JP2019208460A - Novel microorganisms for decomposition of oil and fat - Google Patents

Abstract

To provide microorganisms that have the great power of reducing oil and fat, and can purify wastewater in various water quality environments with differing pHs.SOLUTION: The present invention provides Pseudomonas sp.2-2819 strain (accession number NITE BP-02726) or microorganisms having the same mycological properties as those of it. The present invention provides microorganisms having the same mycological properties as those of the microorganisms described above, and having 16S rDNA base sequence consisting of a specific sequence.SELECTED DRAWING: None

Description

  The present invention relates to a novel degrading microorganism for fats and oils.

  Wastewater (waste water) from kitchens and food factories usually contains garbage and cooking oil. Solids such as raw garbage can be easily removed from the waste water by providing a basket or the like at the drain outlet, but it is not easy to remove liquid substances such as cooking oil. Therefore, in facilities such as kitchens and food factories that discharge wastewater mixed with a large amount of fat and oil, a detoxification facility (for example, a grease trap) is provided to collect the fat and oil and separate and dispose of the fat that has floated on the upper layer. It has been.

  However, the oil accumulated in the grease trap solidifies and remains as scum (lumps of oil) on the water surface of the grease trap, or it accumulates and adheres to the inner wall surface of the grease trap or inside the pipe, thereby closing the pipe. There is. At this time, the accumulated fats and oils may be oxidized and spoiled to cause odors and pests. Moreover, if the accumulated fats and oils are allowed to stand, the grease trap's ability to remove fats and oils decreases, and the fats and oils flow out into sewage and rivers. Therefore, when fats and oils are accumulated in the grease trap, it is necessary to request a specialized supplier to remove the fats and oils by vacuum treatment or high-pressure washing treatment, which increases costs.

  Therefore, a method for efficiently reducing fats and oils in a grease trap, in particular, a method using microorganisms that decompose and assimilate fats and oils has been studied. For example, Patent Document 1 describes Yarrowia lipolytica as a microorganism having an ability to assimilate free fatty acids.

JP2013-14689A

  In the microorganism described in Patent Document 1, an oil decomposition experiment is performed at pH 6 or 7. On the other hand, the water quality such as the pH of the wastewater in the grease trap can vary greatly depending on the food residue discharged. For this reason, the microorganisms used in the grease trap are required to have a characteristic capable of purifying wastewater even in a water quality environment of a wide range of pH (particularly strong alkaline of pH 11 or higher). However, conventionally known microorganisms have not had such characteristics sufficiently.

  Therefore, this invention is made | formed in view of the said situation, and makes it a subject to provide the microorganisms excellent in the reduction effect of the fats and oils in an abatement facility. In particular, an object is to provide a microorganism capable of purifying waste water even in a water environment having a wide pH range (particularly strong alkaline of pH 11 or higher).

  The present inventors have intensively studied to solve the above problems. As a result, the present inventors have found that the above problem can be solved by a microorganism belonging to Pseudomonas and exhibiting a predetermined mycological property, and has completed the present invention.

  ADVANTAGE OF THE INVENTION According to this invention, the microorganisms excellent in the reduction effect of fats and oils in an abatement facility are provided. In particular, according to the present invention, a microorganism capable of purifying wastewater in a water quality environment of a wide range of pH (particularly strong alkaline of pH 11 or higher) is provided.

FIG. 1 schematically shows the mechanism of wastewater treatment by a grease trap. FIG. The 16S rDNA partial base sequence comparison of mossellii CIP105259 ^T (AF072688) is shown. “Query” indicates 2-2919 strain, and “Sbjct” indicates P.I. Shows mosseri. FIG. 2B shows strains 2-2919 and P. pylori. A 16S rDNA partial base sequence comparison of mossellii CIP105259 ^T (AF072688) is shown (continuation of FIG. 2A). “Query” indicates 2-2919 strain, and “Sbjct” indicates P.I. Shows mosseri.

  Embodiments according to one embodiment of the present invention will be described below. The present invention is not limited only to the following embodiments.

  In this specification, “X to Y” indicating a range means “X or more and Y or less”. Unless otherwise specified, measurement of operation and physical properties is performed under conditions of room temperature (20 to 25 ° C.) / Relative humidity 40 to 50% RH.

