier are silicon dioxide (about 55%), calcium oxide (about 16%) and Al ₂ O ₃ (about 14%).

<Microbial growth / desorption test>
Each carrier obtained above was subjected to the test shown in FIG. First, 20 μL of KH-ZF1 was added to each carrier so that OD ₆₀₀ = 1.5. The carrier to which KH-ZF1 was added was put into 20 mL of breeding water and kept warm at 20 ° C. for 24 hours. Then, the breeding water was replaced, and the mixture was kept warm at 20 ° C. for 1 week in newly added 20 mL of breeding water. The carrier after heat insulation for 24 hours and the carrier after heat insulation for 1 week were observed under a microscope. In addition, the breeding water after keeping warm for one week is inoculated on a Pseudomonas isolation agar medium (manufactured by Becton Dickinson) containing kanamycin, which is a selective medium, and cultured at 28 ° C. for one day, and the amount of KH-ZF1 is adjusted to the colony. Observed by formation. These results are shown in FIG.

As shown in FIG. 15, KH-ZF1 was supported on any of the carriers of Test Examples 4 to 8 after 24 hours. In addition, it was observed that the supported KH-ZF1 on each carrier was proliferating after one week. Since all of these carriers have a porous structure, it is considered that they are excellent in the engraftment property of microorganisms, that is, the action of supporting and proliferating microorganisms.

On the other hand, desorption of microorganisms was observed when the carriers of Test Examples 7 to 8 were used. In particular, in the carrier of Test Example 8, a large number of colonies were observed over almost the entire medium on the petri dish. On the other hand, no colonies were observed in Test Examples 4 to 6. From this result, it was found that the carriers of Test Examples 7 to 8 have an action of supporting and proliferating microorganisms and desorbing microorganisms into water, and can be used in the method of the present invention. That is, the carriers of Test Examples 7 to 8 can adhere useful microorganisms, proliferate them, and release them (sustained release). Further, according to the carriers of Test Examples 7 to 8, the microbial activity can be exhibited not only on the carrier but also in the place where the microorganism desorbed from the carrier is newly attached, so that the water treatment capacity of the microorganism can be further improved. Can be done.

<Test for colonization of microorganisms on the epidermis of fish>
A colonization test was performed using a recombinant KH-ZF1 strain into which a fluorescent protein (mCherry) gene was introduced. The aquarium was filled with breeding water and zebrafish. For some zebrafish, the muscle at the base of the dorsal fin was injured with a needle. KH-ZF1 in which mCherry was introduced was added to this breeding water so that OD ₆₀₀ = 0.01, and zebrafish were bred at 20 ° C. A part of the zebrafish was collected and observed under a microscope 6 hours after the exposure to KH-ZF1 and 24 hours after the exposure. The results are shown in FIG.

As shown in FIG. 16, it was confirmed that KH-ZF1 had settled in the vicinity of the wound on the epidermis of the zebrafish 6 hours after the exposure. It was also confirmed that KH-ZF1 was established in other parts as well.

<Infection control test>
200 mL of breeding water (1 L of ultrapure water plus 3 g of instant ocean) was placed in a test container, 5 zebrafish were placed, and the zebrafish were bred at 28 ° C. for 2 days. Then, the breeding water was replaced, KH-ZF1 was added so that OD ₆₀₀ = 0.01, and the animals were bred at 28 ° C. for 24 hours. Then, the zebrafish was anesthetized with an anesthetic solution containing trikine, and the muscle at the base of the dorsal fin of the zebrafish was injured with an injection needle. After the breeding water was replaced, the injured zebrafish was added, KH-ZF1 was added so that OD ₆₀₀ = 0.01, and the animals were bred at 28 ° C for 24 hours and at 20 ° C for 24 hours. The breeding water was replaced again, KH-ZF1 and Yersinia ruckeri (NVH3758) were added so that OD ₆₀₀ = 0.01, respectively, and the zebrafish was allowed to swim at 20 ° C. for 1 hour. Furthermore, the breeding water was replaced, KH-ZF1 was added so that OD ₆₀₀ = 0.01, and the zebrafish was continued to be bred at 20 ° C., and the life and death conditions were followed up. In addition to the above-mentioned treatment group, a control group in which KH-ZF1 was not added at all in the above-mentioned procedure was also tested. In addition, KH-ZF1 and Yersinia ruckeri (NVH3758) were prepared as follows.

KH-ZF1 cultured on a plate was inoculated into a tube containing 2 mL of NB medium, and cultured at 28 ° C. with shaking for 24 hours. 100 μL of this culture solution was inoculated into 10 mL of NB medium and cultured at 28 ° C. with shaking for 24 hours. Then, it was subjected to a centrifugation operation of 8000 × g for 5 minutes to remove the supernatant and collect the bacteria. The cells were resuspended in breeding water, washed, centrifuged at 8000 × g for 5 minutes, and the supernatant was removed to collect bacteria. After repeating this operation three times, it was resuspended in breeding water and OD ₆₀₀ was measured.

