JP7041010B2 - Newly decomposing microorganisms of fats and oils - Google Patents

Description

NPMD NITE BP-02726

The present invention relates to a novel decomposition microorganism of fats and oils.

Wastewater (wastewater) from kitchens and food factories usually contains swill and cooking oil. Solid matter such as swill can be easily removed from the drainage by providing a basket or the like at the drainage port, but it is not easy to remove liquid matter such as cooking oil. Therefore, in facilities such as kitchens and food factories that discharge wastewater mixed with a large amount of fats and oils, a detoxification facility (for example, a grease trap) is provided to collect the fats and oils and separate and dispose of the fats and oils floating on the upper layer. Has been done.

However, the oil and fat accumulated in the grease trap solidifies and remains as a scum (lump of oil) on the water surface of the grease trap, or accumulates and adheres to the inner wall surface of the grease trap or the inside of the pipe to block the pipe. There is. At this time, the accumulated fats and oils may be oxidized and putrefactive, causing stinks and pests. In addition, if the accumulated fats and oils are left unattended, the grease trap's ability to remove the fats and oils deteriorates, causing the fats and oils to flow out into sewage and rivers. Therefore, when oils and fats are accumulated in the grease trap, it is necessary to ask a specialized contractor to remove the oils and fats by vacuum treatment or high-pressure washing treatment, which is costly.

Therefore, in a grease trap, a method for efficiently reducing fats and oils, particularly a method using a microorganism for decomposing and assimilating fats and oils is being studied. For example, Patent Document 1 describes Yarrowia lipolytica as a microorganism having an ability to assimilate free fatty acids.

Japanese Unexamined Patent Publication No. 2013-146689

In the microorganism described in Patent Document 1, an oil decomposition experiment is conducted at pH 6 or 7. On the other hand, the water quality such as the pH of the wastewater in the grease trap may change significantly depending on the discharged food residue and the like. Therefore, the microorganisms used in the grease trap are required to have the property of purifying wastewater even in a water quality environment with a wide range of pH (particularly strong alkaline pH 11 or higher). However, conventionally known microorganisms do not have sufficient such properties.

Therefore, the present invention has been made in view of the above circumstances, and an object of the present invention is to provide a microorganism having an excellent effect of reducing fats and oils in an abatement facility. In particular, it is an object of the present invention to provide a microorganism capable of purifying wastewater even in a water quality environment having a wide range of pH (particularly strong alkaline pH 11 or higher).

The present inventors have conducted diligent research to solve the above problems. As a result, it was found that the above-mentioned problems can be solved by microorganisms belonging to Pseudomonas and exhibiting predetermined mycological properties, and the present invention has been completed.

According to the present invention, a microorganism having an excellent effect of reducing fats and oils in an abatement facility is provided. In particular, according to the present invention, there is provided a microorganism capable of purifying wastewater even in a water quality environment having a wide range of pH (particularly strong alkaline pH 11 or higher).

FIG. 1 schematically shows the mechanism of wastewater treatment by a grease trap. FIG. 2A shows the 2-2819 strain and P. cerevisiae. The 16S rDNA partial base sequence comparison of Mosselii CIP105259 ^T (AF072688) is shown. "Query" indicates a 2-2819 strain, and "Sbjct" is P.I. Indicates mossellii. FIG. 2B shows the 2-2819 strain and P. cerevisiae. A 16S rDNA partial nucleotide sequence comparison of mossellii CIP105259 ^T (AF072688) is shown (continuation of FIG. 2A). "Query" indicates a 2-2819 strain, and "Sbjct" is P.I. Indicates mossellii.

Hereinafter, embodiments according to one embodiment of the present invention will be described. The present invention is not limited to the following embodiments.

In the present specification, "XY" indicating a range means "X or more and Y or less". Unless otherwise specified, the operation and physical properties are measured under the conditions of room temperature (20 to 25 ° C.) / relative humidity of 40 to 50% RH.

<Microorganisms>
One form of the present invention is a microorganism (fat-degrading microorganism) that belongs to Pseudomonas and exhibits the following mycological properties. In one embodiment, a microorganism (fat-degrading microorganism) that belongs to Pseudomonas, exhibits the following mycological properties, and has a 16S rDNA base sequence represented by SEQ ID NO: 1 is provided. In the present specification, a microorganism exhibiting the following mycological properties is also simply referred to as "microorganism according to the present invention".

In a preferred embodiment, the microorganism according to the present invention reduces 1% (w / v) of fats and oils by 50% by weight or more in 24 hours under the condition of pH 4 or higher. The upper limit of pH is not particularly limited, but is, for example, 13 or less. The temperature condition is not particularly limited, but is, for example, 30 ° C.

In a particularly preferred embodiment, the microorganism of this embodiment is Pseudomonas sp. 2-2819 strain (accession number NITE BP-02726).

[screening]
The microorganism according to the present invention was isolated from soil in Tsuruoka City, Yamagata Prefecture by the following screening method.

1. 1. Screening method Add an appropriate amount of a sample collected from soil or grease trap waste liquid, sewage, river water, etc. in Yamagata Prefecture to 5 mL of the primary screening liquid medium prepared by the following method, and incubate at 30 ° C. for one week. 100 μL of the culture solution after culturing is further inoculated into 5 mL of the liquid medium for primary screening, and the cells are cultured again at 30 ° C. for one week.

The liquid medium for primary screening is sterilized at high temperature and high pressure by dissolving each component other than fats and oils in pure water so as to have the composition shown in Table 4 below, adding fats and oils to a final concentration of 0.5 w / v%. To prepare. The fats and oils are prepared by mixing rapeseed oil and soybean oil at a ratio of 1: 1 (w / w).

