JP2022124353A - Microorganisms, oil decomposer and oil decomposing method - Google Patents

Abstract

To provide microorganism that can efficiently decompose oil, an oil decomposer including the microorganism, and an oil decomposition method using the oil decomposer.SOLUTION: Microorganism is provided which belongs to the genus Candida, in which 26S rDNA has a specific base sequence, and which shows predetermined mycological properties, and has oil decomposing activity at a temperature of 20-30°C. There are also provided an oil decomposer containing the microorganisms, and an oil decomposition method in which the oil decomposer is brought into contact with an oil-containing material to decompose oil in the oil-containing material.SELECTED DRAWING: None

Description

TECHNICAL FIELD The present invention relates to a microorganism capable of efficiently decomposing oil, an oil-decomposing agent containing the microorganism, and an oil-decomposing method using the microorganism.

Wastewater containing animal and plant oils is discharged from business facilities such as restaurant kitchens and food factories. Since such wastewater may cause serious water pollution if it is discharged into a river or the like as it is, strict regulation values are set for the amount of oil discharged. In addition, even when waste water is discharged into the sewage system, clogged oil may clog sewage pipes.

Wastewater containing organic pollutants can be purified by the action of aerobic microorganisms in an activated sludge tank. However, if high-concentration oil flows directly into the activated sludge tank, the activity of microorganisms in the activated sludge is inhibited and the settling property of the sludge is impaired, resulting in deterioration of the function of the activated sludge treatment facility.
Therefore, wastewater containing high concentrations of oil is usually pretreated to reduce the oil concentration in large-scale facilities so that it does not flow into the activated sludge tank, and in small-scale facilities, pretreatment is carried out at the business site before it is discharged into sewage. ing.
Conventionally, simple solid-liquid separation is the main method for reducing the oil concentration in waste water. A pressurized flotation device is the main means for removing oil in large-scale facilities, and a grease trap is the main means for removing oil in small-scale facilities. However, these methods have problems such as foul odors and pests caused by the generated oil-containing sludge, the cost of industrial waste treatment of the oil-containing sludge, and the labor and effort of maintenance.

On the other hand, in recent years, biological treatment methods such as an oil decomposition method using a lipase reaction and an oil digestion method using an oil decomposition bacterium have been investigated.

Examples of methods using oil-degrading bacteria include microorganisms belonging to the genus Burkholderia (Patent Document 1), microorganisms belonging to the genus Yarrowia (Patent Document 2), and microorganisms belonging to the genus Pseudomonas (Patent Document 2). Patent Document 3), microorganisms belonging to the genus Serratia (Patent Document 4), Raoultella planticola (Patent Document 5), etc. have been reported.

JP 2014-23474 A JP 2013-146689 A JP 2019-208460 A JP 2011-182782 A JP 2012-105635 A

However, the oil decomposition method by lipase reaction has the problem that the price of the enzyme is high and the running cost tends to be high. In addition, there is also a problem that it is easily deactivated due to the presence of an inhibitor of the enzymatic reaction. The oil-digesting method using oil-degrading bacteria also has problems such as the decomposition speed being slow and not keeping up with the treatment speed required for wastewater treatment. be.
Therefore, there is a demand for a low-cost and efficient method for treating oil by using microorganisms capable of efficiently decomposing and removing oil and an oil-decomposing agent containing the microorganism.

Under such circumstances, an object of the present invention is to provide a microorganism capable of efficiently decomposing oil, an oil decomposing agent containing the microorganism, and an oil decomposing method using the oil decomposing agent.

MEANS TO SOLVE THE PROBLEM As a result of repeating earnest research in order to achieve the said objective, this inventor acquired the novel microorganisms excellent in oil decomposition, and came to the completion of this invention.
That is, the present invention has the following aspects.

[1] A nucleotide sequence belonging to the genus Candida and having a nucleotide sequence represented by SEQ ID NO: 1, or a nucleotide sequence having 90% or more sequence identity with the nucleotide sequence represented by SEQ ID NO: 1 A microorganism that exhibits the following mycological properties and has oil-decomposing activity at a temperature of 20 to 30°C.
(1) Colony morphology (YM agar medium, 27°C, 1 week culture)
Color tone: White to cream Elevation: Cushion-shaped Margin: Entire to wavy Surface shape: Smooth Gloss and properties: Buttery, wet No formation of sexual reproductive organs after 3 weeks of culture [2] The microorganism according to [1] above, which exhibits the following physiological/biochemical properties in a physiological/biochemical property test.
(1) Sugar fermentability test glucose -
galactose -
Maltose -
(2) Carbon resource utilization test Glucose +
Galactose +
L-sorbose -
D-glucosamine +
D-ribose +
D-xylose +
L-arabinose -
D-Arabinose -
L-rhamnose -
Sucrose +
Maltose +
Trehalose +
α-methyl-D-glucoside +
Cellobiose -
salicin -
Melibiose +
Lactose -
Raffinose +
Meletitose +
Inulin -
Soluble starch +
Glycerol +
Erythritol -
Ribitol (Adonitol) +
xylitol -
D-glucitol (sorbitol) +
D-mannitol +
Galactitol (Dulcitol) -
Inositol -
2-keto-D-gluconic acid +
D-gluconic acid +
DL-lactic acid +
Succinate -
Citrate +
Methanol -
Ethanol +
N-acetyl-glucosamine +
Hexadecane +
(3) Nitrogen source utilization Nitrate W
Cadaverine +
(4) Resistance test Growth at 30°C +
Growth at 37°C +
0.01% cycloheximide -
50% (W/V) D-glucose +
10% sodium chloride/5% glucose +
(5) Vitamin auxotrophy test Vitamin-free medium +
(In the above, "+" is positive, "-" is negative, "L" indicates that a positive reaction was rapidly observed after 2 weeks from the start of the test, and "D" indicates that it took more than 1 week after the start of the test. “W” indicates a weak positive reaction, respectively.)
[3] The microorganism according to [1] or [2], which belongs to Candida palmioleophila.

[4] a nucleotide sequence belonging to the genus Candida and having a nucleotide sequence represented by SEQ ID NO: 2, or a nucleotide sequence having 90% or more sequence identity with the nucleotide sequence represented by SEQ ID NO: 2; A microorganism that exhibits the following mycological properties and has oil-decomposing activity at a temperature of 20 to 30°C.
(1) Colony morphology (YM agar medium, 27°C, 1 week culture)
Color tone: White to cream Elevation: Cushion-shaped Margin: Entire to wavy Surface shape: Smooth Gloss and properties: Buttery, wet No formation of sexual reproductive organs after 3 weeks of culture [5] The microorganism according to [4] above, which exhibits the following physiological/biochemical properties in a physiological/biochemical property test.
(1) Sugar fermentability test glucose -
galactose -
Maltose -
(2) Carbon resource utilization test Glucose +
Galactose +
L-sorbose -
D-glucosamine +
D- ribose -
D-xylose +
L-arabinose -
D-Arabinose -
L-rhamnose -
Sucrose +
Maltose +
Trehalose +
α-methyl-D-glucoside +
Cellobiose +
Salicin +
Melibiose +
Lactose -
Raffinose +
Meletitose +
Inulin -
Soluble starch +
Glycerol +
Erythritol -
Ribitol (Adonitol) +
Xylitol D
D-glucitol (sorbitol) +
D-mannitol +
Galactitol (Dulcitol) -
Inositol -
2-keto-D-gluconic acid +
D-gluconic acid +
DL-lactic acid +
Succinate +
Citrate +
Methanol -
Ethanol +
N-acetyl-glucosamine +
Hexadecane +
(3) Nitrogen source utilization Nitrate +
Cadaverine +
(4) Resistance test Growth at 30°C +
Growth at 37°C +
0.01% cycloheximide -
50% (W/V) D-glucose +
10% sodium chloride/5% glucose +
(5) Vitamin auxotrophy test Vitamin-free medium +
(In the above, "+" is positive, "-" is negative, "L" indicates that a positive reaction was rapidly observed after 2 weeks from the start of the test, and "D" indicates that it took more than 1 week after the start of the test. “W” indicates a weak positive reaction, respectively.)
[6] The microorganism according to [4] or [5], which belongs to Candida palmioleophila.

[7] A nucleotide sequence belonging to the genus Candida and having a 26S rDNA nucleotide sequence represented by SEQ ID NO: 3 or having 90% or more sequence identity with the nucleotide sequence represented by SEQ ID NO: 3 A microorganism that exhibits the following mycological properties and has oil-decomposing activity at a temperature of 20 to 30°C.
(1) Colony morphology (YM agar medium, 27°C, 1 week culture)
Color tone: White to cream Elevated state: Cushion-shaped Margin: Thread-like Surface shape: Smooth Gloss and properties: Butter-like, moist No formation of sexual reproductive organs in 3 weeks Formation of pseudohyphae or hyphae is observed [8] The physiological/biochemical property test shows the following physiological/biochemical properties, according to [7] above microbes.
(1) Sugar fermentability test Glucose +
Galactose +
Maltose +
(2) Carbon resource utilization test Glucose +
Galactose +
L-sorbose +
D-Glucosamine W
D- ribose -
D-xylose +
L-arabinose -
D-Arabinose -
L-rhamnose -
Sucrose +
Maltose +
Trehalose +
α-methyl-D-glucoside +
Cellobiose +
Salicin +
Melibiose -
Lactose -
Raffinose -
Meletitose +
Inulin -
Soluble starch +
glycerol -
Erythritol -
Ribitol (Adonitol) +
Xylitol +
D-glucitol (sorbitol) +
D-mannitol +
Galactitol (Dulcitol) -
Inositol -
2-keto-D-gluconic acid +
D-gluconic acid +
DL-lactic acid +
Succinate +
Citrate W
Methanol -
Ethanol +
N-acetyl-glucosamine +
Hexadecane +
(3) Nitrogen source utilization Nitrate W
Cadaverine +
(4) Resistance test Growth at 30°C +
Growth at 37°C +
0.01% cycloheximide +
50% (W/V) D-glucose +
10% sodium chloride/5% glucose +
(5) Vitamin auxotrophy test Vitamin-free medium +
(In the above, "+" is positive, "-" is negative, "L" indicates that a positive reaction was rapidly observed after 2 weeks from the start of the test, and "D" indicates that it took more than 1 week after the start of the test. “W” indicates a weak positive reaction, respectively.)
[9] The microorganism according to the above [7] or [8], which belongs to Candida tropicalis.

