JP7109305B2 - New oil-degrading microorganisms - Google Patents

Description

NPMD NITE BP-02752

TECHNICAL FIELD The present invention relates to novel oil-degrading microorganisms.

Drainage (waste water) from kitchens and food factories usually contains garbage and cooking oil. Solid matter such as food waste 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 containing a large amount of grease, a detoxification facility (for example, a grease trap) is installed in order to separate and dispose of the grease that accumulates and floats to the upper layer. It is

However, the accumulated oil in the grease trap solidifies and remains as scum (oil mass) on the water surface of the grease trap, or accumulates and adheres to the inner wall surface of the grease trap or inside the pipe, clogging the pipe. There is At this time, the accumulated oils and fats may oxidize and putrefy and become a cause of bad smells and pests. In addition, if the accumulated grease is left unattended, the ability of the grease trap to remove grease is reduced, causing the grease to flow into sewage or rivers. Therefore, when the grease accumulates in the grease trap, it is necessary to ask a specialized company to remove the grease by vacuum treatment, high-pressure washing, or the like, which increases costs.

Therefore, in grease traps, methods for efficiently reducing fats and oils, in particular, methods using microorganisms that decompose and assimilate fats and oils are being studied. For example, Patent Document 1 describes Bacillus subtilis BN1001 as a microorganism that can be used to reduce n-hexane extractables in oil-containing wastewater and to decompose scum accumulated in kitchen wastewater tanks. is described.

JP-A-3-236771

However, with conventional microorganisms, it was sometimes difficult to sufficiently reduce fats and oils contained in waste water from abatement facilities. In particular, the water quality such as the pH of the wastewater in the grease trap can change greatly depending on the food residue and the like discharged. For this reason, the microorganisms used in the grease trap are required to have properties capable of purifying wastewater even in water environments with a wide range of pH (eg, pH 5-11). However, conventionally known microorganisms do not have sufficient such characteristics.

Accordingly, the present invention has been made in view of the above circumstances, and an object of the present invention is to provide microorganisms that are excellent in the effect of reducing fats and oils in harm removal facilities. In particular, it is an object of the present invention to provide microorganisms capable of purifying waste water even in water environments with a wide range of pH (eg, pH 5-11).

The present inventors have conducted intensive studies to solve the above problems. As a result, the present inventors have found that the above problems can be solved by a microorganism belonging to Pseudomonas and exhibiting predetermined mycological properties, leading to the completion of the present invention.

INDUSTRIAL APPLICABILITY According to the present invention, microorganisms are provided that are excellent in the effect of reducing fats and oils in detoxification facilities. In particular, the present invention provides microorganisms capable of purifying waste water even in water environments with a wide range of pH (eg, pH 5-11).

FIG. 1 schematically shows the mechanism of wastewater treatment using a grease trap.

An embodiment according to one aspect 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, measurements of operations and physical properties are performed under the conditions of room temperature (20 to 25° C.)/relative humidity of 40 to 50% RH.

<Microbes>
One aspect of the present invention is a microorganism (oil-degrading microorganism) belonging to Pseudomonas and exhibiting the following mycological properties. In one embodiment, a microorganism (oil-degrading microorganism) that belongs to Pseudomonas, exhibits the following mycological properties, and has the 16S rDNA nucleotide sequence shown in SEQ ID NO: 1 is provided. In the present specification, microorganisms exhibiting the following mycological properties are also simply referred to as "microorganisms according to the present invention".

In a preferred embodiment, the microorganism according to the present invention reduces 1% (w/v) fat by 50% or more in 24 hours at pH 5 or higher. Although the upper limit of the pH is not particularly limited, it is, for example, 11 or less. Moreover, although the temperature condition is not particularly limited, it is, for example, 30°C.

In a particularly preferred embodiment, the microorganism of this form is Pseudomonas sp. strain 2-2306 (accession number NITE BP-02752).

[screening]
The microorganisms according to the present invention were isolated from soil in Kahoku City, Ishikawa Prefecture by the following screening method.

