JP7258658B2 - Microorganism, oil decomposition agent, and oil decomposition method - Google Patents

Description

NPMD NITE P-02894

TECHNICAL FIELD The present invention relates to microorganisms, oil decomposition agents, and oil decomposition methods.

Wastewater discharged from food processing factories and cooking facilities such as kitchens often contains oils and fats. Fats and oils cover the top layer of wastewater and not only give off a foul odor, but also require a large amount of oxygen for their oxidative decomposition. Furthermore, when dissolved, they can be a significant source of pollution as they exhibit extremely high BOD values. These oils and fats in the waste water are required to be properly treated to protect the water environment. For example, for wastewater from factories or business establishments with an average daily discharge volume of 50m ³ or more, uniform wastewater standards have been established that limit the content of n-hexane extractable substances to 30mg/L.

In addition, various mineral oils obtained by separating and refining crude oil are mainly used as fossil fuels as major energy sources in modern society, and in the process, they are often mixed with waste water and pollute it. In factories of oil-related industries such as machining, plating, and steel industries, as well as petroleum refining, petrochemicals, and waste oil reclamation, used lubricating oil, cutting oil, hydraulic oil, and heat transfer oil, as well as various other Mineral oil such as waste oil produced as a by-product in refining processes and chemical reaction processes may be mixed into waste water. Wastewater ^containing mineral oil cannot be discharged into the sewage system as it is for the same reason as oils and fats. A uniform wastewater standard has been established with a limit of 5 mg/L as extractable substance content.

Wastewater containing oils such as fats and mineral oils is discharged in larger amounts when the operations (factories, etc.) that discharge them are in full swing. Therefore, such wastewater must be efficiently and quickly decomposed in a limited space without causing secondary pollution, so that the water quality can pass the wastewater quality standards.
BACKGROUND ART Conventionally, pressurized flotation separation has been generally used for treatment of wastewater containing oils such as fats and mineral oils. In this method, the waste water is put into a pressurizing device, pressurized sufficiently, and then transferred to a normal pressure tank. As a result, oil and water, which originally have different specific gravities, separate into two layers, so the oil is separated and removed. be able to. Operational control of the pressurized flotation method is relatively easy, but it is often difficult to keep the oil content of the treated water at a sufficiently low level. Moreover, its implementation requires relatively large equipment and a large amount of energy to pressurize a large volume of water. Moreover, in this method, it is necessary to dispose of the separated oil as industrial waste.

As another method for treating wastewater containing oils such as fats and mineral oils, an inorganic flocculant containing aluminum sulfate, iron salt, etc. or an organic flocculant containing a crosslinkable polymer is added to the wastewater, and the pH value is adjusted. However, in this case, a large amount of oil-containing sludge is generated as a solid phase, and its dehydration and separation is relatively difficult. Oily sludge must be disposed of as industrial waste.

Depending on the wastewater conditions and treatment conditions, the physicochemical treatment methods described above may not sufficiently remove the oil content and may not reduce the oil content to the standard values specified in the Water Pollution Control Law. In some cases, advanced treatment such as adsorption treatment with an adsorbent such as is required. In addition, the conventional physicochemical treatment method has the drawback that the running costs such as equipment costs, use of flocculants and adsorbents, and sludge disposal costs are enormous.

On the other hand, a method of treating wastewater containing oils such as fats and mineral oils directly with an activated sludge method or a biofilm has also been attempted, but these biological treatment methods lack the ability to decompose oil. In addition, the decomposition rate is extremely low, so that excellent treatment effects cannot be obtained, and there is a problem that the treatment equipment becomes long.

Therefore, as a method for advanced treatment of wastewater containing oil, a biological treatment method using activated sludge adapted to decompose oil has been proposed. For example, Patent Literature 1 discloses a treatment method and apparatus for decomposing oil with activated sludge conditioned to decompose oil. The apparatus disclosed in Patent Document 1 contains an acclimatization tank for acclimatizing activated sludge so as to decompose oil, and the activated sludge and diluted wastewater supplied from the acclimatization tank, and removes the oil contained in the wastewater. It comprises a biological treatment tank for decomposing while aerating with activated sludge, and a sedimentation tank for separating the activated sludge from the treated water transferred from the biological treatment tank.