<Microorganism>
One embodiment of the present invention is a microorganism (oil-decomposing microorganism) belonging to Pseudomonas and having the following mycological properties. In one embodiment, a microorganism (oil-decomposing microorganism) belonging to Pseudomonas, having the following mycological properties, and having 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 fat and oil by 50% by weight or more in 24 hours under the condition of pH 4 or higher. Although the upper limit of pH is not specifically limited, For example, it is 13 or less. Further, the temperature condition is not particularly limited, but is 30 ° C., for example.

  In a particularly preferred embodiment, the microorganism of this form is Pseudomonas sp. Strain 2-2919 (Accession No. 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. Screening method An appropriate amount of a sample collected from the waste of Yamagata Prefecture or grease trap wastewater, sewage, river water, etc. is added to 5 mL of the primary screening liquid medium prepared by the following method and cultured at 30 ° C. for one week. 100 μL of the culture solution after the culture is further inoculated into 5 mL of the liquid medium for primary screening and cultured again at 30 ° C. for one week.

  In the liquid medium for primary screening, each component other than fats and oils is dissolved in pure water so as to have the composition shown in Table 4 below. Prepare. In addition, fats and oils are prepared by mixing rapeseed oil and soybean oil at a ratio of 1: 1 (w / w).

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

  The agar medium for secondary screening was prepared by dissolving each component other than fats and fats and agar in pure water so as to have the composition shown in Table 5 below, and fats and oils (rapeseed oil: soybean oil = 1: 1 (w / w)). A final concentration of 0.5 w / v% and agar are added to a final concentration of 2.0 w / v%, and after high-temperature and high-pressure sterilization, they are appropriately dispensed and solidified.

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

  The liquid medium for tertiary screening is prepared by dissolving each component in pure water, adjusting to pH 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 the culture, a normal hexane extract is prepared according to JIS K0102: 2016 revision (industrial wastewater test method). The normal hexane extract was used as the residual amount of oil and fat, and the fat and oil reduction rate was calculated from the following formula (1) using 0.05 g of oil and fat added during the preparation of the test solution and the residual amount of fat and oil (the amount of normal hexane extract (g)). Ask for. As a result, it is possible to isolate a strain having a high fat reduction rate.

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

1. Culture conditions A bacterial strain cultured under the following conditions is used as a test cell.

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.

As a result of BLAST homology search against DNA database DB-BA12.0 (Techno Suruga Lab Co., Ltd.) and international base sequence database (DDBJ / ENA (EMBL) / GenBank) for microorganism identification, 16S rDNA partial base sequence of isolated microorganism Showed a homology of 99.9% with the reference strain CIP 105259 ^T (accession number AF072688) of Pseudomonas mossellii. P.P. Homology with a homology rate of 99% or more was shown for multiple species such as entomophila L48 ^T (AY907756). 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 base 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 phylogenetic branch is the boost trap value, T at the end of the stock name indicates the type strain (Type Strain), and BSL is the biosafety Level (BSL1 * (opportunistic pathogen) or higher is indicated).

In the molecular phylogenetic tree, the isolated microorganism is contained in a cluster constituted by the genus Pseudomonas. Mossellii showed the same molecular phylogenetic position as CIP105259 ^T (AF072688).

As shown in FIG. 2A and FIG. When comparing the 16S rDNA partial base sequence of mossellii CIP105259 ^T (AF072688), a difference of 2 bases is observed, and one base of the difference is due to insertion or deletion (FIG. 2A). Both are considered to be almost identical. However, the remaining 1 base was clearly different (FIG. 2B). Therefore, it is thought that both actually show different molecular phylogenetic positions.

  Therefore, from the results of the 16S rDNA partial nucleotide sequence analysis, the isolated microorganism was found to be P. aeruginosa. Identified as Pseudomonas sp., Closely related to mossellii.

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

  Morphological observation by optical microscope (BX50F4, Olympus, Japan) and the method of Barrow & Feltham (Barrow GI & Feltham RK A. (1993), Cowan and Steel's Manual for the 3rd. Based on edition, Cambridge University Press), catalase reaction, oxidase reaction, acid / gas production from glucose, oxidation / fermentation (O / F) of glucose were tested. Gram staining was performed using a Faber G “Nissui” (Nishisui Pharmaceutical, Japan). The results are shown in Table 12.