1. Screening method Add an appropriate amount of samples collected from soil in Ishikawa Prefecture, waste liquid of grease traps, sewage, river water, etc. to 5 mL of liquid medium for primary screening prepared by the following method, and culture at 30 ° C. for one week. 100 μL of the culture solution after cultivation is further inoculated into 5 mL of liquid medium for primary screening, and cultured again at 30° C. for one week.

The liquid medium for primary screening is prepared 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%, and sterilizing at high temperature and high pressure. to prepare. The oil is prepared by mixing rapeseed oil and soybean oil at a ratio of 1:1 (w/w).

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

The agar medium for secondary screening was prepared by dissolving each component other than oil and agar in pure water so as to have the composition shown in Table 5 below, and adding oil (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, it is appropriately dispensed and solidified to prepare.

Next, 0.05 g of oil (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 1% (w/v)). One platinum loop of each isolated strain obtained in the secondary screening is inoculated into an LB medium prepared by the following method, and cultured with shaking (140 rpm) at 30° C. for 24 hours. 100 μL of the resulting culture solution is inoculated into the test solution prepared by the above method, and cultured with shaking (140 rpm) at 30° C. for 24 hours.

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

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 when preparing the test solution and the residual amount of fats and oils (amount of normal-hexane extract (g)), the fats and oils reduction rate is calculated by the following formula (1). Ask for As a result, it is possible to isolate a strain with a high oil and fat reduction rate.

The 16S rDNA nucleotide sequences and mycological properties of the isolated strains (isolated microorganisms) exhibiting a high rate of fat reduction were determined as follows.

1. Culture conditions Strains cultured under the following conditions are used as test cells.

2.16S rDNA base sequence analysis 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 against DNA database for microorganism identification DB-BA12.0 (Techno Suruga Lab Co., Ltd.) and international nucleotide sequence database (DDBJ/ENA (EMBL)/GenBank), 16S rDNA partial nucleotide sequence of isolated microorganism showed 99.5% homology to the type strain F-279,208 ^T (accession number HF930598) of Pseudomonas soli. Also, P.I. It showed homology with a homology rate of 99% or more to multiple species such as parafulva AJ2129 ^T (AB060132). Table 10 shows the results of BLAST searches against DB-BA12.0, and Table 11 shows the results of BLAST searches against international base sequence databases.

A simplified 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, the T at the end of the strain name indicates the type strain (Type Strain), and BSL indicates biosafety. Levels (expressed as BSL1* (opportunistic pathogen) or higher) are indicated.

In the above molecular phylogenetic tree, the isolated microorganisms are included in a cluster composed of the genus Pseudomonas, P. It was shown to form a cluster with soli F-279,208 ^T (HF930598) and be closely related.

Therefore, from the results of 16S rDNA partial nucleotide sequence analysis, the isolated microorganism is P. It is identified as Pseudomonas sp., which is closely related to soli.

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

Based on morphological observation by an optical microscope (BX50F4, Olympus, Japan) and the method of Barrow & Feltham (Barrow G.I. & Feltham R.K.A. (1993), Cowan and Steel's Manual for the Identification of Medical Bacteria. 3rd edition, Cambridge University Press), catalase reactions, oxidase reactions, acid/gas production from glucose, glucose oxidation/fermentation (O/F) were tested. Gram staining was performed using Favor G “Nissui” (Nissui Pharmaceutical, Japan). Table 12 shows the results.

The following items were then tested using API® 20NE (bioMerieux, France) 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 UK. The following items were tested according to technical cooperation matters with and publicly known technology. Table 14 shows the results.

As shown in Table 12, the isolated microorganism was a motile Gram-negative bacillus that oxidized glucose and was positive for both catalase and oxidase reactions. These properties are considered to match those of the genus Pseudomonas (Barrow G.I. & Feltham R.K.A. (1993). Cowan and Steel's Manual for the Identification of Medical Bacteria. 3rd edition. Cambridge: University Press).

As shown in Table 13, the isolated microorganism did not reduce nitrate; exhibited arginine dihydrolase activity; hydrolyzed gelatin; glucose, D-mannose, D-mannitol, N-acetyl-D-glucosamine, glucone Potassium acid, n-capric acid, dl-malic acid, sodium citrate and phenyl acetate were utilized; L-arabinose, maltose and adipic acid were not utilized.

