JP2020191802A - Microorganisms, agents and methods for decomposing oil components - Google Patents

Abstract

To provide microorganisms, agents and methods for effectively and economically decomposing oil components without sludge generation which requires industrial waste treatment.SOLUTION: The invention provides a microorganism of the genus Candida characterized in that the microorganism comprises a 26S rDNA having a base sequence of SEQ ID NO:1 or a base sequence having 90% or more homology thereto, and the microorganism exhibits an oil decomposing activity in an environment containing 0-5% (w/v) salt. The invention also provides an oil decomposing agent containing the microorganism and a method for decomposing oil using the oil decomposing agent.SELECTED DRAWING: None

Description

The present invention relates to microorganisms, oil decomposing agents, and oil decomposing methods.

Wastewater discharged from cooking facilities such as food processing factories and kitchens often contains oils and fats. Fats and oils not only cover the upper layers of wastewater and give off a foul odor, but also require a large amount of oxygen for their oxidative decomposition. Moreover, when they dissolve, they exhibit extremely high BOD values and can be a significant source of pollution. Appropriate treatment of these oils and fats in wastewater is required to protect the water environment. For example, for wastewater from factories or business establishments with an average daily discharge of 50 m ³ or more, a uniform wastewater standard has been established with a normal hexane extract substance content of up to 30 mg / L.

In addition, various mineral oils obtained by separating and refining from crude oil are mainly used as fossil fuels as a main energy source in modern society, and in the process, they are often mixed with wastewater and pollute it. In addition, in the machining industry, plating industry and steel industry, and in the factories of petroleum-related industries such as petroleum refining, petrochemical and waste oil recycling, used lubricating oil, cutting oil, hydraulic hydraulic oil and heat transfer oil, as well as various types Mineral oil such as waste oil produced as a by-product in the refining process and the chemical reaction process may be mixed in the waste water. Wastewater containing mineral oil cannot be discharged from the sewer as it is for the same reason as in the case of fats and oils. For example, for wastewater from factories or business establishments with an average daily discharge of 50 m ³ or more, normal hexane A uniform wastewater standard has been established with a limit of 5 mg / L as the content of the extracted substance.

Wastewater containing oils such as oils and fats and mineral oils will be discharged in a larger amount if the business (factories, etc.) for discharging them is active. Therefore, these wastewaters must be decomposed efficiently and promptly in a limited space without causing secondary pollution to obtain a water quality that can pass the wastewater quality standards.
For the treatment of wastewater containing oils such as fats and oils and mineral oils, the pressurized flotation separation method has been generally used. In this method, wastewater is put into a pressurizing device to sufficiently pressurize it, and then transferred to a tank at normal pressure to separate two layers of oil and water having different specific gravities. Therefore, the oil is separated and removed. be able to. Although the operation management of the pressurized flotation separation method is relatively easy, it is often difficult to keep the oil content of the treated water at a sufficiently low level. Moreover, in order to do so, a relatively large device and a large amount of energy for pressurizing a large amount of water are required. In addition, in this method, it is necessary to dispose of the separated oil as industrial waste.

As another treatment method for wastewater containing oils such as fats and oils 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. A method of solid-liquid separation of oil may be adopted by adjusting, but in this case, a large amount of oil-containing sludge is generated as a solid phase, and its dehydration separation is relatively difficult, and even if it can be separated, it is still the same. Oil-impregnated sludge must be disposed of as industrial waste.

The above physicochemical treatment method does not sufficiently remove oil depending on the settings of wastewater conditions and treatment conditions, is not reduced to the standard value in the Water Pollution Control Law, further filters the liquid phase, and activates carbon. In some cases, advanced treatment such as adsorption treatment with an adsorbent such as is required. In addition, the conventional physicochemical treatment method has a drawback that the running cost such as equipment cost, use of coagulant and adsorbent, and sludge disposal cost is large.