10 100 μL of the culture solution after the primary screening diluted ⁴ -fold is applied to the agar medium for secondary screening prepared by the following method, and the cells are cultured at 30 ° C. for 48 hours. After culturing, a strain in which the formation of halo by decomposition of fats and oils was confirmed is isolated.

In the agar medium for secondary screening, each component other than fat and oil and agar is dissolved in pure water so as to have the composition shown in Table 5 below, and fat and oil (rapeseed oil: soybean oil = 1: 1 (w / w)) is added. A final concentration of 0.5 w / v% and agar are added so as to have a final concentration of 2.0 w / v%, sterilized at high temperature and high pressure, and then appropriately dispensed and solidified to prepare.

Next, 0.05 g of fat and oil (rapeseed oil: soybean oil = 1: 1 (w / w)) is added to 5 mL of a liquid medium for tertiary screening prepared by the following method to prepare a sterilized test solution (fat and oil). 1% (w / v)). Each isolated strain obtained in the above secondary screening is inoculated into an LB medium prepared by the following method, one loopful at a loop loop, and cultured at 30 ° C. for 24 hours with shaking (140 rpm). 100 μL of the obtained culture solution is inoculated into the test solution prepared by the above method, and shake-cultured (140 rpm) at 30 ° C. for 24 hours.

The liquid medium for tertiary screening is prepared by dissolving each component in pure water, adjusting the pH to 6.0 with hydrochloric acid, and sterilizing at high temperature and high pressure so as to have the composition shown in Table 6 below.

The LB medium is prepared by dissolving each component in pure water and sterilizing at high temperature and high pressure so as to have the composition shown in Table 7 below.

After culturing, a normal hexane extract is prepared according to JIS K0102: 2016 revision (industrial wastewater test method). Using the normal hexane extract as the residual amount of fats and oils, 0.05 g of fats and oils added at the time of preparation of the test solution and the residual amount of fats and oils (amount of normal hexane extract (g)) are used to determine the fat reduction rate according to the following formula (1). Ask for. As a result, a strain having a high fat reduction rate can be isolated.

The 16S rDNA base sequence and mycological properties of the isolated strain (isolated microorganism) having a high fat decomposition rate were determined as follows.

1. 1. Culturing conditions Strains cultivated under the following conditions are used as test cells.

2.16S rDNA nucleotide sequence analysis The operations from PCR amplification to cycle sequencing are performed based on each protocol.

The 16S rDNA base sequence of the isolated microorganism determined above is shown in SEQ ID NO: 1 below.

As a result of BLAST homology search for the DNA database DB-BA12.0 (Technosuruga Lab Co., Ltd.) for microbial identification and the international base sequence database (DDBJ / ENA (EMBL) / GenBank), the 16S rDNA partial base sequence of the isolated microorganism Showed a homology rate of 99.9% with respect to the reference strain CIP 105259 ^T (accession number AF072688) of Pseudomonas mossellii. In addition, P. Homology with a homology rate of 99% or more was shown for multiple species such as entomophila L48 ^T (AY907566). The results of the BLAST search for DB-BA12.0 are shown in Table 10, and the results of the BLAST search for the international nucleotide sequence database are shown in Table 11.

A simple molecular phylogenetic analysis was performed using the shaded sequence data in Table 10. The results are shown below. The upper left line indicates the scale bar, the numerical value located at the branch of the lineage branch is the boost trap value, the T at the end of the strain name indicates the reference strain (Type Strine) of that kind, and the BSL is biosafety. Indicates the level (BSL1 * (opportunistic pathogen) or higher is indicated).

In the above molecular phylogenetic tree, the isolated microorganism is contained in a cluster composed of the genus Pseudomonas, and P. It showed the same molecular phylogenetic position as Mosselii CIP105259 ^T (AF072688).

As shown in FIGS. 2A and 2B, isolated microorganisms and P.I. Comparing the 16S rDNA partial base sequences of Mosselii CIP105259 ^T (AF072688), a difference of 2 bases was observed, and one base of the difference was due to insertion or deletion (FIG. 2A). It can be considered that the two are almost the same. However, the remaining 1 base was clearly different (Fig. 2B). Therefore, they are considered to actually exhibit different molecular phylogenetic positions.

Therefore, from the results of 16S rDNA partial base sequence analysis, the isolated microorganism was P.I. It is identified as Pseudomonas sp., Which is closely related to Mosselii.

3. 3. Mycological properties The mycological properties of the strains obtained by the above screening are shown below.

Morphological observation with an optical microscope (BX50F4, Olympus, Japan) and the method of Barrow & Feltham (Barrow GI & Feltham RKA (1993), Cowan and Steel's Manual Forerti. Based on edition, Cambridge University Press), tests were conducted on catalase reaction, oxidase reaction, acid / gas production from glucose, and oxidation / fermentation (O / F) of glucose. Gram stain was performed using Faber G "Nissui" (Nissui Pharmaceutical, Japan). The results are shown in Table 12.

Next, using API® 20NE (bioMérieux, France), the following items were tested according to the manufacturer's protocol. The results are shown in Table 13.

In addition, Techno Suruga Lab Co., Ltd. and NCIMB Ltd. in the United Kingdom. The following items were tested in accordance with the technical tie-ups with and the publicly known technology. The results are shown in Table 14.

As shown in Table 12, the isolated microorganism was a motility Gram-negative bacillus that oxidized glucose and both catalase and oxidase reactions were positive. These properties are considered to be consistent with those of the genus Pseudomonas (Barrow GI & Feltham R.K.A. (1993). University Press).