As shown in Table 14, the isolated organism grew on 6% NaCl; produced a fluorescent pigment on King's B agar; and did not produce a fluorescent pigment on King's A agar.

These properties are consistent with the results of 16s rDNA partial nucleotide sequence analysis of P. spp. It almost matched the properties of soli. However, the point that it does not grow at 37°C is that P. It was different from soli.

From the above, the properties of the isolated microorganism are included in the genus Pseudomonas, and the known species is P. It is considered the closest relative to soli. However, from 16s rDNA sequence analysis and mycological properties, the isolated microorganism is P. It has been found to have different properties than soli.

Therefore, the isolated strain (isolated microorganism) was determined to be a novel microorganism, and this strain was designated as Pseudomonas sp. ”) was named. In addition, this 2-2306 strain was deposited on July 24, 2018 at the National Institute of Technology and Evaluation Patent Microorganisms Depositary Center (2-5-8 Kazusa Kamatari, Kisarazu City, Chiba Prefecture). Accession number is NITE BP-02752.

[Evaluation of fat reduction effect]
In this specification, reduction of fats and oils is evaluated by the following method. That is, 0.05 g of rapeseed oil: soybean oil = 1: 1 (w / w) was added to a sterile treated oil decomposition evaluation medium (5 mL) that was the same as the above tertiary screening liquid medium except for pH. In addition to prepare a test solution (1% fat (w/v)). At this time, the medium for evaluating oil decomposition used has a pH adjusted in the range of 4 to 11 (for example, medium for evaluating oil decomposition with pH 4, 5, 6, 7, 8, 9, 10 and 11). . The pH is adjusted by using any acid such as inorganic acids such as hydrochloric acid, nitric acid, carbonic acid and sulfuric acid, organic acids such as citric acid and lactic acid, and salts thereof; and/or any acid such as sodium hydroxide, potassium hydroxide and ammonia. alkali; preferably hydrochloric acid (acid side) or sodium hydroxide (alkali side).

Microorganisms cultured on a plate medium (for example, an agar medium for secondary screening) are inoculated into this test solution and cultured with shaking (140 rpm) in an arbitrary temperature zone for 24 hours. The amount of bacteria to be inoculated is about one platinum loop. Microorganisms to be inoculated into the test solution may be those pre-cultured in a liquid medium for tertiary screening or the like. By pre-cultivating, the amount of bacteria to be inoculated can be easily adjusted. When pre-cultured microorganisms are used, they are inoculated at 1.5×10 ⁶ CFU/mL with respect to 1 mL of the test solution. The culture temperature may be set according to the temperature range in which the microbial cells are capable of decomposing and assimilating oils and fats.

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 added at the time of preparation of the test solution (0.05 g) and the residual amount of fats and oils (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 a fat reduction rate of 50% by weight obtained by the above method in all test liquids prepared using a fat decomposition evaluation medium with a pH set in the above range (eg, pH 5 to 11). Anything above that is fine. In a preferred embodiment of the present invention, the oil/fat reduction rate when cultured at 30° C. is 50% by weight or more, more preferably 60% by weight or more, and still more preferably 70% by weight or more. A higher fats and oils reduction rate is more preferable, so the upper limit is not particularly set. The longer the culture, the greater the fat loss. However, since microorganisms are sequentially excreted from the abatement facility, the abatement facility is usually replenished with microorganisms about every 1 to 3 days. Therefore, microorganisms that show a fat reduction rate of 50% by weight or more in a short period of time (for example, within 24 hours) are excellent in terms of practical use.

The water quality environment of the wastewater from the abatement facility can easily change depending on the type of garbage discharged. Therefore, it is preferable for microorganisms used in abatement facilities to be able to purify wastewater in environments with a wide range of pH. The 2-2306 strain is excellent in that it can decompose oils and fats even in a wide range of pH environments (eg, pH 5-11).