[10] A nucleotide sequence belonging to the genus Candida and having a 26S rDNA nucleotide sequence represented by SEQ ID NO: 4 or having 90% or more sequence identity with the nucleotide sequence represented by SEQ ID NO: 4 A microorganism that exhibits the following mycological properties and has oil-decomposing activity at a temperature of 20 to 30°C.
(1) Colony morphology (YM agar medium, 27°C, 1 week culture)
Color tone: White to cream Elevation: Cushion-shaped Periphery: Whole edge Surface shape: Smooth Gloss and properties: Buttery, moist [11] The microorganism according to [10] above, which exhibits the following physiological/biochemical properties in a physiological/biochemical property test: .
(1) Sugar fermentability test Glucose +
Galactose +
Maltose +
(2) Carbon resource utilization test Glucose +
Galactose +
L-sorbose +
D-glucosamine D
D- ribose -
D-xylose +
L-arabinose -
D-Arabinose -
L-rhamnose -
Sucrose +
Maltose +
Trehalose +
α-methyl-D-glucoside +
Cellobiose +
Salicin W
Melibiose -
Lactose -
Raffinose -
Meletitose +
Inulin -
Soluble starch +
glycerol -
Erythritol -
Ribitol (Adonitol) +
xylitol -
D-glucitol (sorbitol) +
D-mannitol +
Galactitol (Dulcitol) -
Inositol -
2-keto-D-gluconic acid +
D-gluconic acid +
DL-lactic acid +
Succinate +
Citrate D
Methanol -
Ethanol +
N-acetyl-glucosamine +
Hexadecane +
(3) Nitrogen source utilization Nitrate W
Cadaverine +
(4) Resistance test Growth at 30°C +
Growth at 37°C +
0.01% cycloheximide +
50% (W/V) D-glucose +
10% sodium chloride/5% glucose +
(5) Vitamin auxotrophy test Vitamin-free medium +
(In the above, "+" is positive, "-" is negative, "L" indicates that a positive reaction was rapidly observed after 2 weeks from the start of the test, and "D" indicates that it took more than 1 week after the start of the test. “W” indicates a weak positive reaction, respectively.)
[12] The microorganism according to the above [10] or [11], which belongs to Candida tropicalis.

[13] A nucleotide sequence belonging to the genus Candida and having a 26S rDNA nucleotide sequence represented by SEQ ID NO: 5 or having 90% or more sequence identity with the nucleotide sequence represented by SEQ ID NO: 5 A microorganism that exhibits the following mycological properties and has oil-decomposing activity at a temperature of 20 to 30°C.
(1) Colony morphology (YM agar medium, 27°C, 1 week culture)
Color tone: white to cream Elevated state: flat peripheral edge, cushion-shaped central edge Peripheral edge: Entire edge Surface shape: smooth Gloss and properties: bright, wet morphology, ovoid, short-rod-shaped Proliferation by budding No formation of sexual reproductive organs after 3 weeks of culture Microorganisms according to.
(1) Sugar fermentability test Glucose +
galactose -
Maltose +
(2) Carbon resource utilization test Glucose +
Galactose +
L-sorbose -
D-glucosamine +
D- ribose -
D-xylose +
L-arabinose -
D-Arabinose -
L-rhamnose -
Sucrose +
Maltose +
Trehalose +
α-methyl-D-glucoside +
Cellobiose +
Salicin +
Melibiose -
Lactose +
Raffinose W
Meletitose +
Inulin -
Soluble starch +
Glycerol +
Erythritol -
Ribitol (Adonitol) +
Xylitol D
D-glucitol (sorbitol) +
D-mannitol +
Galactitol (Dulcitol) -
Inositol -
2-keto-D-gluconic acid +
D-gluconic acid +
DL-lactic acid +
Succinate +
Citrate +
Methanol -
Ethanol +
N-acetyl-glucosamine +
Hexadecane +
(3) Nitrogen source utilization Nitrate W
Cadaverine +
(4) Resistance test Growth at 30°C +
Growth at 37°C +
0.01% cycloheximide +
50% (W/V) D-glucose +
10% sodium chloride/5% glucose +
(5) Vitamin auxotrophy test Vitamin-free medium +
(In the above, "+" is positive, "-" is negative, "L" indicates that a positive reaction was rapidly observed after 2 weeks from the start of the test, and "D" indicates that it took more than 1 week after the start of the test. “W” indicates a weak positive reaction, respectively.)
[15] The microorganism according to the above [13] or [14], which belongs to Candida fluviatilis.

[16] An oil decomposing agent containing the microorganism according to any one of [1] to [15].
[17] An oil decomposition method comprising contacting the oil decomposing agent according to [16] with an oil-containing material to decompose the oil in the oil-containing material.
[18] The oil decomposition method according to the above [17], wherein the oil-containing material is oil-containing wastewater.

ADVANTAGE OF THE INVENTION According to this invention, the microorganisms which can decompose|disassemble oil efficiently, the oil-degrading agent containing the said microorganisms, and the oil-degrading method using the said oil-degrading agent can be provided.

Fig. 10 is an image showing the cell morphology observation results of the MC1 strain on day 5 of culture on the YM plate medium. Fig. 3 is an image showing the results of cell morphology observation of the MC3 strain on day 5 of culture on a YM plate medium. Fig. 4 is an image showing the results of observation of cell morphology of the MC4 strain on day 5 of culture on a YM plate medium. Fig. 10 is an image showing the cell morphology observation results of the MC28 strain on day 5 of culture on the YM plate medium. Fig. 10 is an image showing the cell morphology observation results of the MC29 strain on day 5 of culture on the YM plate medium. It is a molecular phylogenetic tree of the MC1 strain analyzed based on the nucleotide sequences obtained by homology search. It is a molecular phylogenetic tree of the MC3 strain analyzed based on the nucleotide sequences obtained by homology search. It is a molecular phylogenetic tree of the MC4 strain analyzed based on the nucleotide sequences obtained by homology search. It is a molecular phylogenetic tree of the MC28 strain analyzed based on the nucleotide sequences obtained by homology search. It is a molecular phylogenetic tree of the MC29 strain analyzed based on the nucleotide sequences obtained by homology search. FIG. 4 is a diagram showing the oil decomposition rate of each microorganism with respect to various oil components. It is a figure which shows the oil decomposition rate of each microorganism at low temperature (20 degreeC). FIG. 4 is a diagram showing the oil decomposition rate of each microorganism in actual waste water. FIG. 4 is a diagram showing the oil decomposition rate in continuous batch treatment of each microorganism. FIG. 2 is a diagram showing oil decomposition in a continuous treatment test using entrapping immobilization carriers for each microorganism.

Embodiments of the present invention will be described below. The following embodiments are merely examples for explaining the present invention, and are not intended to limit the present invention only to these embodiments. The present invention can be implemented in various forms without departing from its spirit.

As used herein, the term "oil" means an organic oily substance, including fats and mineral oils, with fats being preferred.
The fats and oils refer to substances and fatty acids containing glycerides in which glycerin and 1 to 3 fatty acids are ester-bonded, and in addition to triglycerides (triacylglycerols), which are the main components of fats and oils, diglycerides (diacylglycerols) and monoglycerides. (monoacylglycerol) may be included. In addition, in addition to animal and vegetable oils (animal oils, vegetable oils, fish oils), the oils and fats may contain ingredients other than animal and vegetable oil-derived glycerides (for example, plant sterols, lecithins, antioxidant ingredients, pigment ingredients, etc.). good.

Examples of the fats and oils include olive oil, canola oil, coconut oil, sesame oil, rice oil, rice bran oil, safflower oil, soybean oil, corn oil, rapeseed oil, palm oil, palm kernel oil, sunflower oil, cottonseed oil, and palm oil. Edible oils such as oil, peanut oil, beef tallow, lard, chicken oil, fish oil, whale oil, butter, margarine, fat spread, shortening; and linseed oil, jatropha oil, tall oil, hemana oil, castor oil, jojoba oil Industrial oils and fats such as; preferably edible oils and fats that are frequently discharged from 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.

The microorganisms of the embodiments are particularly capable of efficiently decomposing fats and oils in which 95% by mass or more of the components constituting the fats and oils are glycerides.

In addition, the mineral oil refers to hydrocarbons obtained by refining crude oil, such as hydrocarbons having 4 to 10 carbon atoms (eg, gasoline), hydrocarbons having 9 to 15 carbon atoms ( kerosene), hydrocarbons with 10 to 20 carbon atoms (eg, light oil), hydrocarbons with 17 or more carbon atoms (eg, heavy oil), and hydrocarbons with 15 to 50 carbon atoms (eg, lubricating oils) (eg Sierra terrace oil, engine oil)) and the like.
Gasoline contains a lot of aromatic hydrocarbons, and kerosene, light oil and heavy oil contain a lot of aliphatic hydrocarbons. In particular, the microorganisms of the embodiments can efficiently decompose mineral oils containing a large amount of aliphatic hydrocarbons having 22 to 36 carbon atoms.