However, in this treatment method, since the acclimated activated sludge is added as appropriate during the biological treatment, it is necessary to separately perform the acclimated operation of the activated sludge in parallel with the biological treatment operation. There is a problem that the structure of the apparatus is complicated because it requires three types of tanks consisting of a treatment tank, an acclimatization tank and a sedimentation tank. Furthermore, even when such acclimatized activated sludge is used, the oil decomposition capacity and decomposition rate are low, and there is also the problem that it takes a long time to sufficiently reduce the oil content.

On the other hand, there has been proposed a method of decomposing oil using microorganisms obtained by screening using the activity of decomposing oil as an indicator. For example, methods for purifying contaminated soil and contaminated water using microorganisms of the genus Rhodococcus (Patent Documents 2 to 4), contaminated soil, ocean, groundwater, or wastewater using microorganisms of the genus Azoarcus, etc. purification method (Patent Document 5), cyclic hydrocarbon decomposition method using microorganisms belonging to the genus Gordonia (Patent Document 6), oil and fat decomposition method using microorganisms belonging to the genus Acinetobacter (Patent Document 7) However, even with such methods, there is a problem that the decomposition ability and decomposition rate of the oil cannot be sufficiently improved.

JP-A-2000-271589 JP 2007-135425 A Japanese Patent Application Laid-Open No. 2003-102469 Patent No. 4237998 JP 2007-222004 A Japanese Patent No. 4270837 Japanese Patent No. 4172992

Aiming to reduce oil-containing substances such as oil-containing wastewater such as oil and mineral oil to the discharge standard value, it is effective without generating sludge, etc. that needs to be disposed of as industrial waste. There is a demand for an oil decomposing agent and an oil decomposing method that can effectively and economically decompose the oil in an oil-containing substance.

The present invention has been made to solve such problems, and an oil decomposing agent capable of rapidly and sufficiently decomposing the oil contained in the oil-containing substance and capable of decomposing the oil in a short period of time; An object of the present invention is to provide an oil decomposition method.
As a result of extensive studies, the present inventors have reached the conclusion that it is important to use microorganisms with high oil-degrading ability and to adopt an oil-decomposing method suitable for this purpose. In search of microorganisms, various microorganisms in soil and water are collected, and after a long period of search, we obtain microorganisms that can actively assimilate and decompose oil contained in oil-containing substances, and use the obtained microorganisms. As a result, the inventors found that the oil contained in the oil-containing substance can be decomposed, and completed the present invention.

That is, the present invention has the following aspects.
[1] The nucleotide sequence of 26S rDNA belonging to the genus Candida is the nucleotide sequence represented by SEQ ID NO: 1, or a nucleotide sequence having 90% or more homology with the nucleotide sequence represented by SEQ ID NO: 1. A microorganism characterized by exhibiting the following mycological properties and having oil decomposition activity in an environment containing 0 to 5% (W/V) of salt.
(1) Colony morphology (YM agar medium, 27°C, 1 week culture)
Color tone: White to cream Shape: Circular Elevated state: Flat peripheral part, cushion-shaped central part Peripheral edge: Entire edge Surface shape: Smooth Gloss and properties: Dull, wet ℃
(3) Growth salt concentration: 0 to 5% (W/V)
(4) Observation of cell morphology Vegetative cells are elliptical to oval. Proliferation is by budding. No formation of sexual reproductive organs after 4 weeks of culture. The microorganism according to [1], which shows.
(1) Sugar fermentability test Glucose +
galactose -
Maltose -
Sucrose +
Trehalose -
Raffinose +
(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 L
Arbutin D
Melibiose -
Raffinose +
Meletitose +
Inulin +
Soluble starch -
Glycerol +
Erythritol -
Ribitol (Adonitol) -
xylitol -
D-glucitol (sorbitol) L
D-mannitol D
Galactitol (Dulcitol) -
Inositol -
Succinate -
Citrate D
Ethanol +
(3) Nitrogen source utilization Nitrate +
Nitrite +
L-Lysine +
(4) Resistance test Growth at 25°C +
Growth at 35°C +
Growth at 45°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 (latent)" indicates that a positive reaction was rapidly observed after 2 weeks from the start of the test, and "D (delay)" indicates 1 after the start of the test. indicates that a positive reaction was gradually recognized over a period of more than a week)
[3] The microorganism according to [1] or [2], which belongs to Candida mengyuniae.
[4] Candida mengyuniae strain MC7 (NITE P-02894).
[5] An oil decomposing agent containing the microorganism according to any one of [1] to [4].
[6] An oil decomposition method comprising contacting the oil decomposition agent according to [5] with an oil-containing substance to decompose the oil in the oil-containing substance.
[7] The oil decomposition method according to [6], wherein the oil-containing substance is oil-containing wastewater.