As used herein, "fat" 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 coconut oil. , peanut oil, beef tallow, lard, chicken oil, fish oil, whale oil, butter, margarine, fat spread, shortening; ;, but preferably edible oils and fats that are frequently discharged in restaurants where grease traps are often installed. Examples of fatty acids include, but are not limited to, 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, and lignoceric acid; 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 , docosatetraenoic acid, docosapentaenoic acid, docosapentaenoic acid, and docosahexaenoic acid; Fatty acids may be produced by the decomposition of edible or industrial fats and oils.

[Culturing of microorganisms]
Any method for culturing microorganisms according to the present invention may be used as long as the microorganisms can grow and proliferate. For example, the medium used for culturing microorganisms may be either a solid or liquid medium, and any medium containing a carbon source that can be assimilated by the microorganisms used, an appropriate amount of nitrogen source, inorganic salts and other nutrients. , either synthetic or natural media. Media typically contain a carbon source, a nitrogen source 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 it 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 hydrolysates, molasses, blackstrap molasses , natural products such as barley and rice, alcohols such as glycerol, methanol, and ethanol, organic acids such as acetic acid, phenyl acetate, lactic acid, succinic acid, gluconic acid, capric acid, malic acid, glucuronic acid, pyruvic acid, and citric acid and hydrocarbons such as hexadecane. The carbon source is appropriately selected in consideration of assimilation by the microorganisms to be cultured. For example, when using strain 2-2306, among the above carbon sources, glucose, mannose, mannitol, 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. Also, one or more of the above carbon sources can be selected and used.

Nitrogen sources that can be used in culturing microorganisms according to the present invention include meat extract, fish extract, peptone, polypeptone, tryptone, yeast extract, malt extract, soybean hydrolyzate, soybean powder, casein, milk casein, casamino acid, and glycine. , glutamic acid, various amino acids such as aspartic acid, organic nitrogen sources such as corn steep liquor, other animal, plant, and microbial hydrolysates; ammonia, ammonium salts such as ammonium nitrate, ammonium sulfate, ammonium chloride, nitrates such as sodium nitrate, Examples include nitrites such as sodium nitrite and inorganic nitrogen sources such as urea. The nitrogen source is appropriately selected in consideration of assimilation by the microorganisms to be cultured. For example, when strain 2-2306 is used, among the above nitrogen sources, it is preferable to use fish extract, tryptone, yeast extract, ammonium chloride, and the like. Also, one or more of the above nitrogen sources can be selected and used.

Inorganic substances that can be used in culturing microorganisms according to the present invention include phosphates, hydrochlorides, sulfates, acetates, carbonates and chlorides of magnesium, manganese, calcium, sodium, potassium, copper, iron and zinc. and the like. The inorganic substance is appropriately selected in consideration of the assimilation by the microorganisms to be cultured. Also, one or more of the above inorganic substances can be selected and used. Moreover, a surfactant or the like may be added to the medium, if necessary.

In order to allow the microorganisms according to the present invention to efficiently decompose and assimilate oils and fats or to maintain the ability of microorganisms to decompose and assimilate oils and fats, it is preferable to add oils and fats to the medium. Examples of fats and oils include the above-described edible fats and oils, industrial fats and oils, and fatty acids. The amount of oil to be 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 the oil. Specifically, fat (rapeseed oil: soybean oil=1:1 (w/w)) is preferably added in a concentration of 1 to 30 g, more preferably 5 to 15 g, per 1 L of medium. With such an amount to be added, microorganisms can maintain high ability to decompose and assimilate fats and oils. The oils and fats may be added singly or in the form of a mixture of two or more.

Cultivation of microorganisms according to the present invention can be carried out by conventional methods. For example, microorganisms are cultured under aerobic or anaerobic conditions, depending on the type of microorganism. In the former case, culturing 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 allow the microorganisms according to the present invention to proliferate, and can be appropriately selected according to the type of microorganisms to be cultured. Usually, the culture temperature is preferably 10-35°C, more preferably 15-30°C. In addition, although the pH of the medium suitable for culture is not particularly limited, it is preferably 5 or higher, more preferably 5-11. Furthermore, the culture time is not particularly limited, and varies depending on the type of microorganism to be cultured, the amount of culture medium, culture conditions, and the like. Usually, the culture time is preferably 16-48 hours, more preferably 20-30 hours.