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

  Also, Techno Suruga Lab Co., Ltd. and NCIMB Ltd., UK. The following items were tested in accordance with the technical alliance matters and publicly known technologies. The results are shown in Table 14.

  As shown in Table 12, the isolated microorganism was a gram-negative bacillus having motility, 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 RK (1993). Cowan and Steel's Manual for the Identification of Medical Brit. 3). (University Press).

  As shown in Table 13, the isolated microorganism does not reduce nitrate; exhibits arginine dihydrolase activity; does not hydrolyze esculin; hydrolyzes gelatin; glucose, D-mannose, N-acetyl-D- Glucosamine, potassium gluconate, n-capric acid, adipic acid, dl-malic acid, sodium citrate and phenyl acetate were assimilated; L-arabinose, D-mannitol, maltose and adipic acid were not assimilated.

  As shown in Table 14, the isolated microorganism does not grow at 4 ° C .; grows at 5% NaCl but does not grow at 7% NaCl; exhibits lecithinase activity; exhibits lysine decarboxylase activity and ornithine decarboxylase activity. Not shown; no acetoin produced; fluorescent dye produced on King's B agar.

  From the above, the properties of the isolated microorganism are Almost consistent with the properties of mossellii (Dabbousi F., Hamze M., Singer E., Geoffroy V., Meyer JM & Izard D. (2002). Pseudomonas mosseliiv. isolated from clinical specifications. Int J Syst Evol Microbiol, 52, 363-376.). However, as described above, there is a single base difference in 16S rDNA base sequence analysis.

  Therefore, it is determined that the isolated strain (isolated microorganism) is a novel microorganism, and this strain is referred to as Pseudomonas sp. 2-21919 strain (in this specification, simply “2-21919 strain”). "). In addition, as of May 23, 2018, the 2-2919 shares were deposited with the Patent Microorganism Depositary Center of the National Institute of Technology and Evaluation (2-5-8, Kazusa Kamashichi, Kisarazu City, Chiba Prefecture) The accession number is NITE BP-02726.

[Evaluation of oil reduction effect]
In this specification, the reduction | decrease in fats and oils is evaluated by the following method. That is, 0.05 g of oil / fat rapeseed oil: soybean oil = 1: 1 (w / w) is the same as the liquid medium for tertiary screening described above except for pH, and is a sterile-treated oil / fat decomposition evaluation medium (5 mL) To prepare a test solution (oil 1% (w / v)). As the fat and oil decomposition evaluation medium used at this time, a medium whose pH is 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). Oil and fat degradation evaluation medium). The pH is adjusted by adjusting any acid such as inorganic acid such as hydrochloric acid, nitric acid, carbonic acid and sulfuric acid, organic acid such as citric acid and lactic acid, and salts thereof; and / or any acid such as sodium hydroxide, potassium hydroxide and ammonia. Although it may be carried out by alkali; hydrochloric acid (acid side) or sodium hydroxide (alkali side) is preferable.

The test solution is inoculated with a microorganism cultured on a flat plate medium (for example, an agar medium for secondary screening), and cultured with shaking (140 rpm) at an arbitrary temperature range for 24 hours. The amount of bacteria to be inoculated is about one platinum ear. Microorganisms to be inoculated into the test solution may be those pre-cultured in a liquid medium for tertiary screening. 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 to 1.5 × 10 ⁶ CFU / mL. Although culture | cultivation temperature should just be set according to the temperature range with a high fat-oil decomposition | disassembly and utilization capability of a microbial cell, it is 10-40 degreeC, for example, Preferably it is 20-37 degreeC.

