JP2020000104A - Oil decomposing microorganisms - Google Patents

Abstract

To provide microorganisms that are effective in decomposing mineral oil.SOLUTION: The present invention provides microorganisms that are effective in decomposing mineral oil, the microorganisms belonging to Acinetobacter guillouiae.

Description

  The present invention relates to a novel microorganism belonging to Acinetobacter guillouiae, which is excellent in oil-decomposability, particularly excellent in mineral oil-decomposability. The present invention also relates to a method for decomposing oil, particularly mineral oil, using the microorganism and an oil decomposer (particularly, a mineral oil decomposer).

  Contamination of the natural environment, such as the sea, rivers, and soil, is progressing in inverse proportion to industrial development. The natural environment is a place where many living creatures, including humans, live, and in recent years, the sight of pollutants has become severe. In particular, mineral oils such as petroleum often cause problems in recent years due to crude oil leakage due to tanker aground and soil contamination at gas stations, and there is an increasing demand for safe removal. As a method for removing mineral oil, there are chemical and physical methods, but bioremediation technology for purifying environmental pollution by decomposing pollutants using the action of microorganisms and the like has been attracting attention. Bioremediation using microorganisms has applicability to a variety of pollutants, has a theoretically low input energy, and may possibly have low purification costs. It is considered. For example, Patent Document 1 reports that Acinetobacter baumannii KIM30135 strain can decompose mineral oil such as engine oil even in the absence of a carbon source other than oil.

JP 2006-296382 A

  However, the microorganism described in Patent Literature 1 has a problem that mineral oil degradability is not sufficient.

  Accordingly, the present invention has been made in view of the above circumstances, and has as its object to provide a microorganism having excellent oil (particularly, mineral oil) decomposability.

  The present inventor has made intensive studies to solve the above problems. As a result, they have found that the above-mentioned problems can be solved by an oil-decomposing microorganism belonging to Acinetobacter guillouiae and having predetermined mycological properties, and have completed the present invention.

  ADVANTAGE OF THE INVENTION According to this invention, the microorganisms excellent in oil (especially mineral oil) decomposability are provided.

FIG. 1A shows a comparison of the 16S rDNA partial nucleotide sequences of the isolated microorganism and the reference strain ATCC 11171 ^T (Accession No. X81659) of Acinetobacter guillouiae. FIG. 1B shows a comparison of the 16S rDNA partial nucleotide sequences of the isolated microorganism and the reference strain ATCC 11171 ^T (Accession No. X81659) of Acinetobacter guillouiae.

  Hereinafter, embodiments of the present invention will be described. Note that the present invention is not limited to only the following embodiments.

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

<Oil-degrading microorganism>
According to one aspect of the present invention, an oil-degrading microorganism (particularly a mineral oil-degrading microorganism) belonging to Acinetobacter guillouiae and having the following mycological properties is provided. In one embodiment of the present invention, oil-degrading microorganisms (particularly mineral oil-degrading microorganisms) belonging to Acinetobacter guillouiae, exhibiting the following mycological properties and having the 16S rDNA base sequence represented by SEQ ID NO: 1 Provided. In the present specification, the oil-degrading microorganism belonging to Acinetobacter guillouiae and exhibiting the following mycological properties is also referred to simply as `` the oil-degrading microorganism according to the present invention '' or `` the microorganism according to the present invention ''. Name.

  As a specific example of the oil-degrading microorganism belonging to Acinetobacter guilloiae described above, a strain isolated by the following screening method will be described in detail. The 1-2350 strain according to the present invention was isolated from the soil of Seki City, Gifu Prefecture by the following screening method.

[screening]
An appropriate amount of a sample collected from soil, wastewater, sewage or river water of a waste oil treatment facility in Seki City, Gifu Prefecture is added to a liquid medium for primary screening prepared by the following method, and cultured at 30 ° C. for one week. 100 μL of the culture solution after the culture is further inoculated into 140 mL of the primary screening liquid medium, and cultured again at 30 ° C. for one week. In the present specification, the liquid medium for primary screening prepared by the following method is also simply referred to as “liquid medium for primary screening”.