As shown in Table 13, isolated microorganisms do not reduce nitrates; show arginine dihydrolase activity; do not hydrolyze esculin; hydrolyze gelatin; glucose, D-mannose, N-acetyl-D- Assimilated glucosamine, potassium gluconate, n-capric acid, adipic acid, dl-apple acid, sodium citrate and phenyl acetate; not assimilated L-arabinose, D-mannose, maltose and adipic acid.

As shown in Table 14, isolated microorganisms do not grow at 4 ° C; grow at 5% NaCl but not at 7% NaCl; show recitinase activity; lysine decarboxylase activity and ornithine decarboxylase activity. Not shown; did not produce acetoin; produced fluorescent dye on King's B agar.

From the above, the properties of the isolated microorganisms are described in P.I. Pseudomonas sp. (2002). Pseudomonas sp. (2002). Pseudomonas sp. isolated from clinical specimens. Int J Syst Evol Microbiol, 52, 363-376.). However, as described above, there is a one-base difference in 16S rDNA base sequence analysis.

Therefore, the isolated strain (isolated microorganism) is judged to be a novel microorganism, and this strain is referred to as Pseudomonas sp. 2-2819 strain (in the present specification, simply "2-2819 strain". Also called). In addition, this 2-2819 strain was deposited at the National Institute of Technology and Evaluation Patent Microorganisms Depositary Center (2-5-8 Kazusakamatari, Kisarazu City, Chiba Prefecture) on May 23, 2018. The accession number is NITE BP-02726.

[Evaluation of oil and fat reduction effect]
In the present specification, the reduction of fats and oils is evaluated by the following method. That is, 0.05 g of fat and oil having rapeseed oil: soybean oil = 1: 1 (w / w) is aseptically treated as the liquid medium for tertiary screening except for pH (5 mL). In addition to this, a test solution is prepared (fat and oil 1% (w / v)). As the oil / fat decomposition evaluation medium used at this time, a medium having a pH adjusted in the range of 3 to 13 is used (for example, pH 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 and 13). Medium for fat decomposition evaluation). The pH can be adjusted with any acid such as inorganic acids such as hydrochloric acid, nitrate, carbonic acid, sulfuric acid, organic acids such as citric acid, lactic acid and salts thereof; and / or any acid such as sodium hydroxide, potassium hydroxide, ammonia and the like. Alkaline; may be used, but hydrochloric acid (acidic side) or sodium hydroxide (alkaline side) is preferable.

This test solution is inoculated with microorganisms cultured on a plate medium (for example, agar medium for secondary screening) and cultured with shaking (140 rpm) for 24 hours in an arbitrary temperature range. The amount of bacteria to be inoculated is about one platinum loop. As the microorganism to be inoculated into the test solution, those precultured in a liquid medium for tertiary screening or the like may be used. By pre-culturing, the amount of bacteria to be inoculated can be easily adjusted. When using pre-cultured microorganisms, inoculate 1 mL of the test solution at 1.5 × ¹⁰⁶ CFU / mL. The culture temperature may be set according to a temperature range in which the cells have a high ability to decompose and assimilate fats and oils, and is, for example, 10 to 40 ° C, preferably 20 to 37 ° C.

After culturing, a normal hexane extract is prepared according to JIS K0102: 2016 revision (industrial wastewater test method). Using the normal hexane extract as the residual amount of fats and oils, the fats and oils (0.05 g) added at the time of preparing the test solution and the residual amount of fats and oils (amount of normal hexane extract (g)) are calculated according to the above formula (1). Find the rate of decrease. The microorganism according to the present invention has a fat reduction rate of 50% by weight obtained by the above method in all the test solutions prepared using the medium for fat decomposition evaluation whose pH is set in the above range (for example, pH 4 to 13). Anything above that is fine. In a preferred embodiment of the present invention, the fat reduction rate when cultured at 30 ° C. is 50% by weight or more, more preferably 60% by weight or more. The higher the oil / fat reduction rate is, the more preferable it is. Therefore, the upper limit is not particularly set, but for example, the oil / fat reduction rate measured by the above method is 90% by weight or less. If it is cultured for a long time, the amount of fat reduction will increase. However, since the microorganisms are sequentially excreted from the abatement facility, the microorganisms are usually replenished to the abatement facility about every 1 to 3 days. Therefore, a microorganism showing a fat reduction rate of 50% by weight or more in a short time (for example, within 24 hours) is excellent in practical use.

The water quality environment of the wastewater from the abatement facility can easily change depending on the type of swill discharged. Therefore, it is preferred for microorganisms used in abatement facilities to be able to purify wastewater in a wide range of pH environments. The 2-2819 strain is excellent in that it can decompose fats and oils even in a wide range of pH environments (particularly strong alkalinity of pH 11 or higher).