Yersinia ruckeri cultured on a plate was inoculated into a tube containing 2 mL of LB medium, and cultured at 28 ° C. with shaking for 24 hours. 100 μL of this culture solution was inoculated into 10 mL of LB medium and cultured at 28 ° C. with shaking for 12 hours. Then, it was subjected to a centrifugation operation of 8000 × g for 5 minutes to remove the supernatant and collect the bacteria. The cells were resuspended in breeding water, washed, centrifuged at 8000 × g for 5 minutes, and the supernatant was removed to collect bacteria. After repeating this operation three times, it was resuspended in breeding water and OD ₆₀₀ was measured.

Figure 17 shows the results of the infection control test. As shown in FIG. 17, the treatment group to which KH-ZF1 was added had a significantly increased survival rate up to one week after infection compared to the control group. A logrank test showed P = 0.004 between the treatment group and the control group.

Since the microbial carrier of the present invention is easy to handle and has excellent microbial treatment capacity, it is useful for purifying industrial wastewater such as factory wastewater, domestic wastewater, fish breeding water, and aquatic plant breeding water. In addition, the method for preventing fish and shellfish diseases of the present invention can prevent fish and shellfish diseases sustainably and easily. Therefore, in particular, aquaculture, ornamental, and aquariums for breeding fish and shellfish in a closed environment. It is useful for breeding in such places.

1 Carrier (microbial carrier)
2 Concavities and convexities 3 Thermoplastic resin 4 Inorganic powder 5 Porosity 6 Communication holes 7 Closed pores 11 Extrusion molding machine 12 Hopper 13 Cylinder 14 Heater 15 Screw 16 Cylinder nozzle 17 Die 18 Pellet 19 Foaming agent 21 Biological deodorizing system 22 Biological deodorizing tower 23 Microbial Carrier 24 Sprinkler pipe 25 Drain valve 26 Eliminator 27 Deodorizing fan 28 Activated carbon adsorption tower

Claims

A microbial carrier that carries microorganisms
The microbial carrier is a foamed molded product containing a thermoplastic resin and an inorganic powder, and has irregularities on the surface.
The microbial carrier is characterized in that the inorganic powder is contained in an amount of 15% by mass to 70% by mass with respect to the total amount of the thermoplastic resin and the inorganic powder.
The foam molded product is an extruded foam molded product.
The microbial carrier according to claim 1, wherein the inorganic powder is contained in an amount of 35% by mass to 70% by mass with respect to the total amount of the thermoplastic resin and the inorganic powder.
The inorganic powder includes silicon dioxide, aluminum oxide, calcium silicate hydrate, diatomaceous earth, barley stone, shell, bone meal, calcium carbonate, boehmite, zeolite, apatite, kaolin, mulite, spinel, olivine, sericite, bentonite, etc. The microbial carrier according to claim 1, wherein the microbial carrier is at least one selected from the group consisting of wallastonite, anausite and mica.
The microbial carrier according to claim 1, wherein the specific surface area of the microbial carrier is 1 m 2 / g to 5 m 2 / g.
The method for producing the microbial carrier according to claim 1.
A method for producing a microbial carrier, which comprises kneading at least the thermoplastic resin, the inorganic powder, and a foaming agent to form a foam.
The method for producing a microbial carrier according to claim 5, wherein the production method comprises pelletizing raw materials other than the foaming agent in advance, kneading the obtained pellets with the foaming agent, and performing extrusion foam molding. ..
It is a preventive method to prevent fish and shellfish diseases.
A step of adding a microorganism having an inhibitory effect on the pathogenic microorganism of the disease to a microbial carrier having an desorbing effect of the microorganism, and a step of adding the microorganism.
It has a step of putting the microbial carrier into water in which the fish and shellfish grow, and fixing the microorganism on the epidermis of the fish and shellfish.
A method for preventing diseases of fish and shellfish, wherein the microbial carrier is the microbial carrier according to claim 1.
It is a preventive method to prevent fish and shellfish diseases.
A step of adding a microorganism having an inhibitory effect on the pathogenic microorganism of the disease to a microbial carrier having an desorbing effect of the microorganism, and a step of adding the microorganism.
A method for preventing diseases of fish and shellfish, which comprises a step of putting the microorganism carrier into water in which the fish and shellfish grow, and fixing the microorganism on the epidermis of the fish and shellfish.
The method for preventing diseases of fish and shellfish according to claim 8, wherein the fish and shellfish are farmed fish and shellfish, fish and shellfish bred in an aquarium, ornamental fish, and tropical fish.
The method for preventing a disease of fish and shellfish according to claim 8, wherein the microorganism added to the microorganism carrier is a microorganism derived from the epidermis of the fish and shellfish.
The method for preventing a disease of fish and shellfish according to claim 8, wherein the microorganism is a microorganism of the genus Pseudomonas.