(First microorganism)
The first microorganism (hereinafter also referred to as microorganism 1) of one embodiment of the present invention is
Belonging to the genus Candida, the nucleotide sequence of 26S rDNA is the nucleotide sequence shown by SEQ ID NO: 1, or a nucleotide sequence having 90% or more sequence identity with the nucleotide sequence shown by SEQ ID NO: 1, and has oil-decomposing activity at a temperature of 20-30°C.
(1) Colony morphology (YM agar medium, 27°C, 1 week culture)
Color tone: White to cream Elevation: Cushion-shaped Margin: Entire to wavy Surface shape: Smooth Gloss and properties: Buttery, wet No formation of sexual reproductive organs after 3 weeks of culture

Examples of the microorganism 1 of the embodiment include microorganisms that exhibit the following physiological/biochemical properties in a physiological/biochemical property test.
<Physiological and biochemical property test results>
(1) Sugar fermentability test glucose -
galactose -
Maltose -
(2) Carbon resource utilization test Glucose +
Galactose +
L-sorbose -
D-glucosamine +
D-ribose +
D-xylose +
L-arabinose -
D-Arabinose -
L-rhamnose -
Sucrose +
Maltose +
Trehalose +
α-methyl-D-glucoside +
Cellobiose -
salicin -
Melibiose +
Lactose -
Raffinose +
Meletitose +
Inulin -
Soluble starch +
Glycerol +
Erythritol -
Ribitol (Adonitol) +
xylitol -
D-glucitol (sorbitol) +
D-mannitol +
Galactitol (Dulcitol) -
Inositol -
2-keto-D-gluconic acid +
D-gluconic acid +
DL-lactic acid +
Succinate -
Citrate +
Methanol -
Ethanol +
N-acetyl-glucosamine +
Hexadecane +
(3) Nitrogen source utilization Nitrate W
Cadaverine +
(4) Resistance test Growth at 30°C +
Growth at 37°C +
0.01% cycloheximide -
50% (W/V) D-glucose +
10% sodium chloride/5% glucose +
(5) Vitamin auxotrophy test Vitamin-free medium +
(In the above, "+" is positive, "-" is negative, "L" indicates that a positive reaction was rapidly observed after 2 weeks from the start of the test, and "D" indicates that it took more than 1 week after the start of the test. “W” indicates a weak positive reaction, respectively.)

Microorganisms 1 of the embodiment include, for example, microorganisms belonging to Candida palmioleophila.

(Second microorganism)
The second microorganism (hereinafter also referred to as microorganism 2) of one embodiment of the present invention is
Belonging to the genus Candida, the nucleotide sequence of 26S rDNA is the nucleotide sequence shown by SEQ ID NO: 2, or a nucleotide sequence having 90% or more sequence identity with the nucleotide sequence shown by SEQ ID NO: 2, and has oil-decomposing activity at a temperature of 20-30°C.
(1) Colony morphology (YM agar medium, 27°C, 1 week culture)
Color tone: White to cream Elevation: Cushion-shaped Margin: Entire to wavy Surface shape: Smooth Gloss and properties: Buttery, wet No formation of sexual reproductive organs after 3 weeks of culture

Examples of the microorganism 2 of the embodiment include microorganisms that exhibit the following physiological/biochemical properties in a physiological/biochemical property test.
<Physiological and biochemical property test results>
(1) Sugar fermentability test glucose -
galactose -
Maltose -
(2) Carbon resource utilization test Glucose +
Galactose +
L-sorbose -
D-glucosamine +
D- ribose -
D-xylose +
L-arabinose -
D-Arabinose -
L-rhamnose -
Sucrose +
Maltose +
Trehalose +
α-methyl-D-glucoside +
Cellobiose +
Salicin +
Melibiose +
Lactose -
Raffinose +
Meletitose +
Inulin -
Soluble starch +
Glycerol +
Erythritol -
Ribitol (Adonitol) +
Xylitol D
D-glucitol (sorbitol) +
D-mannitol +
Galactitol (Dulcitol) -
Inositol -
2-keto-D-gluconic acid +
D-gluconic acid +
DL-lactic acid +
Succinate +
Citrate +
Methanol -
Ethanol +
N-acetyl-glucosamine +
Hexadecane +
(3) Nitrogen source utilization Nitrate +
Cadaverine +
(4) Resistance test Growth at 30°C +
Growth at 37°C +
0.01% cycloheximide -
50% (W/V) D-glucose +
10% sodium chloride/5% glucose +
(5) Vitamin auxotrophy test Vitamin-free medium +
(In the above, "+" is positive, "-" is negative, "L" indicates that a positive reaction was rapidly observed after 2 weeks from the start of the test, and "D" indicates that it took more than 1 week after the start of the test. “W” indicates a weak positive reaction, respectively.)

Microorganisms 2 of the embodiment include, for example, microorganisms belonging to Candida palmioleophila.

(Third microorganism)
The third microorganism (hereinafter also referred to as microorganism 3) of one embodiment of the present invention is
Belonging to the genus Candida, the base sequence of 26S rDNA is the base sequence shown in SEQ ID NO: 3, or a base sequence having 90% or more sequence identity with the base sequence shown in SEQ ID NO: 3, and has oil-decomposing activity at a temperature of 20-30°C.
(1) Colony morphology (YM agar medium, 27°C, 1 week culture)
Color tone: White to cream Elevated state: Cushion-shaped Margin: Thread-like Surface shape: Smooth Gloss and properties: Butter-like, moist No formation of sexual reproductive organs at 3 weeks Formation of pseudohyphae or hyphae

Examples of the microorganism 3 of the embodiment include microorganisms that exhibit the following physiological/biochemical properties in a physiological/biochemical property test.
<Physiological and biochemical property test results>
(1) Sugar fermentability test Glucose +
Galactose +
Maltose +
(2) Carbon resource utilization test Glucose +
Galactose +
L-sorbose +
D-Glucosamine W
D- ribose -
D-xylose +
L-arabinose -
D-Arabinose -
L-rhamnose -
Sucrose +
Maltose +
Trehalose +
α-methyl-D-glucoside +
Cellobiose +
Salicin +
Melibiose -
Lactose -
Raffinose -
Meletitose +
Inulin -
Soluble starch +
glycerol -
Erythritol -
Ribitol (Adonitol) +
Xylitol +
D-glucitol (sorbitol) +
D-mannitol +
Galactitol (Dulcitol) -
Inositol -
2-keto-D-gluconic acid +
D-gluconic acid +
DL-lactic acid +
Succinate +
Citrate W
Methanol -
Ethanol +
N-acetyl-glucosamine +
Hexadecane +
(3) Nitrogen source utilization Nitrate W
Cadaverine +
(4) Resistance test Growth at 30°C +
Growth at 37°C +
0.01% cycloheximide +
50% (W/V) D-glucose +
10% sodium chloride/5% glucose +
(5) Vitamin auxotrophy test Vitamin-free medium +
(In the above, "+" is positive, "-" is negative, "L" indicates that a positive reaction was rapidly observed after 2 weeks from the start of the test, and "D" indicates that it took more than 1 week after the start of the test. “W” indicates a weak positive reaction, respectively.)

Microorganisms 3 of the embodiment include, for example, microorganisms belonging to Candida tropicalis.

(Fourth microorganism)
The fourth microorganism (hereinafter also referred to as microorganism 4) of one embodiment of the present invention is
Belonging to the genus Candida, the nucleotide sequence of 26S rDNA is the nucleotide sequence shown by SEQ ID NO: 4, or a nucleotide sequence having 90% or more sequence identity with the nucleotide sequence shown by SEQ ID NO: 4, and has oil-decomposing activity at a temperature of 20-30°C.
(1) Colony morphology (YM agar medium, 27°C, 1 week culture)
Color tone: White to cream Elevation: Cushion-shaped Periphery: Whole edge Surface shape: Smooth Gloss and properties: Buttery, moist No formation of sexual reproductive organs after 3 weeks of culture Formation of pseudohyphae

Examples of the microorganism 4 of the embodiment include microorganisms that exhibit the following physiological/biochemical properties in a physiological/biochemical property test.
<Physiological and biochemical property test results>
(1) Sugar fermentability test Glucose +
Galactose +
Maltose +
(2) Carbon resource utilization test Glucose +
Galactose +
L-sorbose +
D-glucosamine D
D- ribose -
D-xylose +
L-arabinose -
D-Arabinose -
L-rhamnose -
Sucrose +
Maltose +
Trehalose +
α-methyl-D-glucoside +
Cellobiose +
Salicin W
Melibiose -
Lactose -
Raffinose -
Meletitose +
Inulin -
Soluble starch +
glycerol -
Erythritol -
Ribitol (Adonitol) +
xylitol -
D-glucitol (sorbitol) +
D-mannitol +
Galactitol (Dulcitol) -
Inositol -
2-keto-D-gluconic acid +
D-gluconic acid +
DL-lactic acid +
Succinate +
Citrate D
Methanol -
Ethanol +
N-acetyl-glucosamine +
Hexadecane +
(3) Nitrogen source utilization Nitrate W
Cadaverine +
(4) Resistance test Growth at 30°C +
Growth at 37°C +
0.01% cycloheximide +
50% (W/V) D-glucose +
10% sodium chloride/5% glucose +
(5) Vitamin auxotrophy test Vitamin-free medium +
(In the above, "+" is positive, "-" is negative, "L" indicates that a positive reaction was rapidly observed after 2 weeks from the start of the test, and "D" indicates that it took more than 1 week after the start of the test. “W” indicates a weak positive reaction, respectively.)

Microorganisms 4 of the embodiment include, for example, microorganisms belonging to Candida tropicalis.