As used herein, the term "fat and oil" refers to edible or industrial fats and oils rich in glycerides such as triglycerides, diglycerides and monoglycerides, as well as fatty acids. Examples of the fats and oils include olive oil, canola oil, coconut oil, sesame oil, rice oil, rice bran oil, safflower oil, soybean oil, corn oil, rapeseed oil, palm oil, palm kernel oil, sunflower oil, cottonseed oil, and palm oil. , Edible oils such as peanut oil, beef oil, lard, chicken oil, fish oil, whale oil, butter, margarine, fat spread, shortening; However, it is preferably edible oil and fat that is frequently discharged in restaurants and the like where grease traps are often installed. The fatty acid is not particularly limited, and is, for example, butyric acid, hexanoic acid, heptanic acid, octanoic acid, decanoic acid, lauric acid, tridecanoic acid, myristic acid, pentadecanoic acid, pentadecanoic acid, palmitic acid, heptadecanoic acid, and the like. Saturated fatty acids such as stearate, arachidic acid, bechenic acid, lignoseric acid; decenoic acid, myristoleic acid, pentadecenoic acid, palmitreic acid, heptadecenoic acid, oleic acid, icosenoic acid, docosenoic acid, tetracosenoic acid, hexadecadienic acid, hexa Decatrienoic acid, hexadecatetetraenoic acid, linoleic acid, α-linolenic acid, γ-linolenic acid, octadecatetetraenoic acid, icosadienoic acid, icosatorienic acid, icosatetetraenoic acid, arachidonic acid, icosapentaenoic acid, henicosapentaenoic acid, docosadienoic acid , Docosatetraenoic acid, docosapentaenoic acid, docosapentaenoic acid, unsaturated fatty acids such as docosahexaenoic acid; The fatty acid may be produced by decomposing edible or industrial fats and oils.

[Culturing of microorganisms]
The method for culturing a microorganism according to the present invention may be any method as long as the microorganism can grow and proliferate. For example, the medium used for culturing the microorganism may be either a solid or liquid medium, and is a medium containing a carbon source, an appropriate amount of nitrogen source, an inorganic salt and other nutrients that can be assimilated by the microorganism used. , Synthetic medium or natural medium. Medium usually contains carbon sources, nitrogen sources and minerals.

The carbon source that can be used in culturing the microorganism according to the present invention is not particularly limited as long as the strain used can be assimilated. Specifically, in consideration of the assimilation of microorganisms, glucose, mannose, fructose, cellobiose, raffinose, xylose, maltose, galactose, sorbose, glucosamine, ribose, arabinose, ramnorth, sucrose, trehalose, α-methyl-D. -Sugars such as glucoside, salicin, meliviose, lactose, meregitos, inulin, erythritol, rivitol, xylitol, glucitol, mannitol, galactitol, inositol, N-acetyl-D-glucosamine, starch, starch hydrolysate, sugar honey, waste sugar honey, etc. , Natural products such as wheat and rice, alcohols such as glycerol, methanol and ethanol, organic acids such as acetic acid, phenylacetate, lactic acid, succinic acid, gluconic acid, capric acid, malic acid, glucuronic acid, pyruvic acid and citric acid. , Hexadecane and other hydrocarbons. The carbon source is appropriately selected in consideration of the assimilation property of the microorganism to be cultured. For example, when the 2-2819 strain is used, among the above carbon sources, glucose, mannose, N-acetyl-D-glucosamine, gluconic acid, capric acid, malic acid, citric acid, phenyl acetate and salts thereof (for example, for example). It is preferable to use sodium salt, potassium salt) or the like. Further, one type or two or more types of the above carbon sources can be selected and used.

Nitrogen sources that can be used in the cultivation of the microorganism according to the present invention include meat extract, fish meat extract, peptone, polypeptone, tripton, yeast extract, malt extract, soybean hydrolyzate, soybean powder, casein, milk casein, cazamino acid, and glycine. , Various amino acids such as glutamate and aspartic acid, organic nitrogen sources such as corn steep liquor and other animal, plant and microorganism hydrolysates; ammonium salts such as ammonia, ammonium nitrate, ammonium sulfate and ammonium chloride, nitrates such as sodium nitrate, Examples thereof include nitrites such as sodium nitrite and inorganic nitrogen sources such as urea. The nitrogen source is appropriately selected in consideration of the assimilation property of the microorganism to be cultured. For example, when the 2-2819 strain is used, it is preferable to use fish meat extract, tryptone, yeast extract, ammonium chloride and the like among the above nitrogen sources. Further, one type or two or more types of the above nitrogen sources can be selected and used.

Examples of the inorganic substances that can be used in the cultivation of the microorganism according to the present invention include phosphates, hydrochlorides, sulfates, acetates, carbonates and chlorides such as magnesium, manganese, calcium, sodium, potassium, copper, iron and zinc. Such as halides and the like. The above-mentioned inorganic substances are appropriately selected in consideration of the assimilation property by the microorganism to be cultured. Moreover, one kind or two or more kinds of the said inorganic substances can be selected and used. Further, a surfactant or the like may be added to the medium as needed.

It is preferable to add fats and oils to the medium in order to efficiently decompose and assimilate the fats and oils of the microorganism according to the present invention or to maintain the fats and oils decomposition and assimilation ability of the microorganisms. Examples of the fats and oils include the above-mentioned edible fats and oils, industrial fats and oils, and fatty acids. The amount of fats and oils added is not particularly limited, and can be appropriately selected in consideration of the ability of the microorganisms to be cultured to decompose and assimilate fats and oils. Specifically, it is preferable to add oil (rapeseed oil: soybean oil = 1: 1 (w / w)) to 1 L of the medium at a concentration of 1 to 30 g, more preferably 5 to 15 g. With such an addition amount, the microorganism can maintain a high ability to decompose and assimilate fats and oils. The fats and oils may be added alone or in the form of a mixture of two or more kinds.