<Wastewater treatment method>
One embodiment of the present invention relates to a method for treating wastewater, which includes the step of contacting wastewater containing fats and oils with the above microorganisms according to the present invention. The microorganisms according to the present invention are excellent in the effect of reducing fats and oils, and particularly have the property of being able to purify waste water even in water environments with a wide range of pH (eg, pH 5 to 11). Therefore, by bringing the microorganisms (fat-degrading microorganisms) according to the present invention into contact with waste water containing fats and oils, fats and oils can be effectively reduced. Note that the above description of microorganisms can 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. In addition, the waste water 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 typically it is 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 microorganisms according to the present invention can be brought into contact with oil-containing wastewater.

The grease trap 10 may be of any inflow type such as a pipe introduction type or a gutter introduction type. There are no particular restrictions on installation conditions, such as underground installation, slab ceiling installation, cinder installation, and floor installation. In the case of underground burial, the grease trap 10 is buried in a kitchen or a food processing plant, for example, so that the waste water flowing out 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 positioned below the drain of the sink.

In FIG. 1, waste water flows in the direction of the arrow. The introduction of waste water into the grease trap 10 may be performed either batchwise or continuously. 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 fats and oils pass through the residue receiver 3 and flow into the wastewater treatment tank 1. The oil 6 that has flowed into the wastewater treatment tank 1 floats toward the water surface 5 and gathers in the 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 fats and oils 6 gradually aggregate in the space partitioned by the partition plates 2a and 2c to form scum.

When the microorganism according to the present invention is applied to the grease trap 10, in the wastewater treatment tank 1 (mainly in the space partitioned by the partition plates 2a and 2c), the wastewater containing oil and fat and the microorganism according to the present invention are mixed. will come into contact. Since the microorganisms according to the present invention have high activity to decompose fats and oils and have assimilation properties, they can suppress aggregation of the fats and oils 6 and effectively prevent the formation of scum. In particular, the 2-2306 strain has high lipolytic activity even in a wide pH range (eg pH range of 5 to 11). As a result, the oil is prevented from flowing out to the outside environment through the trap tube 4 without depending on the pH of the waste water, which is advantageous from the viewpoint of environmental protection.

In the wastewater treatment method, the microorganisms according to the present invention are in various forms such as suspended in the culture solution, collected as solids from the culture solution, dried, and immobilized on a carrier. can be brought into contact with wastewater at . Microorganisms suspended in the culture medium, recovered as solids from the culture medium, or dried are, for example, added to the waste water and brought into contact with the waste water. The microorganisms immobilized on the carrier may be added to the wastewater. However, the microorganisms and the wastewater are mixed by placing the microorganism-immobilized carrier in a grease trap and passing the wastewater through the microorganism-immobilized carrier. can also be brought into contact with. By placing 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 microorganisms according to the present invention that have been collected as solids from the culture medium, any means known to those skilled in the art can be employed as the collection method. For example, the culture solution of fat-degrading microorganisms cultured by the above method can be solid-liquid separated by centrifugation, filtration, or the like, and the solid content can be recovered. By drying (for example, freeze-drying) this solid content, a dried fat-degrading microorganism can be obtained.

When using a fat-degrading microorganism immobilized on a carrier, the carrier for immobilizing the fat-degrading microorganism is not particularly limited as long as it can immobilize the microorganism. The carrier used to carry out the procedure is used in the same manner or modified as appropriate. For example, a method of entrapping fixation on a gel-like substance such as alginic acid, polyvinyl alcohol, gellan gum, agarose, cellulose, dextran, etc., a method of adsorption fixation on the surface of glass, activated carbon, polystyrene, polyethylene, polypropylene, wood, silica gel, etc., etc. Available.

In addition, the method for immobilizing the lipid-degrading microorganisms on the carrier is also not particularly limited, and a general method for immobilizing microorganisms may be used in the same manner or with appropriate modifications. For example, an immobilization method by pouring a culture solution of microorganisms into a carrier, an immobilization method by placing a carrier under reduced pressure using an aspirator and pouring a culture solution of microorganisms into the carrier, and a medium in which a culture solution of microorganisms is sterilized and a carrier, followed by shaking culture, and the carrier removed from the mixture is air-dried.