(Fifth microorganism)
The fifth microorganism (hereinafter also referred to as microorganism 5) of one embodiment of the present invention is
Belonging to the genus Candida, the nucleotide sequence of 26S rDNA is the nucleotide sequence shown by SEQ ID NO: 5, or a nucleotide sequence having 90% or more sequence identity with the nucleotide sequence shown by SEQ ID NO: 5, and has oil-decomposing activity at a temperature of 20-30°C.
(1) Colony morphology (YM agar medium, 27°C, 1 week culture)
Color tone: white to cream Elevated state: flat peripheral edge, cushion-shaped central edge Peripheral edge: Entire edge Surface shape: smooth Gloss and properties: bright, wet Shape, ovoid, short-rod Proliferation by budding No formation of sexual reproductive organs after 3 weeks of culture

Examples of the microorganism 5 of the embodiment include microorganisms that exhibit the following physiological/biochemical properties in a physiological/biochemical property test.
<Physiological and biochemical property test results>
(1) Sugar fermentability test Glucose +
galactose -
Maltose +
(2) Carbon resource utilization test Glucose +
Galactose +
L-sorbose -
D-glucosamine +
D- ribose -
D-xylose +
L-arabinose -
D-Arabinose -
L-rhamnose -
Sucrose +
Maltose +
Trehalose +
α-methyl-D-glucoside +
Cellobiose +
Salicin +
Melibiose -
Lactose +
Raffinose W
Meletitose +
Inulin -
Soluble starch +
Glycerol +
Erythritol -
Ribitol (Adonitol) +
Xylitol D
D-glucitol (sorbitol) +
D-mannitol +
Galactitol (Dulcitol) -
Inositol -
2-keto-D-gluconic acid +
D-gluconic acid +
DL-lactic acid +
Succinate +
Citrate +
Methanol -
Ethanol +
N-acetyl-glucosamine +
Hexadecane +
(3) Nitrogen source utilization Nitrate W
Cadaverine +
(4) Resistance test Growth at 30°C +
Growth at 37°C +
0.01% cycloheximide +
50% (W/V) D-glucose +
10% sodium chloride/5% glucose +
(5) Vitamin auxotrophy test Vitamin-free medium +
(In the above, "+" is positive, "-" is negative, "L" indicates that a positive reaction was rapidly observed after 2 weeks from the start of the test, and "D" indicates that it took more than 1 week after the start of the test. “W” indicates a weak positive reaction, respectively.)

Microorganisms 5 of the embodiment include, for example, microorganisms belonging to Candida fluviatilis.

In the present specification, "having oil decomposition activity" means that the following oil decomposition rate measured by the following procedure is 5% or more.
Oil is added to the following liquid medium (5 mL), autoclaved, and used as a test solution to determine the oil decomposition rate. A loopful of the strain to be tested is inoculated into the test solution with a platinum loop, and cultured with shaking at 30° C. and 140 rpm for 24 hours.
After culturing, normal-hexane extract is measured according to JIS K0102 (industrial wastewater test method). Using the normal-hexane extract as the remaining oil content, the oil decomposition rate is calculated by the following formula (1) from the amount of oil added during preparation of the test solution and the remaining oil content (amount of normal-hexane extract (g)). .

(Test liquid)
Final concentration of 0.1% (w/v) yeast extract, 1% (w/v) ₍ NH4) _2SO4 , 0.2% ₍ w/v) _K2HPO4 , 0.1% ₍ w/v) /v) _KH2PO4 , 0.5% ₍ w/v) NaCl, 0.002% ₍ w/v) _CaCl2.2H2O , 0.2% ₍ w/v) _MgSO4.7H2O , 0.001% (w/v) FeSO ₄ .7H ₂ O, 0.001% (w/v) CaCl ₂ .2H 2 O, and dispensed 5 mL each into φ24 test tubes. , 0.05 g of oil (rapeseed oil: soybean oil: lard = 2:2:1 (w/w/w)) was added to each test tube and then sterilized under high pressure (121°C, 15 minutes).

The measurement of the normal-hexane extractive substance concentration by the normal-hexane extractive substance-gravimetric method is performed by making the sample slightly acidic, extracting it with normal-hexane, and volatilizing the normal-hexane at 80 ° C. The remaining normal-hexane extractive substance This can be done by determining the weight of

In addition, the oil decomposition rate of the microorganisms of the embodiment is preferably 20% or more, more preferably 30% or more, and even more preferably 40% or more as a decomposition rate of fats and oils under temperature conditions that exhibit the maximum oil decomposition rate.

The identity of nucleotide sequences of microorganisms 1 to 5 can be determined by BLAST search using databases such as GenBank, EMBL, and DDBJ.
The sequence identity is 90% or greater, may be 95% or greater, or may be 98% or greater.

Physiological and biochemical characterization tests are described in Kurtzman C. P., Fell J. W., Boekhout T. & Robert V. (2011). Methods for isolation, phenotypic characterization and maintenance of yeasts. In: Kurtzman C. P., Fell J. W. & Boekhout T. (eds .) The method described in The Yeasts, a taxonomic study, 5th edition. pp. 87-110, Elsevier, Amsterdam.

It is speculated that the microorganisms of the embodiments can hydrolyze fats and oils into glycerin and fatty acids by producing lipase. In addition, the microorganisms of the embodiments can assimilate fatty acids produced by the decomposition of fats and oils. The microorganisms of the embodiments are preferably capable of assimilating glycerol in addition to fatty acids. Fatty acid assimilation prevents fatty acid from accumulating in the wastewater, prevents a decrease in oil decomposition efficiency, and makes it possible to decompose oil more efficiently.
That is, according to the microorganisms of the embodiment, in the oil decomposition, after the oil is decomposed into glycerin and fatty acids by lipase, each of them can be further assimilated, so it is possible to remove glycerin or fatty acids.
Since the microorganisms of the embodiments can efficiently decompose and remove oil, it is possible to remove, for example, oil contained in food factory wastewater and kitchen wastewater, triglycerides contained in biodiesel raw materials, and the like.

<Culturing of microorganisms>
Any method for culturing the microorganisms of the embodiment may be used as long as the microorganisms can grow and proliferate. For example, the medium used for culturing the microorganisms of the embodiment may be either a solid or liquid medium, and a medium containing a carbon source that can be assimilated by the microorganisms used, an appropriate amount of nitrogen source, inorganic salts and other nutrients. It may be either a synthetic medium or a natural medium. Media typically contain a carbon source, a nitrogen source and minerals.

The carbon source that can be used in culturing the microorganisms of the embodiment is not particularly limited as long as it can be assimilated by the strain used. Specifically, considering the assimilation of microorganisms, glucose, fructose, cellobiose, raffinose, xylose, maltose, galactose, sorbose, glucosamine, ribose, arabinose, rhamnose, sucrose, trehalose, α-methyl-D-glucoside , salicin, melibiose, lactose, melezitose, inulin, erythritol, glucitol, mannitol, galactitol, N-acetyl-D-glucosamine, starch, starch hydrolysates, sugars such as molasses and blackstrap molasses, natural products such as wheat and rice , glycerol, methanol, ethanol and other alcohols; acetic acid, lactic acid, succinic acid, gluconic acid, pyruvic acid, citric acid and other organic acids; and hexadecane and other hydrocarbons. The carbon source is appropriately selected in consideration of assimilation by the microorganisms to be cultured. Also, one or more of the above carbon sources can be selected and used.

Nitrogen sources that can be used in culturing the microorganisms of the embodiments include meat extract, peptone, polypeptone, yeast extract, malt extract, soybean hydrolyzate, soybean powder, casein, milk casein, casamino acid, glycine, glutamic acid, asparagine. Organic nitrogen sources such as various amino acids such as acids, corn steep liquor, hydrolysates of other animals, plants, and microorganisms; ammonium salts such as ammonia, ammonium nitrate, ammonium sulfate, and ammonium chloride; nitrates such as sodium nitrite; and inorganic nitrogen sources such as nitrites and urea. The nitrogen source is appropriately selected in consideration of assimilation by the microorganisms to be cultured. Also, one or more of the above nitrogen sources can be selected and used.

Inorganic substances that can be used include halides such as phosphates, hydrochlorides, sulfates, acetates, carbonates, chlorides of magnesium, manganese, calcium, sodium, potassium, copper, iron and zinc. . 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 of the embodiment to efficiently decompose and assimilate oil or to maintain the ability of the microorganisms to decompose and assimilate oil, it is preferable to add oil to the medium. Examples of the oil include the above-mentioned edible oils and fats, industrial oils and fats, 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, oil (rapeseed oil: soybean oil: lard = 2:2:1 (w/w/w)) is added to 1 L of medium at a concentration of 1 to 30 g, more preferably 5 to 15 g. is preferred. With such an amount to be added, the microorganisms can maintain high oil decomposition and assimilation ability. The oils may be added singly or in the form of a mixture of two or more.

Cultivation of the microorganisms of the embodiment can be performed by a normal method. For example, microorganisms are cultured under aerobic or anaerobic conditions, depending on the type of microorganism. Under aerobic conditions, microorganisms are cultured by shaking, aeration, or the like. 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 of the embodiment to grow, and can be appropriately selected according to the type of microorganisms to be cultured.
Usually, the culture temperature is preferably 15-40°C, more preferably 25-35°C. In addition, the pH of the medium suitable for culture is preferably pH 3-11, more preferably pH 4-10.

The culture time is not particularly limited, and can be appropriately selected depending on the type of microorganism to be cultured, the amount of medium, culture conditions, and the like. Usually, the culture time is preferably 16-72 hours, more preferably 20-48 hours.

(Oil decomposition agent)
The oil decomposition agent of the embodiment includes the microorganism of the embodiment or a processed product of the microorganism of the embodiment. Microorganisms of the embodiments include at least one or more of the microorganisms 1 to 5 above.
There are no particular restrictions on the means of treating microorganisms for obtaining a treated product of microorganisms, and known formulation means can be used. Examples of the processed product of the above embodiment include those obtained by collecting the culture solution of the microorganism of the embodiment by centrifugation or the like, suspending it in a medium such as physiological saline or a culture medium, and freeze-drying the suspension.