  After the culture, 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 oil and fat, the oil and fat added at the time of preparing the test liquid (0.05 g) and the residual amount of fat and oil (the amount of normal hexane extract (g)) were calculated according to the above formula (1). Find the rate of decrease. The microorganism according to the present invention has an oil / fat reduction rate of 50% by weight obtained by the above method in all test solutions prepared using the oil / fat decomposition evaluation medium having a pH set in the above range (for example, pH 4 to 13). That is all you need. 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 fat reduction rate, the better. Therefore, the upper limit is not particularly set. For example, the fat reduction rate measured by the above method is 90% by weight or less. If the culture is continued for a long time, the amount of oil reduction increases. However, since 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 / oil 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 facilities can easily vary depending on the type of garbage to be discharged. Therefore, it is preferable that the microorganisms used in the abatement facility can purify the wastewater in a wide range of pH environments. The 2-2919 strain is excellent in that it can decompose oils and fats even in a wide range of pH environments (particularly strong alkaline pH 11 or more).

  As used herein, “oil and fat” refers to edible or industrial fats and oils and fatty acids that are rich in glycerides such as triglycerides, diglycerides and monoglycerides. Examples of the oil 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 tallow, lard, chicken oil, fish oil, whale oil, butter, margarine, fat spread, shortening; and industrial oils such as linseed oil, jatropha oil, tall oil, hamana oil, castor oil, jojoba oil , But is preferably edible oil / fat that is frequently discharged in restaurants and the like where a grease trap is often installed. The fatty acid is not particularly limited, for example, butyric acid, hexanoic acid, heptanoic acid, octanoic acid, decanoic acid, lauric acid, tridecanoic acid, myristic acid, pentadecanoic acid, pentadecanoic acid, palmitic acid, heptadecanoic acid, Saturated fatty acids such as stearic acid, arachidic acid, behenic acid, lignoceric acid; decenoic acid, myristoleic acid, pentadecenoic acid, palmitoleic acid, heptadecenoic acid, oleic acid, icosenic acid, docosenoic acid, tetracosenoic acid, hexadecadienoic acid, hexa Decatrienoic acid, hexadecatetraenoic acid, linoleic acid, α-linolenic acid, γ-linolenic acid, octadecatetraenoic acid, icosadienoic acid, icosatrienoic acid, icosatetraenoic acid, arachidonic acid, icosapentaenoic acid, henicosapentaenoic acid, docosadienoic acid , And unsaturated fatty acids such as docosatetraenoic acid, docosapentaenoic acid, docosapentaenoic acid, and docosahexaenoic acid. Fatty acids may be produced by decomposing edible or industrial fats and oils.

[Culture of microorganisms]
The microorganism culturing method 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 any 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. Either a synthetic medium or a natural medium may be used. Usually, a culture medium contains a carbon source, a nitrogen source, and an inorganic substance.

  The carbon source that can be used in the culture of the microorganism according to the present invention is not particularly limited as long as it is a carbon source that can be assimilated by the strain used. Specifically, considering the assimilation of microorganisms, glucose, mannose, fructose, cellobiose, raffinose, xylose, maltose, galactose, sorbose, glucosamine, ribose, arabinose, rhamnose, sucrose, trehalose, α-methyl-D -Sugars such as glucoside, salicin, melibiose, lactose, melezitose, inulin, erythritol, ribitol, xylitol, glucitol, mannitol, galactitol, inositol, N-acetyl-D-glucosamine, starch, starch hydrolysate, molasses, molasses , Natural products such as wheat, rice, alcohols such as glycerol, methanol, ethanol, acetic acid, phenyl acetate, lactic acid, succinic acid, gluconic acid, capric acid, malic acid, glucuronic acid, pyrpine , Organic acids such as citric acid, and the like hydrocarbons hexadecane and the like. The carbon source is appropriately selected in view of assimilation by the microorganism to be cultured. For example, when using the 2-2819 strain, 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, Sodium salt, potassium salt) and the like are preferably used. Moreover, the said carbon source can be used 1 type or 2 types or more selected.

  Nitrogen sources that can be used in the culture of microorganisms according to the present invention include meat extract, fish extract, peptone, polypeptone, tryptone, yeast extract, malt extract, soybean hydrolysate, soybean powder, casein, milk casein, casamino acid, glycine Organic nitrogen sources such as various amino acids such as glutamic acid and aspartic acid, corn steep liquor, other animals, plants, microbial 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 view of assimilation by the microorganism to be cultured. For example, when 2-219 strain is used, it is preferable to use fish meat extract, tryptone, yeast extract, ammonium chloride, etc. among the nitrogen sources. Further, one or more of the nitrogen sources can be selected and used.