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

Therefore, as a method for highly treating wastewater containing oil, a biological treatment method using activated sludge accustomed to decompose oil has been proposed. For example, Patent Document 1 discloses a treatment method and an apparatus for decomposing oil with activated sludge accustomed to decomposing oil. The apparatus disclosed in Patent Document 1 accommodates a acclimation tank for acclimatizing activated sludge so as to decompose oil, activated sludge supplied from the acclimatization tank and diluted wastewater, and collects oil contained in the wastewater. It is provided with a biological treatment tank that decomposes while being aerated by activated sludge, and a settling tank for separating activated sludge from the treated water transferred from the biological treatment tank.

However, in this treatment method, since the acclimatized activated sludge is appropriately added during the biological treatment, it is necessary to separately perform the acclimatization operation of the activated sludge in parallel with the biological treatment operation, which is troublesome to manage and the organism. There is a problem that the apparatus configuration becomes complicated because three types of tanks including a treatment tank, a acclimatization tank and a settling tank are required. Further, even when the activated sludge acclimatized in this way is used, there is a problem that the decomposition ability and the decomposition rate of the oil content are small, and it takes a long time to sufficiently reduce the oil content.

On the other hand, a method of decomposing oil is proposed by using microorganisms obtained by screening using the activity of decomposing oil as an index. For example, a method for purifying contaminated soil and contaminated water using microorganisms of the genus Rhodococcus (Patent Documents 2 to 4), contaminated soil using microorganisms of the genus Azoarcus, ocean, groundwater, wastewater, etc. (Patent Document 5), Cyclic hydrocarbon decomposition method using microorganisms belonging to the genus Gordonia (Patent Document 6), Oil and fat decomposition treatment method using microorganisms belonging to the genus Acinetobacter (Patent Document 7). There are known methods for treating oils using various microorganisms such as Rhodococcus, but even with such methods, there is a problem that the decomposition ability and decomposition rate of oils cannot be sufficiently improved.

Japanese Unexamined Patent Publication No. 2000-271589 JP-A-2007-135425 Japanese Unexamined Patent Publication No. 2003-102469 Patent No. 4237998 Japanese Unexamined Patent Publication No. 2007-22204 Japanese Patent No. 4270837 Japanese Patent No. 4172992

For the purpose of reducing oil-containing substances such as wastewater containing oils such as oils and mineral oils to their emission standard values, it is effective without generating sludge that needs to be disposed of as industrial waste. There is a demand for an oil-decomposing agent capable of decomposing oil in an oil-containing substance and an oil-decomposing method in a targeted and economical manner.

The present invention has been made to solve such a problem, and is an oil decomposing agent capable of rapidly and sufficiently decomposing oil contained in an oil-containing substance and decomposing oil in a short period of time. It is an object of the present invention to provide an oil decomposition method.
As a result of diligent studies, the present inventors have come to the conclusion that it is important to use a microorganism having a high oil decomposition ability and to further adopt an oil decomposition method suitable for this purpose, which meets the above object. In search of microorganisms, we collected microorganisms from various soils and water areas, and after a long-term search, actively assimilated the oil contained in the oil-containing substances to obtain microorganisms that could decompose, and used the obtained microorganisms. Therefore, they have 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 base sequence of 26S rDNA belonging to the genus Candida is the base sequence shown by SEQ ID NO: 1 or the base sequence having 90% or more homology with the base sequence shown by SEQ ID NO: 1. , A microorganism having 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: White to cream Shape: Circular raised state: Flat at the periphery, cushion at the center Periphery: All edges Surface shape: Smooth Gloss and properties: Dull, damp, (2) Growth temperature: 12-37 ℃
(3) Growth salt concentration: 0 to 5% (W / V)
(4) Observation of cell morphology The vegetative cells are oval to oval. Proliferation is by budding. No sexual reproduction organs are formed in 4 weeks of culture. [2] The microorganism according to [1] shown.
(1) Sugar fermentability test Glucose +
Galactose-
Maltose −
Sucrose +
Trehalose-
Raffinose +
(2) Carbon source 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 +
Melechtose +
Inulin +
Soluble starch-
Glycerol +
Erythritol-
Ribitol (Adonitol)-
Xylitol-
D-Glucitol (sorbitol) L
D-mannitol D
Galactitol (Darcitol)-
Inositol-
Sukusinate-
Citrate D
Ethanol +
(3) Nitrogen source assimilation nitrate +
Nitrite +
L-lysine +
(4) Tolerance 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 requirement test Vitamin-free medium +
(In the above, "+" is positive, "-" is negative, "L (latent)" is a rapid positive reaction 2 weeks after the start of the test, and "D (delay)" is 1 after the start of the test. It shows that a positive reaction was gradually observed over a period of more than a week).
[3] The microorganism according to [1] or [2], which belongs to Candida mengniae.
[4] Candida mengyuniae MC7 strain (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 oils or mineral oil can be treated efficiently at low cost, and in particular, oils in salt-containing wastewater can be treated efficiently at low cost. Microorganisms that can be treated, oil decomposition agents that can decompose oil in oil-containing substances such as rivers, oceans, groundwater, and soil that are contaminated with oil, including the microorganisms, and oil decomposition methods that use the oil decomposition agents Provided.