The oil decomposing agent of the embodiment may be in liquid form or solid (preferably powder form) form. When the oil decomposing agent is solid, the culture solution of microorganisms can be dried by a suitable drying method to be solid. Drying methods include vacuum dryers, fluidized bed dryers, spray dryers, freeze dryers, drum dryers, and the like. An excipient or the like may be added during drying.
Examples of the liquid form include a culture solution of microorganisms themselves, and a product obtained by redispersing microorganisms collected by centrifugation or the like in a medium such as physiological saline or a medium. Alternatively, an appropriately diluted or concentrated culture solution, or a nutrient added as an additive can be exemplified.

The oil-decomposing agent of the embodiment can decompose oil-containing substances. The oil-containing substance is not particularly limited as long as it contains oil and can be decomposed by the microorganisms of the embodiment. Examples include oil-containing soil and oil-containing industrial waste, and oil-containing wastewater is preferred.

Examples of oil-containing water include wastewater discharged from food processing factories, chemical factories, dairy product factories, restaurant kitchens, households, and the like. According to the oil decomposition agent of the embodiment, even for oil-containing water with a high n-hexane extract concentration (for example, oil-containing wastewater with a n-hexane extract concentration of 500 to 10000 mg / L), the treatment time is short (for example, 24 hours), it is possible to obtain clear treated water with little generation of excess sludge. The concentration of normal-hexane extracted substances in oil-containing water is determined by acidifying the sample with hydrochloric acid to a pH of 4 or less, extracting the oil in the sample with hexane, volatilizing the hexane at about 80°C, and measuring the mass of the remaining substance. can be obtained by doing

In addition, hydrolysis of oil and consumption of produced fatty acids can be analyzed and evaluated by thin layer chromatography (TLC). As a specific procedure, a mixed solvent of methanol:methyl-t-butyl ether=1:2 can be used for lipid extraction from specimens. As a developing solvent for TLC, a mixed solvent of n-hexane:methyl-t-butyl ether:acetic acid=85:15:1 can be used. After spraying the phosphomolybdic acid/ethanol solution, the strength and weakness of the spots that appear due to the heat treatment can be analyzed by quantitative analysis by image processing.

In addition, in order to assist the decomposition of oil by microorganisms, the oil-degrading agent of the embodiment contains oil-degrading enzymes such as lipase and phospholipase, other microorganisms, etc., if necessary, in addition to the microorganisms of the embodiment. may

Examples of oil-degrading enzymes include Pseudomonas, Aspergillus, Bacillus, Penicillium, Rhizopus, Rhizomucor, Mucor, Pecilomyces genus, Rhizoctonia genus, Absidia genus, Achromobacter genus, Aeromonas genus, Alternaria genus, Aureobasidium genus, Beauveria ) genus, Chromobacter genus, Coprinus genus, Fusarium genus, Geotricum genus, Humicola genus, Hyphozyma genus, Lactobacillus genus, Metarhizium ) genus, Rhodosporidium genus, Trichoderma genus, Yarrowia genus, Candida genus, Pichia genus, Hansenula genus, Saccharomyces genus, Kluyvelo 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.

(Oil decomposition method)
The oil decomposition method of the embodiment is a method of contacting the oil decomposition agent of the embodiment with the oil-containing substance to decompose the oil in the oil-containing substance. As a method of bringing the oil-containing substance into contact with the oil-decomposing agent of the embodiment, there is a method of directly contacting the oil-decomposing agent of the embodiment with the oil-containing substance. A method of preculturing microorganisms in the oil decomposing agent of the embodiment and adding the obtained culture solution to the oil-containing material, the oil of the embodiment Examples include a method of preculturing microorganisms in the decomposing agent, adding the obtained culture solution to the oil-containing material, and culturing the microorganisms.
Contact between the oil decomposing agent of the embodiment and the oil-containing material can be performed in an oil treatment tank.
Contact between the oil-decomposing agent of the embodiment and the oil-containing material is preferably carried out under aerobic conditions. The microbial culture environment may be aerated to provide aerobic conditions.

The pH at which the oil-decomposing agent of the embodiment is brought into contact with the oil-containing material is not particularly limited, but is preferably pH 4.0 to 9.0, more preferably pH 4.0 to 8.0, and pH 4.0 to 6.0 is more preferred, and pH 5.0 is particularly preferred.
The temperature at which the oil decomposition agent of the embodiment is brought into contact with the oil decomposition product is not particularly limited, but is preferably 10 to 40°C, more preferably 15 to 35°C, further preferably 25 to 35°C, and 30°C. is particularly preferred.
The amount of the oil-decomposing agent when the oil-decomposing agent of the embodiment is brought into contact with the oil-containing material is not particularly limited, but for example, the number of viable microorganisms is about 10 ⁷ to 10 ⁹ /mL. quantity.
The contact time between the oil-decomposing agent of the embodiment and the oil-containing material is not particularly limited, but is preferably 1 hour to 168 hours, more preferably 24 hours to 96 hours, and even more preferably 48 hours to 72 hours.

Preferred conditions for the oil decomposition method of the embodiment include the following.
Preferred temperature range: 10-40°C, more preferably 15-35°C
Preferred pH range: pH 4-9, more preferably pH 5-8
Preferred amount of microorganisms to be used: 1/100 to 1/100,000 of the volume of the reaction vessel into which the oil decomposing agent is charged at a viable cell count concentration of 10 ⁶ to 10 ¹¹ cfu/mL (or g) in the oil decomposing agent. It can be added so that Cfu is an abbreviation for colony forming unit, and is a unit that indicates the number of viable bacteria.

In addition, in the oil decomposition method of the embodiment, in order to assist the decomposition of oil by microorganisms, in addition to the microorganisms of the embodiment, if necessary, oil-degrading enzymes such as lipases and phospholipases exemplified above, other microorganisms, etc. It may be added to the oil treatment tank.

Further, in the oil decomposition method of the embodiment, an oil adsorbent such as an inorganic polymer such as diatomaceous earth or perlite, or an organic polymer such as polyurethane, polyethylene, or melanin resin may be added to the oil treatment tank.

In the oil decomposition method of the embodiment, anionic surfactants such as sodium dodecyl sulfate (SDS), dodecylbenzene sulfonic acid (NaDDBS), ammonium lauryl sulfate, and sodium caseinate are used to prevent oil aggregation and scum formation; cationic surfactants such as group amine salts and quaternary ammonium salts; Surfactants such as non-ionic surfactants such as t-octylphenyl ether (Triton X-100), lecithin; amphoteric surfactants such as betaines, amine oxides, saponins, etc. may be added to the oil treatment tank.

In the oil decomposition method of the embodiment, a fluidized bed carrier may be added to the oil treatment tank in order to improve contact efficiency between oil and microorganisms.
The shape of the fluidized bed carrier may be spherical, pellet-shaped, hollow cylinder-shaped, filamentous, plate-shaped, etc., and the size (diameter) is about 0.1 to 10 mm. The carrier material may be any natural material, inorganic material, polymeric material, or the like, and may be a gel-like substance.
By setting the filling rate of the fluidized bed carrier to 1 to 30%, preferably 5 to 20%, it is possible to efficiently decompose oil even in waste water containing a high concentration of oil. The packing ratio of the carrier represents the ratio of the volume of the carrier to the volume of the liquid in the treatment bath.

By fixing or immobilizing the microorganisms of the embodiments on a carrier and immersing the carrier in an oil-containing substance such as wastewater, or placing the carrier in a position where it can come into contact with the oil-containing substance such as wastewater in the channel, A decomposition reaction of the oil may be caused.
As a method for colonizing or immobilizing microorganisms, conventionally used known methods can be used without particular limitation, as long as the method is such that microorganisms do not flow out from the carrier. Specific methods for colonization or immobilization include, for example, an adherent biofilm method using a carrier to which microorganisms adhere and form a biofilm, a support culture method in which a carrier and a medium are mixed to culture bacteria, A carrier binding method in which bacteria or a composition is bound to a water-insoluble carrier, a method in which microorganisms are enclosed in the pores of the carrier under reduced pressure, and a reagent having two or more functional groups is used to form crosslinks in the cells for immobilization. method, carrier binding method classified into covalent binding method, physical adsorption method, ionic binding method, biochemical specific binding method, etc. according to binding means, and entrapment immobilization that confines microorganisms inside polymer gel or membrane law, etc.

The entrapping immobilization carrier obtained by the entrapping immobilization method polymerizes and gels the immobilizing material in the presence of the microbial cells, entrapping and immobilizing the microbial cells on the immobilizing material (monomer, prepolymer). Formable. Acrylamidemethylenebisacrylamide, triacrylformal, etc. are preferably used as the monomer material used as the immobilizing material. As the prepolymer material, polyethylene glycol diacrylate, polyethylene glycol methacrylate, derivatives thereof, and the like are preferably used. Moreover, it is preferable to contain a cross-linking agent in addition to the immobilizing material. As the cross-linking agent, a monomer or prepolymer that is harmless to the cells and has a low molecular weight, such as ethylene glycol diacrylate and ethylene glycol methacrylate, and derivatives thereof can be preferably used.

The oil-containing treatment tank into which the microorganisms of the embodiment are introduced may be in the form of continuously introducing oil-containing substances such as waste water and continuously discharging oil-containing substances after treatment, or introducing oil-containing substances. However, after collectively processing, the processed oil-containing material may be collectively discharged (batch type).