  Examples of inorganic substances that can be used in culturing microorganisms according to the present invention include phosphates, hydrochlorides, sulfates, acetates, carbonates, chlorides, such as magnesium, manganese, calcium, sodium, potassium, copper, iron, and zinc. And the like. The said inorganic substance is suitably selected in consideration of the assimilation property by the microorganisms to culture. In addition, one or more of the above inorganic materials can be selected and used. Moreover, you may add surfactant etc. in a culture medium as needed.

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

  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 conditions or anaerobic conditions. In the former case, the culture of microorganisms is performed by shaking or aeration stirring. Alternatively, the microorganisms may 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 microorganisms according to the present invention can grow, and can be appropriately selected according to the type of microorganism to be cultured. Usually, culture | cultivation temperature becomes like this. Preferably it is 10-40 degreeC, More preferably, it is 25-37 degreeC. The pH of the medium suitable for culture is not particularly limited, but is preferably 4 or more, more preferably 4 to 13. Furthermore, the culture time is not particularly limited, and varies depending on the type of microorganism to be cultured, the amount of medium, culture 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 wastewater treatment method including a step of bringing a microorganism according to the present invention into contact with wastewater containing fats and oils. The microorganism according to the present invention is excellent in the effect of reducing fats and oils, and has a characteristic capable of purifying wastewater even in a water quality environment having a wide range of pH (particularly strong alkaline of pH 11 or higher). Therefore, fats and oils can be effectively reduced by bringing the microorganisms (fat-decomposing microorganisms) according to the present invention into contact with wastewater containing fats and oils. Note that the above description regarding the microorganism can be modified as needed and applied to the present embodiment.

  Hereinafter, the waste water treatment method according to this aspect will be described in more detail with reference to FIG. In addition, the waste water treatment method of this invention is not limited to FIG.

  FIG. 1 schematically shows a mechanism of waste water treatment (waste water 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 any inflow method such as a pipe introduction type or a side groove introduction type. In addition, the installation conditions are not particularly limited, such as underground burial, slab ceiling, cinder burial, and floor type. 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 wastewater that flows out into 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 positioned below the drainage groove of the sink.

  In FIG. 1, drainage flows in the direction of the arrow. The drainage to the grease trap 10 may be a batch type or a continuous type. The fat and 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 in the residue receiver 3, but most of the oil and fat flows into the waste water treatment tank 1 through the residue receiver 3. The oil 6 that has flowed into the wastewater treatment tank 1 floats toward the water surface 5 and collects in a space partitioned by the partition plates 2a and 2c. Therefore, when the microorganisms according to the present invention are not added to the waste water, the oil 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, in the wastewater treatment tank 1 (mainly in a space partitioned by the partition plates 2a and 2c), the wastewater containing oil and fat and the microorganism according to the present invention are It will come into contact. Since the microorganisms according to the present invention have high fat and oil decomposing activity and have assimilability, aggregation of the oil and fat 6 can be suppressed and scum can be effectively prevented from being formed. In particular, the strain 2-2919 has a high fat-and-oil decomposition activity even in a wide pH range (particularly a strong alkaline range of pH 11 or higher). This prevents the oil and fat from flowing out to the external environment through the trap tube 4 without depending on the pH of the waste water, which is advantageous from the viewpoint of environmental conservation.

  In the wastewater treatment method, the microorganism according to the present invention has various forms such as a state suspended in a culture solution, a state recovered from the culture solution as a solid, a dried state, and a state immobilized on a carrier. Can be brought into contact with drainage. The microorganisms suspended in the culture solution, recovered as a solid content from the culture solution, or dried are added to, for example, waste water and brought into contact with the waste water. Microorganisms in a state of being immobilized on the carrier may be added to the wastewater. However, the carrier on which the microorganisms are immobilized is placed in a grease trap, and the wastewater is allowed to flow through the microorganism-immobilized carrier. Can also be brought into contact with each other. 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 waste water and reducing the number of bacteria.

  When using the microorganism according to the present invention recovered as a solid content from the culture solution, 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 microorganisms cultured by the above-described method can be obtained by solid-liquid separation by centrifugation, filtration, etc., and collecting the solid content. If this solid content is dried (for example, freeze-dried), a dried oil-degrading microorganism can be obtained.