The microorganism according to the present invention can be cultured by a usual method. For example, depending on the type of microorganism, the microorganism is cultured under aerobic or anaerobic conditions. In the former case, the cultivation of microorganisms is carried out by shaking or aeration stirring. Microorganisms may also be cultured continuously or in batches. The culture conditions are appropriately selected depending on the composition of the medium and the culture method, and are not particularly limited as long as the conditions are such that the microorganism according to the present invention can grow, and can be appropriately selected depending on the type of the microorganism to be cultured. Usually, the culture temperature is preferably 10 to 40 ° C, more preferably 25 to 37 ° C. The pH of the medium suitable for culturing is not particularly limited, but is preferably 4 or more, more preferably 4 to 13. Further, the culturing time is not particularly limited and varies depending on the type of microorganism to be cultivated, the amount of medium, the culturing conditions and the like. Usually, the culture time is preferably 16 to 48 hours, more preferably 20 to 30 hours.

<Wastewater treatment method>
One embodiment of the present invention relates to a method for treating wastewater, which comprises a step of contacting a wastewater containing fats and oils with the microorganism according to the present invention. The microorganism according to the present invention has an excellent effect of reducing fats and oils, and has a property of being able to purify wastewater even in a water quality environment having a wide range of pH (particularly, a strong alkaline pH of 11 or higher). Therefore, the oil and fat can be effectively reduced by contacting the wastewater containing the oil and fat with the microorganism (oil-decomposing microorganism) according to the present invention. The above description of microorganisms may be modified as necessary and applied to the present embodiment.

Hereinafter, the wastewater treatment method according to this aspect will be described in more detail with reference to FIG. 1. The wastewater treatment method of the present invention is not limited to FIG.

FIG. 1 schematically shows the mechanism of wastewater treatment (wastewater treatment) by the grease trap 10. In the wastewater treatment method, the microorganism according to the present invention may be added in advance to the wastewater before being discharged to the grease trap 10, but is typically added to the wastewater in the wastewater treatment tank 1. However, the wastewater treatment method according to the present invention is not particularly limited as long as the microorganism according to the present invention can be brought into contact with the oil-containing wastewater.

The grease trap 10 may be of any inflow method such as a pipe introduction type or a gutter introduction type. In addition, installation conditions such as underground burial, slab ceiling, burial in a cinder, and floor-standing type are not particularly limited. In the case of underground burial, for example, in a kitchen or a food processing plant, the grease trap 10 is buried so that the drainage discharged to the drainage channel is poured into the residue receiver 3. In the case of the movable type, for example, the grease trap 10 is installed so that the residue receiver 3 is located at the lower part of the drainage groove of the sink.

In FIG. 1, the drainage flows in the direction of the arrow. The drainage to the grease trap 10 may be input to the grease trap 10 by a batch type or a continuous type. The oil-containing wastewater flows into the wastewater treatment tank 1 through the residue receiver 3. At this time, all or part of the residue such as garbage is collected by the residue receiver 3, but most of the fats and oils pass through the residue receiver 3 and flow into the wastewater treatment tank 1. The oil and fat 6 that has flowed into the wastewater treatment tank 1 rises toward the water surface 5 and gathers in the space partitioned by the partition plates 2a and 2c. Therefore, when the microorganism according to the present invention is not added to the wastewater, the oil and fat 6 gradually aggregates in the space partitioned by the partition plates 2a and 2c to form a scum.

When the microorganism according to the present invention is applied to the grease trap 10, the wastewater containing oil and fat and the microorganism according to the present invention are separated in the wastewater treatment tank 1 (mainly in the space partitioned by the partition plates 2a and 2c). It will come into contact. Since the microorganism according to the present invention has high fat-decomposing activity and assimilation, it can suppress the aggregation of fats and oils 6 and effectively prevent the formation of scum. In particular, the 2-2819 strain has high oil-decomposing activity even in a wide pH range (particularly in a strongly alkaline region having a pH of 11 or higher). This prevents fats and oils from flowing out to the external environment through the trap pipe 4 regardless of the pH of the wastewater, which is also advantageous from the viewpoint of environmental protection.

In the wastewater treatment method, the microorganism according to the present invention has various forms such as a state of being suspended in a culture solution, a state of being recovered as solid content from the culture solution, a state of being dried, and a state of being immobilized on a carrier. Can be brought into contact with drainage. Microorganisms suspended in the culture medium, recovered as solids from the culture medium, or dried are added, for example, into the wastewater and brought into contact with the wastewater. The microorganisms immobilized on the carrier may be added to the wastewater, but the microorganisms and the wastewater are discharged by installing the carrier on which the microorganisms are immobilized in a grease trap and allowing the wastewater to pass through the microorganism-immobilized carrier. Can also be contacted with. By installing the microorganisms immobilized on the carrier in the grease trap, it is possible to prevent the microorganisms from flowing out together with the drainage and reducing the number of microorganisms.

When the microorganism according to the present invention recovered from the culture solution as a solid content is used, any means known to those skilled in the art can be adopted as the recovery method. For example, the culture solution of the oil-degrading microorganism cultivated by the above-mentioned method can be obtained by solid-liquid separation by centrifugation, filtration, or the like, and the solid content can be recovered. If this solid content is dried (for example, freeze-dried), a dried oil-decomposing microorganism can be obtained.

When an oil-degrading microorganism immobilized on a carrier is used, the carrier for immobilizing the oil-degrading microorganism is not particularly limited as long as it can immobilize the microorganism, and generally the microorganism is immobilized. The carrier used to do so is used in the same manner or with appropriate modifications. For example, a method of comprehensively fixing to a gel-like substance such as alginic acid, polyvinyl alcohol, gellan gum, agarose, cellulose, dextran, or a method of adsorbing and fixing to the surface of glass, activated carbon, polystyrene, polyethylene, polypropylene, wood, silica gel, etc. Can be used.