In the oil decomposition method of the embodiment, the microorganism of the embodiment can be used in combination with the membrane separation activated sludge process (MBR). When treating oil-containing substances, if oil adheres to the separation membrane, the separation performance of the membrane decreases. After performing pretreatment, it is generally supplied to a membrane separation activated sludge treatment apparatus. By combining the microorganisms of the embodiment and the membrane separation activated sludge method (MBR), even if the membrane separation activated sludge method (MBR) and the oil-water separation are insufficient, or if a large amount of oil flows in due to an operation error, etc. Since the influence on the membrane can be suppressed and the outflow of microorganisms can be prevented, it is possible to exhibit stable treatment capacity.

The oil decomposition method of the embodiment is suitable for use in wastewater treatment. By applying this method to wastewater treatment, compared with conventional wastewater treatment using microorganisms, it is possible to greatly improve the treatment efficiency of oil and reduce the burden on the environment.

EXAMPLES The present invention will be described in more detail below with reference to Examples, but the present invention is not limited to these Examples.

[Experiment 1] Screening of oil-degrading bacteria 1. Screening Method Environmental samples such as oil, sludge, and soil in wastewater treatment facilities were added in appropriate amounts to primary screening liquid media prepared by the following method, and cultured with shaking at 30° C. and 120 rpm for two weeks. After culturing, 1 mL of the culture solution was further inoculated into the liquid medium for primary screening, and cultured again with shaking at 30° C. and 120 rpm for 2 weeks. This operation was repeated two more times.

(Method for preparing liquid medium for primary screening)
0.9% (w/v) _KH2PO4 , ₁ % (w/v) ₍ NH4) _2SO4 , 0.1% ₍ w/v) _MgSO4.7H2O , 0.9% ₍ w/v) final concentration. 005% (w/v) _FeSO4.7H2O , 0.01% ₍ w/v) _CaCl2.2H2O , 0.05% ₍ w/v) NaCl, 0.01% (w/v) The yeast extract was dissolved in pure water, and 100 mL portions were dispensed into 500 mL baffled flasks. After adding 20 g of oil (rapeseed oil: soybean oil: lard = 2: 2: 1 (w / w / w)) to each flask, high pressure sterilization (121 ° C., 15 minutes) was used as the liquid medium for primary screening. did.

100 μL of the culture medium after the primary screening was smeared on an agar medium for secondary screening prepared by the following method, and cultured at 30° C. for 1 week. After culturing, strains in which halos due to oil decomposition were confirmed were smeared again on the agar medium for secondary screening to purify and isolate.

(Method for preparing agar medium for secondary screening)
0.9% (w/v) _KH2PO4 , ₁ % (w/v) ₍ NH4) _2SO4 , 0.1% ₍ w/v) _MgSO4.7H2O , 0.9% ₍ w/v) final concentration. 005% (w/v) _FeSO4.7H2O , 0.01% ₍ w/v) _CaCl2.2H2O , 0.05% ₍ w/v) NaCl, 0.01% (w/v) Yeast extract, 2% (w/v) Triton (registered trademark) X-100, 3% (w/v) agar, 1% (w/v), oil (rapeseed oil: soybean oil: pork fat = 2:2: 1 (w/w/w)), autoclaved (121°C, 15 minutes), and dispensed into petri dishes in 20 mL increments to prepare agar media for secondary screening.

Next, oils and fats were added to a tertiary screening liquid medium (5 mL) prepared by the following method, and the resulting mixture was sterilized under high pressure to obtain the oil decomposition rate. One platinum loop of each isolated strain obtained in the secondary screening was inoculated into the test solution, and cultured with shaking at 30° C. and 140 rpm for 24 hours.

(Method for preparing liquid medium for tertiary screening)
Final concentration of 0.1% (w/v) yeast extract, 1% (w/v) ₍ NH4) _2SO4 , 0.2% ₍ w/v) _K2HPO4 , 0.1% ₍ w/v) /v) _KH2PO4 , 0.5% ₍ w/v) NaCl, 0.002% ₍ w/v) _CaCl2.2H2O , 0.2% ₍ w/v) _MgSO4.7H2O , 0.001% (w/v) FeSO ₄ .7H ₂ O and 0.001% (w/v) CaCl ₂ .2H 2 O, and dispensed 5 mL each into φ24 test tubes. . After adding 0.05 g of oil (rapeseed oil: soybean oil: lard = 2: 2: 1 (w / w / w)) to each test tube, sterilize under high pressure (121 ° C., 15 minutes) for tertiary screening A liquid medium was used.

After culturing, normal-hexane extract was measured according to JIS K0102 (industrial wastewater test method). Using the normal-hexane extract as the remaining oil content, the oil decomposition rate is calculated by the following formula (1) from the amount of oil added during preparation of the test solution and the remaining oil content (amount of normal-hexane extract (g)). rice field. As a result, 5 strains (MC1 strain, MC3 strain, MC4 strain, MC28 strain, MC29 strain) with high oil decomposition rate were isolated.

FIG. 1 shows the cell morphology observation results of the MC1 strain on the 5th day of culture on the YM plate medium.
FIG. 2 shows the cell morphology observation results of the MC3 strain on the 5th day of culture on the YM plate medium.
FIG. 3 shows the cell morphology observation results of the MC4 strain on the 5th day of culture on the YM plate medium. Arrows in FIG. 3 indicate the formation of pseudohyphae or mycelia.
FIG. 4 shows the results of observation of cell morphology of the MC28 strain on day 5 of culture on the YM plate medium. Arrows in FIG. 3 indicate the formation of pseudohyphae.
FIG. 5 shows the cell morphology observation results of the MC29 strain on the 5th day of culture on the YM plate medium.

The nucleotide sequence of the 26S rDNA-D1/D2 region of the isolated strain was determined as follows.
1. Culture conditions Strains cultured under the following conditions were used as test cells.
・Medium: Difco YM Broth (Becton Dickinson, MD, USA) + agar medium (YM agar)
・Incubation temperature: 30℃
・Culturing time: 1 day ・Other conditions: Aerobic culture

2.26S rDNA-D1/D2 base sequence analysis
Operations from PCR amplification to cycle sequencing were based on each protocol.
・DNA extraction: physical disruption and modification of Marmur ・PCR amplification: PrimeSTAR HS DNA Polymerase (Takara Bio)
・Cycle sequence:
BigDye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems, CA, USA)
・Primers used: PCR amplification; ITS5, NL4
Sequence; NL1, NL4
・Sequence:
ABI PRISM 3130xlGenetic Analyzer System (Applied Biosystems,CA,USA)
・Nucleotide sequencing: ChromasPro1.7 (Technelysium Pty Ltd., Tewantin, AUS)

The 26S rDNA-D1/D2 base sequences of the isolated microorganisms determined above are shown in SEQ ID NOS: 1-5 below.
SEQ ID NO: 1: MC1 strain 26S rDNA-D1/D2 regions SEQ ID NO: 2: MC3 strain 26S rDNA-D1/D2 regions SEQ ID NO: 3: MC4 strain 26S rDNA-D1/D2 regions SEQ ID NO: 4: MC28 strain 26S rDNA-D1/ D2 region SEQ ID NO: 5: MC29 strain 26S rDNA-D1/D2 regions

In addition, a BLAST search (homology search) was performed on the nucleotide sequences obtained as follows.
・Database used:
DB-FU10.0 (Technosuruga Laboratory)
International Nucleotide Sequence Database (GenBank/DDBJ/ENA(EMBL))

As a result, the base sequences of the 26S rDNA-D1/D2 regions of the isolated microorganisms were respectively MC1 and MC3 strains: Candida palmioleophila, MC4 and MC28 strains: Candida tropicalis, Strain MC29: showed a high identity of 99.8-100% to the type strain of Candida fluviatilis.
6 to 10 show molecular phylogenetic trees analyzed based on the nucleotide sequences obtained by homology search.
MC1 strain: SIID31647-01-26S
MC3 strain: SIID31647-02-26S
MC4 strain: SIID31647-03-26S
MC28 strain: SIID31647-04-26S
MC29 strain: SIID31647-05-26S
The phylogenetic tree was estimated using the neighbor-joining method, the base substitution model using Kimura-2-parameter, and the bootstrap method (1000 repetitions) to evaluate the reliability of the tree shape. Numbers located at branches of phylogenetic branches represent bootstrap values. The T at the end of the strain name represents the type strain of that species.
According to the above nucleotide identity and phylogenetic analysis, the isolated microorganisms are, respectively, MC1 and MC3 strains of Candida palmioleophila, MC4 and MC28 strains of Candida tropicalis, and MC29 strains. attributed to Candida fluviatilis.

[Experiment 2] Oil decomposition test in model liquid Microorganisms (MC1 strain, MC3 strain, MC4 strain, MC28 strain, and MC29 strain) were evaluated for oil decomposition by the following method. As a comparative example, a budding yeast strain (Saccharomyces cerevisiae S288C) different from the genus Candida and a microbial preparation A (trade name “BioRemove 3200”, manufactured by Novozymes) were evaluated for oil decomposition by the following method.

Using Soybean-Casein Digest (SCD) medium (manufactured by Wako Pure Chemical Industries, Ltd.) as a pre-culture medium, appropriate amounts of each of the above microorganisms and microbial preparations were inoculated and pre-cultured at 30° C. and 120 rpm for 24 hours. Subsequently, the bacterial cell concentration in the preculture was measured by the dry weight method. Subsequently, each preculture solution was added to a liquid medium (100 mL) for oil decomposition evaluation prepared by the following method so that the final cell concentration was 100 mg / L, and the mixture was heated at 30 ° C. and 120 rpm for 24 hours. Cultured with shaking.