It is a figure which showed the molecular phylogenetic tree of MC7 strain analyzed based on the base sequence obtained by the homology search. It is a figure which showed the decomposition rate of the mineral oil at pH 7 and a temperature of 15-35 degreeC of MC7 strain. It is a figure which showed the decomposition rate of the mineral oil at the temperature of 30 degreeC, pH 4-8 of MC7 strain. It is a figure which showed the decomposition rate of fats and oils at pH 7 and a temperature of 15-35 degreeC of MC7 strain. It is a figure which showed the decomposition rate of fats and oils at a temperature of 30 degreeC, pH 4-8 of MC7 strain. It is a figure which showed the decomposition rate of fats and oils at pH 7, temperature 15-35 ° C. of Candida albicans ATCC90029 strain. It is a figure which showed the decomposition rate of the mineral oil at the temperature of 30 degreeC, and the pH 4-8 of the Candida albicans ATCC90029 strain.

Hereinafter, embodiments of the present invention will be described. The following embodiments are merely exemplary to illustrate the invention and are not intended to limit the invention to this embodiment alone. The present invention can be implemented in various aspects as long as it does not deviate from the gist thereof.

As used herein, the term "oil" means an organic oily substance, and includes fats and oils and mineral oils.
The fat and oil means a substance containing a glyceride in which glycerin and 1 to 3 fatty acids are ester-bonded, and in addition to triglyceride (triacylglycerol) which is a main component of fat and oil, diglyceride (diacylglycerol) and monoglyceride ( It may contain monoacylglycerol). Further, the fats and oils may contain components other than animal and vegetable oil-derived glycerides (for example, plant sterols, lecithin, antioxidant components, pigment components, etc.) in addition to animal and vegetable oils and fats (animal oil, vegetable oil, fish oil). 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.

The mineral oil refers to hydrocarbons obtained by refining crude oil, for example, hydrocarbons having 4 to 10 carbon atoms (for example, gasoline) and hydrocarbons having 9 to 15 carbon atoms (for example, gasoline). For example, 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 oil). (For example, hydrocarbon oil, engine oil)) and the like.
Gasoline contains a large amount of aromatic hydrocarbons, and kerosene, light oil and heavy oil contain a large amount 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.

(Microorganisms)
The microorganism of the present invention belongs to the genus Candida, and the base sequence of 26S rDNA is 90% or more, preferably 95% or more of the base sequence shown by SEQ ID NO: 1 or the base sequence shown by SEQ ID NO: 1. , More preferably 98% or more, still more preferably 99% or more homology, exhibiting the following mycological properties, and decomposing oil in an environment containing 0 to 5% (W / V) of salt. It is characterized by having activity.
(1) Colony morphology (YM agar medium, 27 ° C., 1 week culture)
Color: White to cream Shape: Circular raised state: Flat at the periphery, cushion at the center Periphery: All edges Surface shape: Smooth Gloss and properties: Dull, damp, (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 Nutrient cells are oval to oval. Proliferation is by budding. No sexual reproductive organs are formed in 4 weeks of culture.