In the method according to the present invention, when the fat-degrading microorganisms are added to the wastewater and brought into contact with the wastewater, the amount of microorganisms to be added can be set arbitrarily. The amount of bacteria added to waste water is not particularly limited, but is, for example, 1×10 ⁴ to 1×10 ¹² CFU, preferably 1×10 ⁵ to 1×10 CFU, per 1 g of oil and fat contained in waste water. ¹¹ CFU. Alternatively, it is, 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 ( dry cell weight). Alternatively, the amount may be such that the amount of wastewater in the grease trap is, for example, 1×10 ⁶ to 1×10 ¹² CFU/L, more preferably 1×10 ⁷ to 1×10 ¹¹ CFU/L. . 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, relative to the waste water in the grease trap. When two or more types of microorganisms are used in combination, the total amount is meant. It should be noted that pre-cultured microorganisms may be used as the microorganisms added to the waste water. By pre-cultivating, the amount of bacteria to be inoculated can be easily adjusted.

When the wastewater is discharged to the external environment, the fat-degrading microorganisms that are not immobilized on the carrier are discharged out of the grease trap together with the wastewater. preferably. The interval of addition is not particularly limited, but, for example, it is preferable to add once every 3 hours, once every 24 hours, or once every 2 to 3 days. The method of addition is not particularly limited, and when the waste water continuously flows into the grease trap, it may be added by mixing with the waste water, or may be added directly to the waste water in the grease trap. If the microorganisms are added from a kitchen sink or other drainage outlet, the microorganisms can be introduced into the grease trap along with the wastewater discharged from cleaning.

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 reducing fats and oils more efficiently. Other components include, for example, microorganisms described in JP-A-2017-136033, oil-degrading enzymes such as lipase, pH adjusters, fat adsorbents, surfactants, and the like.

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, and the genus Kluybero. Kluyveromyces, Trichosporon, Bacillus, Lactobacillus, Aspergillus, Rhodococcus, Enterococcus, Sphingomonas , Enterobacter genus, Burkholderia genus, Pseudomonas genus, Penicillium genus, Staphylococcus genus, Rhizopus genus, Rhizobium genus, Acinetobacter ( Acinetobacter, Alcaligenes, Fusarium, Serratia, Tetrasphaera, Humicola, and 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 their high ability to decompose oils and fats.

Examples of microorganisms of the genus Yarrowia include Yarrowia lipolytica ATCC48436, Yarrowia lipolytica NBRC1548, Yarrowia lipolytica LM02-011 (accession number NITE P-01813), Yarrowia lipolytica NBRC0746, and Yarrowia lipolytica such as Yarrowia lipolytica NBRC1209. ), Yarrowia sp. such as Yarrowia YH-01 can be exemplified, but Yarrowia lipolytica is more preferable, and Yarrowia lipolytica LM02-011 (LM02-011 stock is an independent administrative agency product Evaluation Technology Platform Organization Patent Microorganism Depositary Center (2-5-8 Kazusa Kamatari, Kisarazu City, Chiba Prefecture, Japan 292-0818, deposited as accession number NITE P-01813 on March 6, 2014) More preferred.

As a microorganism of the genus Enterobacter, Enterobacter sp. LM02-030 strain (LM02-030 strain is an independent administrative agency Product evaluation technology base Patent microorganism depositary center (〒 292-0818 Kisarazu, Chiba prefecture, Japan Ichi Kazusa Kamatari 2-5-8), deposited as accession number NITE P-02048 on May 12, 2015).

Examples of microorganisms of the genus Sphingomonas include, for example, Sphingomonas sp. 2629-3b described in JP-A-2006-166874, Sphingomonas sp. The strain was deposited at the Patent Microorganism Depositary Center, National Institute of Technology and Evaluation (2-5-8 Kazusa Kamatari, Kisarazu City, Chiba Prefecture, 292-0818, Japan) with accession number NITE P-02069 on June 19, 2015. and Sphingomonas sp.) can be exemplified.