According to the present invention, oil-containing substances such as wastewater containing oils such as fats and mineral oils can be treated efficiently at low cost. A microorganism that can be treated, an oil-decomposing agent capable of decomposing oil in oil-containing substances such as oil-contaminated rivers, oceans, groundwater or soil containing the microorganism, and an oil-decomposing method using the oil-decomposing agent. provided.

FIG. 2 is a diagram showing a molecular phylogenetic tree of the MC7 strain analyzed based on the nucleotide sequences obtained by homology search. FIG. 10 is a diagram showing the mineral oil decomposition rate of MC7 strain at pH 7 and temperature 15 to 35°C. FIG. 10 is a diagram showing the mineral oil decomposition rate of MC7 strain at a temperature of 30° C. and a pH of 4 to 8. FIG. FIG. 2 is a diagram showing the fat and oil decomposition rate of MC7 strain at pH 7 and temperature 15 to 35° C. FIG. FIG. 3 is a diagram showing the decomposition rate of oils and fats of MC7 strain at a temperature of 30° C. and a pH of 4 to 8. FIG. FIG. 2 is a diagram showing the oil and fat decomposition rate of Candida albicans ATCC90029 strain at pH 7 and temperature 15 to 35°C. FIG. 3 is a diagram showing the decomposition rate of mineral oil of Candida albicans ATCC90029 strain at a temperature of 30° C. and pH of 4-8.

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 oils and mineral oils.
The term “fat and oil” refers to a substance containing a glyceride in which glycerin and 1 to 3 fatty acids are ester-bonded. monoacylglycerol). 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.

In particular, the microorganism of the present invention can efficiently decompose 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 microorganism of the present invention can efficiently decompose mineral oil containing a large amount of aliphatic hydrocarbons having 22 to 36 carbon atoms.

(Microorganism)
The microorganism of the present invention belongs to the genus Candida, and the base sequence of 26S rDNA is the base sequence shown by SEQ ID NO: 1, or 90% or more, preferably 95% or more, of the base sequence shown by SEQ ID NO: 1. , more preferably 98% or more, more preferably 99% or more homology, exhibits the following mycological properties, and decomposes oil in an environment containing 0 to 5% salt (W / V) It is characterized by having activity.
(1) Colony morphology (YM agar medium, 27°C, 1 week culture)
Color tone: White to cream Shape: Circular Elevated state: Flat peripheral part, cushion-shaped central part Peripheral edge: Entire edge Surface shape: Smooth Gloss and properties: Dull, moist (2) Growth temperature: 12-37 ℃
(3) Growth pH: 4.0-9.0
(4) Growth salt concentration: 0 to 5% (W/V)
(5) Observation of cell morphology Vegetative cells are oval to oval. Growth is by budding. No formation of sexual reproductive organs after 4 weeks of culture.

The nucleotide sequence homology can be determined by BLAST search using the GenBank/EMBL/DDBJ database.

The microorganism of the present invention exhibits growth ability even in a wide pH range of pH 4.0 to 9.0, a wide temperature range of 12 ° C. to 37 ° C., a salt concentration of 0 to 5% (W / V), and a salinity of 0 to 5. % (W/V), it has excellent oil decomposition activity.

Microorganisms of the present invention include, for example, microorganisms belonging to the genus Candida mengyuniae.

Examples of the microorganisms of the present invention include microorganisms that exhibit the following physiological/biochemical properties in physiological/biochemical property tests.
In addition, in the present invention, the physiological / biochemical property test, Kurtzman CP, Fell JW, Boekhout T. & Robert V. (2011). Methods for isolation, phenotypic characterization and maintenance of yeasts. In: Kurtzman CP, Fell JW & Boekhout T. (eds.) The Yeasts, a taxonomic study, 5th edition. pp. 87-110, Elsevier, Amsterdam.
<Physiological and biochemical property test results>
(1) Sugar fermentability test Glucose +
galactose -
Maltose -
Sucrose +
Trehalose -
Raffinose +
(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 L
Arbutin D
Melibiose -
Raffinose +
Meletitose +
Inulin +
Soluble starch -
Glycerol +
Erythritol -
Ribitol (Adonitol) -
xylitol -
D-glucitol (sorbitol) L
D-mannitol D
Galactitol (Dulcitol) -
Inositol -
Succinate -
Citrate D
Ethanol +
(3) Nitrogen source utilization Nitrate +
Nitrite +
L-Lysine +
(4) Resistance test Growth at 25°C +
Growth at 35°C +
Growth at 45°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 (latent)" indicates that a positive reaction was rapidly observed after 2 weeks from the start of the test, and "D (delay)" indicates 1 after the start of the test. indicates that a positive reaction was gradually recognized over a period of more than a week)