  In the liquid medium for primary screening, each component other than mineral oil is dissolved in pure water so as to have the composition shown in Table 4 below, so that the final concentration of mineral oil becomes 0.5 w / v% (0.5 g / 100 mL). And sterilized by high temperature and high pressure. The mineral oil is an equal weight ratio of diesel engine oil, gasoline engine oil, machine oil, gear oil and cutting oil (that is, a mixture ratio (weight ratio) of diesel engine oil: gasoline engine oil: machine oil: gear oil: cutting oil). 1: 1: 1: 1: 1). Here, as a diesel engine oil, a trade name of CF Kodiesel manufactured by Fujikosan Co., Ltd., and as a gasoline engine oil, a trade name of Sanyo Chemical Industry Co., Ltd .: 4-cycle gasoline engine oil for general-purpose machines, as machine oil Trade name of Azzet Co., Ltd .: machine oil, trade name of Trusco Nakayama Co., Ltd. as gear oil: industrial gear oil ISO VG220, and trade name of Azzet Co., Ltd. as cutting oil: tap & drill oil cutting oil, Use each one. The pH of the primary screening liquid medium was not adjusted.

10 ⁴ fold culture 100μL after primary screening were diluted and applied to an agar medium for secondary screening was prepared in the following manner, and cultured for 1 week at 30 ° C.. After culturing, a strain whose growth on agar has been confirmed is isolated. In addition, in this specification, the agar medium for secondary screening produced by the following method is also simply referred to as "agar medium for secondary screening".

  The secondary screening agar medium was prepared by dissolving each component other than mineral oil and agar in pure water so as to have the composition shown in Table 5 below, and the final concentration of mineral oil was 0.5 w / v% (0.5 g / 100 mL). ) And agar are added to obtain a final concentration of 2.0 w / v% (2.0 g / 100 mL), and mineral oil and agar are added thereto. In Table 5 below, the same mineral oil as used in the above-described liquid medium for primary screening is used as the mineral oil. The pH of the agar medium for secondary screening was not adjusted.

  Next, 0.05 g of mineral oil is added to 5 mL of the liquid medium for tertiary screening prepared by the following method to prepare a sterilized test solution (mineral oil concentration: 1% (w / v)). Each of the isolated strains obtained in the above secondary screening is inoculated with a platinum loop, one platinum loop at a time, into the test solution prepared by the above method, and cultured with shaking (rotational speed: 140 rpm) at 30 ° C. for 24 hours. In the present specification, the liquid medium for tertiary screening prepared by the following method is also simply referred to as “liquid medium for tertiary screening”.

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

  After the culture, a normal hexane extract is prepared according to JIS K0102: 2016 revision (industrial wastewater test method). Using the normal hexane extract as the residual amount of mineral oil, 0.05 g of the mineral oil added at the time of preparing the test solution and the residual amount of mineral oil (g) (the amount of normal hexane extract (g)) were calculated by the following formula (1). Calculate the mineral oil reduction rate. As a result, a strain having a high mineral oil reduction rate can be isolated.

  As a result, the strain with the highest reduction rate of mineral oil was isolated from the soil in Seki City, Gifu Prefecture. This isolated strain (isolated microorganism) was identified as follows.

[Strain identification (classification)]
The isolated microorganism was subjected to aerobic culture at 30 ° C. for 24 hours using an LB agar medium (Becton Dickinson, USA), and subjected to the following strain identification (classification).

1.16S rDNA nucleotide sequence analysis The 16S rDNA nucleotide sequence of the isolated microorganism was analyzed based on the following protocol (operation from PCR amplification to cycle sequence). The nucleotide sequence is determined after visually confirming raw data (electropherogram) from the sequencer and performing correction.

  The 16S rDNA base sequence of the isolated microorganism determined above is shown in SEQ ID NO: 1 below.

As a result of a BLAST homology search against the DNA database for microorganism identification DB-BA12.0 (Techno Suruga Lab Co., Ltd.) and the international base sequence database (DDBJ / ENA (EMBL) / GenBank), a partial base sequence of 16S rDNA of the isolated microorganism was obtained. Showed a homology of 99.2% to the reference strain ATCC 11171 ^T (Accession No. X81659) of Acinetobacter guillouiae (see Tables 7 and 8 below). Table 7 shows the results of homology search for the microorganism identification DNA database DB-BA12.0. Table 8 shows the results of homology search against the international base sequence database (DDBJ / ENA (EMBL) / GenBank). In the following table, “BSL” means the biosafety level, and the biosafety level (BSL) complies with the Bacteriological Society of Japan Biosafety Guideline “BSL Level of Pathogenic Bacteria” (Level 1: Causes human disease or Not veterinarily significant in animals (including opportunistic infections), Level 2: Pathogenic to humans or animals, but not available to laboratory personnel, local communities, livestock, environment, etc. Not serious catastrophic, could cause serious infection if exposed in laboratory, but has effective treatment, prevention, low likelihood of transmission, level 3: human transmission This can cause serious illness but is unlikely to spread to other individuals). In the table below, opportunistic pathogens are indicated as "BSL1 *". Shaded cells indicate sequence data subjected to simple molecular phylogenetic analysis.