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

The microorganism of the present invention exhibits viability even in a wide pH range of pH 4.0 to 9.0, a wide temperature range of 12 ° C. to 37 ° C., and a salt concentration of 0 to 5% (W / V), and has a salt content of 0 to 5. Has excellent oil decomposition activity in an environment containing% (W / V).

Examples of the microorganism of the present invention include microorganisms belonging to the genus Candida mengyuniae.

Examples of the microorganism of the present invention include microorganisms exhibiting the following physiological / biochemical properties in a physiological / biochemical property test.
In the present invention, the physiological and biochemical property tests are performed by 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 / biochemical property test results>
(1) Sugar fermentability test Glucose +
Galactose-
Maltose −
Sucrose +
Trehalose-
Raffinose +
(2) Carbon source 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 +
Melechtose +
Inulin +
Soluble starch-
Glycerol +
Erythritol-
Ribitol (Adonitol)-
Xylitol-
D-Glucitol (sorbitol) L
D-mannitol D
Galactitol (Darcitol)-
Inositol-
Sukusinate-
Citrate D
Ethanol +
(3) Nitrogen source assimilation nitrate +
Nitrite +
L-lysine +
(4) Tolerance 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 requirement test Vitamin-free medium +
(In the above, "+" is positive, "-" is negative, "L (latent)" is a rapid positive reaction 2 weeks after the start of the test, and "D (delay)" is 1 after the start of the test. It shows that a positive reaction was gradually observed over a period of more than a week).

Examples of the microorganism belonging to the genus Candida mengniae of the present invention include the Candida menguniae MC7 strain. Candida mengyuniae MC7 strain was transferred to the National Institute of Technology and Evaluation Patent Microorganisms Depositary Center (NPMD) (2-5-8 Kazusakamatari, Kisarazu City, Chiba Prefecture) on February 26, 2019. It has been deposited domestically under the accession number NITE P-02894.

The microorganism of the present invention has oil decomposition activity even in an environment containing salt, for example, has oil decomposition activity in an environment having 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.
Step 1: Add 100 μL of 1 loopful or glycerol stock strain to 50 mL of SCD medium (15 g / L casein peptone, 5 g / L soybean peptone, 5 g / L sodium chloride) and overnight at 30 ° C. Shake and pre-culture.
Step 2: Add 100 μL of the preculture solution to 150 mL of 0% salt (W / V) decomposition test medium (10 g / L mineral oil or fat, 1 g / L corn steep liquor, 0.5 g / L urea), and pH 7 , Shake at 30 ° C. for 24 hours to carry out an oil decomposition reaction.
Step 3: The concentration of the normal hexane extract before and after the reaction is measured by the normal hexane extract-weight method (JIS K102), and the concentration of the normal hexane extract after the reaction is subtracted from the concentration of the normal hexane extract before the reaction. The oil decomposition rate is determined by dividing by the previous concentration of the normal hexane extractant.

In the measurement of the concentration of the normal hexane extract by the normal hexane extract-weight method, specifically, the sample is made slightly acidic, extracted with normal hexane, and the normal hexane is volatilized at 80 ° C. to leave the normal hexane extract. It can be done by determining the weight of.

The oil decomposition rate of the microorganism of the present invention is preferably 10% or more, more preferably 15% or more, still more preferably 20% or more as the decomposition rate with respect to mineral oil under the temperature condition showing the maximum oil decomposition rate.
The oil decomposition rate of the microorganism of the present invention is preferably 20% or more, more preferably 30% or more, still more preferably 40% or more, as the decomposition rate for fats and oils under the temperature condition showing the maximum oil decomposition rate.