Microorganisms belonging to the genus Candida mengyuniae of the present invention include, for example, Candida mengyuniae MC7 strain. Candida mengyuniae MC7 strain was deposited on February 26, 2019 at the National Institute of Technology and Evaluation Patent Microorganism Depositary Center (NPMD) (2-5-8 Kazusa Kamatari, Kisarazu City, Chiba Prefecture). It has been deposited in Japan under accession number NITE P-02894.

The microorganism of the present invention has oil decomposition activity even in an environment containing salt, for example, in an environment with a salt concentration of 0 to 5% (W/V).
Here, "having oil decomposition activity" means that the oil decomposition rate measured by the following procedure is 5% or more.
Procedure 1: To 50 mL of SCD medium (15 g/L casein peptone, 5 g/L soybean peptone, 5 g/L sodium chloride), add 1 platinum loop of the strain on the agar plate or 100 μL of the glycerol stock strain, overnight at 30°C, Shake and pre-incubate.
Procedure 2: Add 100 μL of pre-culture solution to 150 mL of decomposition test medium (10 g/L mineral oil or oil, 1 g/L corn steep liquor, 0.5 g/L urea) with 0% (W/V) salt content, pH 7 , and shake at 30°C for 24 hours to perform an oil decomposition reaction.
Procedure 3: Normal-hexane extractable substance - by weight method (JIS K102), measure the normal-hexane extractive substance concentration before and after the reaction, subtract the normal-hexane extractable substance concentration after the reaction from the normal-hexane extractable substance concentration before the reaction, The oil decomposition rate is obtained by dividing by the previous n-hexane extract concentration.

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

The oil decomposition rate of the microorganisms of the present invention is preferably 10% or more, more preferably 15% or more, and even more preferably 20% or more as a decomposition rate of mineral oil under temperature conditions showing the maximum oil decomposition rate.
In addition, the oil decomposition rate of the microorganism of the present invention 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.

(Oil decomposition agent)
The oil decomposing agent of the present invention contains the microorganism of the present invention or a processed product of the microorganism of the present invention. Examples of the treated microorganisms include those obtained by suspending the culture solution of the microorganism of the present invention 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 present invention may be in liquid or solid form. 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. Solid forms are exemplified by, for example, freeze-dried cultured cells of microorganisms. The solid form may be, for example, granulated microorganisms by the method described in "Industrial Enzyme, Takayuki Ueshima, Maruzen, 1995". Also, microorganisms immobilized on various carriers may be used.

The oil-decomposing agent of the present invention 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 present invention. Examples include soil and oil-containing industrial waste, and oil-containing wastewater is preferred.

(Oil decomposition method)
The oil decomposition method of the present invention is a method of contacting the oil decomposition agent of the present invention 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 present invention, there is a method of bringing the oil-decomposing agent of the present invention into direct contact with the oil-containing substance. A method of preculturing microorganisms in the oil decomposing agent of the present invention and adding the obtained culture solution to the oil-containing substance, the oil of the present invention Examples include a method of preculturing microorganisms in a decomposing agent, adding the obtained culture solution to the oil-containing substance, and culturing the microorganisms.
Contact between the oil-decomposing agent of the present invention and the oil-containing substance is preferably carried out under aerobic conditions.

The pH at which the oil-decomposing agent of the present invention is brought into contact with the oil-containing substance 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 4.0. 6.0 is more preferred, and pH 5.0 is particularly preferred.
The temperature at which the oil decomposing agent of the present invention and the oil decomposing substance are brought into contact is not particularly limited, but is preferably 12 to 37°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 present invention is brought into contact with the oil-containing substance 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 present invention and the oil-containing substance 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.