In addition, as a result of analyzing a molecular phylogenetic tree based on a base sequence obtained by homology search with respect to a DNA database for microorganism identification DB-BA12.0 (Techno Suruga Lab Co., Ltd.), isolated microorganisms belonging to the genus Acinetobacter It was included in the constituting cluster and showed the same molecular phylogenetic position as the reference strain ATCC 11171 ^T (Accession No. X81659) of Acinetobacter guillouiae (see the phylogenetic tree below). In the following phylogenetic tree, the upper left line shows the scale bar, the number located at the branch of the phylogenetic branch shows the bootstrap value, the T at the end of the strain name shows the reference strain (Type strain) of the species, and the BSL shows the biotype strain. The safety levels (BSL1 * (opportunistic pathogen) and above) are shown, respectively.

As shown in FIGS. 1A and 1B, 11 base differences between the 16S rDNA partial base sequences of the isolated microorganism and the reference strain ATCC 11171 ^T (Accession No. X81659) of Acinetobacter guillouiae (see FIG. 1). 1A). In FIG. 1, D = A, G or T, R = G or A, Y = C or T, K = T or G, M = C or A (IUB code). In FIG. 1A and FIG. 1B, “Query” indicates a partial base sequence of 16S rDNA of the isolated microorganism, and “Sbjct” indicates 16S rDNA of Acinetobacter guillouiae reference strain ATCC 11171 ^T (Accession No. X81659). The rDNA partial base sequences are shown respectively. In FIG. 1A and FIG. 1B, a vertical line is drawn between each base at a position where the sequences match.

  From the above, it is presumed that the isolated microorganism belongs to Acinetobacter guillouiae.

2. Mycological properties The mycological properties of the strain (isolated microorganism) isolated by the above screening are shown below. Morphological observation was performed using an optical microscope (BX50F4, Olympus, Japan). Gram staining was performed using Faber G "Nissui" (Nissui Pharmaceutical, Japan). Tests for catalase reaction, oxidase reaction, acid / gas production from glucose, and glucose oxidation / fermentation (O / F) were conducted according to the method of Barrow & Feltham (Barrow GI & Feltham RKA (1993), Cowan and Steel's Manual for the Identification of Medical Bacteria. 3rd edition, Cambridge University Press).

  From the results in Table 9 above, the strain (isolated microorganism) isolated by the above screening was a gram-negative (indeterminate) bacillus having no motility, did not oxidize glucose, had a positive catalase reaction, and had a positive oxidase reaction. Showed negative. These properties are considered consistent with those of the genus Acinetobacter (Barrow G.I. & Feltham R.K.A. (1993), Cowan and Steel's Manual for the Identification of Medical Bacteria. 3rd edition, Cambridge University Press).

  Next, the following items were tested using API® 20NE (bioMerieux, France) according to the manufacturer's protocol. Table 10 shows the results.

  In addition, Techno Suruga Lab, Inc. and NCIMB Ltd., UK Technical collaborations and related technologies for classification and identification (Nemec A., Musilek M., Sedo O., De Baere T., Maixnerova M., Van Der Reijden TJK, Zdrahal Z., Vaneechoutte M. & Dijkshoorn L. Acinetobacter bereziniae sp.nov. And Acinetobacter guillouiae sp.nov., To accommodate Acinetobacter genomic species 10 and 11, respectively.Int.J. Syst. Evol. Microbiol., 60, 896-903) Items were tested. Table 11 shows the results.