Oil-degrading enzymes, such as lipases and phospholipases, may be added to the waste water to aid in the decomposition of mineral oil by the microorganisms used in the method of the present invention. Examples of oil-degrading enzymes include Pseudomonas genus, Aspergillus genus, Bacillus genus, Penicillium genus, Rhizopus genus, Rhizomucor genus, Mucor genus , Paecilomyces genus, Rhizoctonia genus, Absidia genus, Achromobacter genus, Aeromonas genus, Alternaria genus, Aureobasidium genus, Beauveria ( Beauveria) genus, Chromobacter genus, Coprinus genus, Fusarium genus, Geotricum genus, Humicola genus, Hyphozyma genus, Lactobacillus genus, Metalidium ( Metarhizium genus, Rhodosporidium genus, Trichoderma genus, Yarrowia genus, Candida genus, Pichia genus, Hansenula genus, Saccharomyces genus, Kur It can be obtained from the genera Kluyveromyces and/or Trichosporon.

Commercially available oil-degrading enzymes include Lipase MY, Lipase OF, Lipase PL, Lipase QLM (Meito 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 Nippon Chemical Industry Co., Ltd.); Relipase (registered trademark) A-10D, Relipase (registered trademark) AF-5, PLA2 Nagase (Nagase Chemtex Co., Ltd.); Enchilon AKG-2000, Enchilon LP, Enchilon LPG (Rakuto Kasei Kogyo Co., Ltd.); registered trademark) 100L, Lipozyme (registered trademark) RMIM, Lipozyme (registered trademark) TLIM, Novozyme (registered trademark) 435FG (manufactured by Novozymes); Picantase A, Picantase R800 (manufactured by DM Japan), etc. . 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. 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, based on the capacity of the grease trap. The activity unit (U) of the oil-degrading enzyme is the amount of the enzyme that liberates 1 μmole of fatty acid per minute at 37° C. and pH 7.

In the method according to the present invention, shirasu balloon described in JP-A-2012-206084, diatomaceous earth, inorganic polymers such as perlite, organic polymers such as polyurethane, polyethylene, melanin resin, etc., oil adsorbents are used. May be added to waste water.

In the method according to the present invention, anionic surfactants such as sodium dodecyl sulfate (SDS), dodecylbenzene sulfonic acid (NaDDBS), ammonium lauryl sulfate, sodium caseinate, etc.; 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 Surfactants such as non-ionic surfactants such as octylphenyl ether (Triton X-100), lecithin; amphoteric surfactants such as betaine, amine oxides, saponins may be added to the waste water.

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

Moreover, in the wastewater treatment method according to the present invention, the temperature at which the fat-degrading microorganisms and fats are brought into contact, that is, the temperature of the wastewater in the grease trap, can be set arbitrarily. In addition, the pH at which the fat-decomposing microorganisms and fats are brought into contact, that is, the pH of the waste water in the grease trap, can be set arbitrarily. Generally, the temperature is, for example, 5 to 35°C, preferably 10 to 35°C, more preferably 15 to 30°C. The pH is, for example, 5 or higher, preferably 5-11. Furthermore, if necessary, the waste water may be aerated by aeration or the like.

<Wastewater treatment agent>
In one embodiment of the present invention, there is provided a wastewater treatment agent containing the microorganisms according to the present invention. The microorganisms according to the present invention are excellent in the effect of reducing fats and oils, and particularly have the property of being able to purify waste water even in water environments with a wide range of pH (eg, pH 5 to 11). Therefore, by using the wastewater treatment agent containing microorganisms according to the present invention in wastewater treatment facilities (harm abatement facilities) such as grease traps, oils and fats can be effectively reduced. Note that the above explanations regarding microorganisms and wastewater treatment methods can be modified as necessary and applied to the present embodiment.

The waste water treatment agent may be in either a dry form or a liquid form, but a dry form such as powder, granules, pellets, tablets, etc. is preferable from the viewpoint of storage. Microorganisms according to the present invention that are used in such a dried wastewater treatment agent include bacterial powder obtained by drying a culture solution by spray-drying, freeze-drying, or the like, or bacteria immobilized on a carrier as described above. It may be in the form of a body, and may be molded into powder, granules, pellets, or tablets. Alternatively, the cells or culture solution may be encapsulated with hydroxypropylmethylcellulose, gelatin, or the like. Wastewater treatment agents may also include excipients such as hydroxypropylcellulose, dextrin, lactose, starch, and the like.