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

(Example 1)
<Isolation of MC7 strain>
In an L-shaped test tube, 10 mL of an inorganic medium [(NH ₄ ) ₂ SO ₄ , NaNO ₃ and K ₂ HPO ₄ were mixed in an arbitrary ratio, and the precipitate was filtered through a 10 μm filter], and 10 μL of rapeseed oil as fats and oils. , and 1 g of sludge were added and cultured with oblique shaking at 30° C. until suspended. 100 μL of the resulting suspension was added to an L-shaped test tube to which 10 mL of an inorganic medium and 10 μL of rapeseed oil were similarly added, and cultured with shaking until the inorganic medium was suspended. After repeating this procedure six times, 100 μL of the stepwise diluted suspension was dropped onto an agar plate medium, smeared with a Conlarge stick, and cultured at 30° C. for 24 hours. An inorganic agar medium coated with rapeseed oil is used as the agar medium, and a culture petri dish on which colonies are appropriately formed is selected. bottom. Using such a method, 50 strains having lipolytic activity were obtained.

Subsequently, 10 mL of an inorganic medium, 10 μL of Shell Tellus Oil C (manufactured by Showa Shell Sekiyu K.K.) as a mineral oil, and colonies of the above 50 strains were added to an L-shaped test tube and cultured with oblique shaking at 30°C. Shell Tellus Oil C is an industrial lubricating oil composed of 97% by mass or more of base oil and 3% by mass or less of additives. Of the 50 strains, for 1 strain in which suspension was observed, 100 μL of the suspension was taken, and similarly added to an L-shaped test tube containing 10 mL of inorganic medium and 10 μL of shell terrace oil C, and the inorganic medium was added. It was cultured with shaking until it was suspended. After repeating this procedure six times, 100 μL of the stepwise diluted suspension was dropped onto an agar plate medium, smeared with a Conlarge stick, and cultured at 30° C. for 24 hours. An inorganic agar medium coated with Shell Terrace Oil C is used as the agar medium, and a culture petri dish on which colonies are appropriately formed is selected. separated the body. The strain isolated in this manner was named MC7 strain.

<Identification of MC7 strain>
Base sequence analysis of 26S rDNA was performed on the MC7 strain obtained above. For PCR amplification, ITS5 and NL4 primers were used to amplify the DNA of the target region, and then NL1 and NL4 primers were used for sequence amplification to determine the base sequence. It was confirmed that the determined 26S rDNA of the MC7 strain consisted of the base sequence shown in SEQ ID NO:1.
As a result of BLAST homology search of the obtained nucleotide sequence against the international nucleotide sequence database (DDBJ/EMBL/GenBank), the nucleotide sequence of 26S rDNA of the MC7 strain was found to be Candida mengyuniae CBS 10845T, which is a kind of ascomycete yeast. On the other hand, it showed a homology with a homology rate of 100%. FIG. 1 shows a molecular phylogenetic tree analyzed based on the nucleotide sequences obtained by homology search. In this molecular phylogenetic tree, the MC7 strain is included in the phylogenetic group composed of the genera Candida and Cyberlndnera, and has the same molecular phylogenetic position as Candida mengyuniae CBS 10845T (accession number EU043158). Indicated. From the above, the MC7 strain was presumed to belong to Candida mengyuniae.

The mycological properties of the MC7 strain are shown below.
(1) Colony morphology (YM agar medium, 27°C, 1 week culture)
Color tone: White to cream Shape: Circular Elevated state: Flat peripheral part, cushion-shaped central part Peripheral edge: Entire edge Surface shape: Smooth Gloss and properties: Dull, moist (2) Growth temperature: 12-37 °C (optimal temperature: 30 °C)
(3) Growth pH: 4.0 to 9.0 (optimal pH: 5.0)
(4) Growth salt concentration: 0 to 5% (W/V)
(5) Observation of cell morphology Vegetative cells are oval to oval. Growth is by budding. No formation of sexual reproductive organs after 4 weeks of culture.

The MC7 strain exhibits the following physiological and biochemical properties in the physiological and biochemical property tests.
<Physiological and biochemical property test results>
(1) Sugar fermentability test Glucose +
galactose -
Maltose -
Sucrose +
Trehalose -
Raffinose +
(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 L
Arbutin D
Melibiose -
Raffinose +
Meletitose +
Inulin +
Soluble starch -
Glycerol +
Erythritol -
Ribitol (Adonitol) -
xylitol -
D-glucitol (sorbitol) L
D-mannitol D
Galactitol (Dulcitol) -
Inositol -
Succinate -
Citrate D
Ethanol +
(3) Nitrogen source utilization Nitrate +
Nitrite +
L-Lysine +
(4) Resistance test Growth at 25°C +
Growth at 35°C +
Growth at 45°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 (latent)" indicates that a positive reaction was rapidly observed after 2 weeks from the start of the test, and "D (delay)" indicates 1 after the start of the test. indicates that a positive reaction was gradually recognized over a period of more than a week)

The mycological properties of strain MC7 were largely consistent with the characteristics of Candida mengyuniae. The physiological and biochemical properties of the MC7 strain and the physiological and biochemical properties of the known Candida mengyuniae (Chen B., HuangX., Zheng J.-W., Li S.-P. & He J. (2009). Candida mengyuniae sp. nov., a metsulfuronmethyl-resistant yeast. Int J Syst Envol Microbiol 59, 1237-1241.). and melezitose assimilation, resistance test at 45°C, and growth at 10% sodium chloride/5% glucose. ) showed similar physiological and biochemical properties.