  According to one aspect of the present invention, there is provided an oil-degrading microorganism (particularly a mineral oil-degrading microorganism) belonging to Acinetobacter guillouiae and exhibiting the above-mentioned mycological properties. In one embodiment of the present invention, an oil-degrading microorganism (particularly a mineral oil-degrading microorganism) belonging to Acinetobacter guillouiae, exhibiting the above-described mycological properties, and having the 16S rDNA base sequence represented by SEQ ID NO: 1 ) Is provided.

From the results in Table 10 above, the strain (isolated microorganism) isolated by the above screening does not reduce nitrate, does not hydrolyze gelatin, and does not show assimilation ability to glucose and L-arabinose. However, it showed assimilation ability to n-capric acid and phenyl acetate. In addition, from the results in Table 11, the bacterial strain (isolated microorganism) isolated by the above-mentioned screening uses citric acid (Simons method), does not use an inorganic nitrogen source (NH ₄ Cl), and shows hemolysis. Did not. These properties almost coincide with the properties of Acinetobacter guillouiae whose assignment was suggested in the above “1.16S rDNA nucleotide sequence analysis” (Nemec A., Musilek M., Sedo O., De Baere T., Maixnerova M., Van Der Reijden TJK, Zdrahal Z., Vaneechoutte M. & Dijkshoorn L. (2010) .Acinetobacter bereziniae sp. Nov. And Acinetobacter guillouiae sp. Int. J. Syst. Evol. Microbiol., 60, 896-903), as described above, differs in base sequence from the conventionally known Acinetobacter guillouiae.

  Therefore, the isolated strain (isolated microorganism) was found to have different properties from the conventionally known Acinetobacter guillouiae, and was determined to be a novel microorganism. Was named Acinetobacter guillouiae 1-2350 (hereinafter also simply referred to as “strain 1-2350”). The 1-2350 strain was deposited on June 20, 2018 with the Patent Evaluation and Microorganisms Depositary Center (NPMD) of the National Institute of Technology and Evaluation (NPMD) (2-5-8 Kazusa Kamatari, Kisarazu-shi, Chiba). And its receipt number is NITE BP-02734. That is, one aspect of the present invention relates to an oil-degrading microorganism (particularly a mineral oil-degrading microorganism) specified by Acinetobacter guillouiae strain 1-2350 (accession number: NITE BP-027334).

  The oil-degrading microorganisms (especially strain 1-2350) exhibiting the above microbiological properties are excellent in decomposing power of mineral oil (hydrocarbon), and can be used in a wide range of mineral oil concentrations including unsaturated hydrocarbons and polycyclic aromatics. It has the property of purifying wastewater. For this reason, oil-degrading microorganisms (especially strain 1-2350) exhibiting the above microbiological properties can be removed from industrial wastewater containing mineral oil, abatement facilities, maintenance factories, and parking lots for treating industrial wastewater containing mineral oil. By adding oil traps and gas traps installed in car wash stations and gas stations, and to seas, rivers, and soil where mineral oil has leaked, mineral oil can be efficiently decomposed to prevent and suppress pollution of the natural environment. it can. In addition, the oil-decomposing microorganisms (especially strain 1-2350) exhibiting the above mycological properties are microorganisms originally existing in nature. Therefore, the addition of the microorganism according to the present invention has little or no adverse effect on the natural environment. Therefore, according to the microorganism of the present invention, mineral oil can be safely removed. In addition, since there is no need to separately collect the microorganisms according to the present invention after mineral oil decomposition, there is an advantage that purification costs can be reduced.

  Here, the mineral oil may be unused or used. Specific examples of such mineral oil include, but are not limited to, diesel engine oil, gasoline engine oil, machine oil, gear oil, cutting oil, and the like. Oil-degrading microorganisms (in particular, mineral oil-degrading microorganisms such as strain 1-2350) exhibiting the above-described mycological properties exhibit high oil-decomposability (particularly, mineral-oil-decomposability) for such a wide range of mineral oils. In the present specification, "machine oil" is a lubricating oil that reduces friction that occurs when metals come into contact with each other. Depending on the machine part or application to be used, machine oil, turbine oil, spindle oil, dynamo oil , Cylinder oil, bearing oil, refrigerator oil, hydraulic oil, gear oil, compressor oil, sliding surface oil, etc. In this specification, "gear oil" is oil mainly used for lubrication and cooling of engines and gears of cars and motorcycles. Gear oils may include additives such as antioxidants, antiwear agents, detergents and dispersants, viscosity index improvers, defoamers, pour point depressants, etc., to improve the desired properties. Further, in this specification, “cutting oil” is a cutting oil that plays a role of lubrication, cooling, and removal of cutting in machining such as cutting, turning, milling, and tapping.