Further, the method for immobilizing the oil-degrading microorganism on the carrier is also not particularly limited, and a general method for immobilizing the microorganism is used in the same manner or appropriately modified. For example, an immobilization method by pouring a microbial culture solution into a carrier, an immobilization method by placing the carrier under reduced pressure using an aspirator and pouring the microbial culture solution into the carrier, and a medium in which the microbial culture medium is sterilized. Examples thereof include a method of pouring into a mixture of the carrier and the carrier, culturing with shaking, and naturally drying the carrier taken out from the mixture.

In the method according to the present invention, when oil-degrading microorganisms are added to the wastewater and brought into contact with the wastewater, the amount of the added bacteria can be arbitrarily set. The amount of bacteria added to the wastewater is not particularly limited, but is, for example, 1 × 10 ⁴ to 1 × 10 ¹² CFU, preferably 1 × 10 ⁵ to 1 × 10 per 1 g of oil / fat contained in the waste water. ¹¹ CFU. Alternatively, for 1 g of fat and oil contained in the wastewater, for example, it is 0.1 mg to 5 g (dry cell weight), preferably 1 mg to 1.5 g (dry cell weight), and more preferably 10 mg to 150 mg (dry cell weight). Dry cell weight). Alternatively, the amount may be, for example, 1 × 10 ⁶ to 1 × 10 ¹² CFU / L, more preferably 1 × 10 ⁷ to 1 × 10 ¹¹ CFU / L with respect to the drainage in the grease trap. .. Alternatively, it is, for example, 10 mg to 15 g (dry cell weight) / L, preferably 0.1 g to 1.5 g (dry cell weight) / L with respect to the drainage in the grease trap. When two or more kinds of microorganisms are used in combination, it means the total amount. As the microorganism added to the wastewater, pre-cultured microorganisms may be used. By pre-culturing, the amount of bacteria to be inoculated can be easily adjusted.

When the wastewater is discharged to the external environment, the oil-decomposing microorganisms that are not immobilized on the carrier are discharged to the outside of the grease trap together with the wastewater. Therefore, in the present invention, the oil-decomposing microorganisms are periodically added to the grease trap (drainage). It is preferable to do. The interval of addition is not particularly limited, but it is preferable to add at intervals of, for example, once / three hours, once / 24 hours, or once every 2 to 3 days. The method of addition is not particularly limited, and when the wastewater continuously flows into the grease trap, it may be added mixed with the wastewater or may be added directly to the wastewater in the grease trap. If microorganisms are added from a drain outlet such as a kitchen sink, the microorganisms can be introduced into the grease trap together with the drainage discharged by washing.

In the wastewater treatment method, in addition to the microorganism according to the present invention, other components may be added to the wastewater from the viewpoint of more efficiently reducing fats and oils. Examples of other components include microorganisms described in JP-A-2017-136033, oil-degrading enzymes such as lipase, pH adjusters, oil-and-fat adsorbents, and surfactants.

Examples of other microorganisms capable of coexisting with the microorganism according to the present invention include the genus Yarrowia, the genus Candida, the genus Pichia, the genus Hansenula, the genus Saccharomyces, and the genus Kluybello. Genus Kluyveromyces, genus Trichosporon, genus Bacillus, genus Lactobacillus, genus Aspergillus, genus Rhodococcus, genus Rhodococcus, genus Enteloccus , Enterobacter, Burkholderia, Pseudomonas, Penicillium, Staphylococcus, Staphylococcus, Rhizopsis, Rhizo Examples include the genus Acinetobacter, the genus Alcaligenes, the genus Fusarium, the genus Serratia, the genus Tetrasphaera, the genus Humicola, and the genus Stenotrophomonas. These microorganisms may be obtained from culture collections such as ATCC, NBRC, DSMZ and the like. Among these microorganisms, it is preferable to use microorganisms of the genus Yarrowia, Enterobacter or Sphingomonas because of the high resolution of fats and oils.

Microorganisms of the genus Yarrowia include Yarrowia lipolytica ATCC48436, Yarrowia lipolytica NBRC1548, Yarrowia lipolytica LM02-011 (accession number NITE P-01813), Yarrowia lipolytica NBRC0746, Yarrowia lya ), Yarrowia Species such as Yarrowia YH-01, etc., but Yarrowia lipolytica is more preferable, and Yarrowia lipolytica LM02-011 (LM02-011 strain) is an independent administrative product. Evaluation Technology Infrastructure Organization Patented Microbial Deposit Center (2-5-8 Kazusakamatari, Kisarazu City, Chiba Prefecture, Japan 292-0818) has been deposited under the accession number NITE P-01813 on March 6, 2014). More preferred.

As for the microorganisms of the genus Enterobacter, Enterobacter sp. LM02-030 strain (LM02-030 strain is the National Institute of Technology and Evaluation Patent Microorganisms Depositary Center (Kisarazu, Chiba Prefecture, Japan 292-0818) (Deposited as the accession number NITE P-02048 on May 12, 2015) to 2-5-8) Kazusaka Ichi, etc. can be exemplified.

Examples of the microorganisms of the genus Sphingomonas include Sphingomonas sp. 2629-3b described in JP-A-2006-166874 and Sphingomonas sp. LM02-032 strain (LM02-032) described in JP-A-2017-136033. The stock was sent to the National Institute of Technology and Evaluation Patent Microbial Deposit Center (2-5-8 Kazusakamatari, Kisarazu City, Chiba Prefecture, Japan 292-0818) on June 19, 2015, with the accession number NITE P-02869. (Deposited as), Sphingomonas sp., Etc. can be exemplified.