(Method for preparing liquid medium for oil decomposition evaluation)
Final concentration of 0.1% (w/v) yeast extract, 1% (w/v) ₍ NH4) _2SO4 , 0.2% ₍ w/v) _K2HPO4 , 0.1% ₍ w/v) /v) _KH2PO4 , 0.5% ₍ w/v) NaCl, 0.002% ₍ w/v) _CaCl2.2H2O , 0.2% ₍ w/v) _MgSO4.7H2O , 0.001% (w/v) FeSO ₄ .7H ₂ O, and 0.001% (w/v) CaCl ₂ .2H ₂ O. 100 mL of the medium was dispensed into 500 mL baffled Erlenmeyer flasks. After 1 g of oil was added to each flask, the mixture was sterilized under high pressure (121° C., 15 minutes) and used as a liquid medium for oil decomposition evaluation. Rapeseed oil, palm oil, soybean oil, fish oil, lard, beef tallow, and fatty acid (oleic acid) were used as the oil.
After completion of the culture, the fat and oil components remaining in the culture medium were quantified. The quantification method of the fat and oil component was determined as the amount of normal-hexane extract according to the method of JIS K0102. As a control, the amount of n-hexane extract was measured under the same conditions as above, except that no microorganism or microbial preparation was inoculated. Each oil component decomposition rate was determined by the above formula (1).

The results obtained are shown in FIG. From these results, it was confirmed that the microorganisms belonging to the genus Candida (strains MC1, MC3, MC4, MC28, and MC29) have a high ability to decompose oil.

[Experiment 3] Oil decomposition test at low temperature in model liquid The microorganisms and microbial preparations shown in Experiment 2 were evaluated for oil decomposition at low temperature (20°C). The evaluation method was the same as in Experiment 2, except that the culture temperature in the culture using the oil decomposition evaluation liquid medium was 20° C. and the oil was rapeseed oil.
The results obtained are shown in FIG. From these results, it was confirmed that the microorganisms belonging to the genus Candida (strains MC1, MC3, MC4, MC28, and MC29) have high oil-degrading ability even at low temperatures.

[Experiment 4] Oil decomposition test in actual wastewater The microorganisms and microbial preparations shown in Experiment 2 were evaluated for oil decomposition in actual wastewater. The evaluation method was the same as in Experiment 2, except that the liquid medium for evaluation of actual wastewater oil decomposition was used.
(Method for preparing liquid medium for evaluation of decomposition of oil in actual wastewater)
Raw water and floating oil were collected from the raw water tank of the wastewater treatment facility of a food factory in Kanagawa Prefecture. They were dissolved in raw water to final concentrations of 0.1% (w/v) corn steep liquor and 0.05% (w/v) urea, and 100 mL portions were dispensed into 500 mL baffled Erlenmeyer flasks. 1 g of floating oil was added to each flask to prepare a liquid medium for evaluation of decomposition of oil in actual waste water.

The results obtained are shown in FIG. From these results, the microorganisms belonging to the genus Candida (strains MC1, MC3, MC4, MC28, and MC29) were found to have high oil-decomposing ability even in actual wastewater.

[Experiment 5] Continuous batch treatment test In Experiment 2-4, microorganisms belonging to the genus Candida (strains MC1, MC3, MC4, MC28, and MC29) can exhibit high oil decomposition treatment ability. It was revealed. Therefore, whether or not the oil decomposition processing ability is continuously observed in batch culture was examined using (1) the model solution described in Experiment 3 and (2) the actual wastewater described in Experiment 4. Candida palmi as a microorganism Investigation was carried out using oleophila (Candida palmioleophila) MC1 strain and Candida tropicalis (Candida tropicalis) MC28 strain.

Specifically, first, using Soybean-Casein Digest (SCD) medium (manufactured by Wako Pure Chemical Industries, Ltd.) as a pre-culture medium, an appropriate amount of microorganisms belonging to the genus Candida (MC1 strain or MC28 strain) was inoculated, and Preculture was performed at 120 rpm for 24 hours. Subsequently, as the main culture medium, (1) the model solution (100 mL) described in Experiment 3 and (2) the actual wastewater (100 mL) described in Experiment 4 were added so that the oil content was 1% (v / v), respectively. was added, and cultured with shaking at 30° C. and 120 rpm for 24 hours. After culturing, the culture solution was further added to the main culture medium so that the oil content was 1% (v/v), and cultured with shaking at 30° C. and 120 rpm for 24 hours. This operation was repeated to continuously perform batch culture for a total of 35 days.
After completion of each culture, the fat and oil components remaining in the culture medium were quantified under the same conditions as above to determine the oil decomposition rate. As a control, the test was conducted under the same conditions as above, except that no microorganism or microbial preparation was inoculated.

The results obtained are shown in FIG. From these results, it was confirmed that the Candida genus microorganisms (MC1 strain and MC28 strain) can remove oil even if batch culture is repeated in both the model solution and the actual wastewater as the main culture medium. rice field.

[Experiment 6] Continuous treatment test using entrapping immobilization carriers (cultivation of microorganisms)
Soybean-Casein Digest (SCD) medium (manufactured by Wako Pure Chemical Industries, Ltd.) is used as a pre-culture medium, and an appropriate amount of Candida palmioleophila MC1 strain or Candida tropicalis MC28 strain is inoculated, Preculture was performed at 30° C. and 120 rpm for 24 hours. After culturing, the cells were collected by centrifugation and purely suspended to prepare suspensions of MC1 strain and MC28 strain, respectively.

(Manufacturing method of comprehensively immobilized simple substance)
After adding a polymerization accelerator (N,N,N',N'-tetramethylenediamine) to polyethylene glycol, sulfuric acid was used to adjust the pH of the mixture to 8.0. After mixing well, the MC1 strain and MC28 strain suspensions were added and mixed. After that, a polymerization initiator (potassium persulfate) was added, and the mixture was poured into a pre-prepared frame so as to form a sheet. After about 1 minute, the polymerization was completed and a sheet-like gel was obtained. This was shaped into a cube of 4 mm square, and used as entrapping immobilization carriers for MC1 and MC28 strains for testing. The composition of each drug is polyethylene glycol 10% (w/v), polymerization accelerator 0.5% (w/v), polymerization initiator 0.25% (w/v), MC1 strain or MC28 strain 0.4 % (w/v).
[Continuous treatment test]
Using a 2 L reactor, a continuous treatment test was performed on the entrapping immobilization pellets. The filling rate of the carrier was 10 (v/v)% of the reactor capacity. Continuous treatment was performed using a liquid medium for continuous treatment having the following composition as raw water to be treated. As for the condition of the continuous treatment, 0.1 N hydrochloric acid and 0.1 N sodium hydroxide aqueous solution were used to constantly adjust the pH in the reactor to around 7.2. The water temperature was adjusted to 25° C., and the raw water was adjusted to the retention time to adjust the influent load per unit volume to 0.5 to 1.0 kg-n-hexane/m ³ ·day. The influent water load was 0.5 kg-n-hexane/m ³ ·day for days 1-20, and 1.0 kg-n-hexane/m ³ ·day for days 21-40. Aeration was performed from below, and an air pump (maximum air discharge rate: 2 L/min) generally used for aquarium fish was used as the apparatus. As a control, a continuous treatment test was performed under the same conditions as above, except that the entrapping immobilization carrier was not added. The amount of n-hexane extract in the treated water was measured daily.

(Method for preparing liquid medium for continuous treatment)
Final concentration of 0.17% (w/v) casein peptone, 0.03% (w/v) soy peptone, 0.025% (w/v) glucose, 0.025% (w/v) _{K2HPO 4} , dissolved in pure water to give 0.05% (w/v) sodium chloride and 0.1% (w/v) rapeseed oil.

The results obtained are shown in FIG. From these results, it was confirmed that oil can be efficiently removed even in continuous treatment using entrapping immobilization carriers of microorganisms belonging to the genus Candida (strains MC1 and MC28).

From the above, it was revealed that the five types of microorganisms obtained in the examples are excellent in oil decomposition efficiency. It is also worth noting that among these microorganisms, there are microorganisms that are particularly difficult to decompose by microorganisms, and that have remarkably high degradability under low-temperature conditions or animal oil components.

Each configuration and combination thereof in each embodiment described above is an example, and addition, omission, replacement, and other modifications of the configuration are possible without departing from the scope of the present invention. Moreover, the present invention is not limited by each embodiment, but is limited only by the scope of the claims.

Claims (18)