  The oil-degrading microorganisms (especially strain 1-2350) of the present invention belonging to Acinetobacter guilloiae exhibiting the above-mentioned mycological properties show high resolution for a wide range of concentrations of mineral oil. Specifically, the oil-decomposing microorganism according to the present invention has a mineral oil reduction rate of at least one mineral oil concentration within a range of 1,000 to 10,000 ppm, measured by the method described below, of more than 35% by weight, preferably 40% by weight. %, More preferably at least 50% by weight, particularly preferably more than 55.0% by weight (upper limit: 100% by weight). Preferably, the oil-degrading microorganism according to the present invention has a mineral oil reduction rate of more than 30% by weight, more preferably more than 40% by weight, at a total mineral oil concentration of 1,000 to 10,000 ppm as measured by the method described below. It is particularly preferably at least 45% by weight (upper limit: 100% by weight). In addition, in this specification, "mineral oil reduction rate in the range of 1,000 to 10,000 ppm of mineral oil measured by the method described later" is simply referred to as "mineral oil reduction rate". That is, in a preferred embodiment of the present invention, the oil-degrading microorganism according to the present invention has a mineral oil reduction rate of more than 40% by weight at at least one mineral oil concentration in the range of 1,000 to 10,000 ppm measured by the following method ( It is more preferably at least 50% by weight, particularly preferably more than 55.0% by weight (upper limit: 100% by weight). In a preferred embodiment of the present invention, the oil-decomposing microorganism according to the present invention has a mineral oil reduction rate of more than 30% by weight at a total mineral oil concentration in a range of 1,000 to 10,000 ppm measured by the following method ( It is more preferably more than 40% by weight, particularly preferably 45% by weight or more (upper limit: 100% by weight). Normally, when oil-degrading microorganisms are cultured with oil for a long time, the oil gradually decomposes (mineral oil reduction rate increases). However, for example, when the microorganisms are added to the detoxification facility, the microorganisms are sequentially discharged from the detoxification facility. Therefore, the microorganisms are usually supplied to the detoxification facility about every 1 to 3 days. On the other hand, the oil-decomposing microorganisms according to the present invention show a high mineral oil reduction rate in a short time of 24 hours, as is apparent from the following method. Excellent. In addition, in this specification, "mineral oil reduction rate" is a value measured by the following method.

[Evaluation of mineral oil reduction effect (measurement of mineral oil reduction rate)]
In the present specification, the effect of reducing mineral oil is evaluated based on the rate of reduction of mineral oil by the following method. That is, mineral oil is added to a sterile-treated oil decomposition evaluation medium (5 mL), which is the same as the tertiary screening liquid medium described above, so that the mineral oil concentration becomes 1,000 to 10,000 ppm, and the test liquid is added. Prepare (oil: 0.1-1% (w / v) (1 g / L-10 g / L)). The test liquid is inoculated with a microorganism cultured on a plate medium (eg, an agar medium for secondary screening), and cultured at an arbitrary temperature zone (eg, 30 ° C.) for 24 hours with shaking (140 rpm). The amount of inoculated bacteria is about one platinum loop. As the microorganism to be inoculated into the test solution, a microorganism pre-cultured in a liquid medium for tertiary screening or the like may be used. By pre-culturing, the amount of bacteria to be inoculated can be easily adjusted. When using a precultured microorganism, inoculate the test solution at a concentration of 1.5 × 10 ⁶ CFU / ml with respect to 1 mL of the test solution. The cultivation temperature may be set in accordance with the temperature range in which the cells have a high ability to decompose and assimilate the oil.

  After the culture, a normal hexane extract is prepared according to JIS K0102: 2016 revision (industrial wastewater test method). The normal hexane extract was defined as the residual amount of mineral oil, and the mineral oil (g) added during the preparation of the test solution and the residual amount of mineral oil (g) (the amount of normal hexane extract (g)) were calculated by the following equation (1). The mineral oil reduction rate is calculated.