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

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

In the method according to the present invention, when an oil-degrading microorganism is added to the waste water and brought into contact therewith, the amount of bacteria added 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 fats and oils contained in the wastewater. ¹¹ CFU. Alternatively, for example, 0.1 mg to 5 g (dry cell weight), preferably 1 mg to 1.5 g (dry cell weight), more preferably 10 mg to 150 mg (1 g of fat and oil contained in the waste water. 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 wastewater in the grease trap. . Or it is 10 mg-15 g (dry cell weight) / L with respect to the waste_water | drain in a grease trap, for example, Preferably it is 0.1g-1.5g (dry cell weight) / L. In addition, when using combining 2 or more types of microorganisms, the total amount is meant. In addition, you may use the pre-cultured microorganisms added to waste water. By pre-culturing, the amount of bacteria to be inoculated can be easily adjusted.

  When the wastewater is discharged to the outside environment, the oil-decomposing microorganisms that are not immobilized on the carrier are discharged together with the wastewater to the outside of the grease trap. Therefore, in the present invention, the oil-decomposing microorganisms are periodically added to the grease trap (drainage). It is preferable to do this. The addition interval is not particularly limited, but for example, it is preferably added once every 3 hours, once every 24 hours, or once every 2 to 3 days. The method of adding is not particularly limited, and when the wastewater flows continuously into the grease trap, it may be added to 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 wastewater discharged by washing.

  In the wastewater treatment method, in addition to the microorganisms according to the present invention, other components may be added to the wastewater from the viewpoint of more effectively 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 adsorbents, and surfactants.

  Other microorganisms that can coexist with the microorganisms of the present invention include, for example, the genus Yarrowia, the genus Candida, the genus Pichia, the genus Hansenula, the genus Saccharomyces, the genus Kluyvero Myces (Kluyveromyces) genus, Trichosporon genus, Bacillus genus, Lactobacillus genus, Aspergillus genus, Rhodococcus genus termocos genus, Enterococcus cc , Enterobacter genus, Burkholderia (B genus rkholderia, genus Pseudomonas, genus Penicillium, genus Staphylococcus, genus Rhizopus, genus Rhizobium, genus Acineto Examples include 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. Among these microorganisms, microorganisms belonging to the genus Yarrowia, Enterobacter or Sphingomonas are preferably used because of the high resolution of fats and oils.

  The microorganisms belonging to the genus Yarrowia include Yarrowia lipolytica ATCC 48436, Yarrowia lipolytica NBRC1548, Yarrowia lipolytica LM02-011 (Accession number NITE P-01813), Yarrowia lipolytica NBRC0746, Yarrowia lipolytica NBRC0746 ), Yarrowia sp. Such as Yarrowia YH-01 can be exemplified, but Yarrowia lipolytica is more preferable, and Yarrowia lipolytica LM02-011 (LM02-011 strain is an independent administrative corporation product) National Institute for Evaluation Technology Patent Microorganism Depositary Center (〒29 To -0818 Japan Chiba Prefecture Kisarazu Kazusa legs bowed in 2-5-8), has been deposited as accession number NITE P-01813 in the March 6 date 2014) it is more preferred.

  Enterobacter sp. LM02-030 strain (LM02-030 strain is the National Institute for Product Evaluation Technology Patent Microorganism Depositary Center, Kisarazu, Chiba Prefecture, Japan 292-0818). (Kazusa City Kamashita 2-5-8) is deposited on May 12, 2015 as deposit number NITE P-0208).

  Examples of microorganisms belonging to the genus Sphingomonas include, for example, Sphingomonas sp. 2629-3b described in JP-A No. 2006-166874 and Sphingomonas sp. LM02-032 strain (LM02-032 described in JP-A No. 2017-136033). The stock is registered with the National Institute of Technology and Evaluation Patent Microorganisms Depositary Center (2-5-8 Kazusa-Kamashita, Kisarazu City, Chiba Prefecture, Japan 292-0818), with accession number NITE P-02069 on June 19, 2015. Sphingomonas sp., Etc. (deposited as