The microorganisms according to the present invention contained in the wastewater treatment agent may be dead or viable, but are preferably viable from the viewpoint of durability of 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 of the solid content of the wastewater treatment agent. Alternatively, the amount of the microorganisms according to the present invention contained in the wastewater treatment agent is, for example, 1×10 ² to 1×10 ¹⁰ CFU/g of the entire wastewater treatment agent. In addition, the waste water treatment agent is a group consisting of other microorganisms capable of coexisting with the above microorganisms according to the present invention, a fat-degrading enzyme, a fat-adsorbent, and a surfactant, as long as the intended effect of the present invention is achieved. It may contain additives such as one or more selected from. Other symbiotic microorganisms, lipolytic enzymes, lipid adsorbents, and surfactants can be used, for example, those described in JP-A-2017-136033.

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 Kahoku City, Ishikawa Prefecture was inoculated into a liquid medium for primary screening by the method described above and cultured at 30° C. for one week. After culturing, 100 μL of the culture solution was further inoculated into 5 mL of liquid medium for primary screening, and cultured again at 30° C. for one week.

100 μL of the 10 ⁴ -fold diluted culture solution after the primary screening was spread on the secondary screening agar medium prepared by the above method, and cultured at 30° C. for one week. After culturing, strains were isolated in which the formation of halos due to the decomposition of fats and oils was confirmed.

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

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 when preparing the test solution and the residual amount of fats and oils (amount of normal-hexane extract (g)), the fats and oils reduction rate is calculated by the following formula (1). asked for As a result, we isolated a strain with a high rate of fat reduction.

The isolated strain was named Pseudomonas sp. 2-2306 strain, and was deposited at the National Institute of Technology and Evaluation Patent Microorganisms Depositary (accession number NITE BP-02752).

Example 2: Evaluation of Oil/Fat Reduction Rate 0.05 g of oil/fat was added to 5 mL of the liquid medium for the tertiary screening, the pH of which was adjusted in the range of 3 to 13 using hydrochloric acid or sodium hydroxide, to prepare a sterilized test solution. One platinum loop of the isolated strain cultured on the secondary screening agar medium was inoculated into the test solution prepared above, and cultured with shaking (140 rpm) at 30° C. for 24 hours.

In addition, to the test solution prepared above, 0.75 mg of "Grease Guard (registered trademark) D Lipase" (Novozymes) or "BN clean (powder)" (Meiji Food Materia Co., Ltd.; Bacillus subtilis BN1001 (containing Bacillus subtilis BN1001)) was added and shaken at 30° C. for 24 hours.

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 when preparing the test solution and the residual amount of fats and oils (amount of normal-hexane extract (g)) were used to calculate the fats and oils reduction rate according to the above formula (1). asked. The results are shown in Table 15 below.

As shown in Table 15, it can be seen that the 2-2306 strain reduced 1% (w/v) fat by 50 wt% or more in 24 hours under conditions of 30°C and pH 5 to 11. In other words, it can be seen that the 2-2306 strain has excellent lipolytic activity even in water environments with a wide range of pH.

1 wastewater treatment tank,
2a, 2b, 2c partition plate,
3 residue receiving,
4 trap tube,
5 water surface,
6 oils and fats,
10 Grease trap.

Claims (5)

A microorganism identified by Pseudomonas sp. strain 2-2306 (accession number NITE BP-02752). 2. A microorganism according to claim 1 , belonging to Pseudomonas and exhibiting the following mycological properties.

3. The microorganism according to claim 1, which has the 16S rDNA nucleotide sequence shown in SEQ ID NO:1. A method for treating wastewater, comprising the step of bringing the microorganism according to any one of claims 1 to 3 into contact with wastewater containing fats and oils. A wastewater treatment agent comprising the microorganism according to any one of claims 1 to 3 .

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