[Culture of microorganism]
The method for culturing an oil-degrading microorganism belonging to Acinetobacter guillouiae (hereinafter, also simply referred to as “microorganism”) according to the present invention may be any method as long as the microorganism can grow and proliferate. . For example, the medium used for culturing the microorganism of the present invention may be either a solid or liquid medium, and a medium containing a carbon source, an appropriate amount of nitrogen source, an inorganic salt and other nutrients that the microorganism used can utilize. If so, either a synthetic medium or a natural medium may be used. Usually, the medium contains a carbon source, a nitrogen source and a mineral.

  The carbon source that can be used in the culture of the microorganism of the present invention is not particularly limited as long as the strain to be used can utilize the carbon source. Specifically, considering the assimilation of microorganisms, glucose (glucose), fructose, cellobiose, raffinose, xylose, galactose, sorbose, glucosamine, ribose, rhamnose, sucrose, trehalose, α-methyl-D-glucoside, Salicin, melibiose, lactose, melezitose, inulin, erythritol, ribitol, xylitol, glucitol, galactitol, inositol, sorbitol, amidagulin, starch, starch hydrolyzate, sucrose, molasses, molasses, and other natural sugars, wheat, rice, etc. Substances, alcohols such as glycerol, methanol, ethanol, etc., organic acids such as acetic acid, phenyl acetate, lactic acid, succinic acid, gluconic acid, capric acid, adipic acid, pyruvic acid, citric acid, malic acid and salts thereof, Such as a hydrocarbon such as Sadekan like. The carbon source is appropriately selected in consideration of assimilation by the microorganism to be cultured. For example, when using strain 1-2350, glucose, capric acid, adipic acid, malic acid, citric acid and phenyl acetate and salts thereof (eg, sodium salt, potassium salt) and the like are used among the above carbon sources. Is preferred. Further, one or more of the above carbon sources can be selected and used. The carbon source is appropriately selected in consideration of assimilation by the microorganism to be cultured.

  The nitrogen source that can be used in the culture of the oil-decomposing microorganism according to the present invention includes meat extract, fish meat extract, peptone, polypeptone, tryptone, yeast extract, malt extract, soybean hydrolyzate, soybean powder, casein, milk casein, Various amino acids such as casamino acid, glycine, glutamic acid, and aspartic acid, corn steep liquor, and other organic nitrogen sources such as hydrolysates of animals, plants, and microorganisms; ammonium salts such as ammonia, ammonium nitrate, ammonium sulfate, and ammonium chloride; sodium nitrate And the like, nitrites such as sodium nitrite, and inorganic nitrogen sources such as urea. The nitrogen source is appropriately selected in consideration of assimilation by the microorganism to be cultured. For example, when the 1-2350 strain is used, it is preferable to use tryptone, yeast extract, ammonium chloride and the like among the above nitrogen sources. In addition, one or more of the above nitrogen sources can be selected and used.

  Examples of the inorganic substance that can be used in the present invention include halides such as phosphate, hydrochloride, sulfate, acetate, carbonate, and chloride such as magnesium, manganese, calcium, sodium, potassium, copper, iron, and zinc. Is mentioned. The inorganic substance is appropriately selected depending on the microorganism to be cultured. In addition, one or more of the above inorganic substances can be selected and used. Further, a surfactant or the like may be added to the medium as needed.

  In order for the microorganisms according to the present invention to efficiently decompose and assimilate oil (particularly mineral oil), or to maintain the ability of the microorganisms according to the present invention to decompose and assimilate oil (particularly mineral oil decomposing and assimilating ability), a medium is required. It is preferable to add an oil component (particularly, mineral oil) therein. Examples of the oil component include mineral oils (hydrocarbons) such as the aforementioned diesel engine oil, gasoline engine oil, machine oil, gear oil, and cutting oil; edible fats and oils, industrial fats and oils, and fatty acids. The amount of the oil added is not particularly limited, and may be appropriately selected in consideration of the oil content resolution by the microorganism to be cultured. Specifically, it is preferable to add oil at a concentration of 0.5 to 50 g, more preferably 1 to 30 g, and particularly preferably 5 to 15 g per liter of medium. With such an addition amount, the microorganism can maintain a high oil content resolution. In particular, when using mineral oil, mineral oil (diesel engine oil: gasoline engine oil: machine oil: gear oil: cutting oil = 1: 1: 1: 1: 1 (w / w / w / w / w)) is used. A concentration of 0.5 to 50 g (500 to 50,000 ppm), more preferably 1 to 30 g (1,000 to 30,000 ppm), and particularly preferably 3 to 20 g (3,000 to 20,000 ppm) in 1 L of the medium. It is preferable to add in. With such an addition amount, the microorganism can maintain a high mineral oil decomposition / assimilation ability. The oil component (mineral oil) may be added alone or in the form of a mixture of two or more.