In order to assist the decomposition of mineral oil by microorganisms used in the method according to the present invention, an oil-degrading enzyme such as lipase or phospholipase may be added to wastewater or the like. Examples of oil-degrading enzymes include genus Pseudomonas, genus Aspergillus, genus Bacillus, genus Penicillium, genus Rhizopus, and genus Rhizomu. , Pecilomyces, Rhizoctonia, Absidia, Achromobacter, Aeromonas, Alternaria, Alternaria Beauveria, Chromobacter, Coprinus, Fusarium, Geotricum, Humicola, Hyphozima, Hyphozyma Genus Metalhizium, genus Rhodospolidium, genus Trichoderma, genus Yarrowia, genus Candida, genus Pichia, genus Hansenula, genus Hansenula, genus Hansenula It can be obtained from the genus Kluyveromyces and / or the genus Trichosporon.

Commercially available oil-degradable enzymes include Lipase MY, Lipase OF, Lipase PL, Lipase QLM (Meishu Sangyo Co., Ltd.); Lipase A "Amano (registered trademark)" 6, Lipase DF "Amano (registered trademark)" 15, Lipase G "Amano (registered trademark)" 50, Lipase AY "Amano (registered trademark)" 30SD, Lipase R "Amano (registered trademark)", Lipase MER "Amano (registered trademark)", Neurase (registered trademark) F ( Amano Enzyme Co., Ltd.); Sumiteam (registered trademark) NLS, Sumiteam (registered trademark) RLS (Shin Nihon Kagaku Kogyo Co., Ltd.); Lillipase (registered trademark) A-10D, Lillipase (registered trademark) AF-5, PLA2 Nagase (Nagase) Chemtex Co., Ltd.); Entilon AKG-2000, Entilon LP, Entilon LPG (Rakuto Kasei Kogyo Co., Ltd.); Examples thereof include 100L (registered trademark), Lipozyme (registered trademark) RMIM, Lipozyme (registered trademark) TLIM, Novozyme (registered trademark) 435FG (manufactured by Novozymes); Picantase A, Picantase R800 (manufactured by DSM Japan), and the like. .. It is also possible to use two or more of these in combination.

The amount of the oil-degradable enzyme is not particularly limited as long as the enzyme can react with the fat and oil, but it is preferably used at 10 to 2,000 U with respect to 1 g of the fat and oil contained in wastewater or the like. It is more preferably 50 to 1,500 U, still more preferably 100 to 1,000 U. Alternatively, the amount may be preferably 1,000 to 100,000 U / L, more preferably 2,000 to 80,000 U / L with respect to the capacity of the grease trap. The active unit (U) of the oil-degrading enzyme is the amount of the enzyme that liberates 1 μmol of fatty acid per minute under the conditions of 37 ° C. and pH 7.

In the method according to the present invention, an oil / fat adsorbent such as a silas balloon, diatomaceous earth, an inorganic polymer such as pearlite, or an organic polymer such as polyurethane, polyethylene, or melanin resin described in JP2012-206084 is used. It may be added to the wastewater.

In the method according to the present invention, anionic surfactants such as sodium dodecylsulfate (SDS), dodecylbenzenesulfonic acid (NaDDBS), ammonium laurylsulfate, sodium caseinate, etc. in order to prevent the aggregation of fats and oils and the formation of scum; Cationic surfactants such as amine salts and quaternary ammonium salts; fatty acid esters (monoglycerin fatty acid ester, diglycerin fatty acid ester, sorbitan fatty acid ester, propylene glycol fatty acid ester, sucrose fatty acid ester), polyethylene glycol, polyethylene glycol-t. -Nonionic surfactants such as octylphenyl ether (Triton X-100), lecithin; surfactants such as amphoteric surfactants such as betaine, amine oxide and saponin may be added to the wastewater.

The grease trap may be in the form of continuously introducing oil-containing wastewater and continuously discharging the treated wastewater, or after introducing oil-containing wastewater and collectively treating the wastewater, the treated wastewater. May be in the form of discharging all at once.

Further, in the wastewater treatment method according to the present invention, the temperature at which the oil-decomposing microorganism and the oil-fat are brought into contact with each other, that is, the temperature of the wastewater in the grease trap can be arbitrarily set. Further, the pH at the time of contacting the oil-decomposing microorganism with the oil-fat, that is, the pH of the waste water in the grease trap can be arbitrarily set. Generally, the temperature is, for example, 5 to 4 ° C, preferably 10 to 40 ° C, more preferably 20 to 37 ° C. The pH is, for example, 4 or more, preferably 4 to 13. Further, if necessary, the wastewater may be aerated by aeration or the like.

<Wastewater treatment agent>
In one embodiment of the present invention, a wastewater treatment agent containing the above-mentioned microorganism according to the present invention is provided. The microorganism according to the present invention has an excellent effect of reducing fats and oils, and has a property of being able to purify wastewater even in a water quality environment having a wide range of pH (particularly, a strong alkaline pH of 11 or higher). Therefore, by using the wastewater treatment agent containing microorganisms according to the present invention in a wastewater treatment facility (harmfulness facility) such as a grease trap, fats and oils can be effectively reduced. The above description of the microorganism and the wastewater treatment method may be modified as necessary and applied to the present embodiment.

The wastewater treatment agent may be in either a dry form or a liquid form, but a dry form such as powder, granules, pellets or tablets is preferable from the viewpoint of storage stability. As the microorganism according to the present invention used for such a dry form of wastewater treatment agent, the bacterial cell powder obtained by spray-drying or freeze-drying the culture solution, or the microorganism in a state of being immobilized on a carrier as described above. It may be body-based, and may be further molded into powder, granules, pellets, or tablets. Alternatively, the cells or the culture solution may be encapsulated with hydroxypropylmethylcellulose, gelatin or the like. The wastewater treatment agent may also contain excipients such as hydroxypropyl cellulose, dextrin, lactose, starch and the like.