(Oil decomposition agent)
The oil decomposing agent of the present invention includes the microorganism of the present invention or a processed product of the microorganism of the present invention. Examples of the processed product of the microorganism include a product obtained by collecting 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 medium, and freeze-drying it.
The oil decomposing agent of the present invention may be in the form of a liquid or a solid. Examples of the liquid form include a culture solution of microorganisms itself, a substance obtained by collecting microorganisms by centrifugation or the like, and redispersing the cells in a medium such as physiological saline or a medium. Examples of the solid form include freeze-dried cultured cells of microorganisms. The solid form may be obtained by granulating the microorganism by the method described in, for example, "Industrial Enzyme, by Takayuki Ueshima, Maruzen, 1995". Further, the microorganism may be immobilized on various carriers.

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 microorganism of the present invention. For example, oil-containing water (wastewater, river water, groundwater, seawater, etc.) and oil-containing substance are contained. Examples thereof include soil and industrial waste containing oil, but oil-containing wastewater is preferable.

(Oil decomposition method)
The oil decomposition method of the present invention is a method of bringing the oil decomposition agent of the present invention into contact with the oil-containing substance to decompose the oil in the oil-containing substance. As a method of bringing the oil-decomposing agent of the present invention into contact with the oil-containing substance, a method of directly contacting the oil-decomposing agent of the present invention with the oil-containing substance, and using the oil-decomposing agent of the present invention as the oil-containing substance. A method of adding and culturing the microorganisms in the oil-decomposing agent, a method of pre-culturing the microorganisms in the oil-decomposing agent of the present invention, and adding the obtained culture solution to the oil-containing substance, the oil content of the present invention. Examples thereof include a method of pre-culturing the microorganism in the decomposing agent and adding the obtained culture solution to the oil-containing substance to cultivate the microorganism.
The 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 the time of contacting the oil decomposing agent of the present invention 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 with each other 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 preferable.
The amount oil decomposition agent upon contacting the oil-containing material, oil degradation agent of the present invention is not particularly limited, for example, as a viable cell count of a microorganism, the 10 ⁷ to 10 about ⁹ cells / mL The amount.
The time for contacting the oil-decomposing agent of the present invention with 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.

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)
<Separation of MC7 strains>
Inorganic medium [(NH ₄ ) ₂ SO ₄ , NaNO ₃ , and K ₂ HPO ₄ are mixed in an L-shaped test tube at an arbitrary ratio, and the precipitate is filtered through a 10 μm filter] 10 mL, rapeseed oil 10 μL as fat and oil. , And 1 g of sludge were added, and the mixture was cultured with oblique shaking at 30 ° C. until suspended. 100 μL of the obtained suspension was taken, 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 operation 6 times, 100 μL of the gradually diluted suspension was added dropwise to the agar plate medium, smeared with a spreader, and cultured at 30 ° C. for 24 hours. As the agar medium, use an inorganic agar medium coated with rapeseed oil, select a culture dish in which colonies are appropriately formed on these media, and separate the well-shaped colonies into a new LB agar medium to separate the cells. did. Using such a method, 50 strains having fat-decomposing activity were obtained.

Subsequently, 10 mL of an inorganic medium, 10 μL of Shell Terrace Oil C (manufactured by Showa Shell Sekiyu Co., Ltd.) as a mineral oil, and colonies of the 50 strains were added to an L-shaped test tube, and the cells were cultured with oblique shaking at 30 ° C. Shell terrace oil C is an industrial lubricating oil composed of 97% by mass or more of a base oil and 3% by mass or less of an additive. Of the 50 strains, 100 μL of the suspension was dispensed from one strain in which suspension was observed, and 10 mL of the inorganic medium and 10 μL of shell terrace oil C were similarly added to an L-shaped test tube to prepare the inorganic medium. The cells were shake-cultured until suspended. After repeating this operation 6 times, 100 μL of the gradually diluted suspension was added dropwise to the agar plate medium, smeared with a spreader, and cultured at 30 ° C. for 24 hours. As the agar medium, use an inorganic agar medium coated with Shell Terrace Oil C, select a culture dish in which colonies are appropriately formed on these media, and divide the well-shaped colonies into a new LB agar medium for bacteria. The body was separated. The strain isolated in this way was named MC7 strain.