Belonging to the genus Candida, the nucleotide sequence of 26S rDNA is the nucleotide sequence shown by SEQ ID NO: 1, or a nucleotide sequence having 90% or more sequence identity with the nucleotide sequence shown by SEQ ID NO: 1, and have oil-decomposing activity at a temperature of 20-30°C.
(1) Colony morphology (YM agar medium, 27°C, 1 week culture)
Color tone: White to cream Elevation: Cushion-shaped Margin: Entire to wavy Surface shape: Smooth Gloss and properties: Buttery, wet No formation of sexual reproductive organs after 3 weeks of culture 2. The microorganism according to claim 1, which exhibits the following physiological/biochemical properties in a physiological/biochemical property test.
(1) Sugar fermentability test glucose -
galactose -
Maltose -
(2) Carbon resource utilization test Glucose +
Galactose +
L-sorbose -
D-glucosamine +
D-ribose +
D-xylose +
L-arabinose -
D-Arabinose -
L-rhamnose -
Sucrose +
Maltose +
Trehalose +
α-methyl-D-glucoside +
Cellobiose -
salicin -
Melibiose +
Lactose -
Raffinose +
Meletitose +
Inulin -
Soluble starch +
Glycerol +
Erythritol -
Ribitol (Adonitol) +
xylitol -
D-glucitol (sorbitol) +
D-mannitol +
Galactitol (Dulcitol) -
Inositol -
2-keto-D-gluconic acid +
D-gluconic acid +
DL-lactic acid +
Succinate -
Citrate +
Methanol -
Ethanol +
N-acetyl-glucosamine +
Hexadecane +
(3) Nitrogen source utilization Nitrate W
Cadaverine +
(4) Resistance test Growth at 30°C +
Growth at 37°C +
0.01% cycloheximide -
50% (W/V) D-glucose +
10% sodium chloride/5% glucose +
(5) Vitamin auxotrophy test Vitamin-free medium +
(In the above, "+" is positive, "-" is negative, "L" indicates that a positive reaction was rapidly observed after 2 weeks from the start of the test, and "D" indicates that it took more than 1 week after the start of the test. “W” indicates a weak positive reaction, respectively.) 3. The microorganism according to claim 1 or 2, belonging to Candida palmioleophila. Belonging to the genus Candida, the nucleotide sequence of 26S rDNA is the nucleotide sequence shown by SEQ ID NO: 2, or a nucleotide sequence having 90% or more sequence identity with the nucleotide sequence shown by SEQ ID NO: 2, and have oil-decomposing activity at a temperature of 20-30°C.
(1) Colony morphology (YM agar medium, 27°C, 1 week culture)
Color tone: White to cream Elevation: Cushion-shaped Margin: Entire to wavy Surface shape: Smooth Gloss and properties: Buttery, wet No formation of sexual reproductive organs after 3 weeks of culture 5. The microorganism according to claim 4, which exhibits the following physiological/biochemical properties in a physiological/biochemical property test.
(1) Sugar fermentability test glucose -
galactose -
Maltose -
(2) Carbon resource utilization test Glucose +
Galactose +
L-sorbose -
D-glucosamine +
D- ribose -
D-xylose +
L-arabinose -
D-Arabinose -
L-rhamnose -
Sucrose +
Maltose +
Trehalose +
α-methyl-D-glucoside +
Cellobiose +
Salicin +
Melibiose +
Lactose -
Raffinose +
Meletitose +
Inulin -
Soluble starch +
Glycerol +
Erythritol -
Ribitol (Adonitol) +
Xylitol D
D-glucitol (sorbitol) +
D-mannitol +
Galactitol (Dulcitol) -
Inositol -
2-keto-D-gluconic acid +
D-gluconic acid +
DL-lactic acid +
Succinate +
Citrate +
Methanol -
Ethanol +
N-acetyl-glucosamine +
Hexadecane +
(3) Nitrogen source utilization Nitrate +
Cadaverine +
(4) Resistance test Growth at 30°C +
Growth at 37°C +
0.01% cycloheximide -
50% (W/V) D-glucose +
10% sodium chloride/5% glucose +
(5) Vitamin auxotrophy test Vitamin-free medium +
(In the above, "+" is positive, "-" is negative, "L" indicates that a positive reaction was rapidly observed after 2 weeks from the start of the test, and "D" indicates that it took more than 1 week after the start of the test. “W” indicates a weak positive reaction, respectively.) 6. The microorganism according to claim 4 or 5, belonging to Candida palmioleophila. Belonging to the genus Candida, the base sequence of 26S rDNA is the base sequence shown in SEQ ID NO: 3, or a base sequence having 90% or more sequence identity with the base sequence shown in SEQ ID NO: 3, and have oil-decomposing activity at a temperature of 20-30°C.
(1) Colony morphology (YM agar medium, 27°C, 1 week culture)
Color tone: White to cream Elevated state: Cushion-shaped Margin: Thread-like Surface shape: Smooth Gloss and properties: Butter-like, moist No formation of sexual reproductive organs at 3 weeks Formation of pseudohyphae or hyphae 8. The microorganism according to claim 7, which exhibits the following physiological/biochemical properties in a physiological/biochemical property test.
(1) Sugar fermentability test Glucose +
Galactose +
Maltose +
(2) Carbon resource utilization test Glucose +
Galactose +
L-sorbose +
D-Glucosamine W
D- ribose -
D-xylose +
L-arabinose -
D-Arabinose -
L-rhamnose -
Sucrose +
Maltose +
Trehalose +
α-methyl-D-glucoside +
Cellobiose +
Salicin +
Melibiose -
Lactose -
Raffinose -
Meletitose +
Inulin -
Soluble starch +
glycerol -
Erythritol -
Ribitol (Adonitol) +
Xylitol +
D-glucitol (sorbitol) +
D-mannitol +
Galactitol (Dulcitol) -
Inositol -
2-keto-D-gluconic acid +
D-gluconic acid +
DL-lactic acid +
Succinate +
Citrate W
Methanol -
Ethanol +
N-acetyl-glucosamine +
Hexadecane +
(3) Nitrogen source utilization Nitrate W
Cadaverine +
(4) Resistance test Growth at 30°C +
Growth at 37°C +
0.01% cycloheximide +
50% (W/V) D-glucose +
10% sodium chloride/5% glucose +
(5) Vitamin auxotrophy test Vitamin-free medium +
(In the above, "+" is positive, "-" is negative, "L" indicates that a positive reaction was rapidly observed after 2 weeks from the start of the test, and "D" indicates that it took more than 1 week after the start of the test. “W” indicates a weak positive reaction, respectively.) 9. The microorganism according to claim 7 or 8, belonging to Candida tropicalis. Belonging to the genus Candida, the nucleotide sequence of 26S rDNA is the nucleotide sequence shown by SEQ ID NO: 4, or a nucleotide sequence having 90% or more sequence identity with the nucleotide sequence shown by SEQ ID NO: 4, and have oil-decomposing activity at a temperature of 20-30°C.
(1) Colony morphology (YM agar medium, 27°C, 1 week culture)
Color tone: White to cream Elevation: Cushion-shaped Periphery: Whole edge Surface shape: Smooth Gloss and properties: Buttery, moist No formation of sexual reproductive organs after 3 weeks of culture Formation of pseudohyphae 11. The microorganism according to claim 10, which exhibits the following physiological/biochemical properties in a physiological/biochemical property test.
(1) Sugar fermentability test Glucose +
Galactose +
Maltose +
(2) Carbon resource utilization test Glucose +
Galactose +
L-sorbose +
D-glucosamine D
D- ribose -
D-xylose +
L-arabinose -
D-Arabinose -
L-rhamnose -
Sucrose +
Maltose +
Trehalose +
α-methyl-D-glucoside +
Cellobiose +
Salicin W
Melibiose -
Lactose -
Raffinose -
Meletitose +
Inulin -
Soluble starch +
glycerol -
Erythritol -
Ribitol (Adonitol) +
xylitol -
D-glucitol (sorbitol) +
D-mannitol +
Galactitol (Dulcitol) -
Inositol -
2-keto-D-gluconic acid +
D-gluconic acid +
DL-lactic acid +
Succinate +
Citrate D
Methanol -
Ethanol +
N-acetyl-glucosamine +
Hexadecane +
(3) Nitrogen source utilization Nitrate W
Cadaverine +
(4) Resistance test Growth at 30°C +
Growth at 37°C +
0.01% cycloheximide +
50% (W/V) D-glucose +
10% sodium chloride/5% glucose +
(5) Vitamin auxotrophy test Vitamin-free medium +
(In the above, "+" is positive, "-" is negative, "L" indicates that a positive reaction was rapidly observed after 2 weeks from the start of the test, and "D" indicates that it took more than 1 week after the start of the test. “W” indicates a weak positive reaction, respectively.) 12. The microorganism according to claim 10 or 11, belonging to Candida tropicalis. Belonging to the genus Candida, the nucleotide sequence of 26S rDNA is the nucleotide sequence shown by SEQ ID NO: 5, or a nucleotide sequence having 90% or more sequence identity with the nucleotide sequence shown by SEQ ID NO: 5, and have oil-decomposing activity at a temperature of 20-30°C.
(1) Colony morphology (YM agar medium, 27°C, 1 week culture)
Color tone: white to cream Elevated state: flat peripheral edge, cushion-shaped central edge Peripheral edge: Entire edge Surface shape: smooth Gloss and properties: bright, wet Shape, ovoid, short-rod Proliferation by budding No formation of sexual reproductive organs after 3 weeks of culture 14. The microorganism according to claim 13, which exhibits the following physiological/biochemical properties in a physiological/biochemical property test.
(1) Sugar fermentability test Glucose +
galactose -
Maltose +
(2) Carbon resource utilization test Glucose +
Galactose +
L-sorbose -
D-glucosamine +
D- ribose -
D-xylose +
L-arabinose -
D-Arabinose -
L-rhamnose -
Sucrose +
Maltose +
Trehalose +
α-methyl-D-glucoside +
Cellobiose +
Salicin +
Melibiose -
Lactose +
Raffinose W
Meletitose +
Inulin -
Soluble starch +
Glycerol +
Erythritol -
Ribitol (Adonitol) +
Xylitol D
D-glucitol (sorbitol) +
D-mannitol +
Galactitol (Dulcitol) -
Inositol -
2-keto-D-gluconic acid +
D-gluconic acid +
DL-lactic acid +
Succinate +
Citrate +
Methanol -
Ethanol +
N-acetyl-glucosamine +
Hexadecane +
(3) Nitrogen source utilization Nitrate W
Cadaverine +
(4) Resistance test Growth at 30°C +
Growth at 37°C +
0.01% cycloheximide +
50% (W/V) D-glucose +
10% sodium chloride/5% glucose +
(5) Vitamin auxotrophy test Vitamin-free medium +
(In the above, "+" is positive, "-" is negative, "L" indicates that a positive reaction was rapidly observed after 2 weeks from the start of the test, and "D" indicates that it took more than 1 week after the start of the test. “W” indicates a weak positive reaction, respectively.) 15. Microorganism according to claim 13 or 14, belonging to Candida fluviatilis. An oil decomposition agent containing the microorganism according to any one of claims 1 to 15. An oil decomposition method comprising contacting the oil decomposition agent according to claim 16 with an oil-containing material to decompose the oil in the oil-containing material. 18. The oil decomposition method according to claim 17, wherein the oil-containing substance is oil-containing wastewater.

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