The microorganism according to the present invention contained in the wastewater treatment agent may be a dead bacterium or a live bacterium, but is preferably a live bacterium from the viewpoint of sustainability of fat decomposition activity.

The amount of the microorganism according to the present invention contained in the wastewater treatment agent is, for example, 10 to 100% by weight in the solid content of the wastewater treatment agent. Alternatively, the amount of the microorganism according to the present invention contained in the wastewater treatment agent is, for example, an amount of 1 × 10 ² to 1 × 10 ¹⁰ CFU / g with respect to the entire wastewater treatment agent. Further, the wastewater treatment agent is a group consisting of other microorganisms capable of coexisting with the above-mentioned microorganism according to the present invention, a fat-degrading enzyme, a fat-adsorbing agent, and a surfactant as long as the object effect of the present invention is achieved. It may contain one or more additives selected from the above. As other microorganisms capable of symbiosis, a fat-degrading enzyme, a fat-adsorbing agent, and a surfactant, for example, those described in JP-A-2017-136033 can be used.

The effects of the present invention will be described with reference to the following examples and comparative examples. However, the technical scope of the present invention is not limited to the following examples.

Example 1: Isolation of microorganisms A sample collected from soil in Tsuruoka City, Yamagata Prefecture was inoculated into a liquid medium for primary screening by the above method and cultured at 30 ° C. for one week. 100 μL of the culture solution after culturing was further inoculated into 5 mL of the liquid medium for primary screening, and the cells were cultured again at 30 ° C. for one week.

100 μL of the culture solution after the primary screening diluted 10 ^- fold was applied to the agar medium for secondary screening prepared by the above method, and cultured at 30 ° C. for one week. After culturing, a strain in which the formation of halo by decomposition of fats and oils was confirmed was isolated.

Next, 0.05 g of fat and oil (rapeseed oil: soybean oil = 1: 1 (w / w)) was added to 5 mL of the liquid medium for tertiary screening prepared by the above method to prepare a sterilized test solution (fat and oil). 1% (w / v)). Each isolated strain obtained in the above secondary screening was inoculated into the test solution prepared by the above method one by one with a platinum loop, and cultured with shaking at 30 ° C. for 24 hours (140 rpm).

After culturing, a normal hexane extract was prepared according to JIS K0102: 2016 revision (industrial wastewater test method). Using the normal hexane extract as the residual amount of fats and oils, 0.05 g of fats and oils added at the time of preparation of the test solution and the residual amount of fats and oils (amount of normal hexane extract (g)) are used to determine the fat reduction rate according to the following formula (1). Asked. As a result, a strain having a high fat reduction rate was isolated.

The isolated strain was named Pseudomonas sp. 2-2819 strain and deposited at the National Institute of Technology and Evaluation Patent Microorganisms Depositary (accession number NITE BP-02726).

Example 2: Evaluation of oil and fat reduction rate Using the 2-2819 strain and a conventionally known oil and fat decomposing microorganism, the oil and fat reduction rate was evaluated by the following method. As a conventionally known oil-degrading microorganism, Yarrowia lipolytica NBRC0746 strain (standard strain of Yarrowia lipolytica; hereinafter, also simply referred to as “NBRC0746 strain”) purchased from the National Institute of Technology and Evaluation of Incorporated Administrative Agency was used. ..

A sterilized test solution was prepared by adding 0.05 g of fat and oil to 5 mL of a liquid medium for tertiary screening whose pH was adjusted in the range of 3 to 13 using hydrochloric acid or sodium hydroxide. The isolated strains cultured on the agar medium for secondary screening were inoculated into a loopful with a loopful and the test solution prepared above, and cultured with shaking at 30 ° C. for 24 hours (140 rpm).

After culturing, a normal hexane extract was prepared according to JIS K0102: 2016 revision (industrial wastewater test method). Using the normal hexane extract as the residual amount of fats and oils, 0.05 g of the fats and oils added at the time of preparation of the test solution and the residual amount of fats and oils (amount of normal hexane extract (g)) are used to determine the fat reduction rate by the above formula (1). I asked. The results are shown in Table 15 below.

As shown in Table 15, it can be seen that the 2-2819 strain reduced 1% (w / v) of fats and oils by 50% by weight or more in 24 hours under the conditions of 30 ° C. and pH 4 to 13. That is, it can be seen that the 2-2819 strain is excellent in fat-decomposing ability even in a water quality environment of a wide range of pH (particularly strong alkaline with pH 11 or higher).

1 Wastewater treatment tank,
2a, 2b, 2c dividers,
3 Residue receiving,
4 Trap tube,
5 water surface,
6 fats and oils,
10 Grease trap.

Claims (6)

Microorganism deposited as Pseudomonas sp. 2-2819 strain (accession number NITE BP-02726). The microorganism according to claim 1 , which exhibits the following mycological properties.

The microorganism according to claim 1 or 2 , which has the 16S rDNA base sequence represented by SEQ ID NO: 1. The microorganism according to any one of claims 1 to 3, which reduces 1% (w / v) of fats and oils by 50% by weight or more in 24 hours under the condition of pH 4 or higher. A method for treating wastewater, which comprises a step of contacting the wastewater containing oil and fat with the microorganism according to any one of claims 1 to 4. A wastewater treatment agent containing the microorganism according to any one of claims 1 to 4.

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