Based on the base sequence analysis, mycological properties, and physiological/biochemical properties of the 26S rDNA described above, the MC7 strain was identified as Candida mengyuniae. And as shown in Comparative Example 1, compared with conventional microorganisms belonging to the genus Candida, it was judged to be a novel microorganism because it had superior oil-decomposing activity.

(Example 2)
<Degradation of mineral oil using MC7 strain>
Add 50 mL of SCD medium (15 g/L casein peptone, 5 g/L soybean peptone, 5 g/L sodium chloride) and 1 platinum loop of MC7 strain on an agar plate to a 300 mL Erlenmeyer flask, shake overnight at 30 ° C. cultured. In the oil decomposition test, 150 mL of the decomposition test medium (10 g / L shell terrace oil C, 1 g / L corn steep liquor, 0.5 g / L urea) and 100 μL of the pre-culture solution are added to a 500 mL Erlenmeyer flask, and the predetermined pH, temperature shaken with FIG. 2 shows the results of decomposition at pH 7 at 15° C. to 35° C. for 24 hours. The MC7 strain showed a maximum degradation rate of 23% at 30°C. FIG. 3 shows the results of decomposition at pH 4 to 8 at 30° C. for 24 hours. Strain MC7 showed a maximum degradation rate of 29% at pH5.

(Example 3)
<Decomposition of oils and fats using MC7 strain>
Add 50 mL of SCD medium (15 g/L casein peptone, 5 g/L soybean peptone, 5 g/L sodium chloride) and 1 platinum loop of MC7 strain on an agar plate to a 300 mL Erlenmeyer flask, shake overnight at 30 ° C. cultured. In the oil decomposition test, 150 mL of the decomposition test medium (10 g / L rapeseed oil, 1 g / L corn steep liquor, 0.5 g / L urea) and 100 μL of the preculture solution are added to a 500 mL Erlenmeyer flask, and shaken at a predetermined pH and temperature. bottom. FIG. 4 shows the results of decomposition at pH 7 at 15° C. to 35° C. for 24 hours. The MC7 strain showed a maximum degradation rate of 54% at 30°C. 5 shows the results of decomposition at pH 4-8 at 30° C. for 24 hours. The MC7 strain showed the highest degradation rate of 67% at pH5.

(Comparative example 1)
50 mL of SCD medium (15 g/L casein peptone, 5 g/L soybean peptone, 5 g/L sodium chloride) and 1 platinum loop of Candida albicans ATCC90029 strain on an agar plate were added to a 300 mL Erlenmeyer flask and incubated at 30°C. Shake and pre-incubate overnight. The mineral oil decomposition test was performed by the same method as in Example 2, and the fat decomposition test was performed by the same method as in Example 3. FIG. 6 shows the results of decomposing oils and fats at pH 7 at 15° C. to 35° C. for 24 hours. Fig. 7 shows the results of decomposition of mineral oil at pH 4 to 8 at 30°C for 24 hours. The decomposition rate of Candida albicans ATCC90029 strain was inferior to that of MC7 strain for both oil components.

The microorganism of the present invention has a high ability to decompose oil. Moreover, the microorganism of the present invention can grow in a wide temperature range, pH range, and high salt concentration. Therefore, the microorganism of the present invention and the oil-decomposing agent containing the microorganism of the present invention can efficiently decompose oil in oil-containing substances such as oil-containing wastewater.

Claims (4)

Candida mengyuniae strain MC7 deposited under accession number NITE P-02894 . An oil degrading agent containing the Candida mengyuniae MC7 strain according to claim 1 . A method for decomposing oil, comprising contacting the oil decomposing agent according to claim 2 with an oil-containing substance to decompose the oil in the oil-containing substance. 4. The oil decomposition method according to claim 3 , wherein the oil-containing substance is oil-containing wastewater.

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