  In order to assist the decomposition of the mineral oil by the microorganism used in the method according to the present invention, an oil-degrading enzyme such as lipase or phospholipase may be added to the waste water. Examples of the oil-degrading enzyme include the genus Pseudomonas, the genus Aspergillus, the genus Bacillus, the genus Penicillium, the genus Rhizopus, and the genus Rhizomucor. , Genus Paecilomyces, genus Rhizoctonia, genus Absidia, genus Achromobacter, genus Aeromonas, genus Alternaria, i. Beauveria), Chromobacte ), Coprinus, Fusarium, Geotricum, Humicola, Hyphozyma, Lactobacillus, Lactobacillus, Metalliz Rhodosporidium, Trichoderma, Yarrowia, Candida, Pichia, Hansenula, Saccharomyces, Kaccharomyces And / or from the genus Trichosporon Door can be.

  Commercially available oil-degrading enzymes include lipase MY, lipase OF, lipase PL, lipase QLM (name sugar industry); 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 (New Nippon Chemical Industry Co., Ltd.); Chemtex Co., Ltd.); Entilon AKG-2000, Entilon LP, Entilon LPG (Santo Kasei Kogyo Co., Ltd.) Company); Lipolase (registered trademark) 100T, Lipolase (registered trademark) 100L, Palatase 20000L, Lipex (registered trademark) 100T, Lipex (registered trademark) 100L, 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. Two or more of these may be used in combination.

  The amount of the oil-degrading enzyme is not particularly limited as long as the enzyme can react with fats and oils, but it is preferably used at 10 to 2,000 U with respect to 1 g of fats and oils contained in drainage. More preferably, it is 50-1,500U, More preferably, it is 100-1,000U. Alternatively, it may be an amount that is 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 activity unit (U) of the oil-degrading enzyme is the amount of 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 and fat adsorbent such as an inorganic polymer such as shirasu balloon, diatomaceous earth and perlite, an organic polymer such as polyurethane, polyethylene and melanin resin described in JP2012-206084A is used. It may be added to the waste water.

  In the method according to the present invention, an anionic surfactant such as sodium dodecyl sulfate (SDS), dodecyl benzene sulfonic acid (NaDDBS), ammonium lauryl sulfate, sodium caseinate, etc .; aliphatic to prevent oil aggregation and scum formation 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) and lecithin; surfactants such as amphoteric surfactants such as betaine, amine oxide and saponin may be added to the waste water.

  The grease trap may be configured to continuously introduce fat and oil-containing wastewater and continuously discharge the treated wastewater, or after introducing fat and oil-containing wastewater and treating it together, the treated wastewater. May be discharged in a batch.

  Moreover, in the waste water treatment method according to the present invention, the temperature at which the oil-degrading microorganisms and the oil are brought into contact with each other, that is, the temperature of the waste water in the grease trap can be arbitrarily set. Moreover, it can also set arbitrarily as pH at the time of contacting an oil-fat decomposition microorganism and fats, ie, pH of the waste_water | drain in a grease trap. Generally, temperature is 5-4 degreeC, for example, 10-40 degreeC is preferable and 20-37 degreeC is more preferable. pH is 4 or more, for example, Preferably it is 4-13. Further, the waste water may be aerated by aeration or the like as necessary.

<Wastewater treatment agent>
In one Embodiment of this invention, the waste water treatment agent containing the microorganisms which concern on the said invention is provided. The microorganism according to the present invention is excellent in the effect of reducing fats and oils, and has a characteristic capable of purifying wastewater even in a water quality environment having a wide range of pH (particularly strong alkaline of pH 11 or higher). Therefore, fats and oils can be effectively reduced by using the wastewater treatment agent containing microorganisms according to the present invention for wastewater treatment facilities (abatement facilities) such as grease traps. In addition, the description regarding the above-described microorganisms 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, granule, pellet, tablet or the like is preferable from the viewpoint of storage stability. The microorganism according to the present invention used for such a wastewater treatment agent in a dry form includes a bacterial powder obtained by drying a culture solution by spray drying, freeze drying, or the like, or a bacterium in a state of being immobilized on a carrier as described above It may be a body, and may be formed into a powder, granule, pellet, or tablet. Alternatively, the bacterial cells and the culture solution may be encapsulated with hydroxypropylmethylcellulose, gelatin, or the like. The wastewater treatment agent may also contain excipients such as hydroxypropylcellulose, dextrin, lactose, starch and the like.