<Identification of MC7 strain>
Nucleotide sequence analysis of 26S rDNA was performed on the MC7 strain obtained above. In PCR amplification, DNA in the target region was amplified using ITS5 and NL4 primers, and then the sequence was amplified using NL1 and NL4 primers to determine the base sequence. It was confirmed that the 26S rDNA of the determined MC7 strain consisted of the nucleotide sequence shown in SEQ ID NO: 1.
As a result of performing a BLAST homology search on the obtained nucleotide sequence in the international nucleotide sequence database (DDBJ / EMBL / GenBank), the nucleotide sequence of 26S rDNA of the MC7 strain was found in Candida mengiuniae CBS 10845T, which is a kind of ascospore yeast. On the other hand, the homology was 100%. FIG. 1 shows a molecular phylogenetic tree analyzed based on the nucleotide sequence obtained by the homology search. In this molecular phylogenetic tree, the MC7 strain is included in the phylogenetic group composed of the genera Candida and Cyberndnera, and has the same molecular phylogenetic position as Candida albicans CBS 10845T (Axion No. EU043158). Indicated. From the above, it was presumed that the MC7 strain belongs 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: White to cream Shape: Circular raised state: Flat at the periphery, cushion at the center Periphery: All edges Surface shape: Smooth Gloss and properties: Dull light, wet (2) Growth temperature: 12-37 ℃ (optimum temperature: 30 ℃)
(3) Growth pH: 4.0-9.0 (optimal pH: 5.0)
(4) Growth salt concentration: 0 to 5% (W / V)
(5) Observation of cell morphology Nutrient cells are oval to oval. Proliferation is by budding. No sexual reproductive organs are formed in 4 weeks of culture.

The MC7 strain exhibits the following physiological and biochemical properties in a physiological and biochemical property test.
<Physiological / biochemical property test results>
(1) Sugar fermentability test Glucose +
Galactose-
Maltose −
Sucrose +
Trehalose-
Raffinose +
(2) Carbon source 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 +
Melechtose +
Inulin +
Soluble starch-
Glycerol +
Erythritol-
Ribitol (Adonitol)-
Xylitol-
D-Glucitol (sorbitol) L
D-mannitol D
Galactitol (Darcitol)-
Inositol-
Sukusinate-
Citrate D
Ethanol +
(3) Nitrogen source assimilation nitrate +
Nitrite +
L-lysine +
(4) Tolerance 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 requirement test Vitamin-free medium +
(In the above, "+" is positive, "-" is negative, "L (latent)" is a rapid positive reaction 2 weeks after the start of the test, and "D (delay)" is 1 after the start of the test. It shows that a positive reaction was gradually observed over a period of more than a week).

The mycological properties of the MC7 strain were in good agreement with the characteristics of Candida mengyuniae. Physiological and biochemical properties of MC7 strain and known physiological and biochemical properties of Candida mengniae (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 the assimilation of meletitose, the resistance test at 45 ° C., and the growth at 10% sodium chloride / 5% glucose were found to be different, but for other items, known Candida chemyuniae ) Showed similar physiological and biochemical properties.

From the above-mentioned nucleotide sequence analysis of 26S rDNA, mycological properties, and physiological / biochemical properties, the MC7 strain was identified as Candida microbial strain, but the MC7 strain was identified in Examples 2 and 3 below. And, as shown in Comparative Example 1, it was judged to be a novel microorganism because it has excellent oil decomposition activity as compared with the conventional microorganism belonging to the genus Candida.

(Example 2)
<Decomposition 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 loopful of MC7 strain on an agar plate to a 300 mL Erlenmeyer flask, shake at 30 ° C. overnight, and shake before. Culture was performed. In the oil decomposition test, 150 mL of the medium for decomposition test (10 g / L shell terrace oil C, 1 g / L corn steep liquor, 0.5 g / L urea) and 100 μL of the preculture solution were added to a 500 mL Erlenmeyer flask, and the predetermined pH and temperature were added. I shook it. The results of decomposition at pH 7 at 15 ° C. to 35 ° C. for 24 hours are shown in FIG. The MC7 strain showed a maximum degradation rate of 23% at 30 ° C. The results of decomposition at 30 ° C. for 24 hours at pH 4 to 8 are shown in FIG. The MC7 strain showed a maximum degradation rate of 29% at pH 5.

(Example 3)
<Decomposition of fats and oils 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 loopful of MC7 strain on an agar plate to a 300 mL Erlenmeyer flask, shake at 30 ° C. overnight, and shake before. Culture was performed. For the oil decomposition test, add 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 to a 500 mL Erlenmeyer flask, and shake at a predetermined pH and temperature. did. The results of decomposition at pH 7 at 15 ° C. to 35 ° C. for 24 hours are shown in FIG. The MC7 strain showed a maximum degradation rate of 54% at 30 ° C. The results of decomposition at 30 ° C. for 24 hours at pH 4 to 8 are shown in FIG. The MC7 strain showed the maximum degradation rate of 67% at pH 5.

(Comparative Example 1)
Add 50 mL of SCD medium (15 g / L casein peptone, 5 g / L soybean peptone, 5 g / L sodium chloride) and 1 Candida albicans ATCC90029 strain on an agar plate to a 300 mL Erlenmeyer flask at 30 ° C. It was shaken overnight and precultured. The mineral oil decomposition test was carried out by the same method as in Example 2, and the oil and fat decomposition test was carried out by the same method as in Example 3. The results of decomposing the fats and oils at pH 7 at 15 ° C. to 35 ° C. for 24 hours are shown in FIG. Further, FIG. 7 shows the results of decomposing mineral oil at 30 ° C. at pH 4 to 8 for 24 hours. The degradation rate of the Candida albicans ATCC90029 strain was inferior to that of the MC7 strain for any of the oils.

The microorganism of the present invention has a high resolution for oil content. In addition, the microorganism of the present invention can grow in a wide temperature range, pH range, and high salt concentration. Therefore, according to the microorganism of the present invention and the oil decomposing agent containing the microorganism of the present invention, oil in an oil-containing substance such as oil-containing wastewater can be efficiently decomposed.

Claims (7)

It belongs to the genus Candida, and the base sequence of 26S rDNA is the base sequence shown by SEQ ID NO: 1 or the base sequence having 90% or more homology with the base sequence shown by SEQ ID NO: 1, and is as follows. A genus that exhibits mycological properties and has 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: White to cream Shape: Circular raised state: Flat at the periphery, cushion at the center Periphery: All edges Surface shape: Smooth Gloss and properties: Dull, damp, (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 Nutrient cells are oval to oval. Proliferation is by budding. No sexual reproductive organs are formed in 4 weeks of culture. 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 −
Sucrose +
Trehalose-
Raffinose +
(2) Carbon source 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 +
Melechtose +
Inulin +
Soluble starch-
Glycerol +
Erythritol-
Ribitol (Adonitol)-
Xylitol-
D-Glucitol (sorbitol) L
D-mannitol D
Galactitol (Darcitol)-
Inositol-
Sukusinate-
Citrate D
Ethanol +
(3) Nitrogen source assimilation nitrate +
Nitrite +
L-lysine +
(4) Tolerance 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 requirement test Vitamin-free medium +
(In the above, "+" is positive, "-" is negative, "L" is a rapid positive reaction 2 weeks after the start of the test, and "D" is 1 week or more after the start of the test. (Indicates that a positive reaction was gradually observed) The microorganism according to claim 1 or 2, which belongs to Candida mengyuniae. Candida mengyuniae MC7 strain (NITE P-02894). An oil decomposing agent containing the microorganism according to any one of claims 1 to 4. An oil decomposition method according to claim 5, wherein the oil-decomposing agent is brought into contact with the oil-containing substance to decompose the oil in the oil-containing substance. The oil decomposition method according to claim 6, wherein the oil-containing substance is oil-containing wastewater.

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