  The microorganism according to the present invention contained in the wastewater treatment agent may be dead or live, but is preferably live from the viewpoint of the persistence of the oil decomposing 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. Or, the amount of the microorganism according to the present invention contained in the waste water treatment agent, for example, for the entire waste water treatment agent is an amount that a ^{1 × 10 2 ~1 × 10 10} CFU / g. Further, the wastewater treatment agent is a group consisting of other microorganisms capable of symbiosis with the microorganisms according to the present invention, an oil-degrading enzyme, an oil-and-fat adsorbent, and a surfactant as long as the objective effect of the present invention is achieved. An additive such as one or more selected from may be included. As other symbiotic microorganisms, fat-and-oil degrading enzymes, fat and oil adsorbents, and surfactants, for example, those described in JP-A-2017-136033 can be used.

  The effects of the present invention will be described using the following examples and comparative examples. However, the technical scope of the present invention is not limited only 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 the culture was further inoculated into 5 mL of the liquid medium for primary screening, and again cultured at 30 ° C. for one week.

100 μL of the culture solution after the primary screening diluted 10 ⁴ times was applied to the agar medium for secondary screening prepared by the above method and cultured at 30 ° C. for one week. After the cultivation, a strain was confirmed that was able to confirm the formation of halo due to the decomposition of fats and oils.

  Next, 0.05 g of oil / fat (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 (oil / fat 1% (w / v)). Each isolated strain obtained by the secondary screening was inoculated with a platinum loop one by one into the test solution prepared by the above method, and cultured with shaking (140 rpm) at 30 ° C. for 24 hours.

  After the culture, a normal hexane extract was prepared according to JIS K0102: 2016 revision (industrial wastewater test method). The normal hexane extract was used as the residual amount of oil and fat, and the fat and oil reduction rate was calculated from the following formula (1) using 0.05 g of oil and fat added during the preparation of the test solution and the residual amount of fat and oil (the amount of normal hexane extract (g)). Asked. As a result, a strain having a high rate of fat reduction was isolated.

  The isolated strain was named Pseudomonas sp. 2-2919 and deposited with the Patent Microorganism Depositary, National Institute of Technology and Evaluation (Accession Number NITE BP-02726).

Example 2: Evaluation of oil / fat reduction rate The oil / fat reduction rate was evaluated by the following method using 2-2919 strain and conventionally known oil-degrading microorganisms. As a conventionally known oil-degrading microorganism, Yarrowia lipolytica NBRC0746 strain (standard strain of Yarrowia lipolytica; hereinafter simply referred to as “NBRC0746 strain”) purchased from the National Institute for Product Evaluation Technology was used. .

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

  After the culture, 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 fat and oil, the oil / fat reduction rate was calculated by the above formula (1) from 0.05 g of the fat and oil added at the time of preparing the test solution and the residual amount of fat (normal hexane extract amount (g)). Asked. The results are shown in Table 15 below.

  As shown in Table 15, it can be seen that 2-2919 strain reduced 1% (w / v) of fat and oil 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 strain 2-2919 is excellent in oil and fat decomposing ability even in a water environment of a wide range of pH (particularly strong alkaline of pH 11 or higher).

1 Wastewater treatment tank,
2a, 2b, 2c partition plate,
3 Receiving residue
4 Trap tube,
5 Water surface
6 Oils and fats,
10 Grease trap.

Claims (6)

A microorganism belonging to Pseudomonas and exhibiting the following mycological properties.
  The microorganism according to claim 1, which has a 16S rDNA base sequence represented by SEQ ID NO: 1.   The microorganism according to claim 1 or 2, which reduces 1% (w / v) of fat and oil by 50% by weight or more in 24 hours under a condition of pH 4 or more.   The microorganism according to any one of claims 1 to 3, which is specified by Pseudomonas sp. 2-2919 strain (Accession No. NITE BP-02726).   The wastewater processing method including the process of making the microorganisms of any one of Claims 1-4 contact the wastewater containing fats and oils.   Waste water treatment agent containing the microorganism of any one of Claims 1-4.