  Cultivation of the microorganism can be performed by a usual method. For example, the culture is performed under aerobic or anaerobic conditions depending on the type of microorganism. In the former case, the cultivation of the microorganism is performed by shaking or aeration stirring. Also, the microorganisms may be cultured continuously or in batches. Culture conditions are appropriately selected depending on the composition of the medium and the culture method, and are not particularly limited as long as the microorganisms of the present invention can grow, and may be appropriately selected depending on the type of microorganism to be cultured. Usually, the culture temperature is preferably 10 ° C or higher and lower than 40 ° C, and more preferably 15 to 35 ° C. Further, the pH of the medium suitable for culture is preferably 5 to 8, more preferably 5.5 to 7.5. The culturing time is not particularly limited, either, and varies depending on the type of microorganism to be cultured, the amount of medium, the culturing conditions, and the like. Usually, the culture time is preferably 16 to 48 hours, more preferably 20 to 30 hours.

<How to decompose oil>
In one aspect of the present invention, a method for decomposing oil, comprising the step of contacting the oil-degrading microorganisms according to the present invention with oil, or the step of treating wastewater, comprising the step of contacting the oil-degrading microorganisms according to the present invention with wastewater containing oil. A method is provided. In a preferred embodiment of the present invention, the method for treating wastewater includes the step of contacting the oil-degrading microorganisms according to the present invention with mineral oil, the method for decomposing mineral oil or the step of contacting the oil-degrading microorganisms according to the present invention with wastewater containing mineral oil. A method is provided. Here, the object with which the oil-decomposing microorganism according to the present invention comes into contact is not particularly limited as long as it contains oil. In addition to the above-described mineral oil itself, industrial wastewater containing oil (particularly mineral oil), oil ( Oil traps and gasoline traps installed in wastewater treatment facilities (in particular, wastewater treatment facilities) for the treatment of industrial wastewater (including mineral oil), maintenance shops, parking lots, car wash stations and gas stations, and There may be seas, rivers and soils where oil (especially mineral oil) has leaked. The oil-degrading microorganism (especially strain 1-2350) according to the present invention has a high resolution for oil, particularly mineral oil (hydrocarbon), and can efficiently remove oil in a water environment with a wide range of oil concentrations. Therefore, according to the method of the present invention, the oil-degrading microorganisms (especially strain 1-2350) of the present invention are drained, wastewater treatment facilities, oil traps and gasoline traps, seas, rivers, and soils from which oil (oil) has leaked. By adding to oils and the like, oil (particularly mineral oil) can be efficiently decomposed and pollution of the natural environment can be effectively prevented or suppressed. The oil-degrading microorganism (especially strain 1-2350) according to the present invention is a microorganism originally existing in nature. Therefore, the method of the present invention has little or no adverse effect on the natural environment (the oil can be safely removed). In addition, since it is not necessary to separately collect the microorganisms of the present invention after decomposing the oil (particularly mineral oil), the method of the present invention has a low purification cost and is economically preferable.

  Here, the method of contacting the oil-decomposing microorganism according to the present invention with an oil (oil component) (particularly, mineral oil) is not particularly limited as long as the oil-decomposed microorganism according to the present invention can be brought into contact with the oil. For example, the oil-degrading microorganisms of the present invention can be placed in a desired location (for example, mineral oil, wastewater containing mineral oil, wastewater treatment facilities, oil traps and gasoline traps, and seas, rivers, and soil from which mineral oil has leaked; It may be added directly to “drain water”. Alternatively, a carrier or the like on which the oil-decomposing microorganism according to the present invention is immobilized may be installed in a route (pipe) or a storage tank, and the microorganism may be brought into contact with the microorganism by flowing oil therethrough.

  The oil-degrading microorganism used in the method according to the present invention may be variously suspended in a culture solution, recovered as a solid from the culture solution, dried, immobilized on a carrier, etc. Can be brought into contact with drainage or the like in any suitable form. The oil-degrading microorganism suspended in the culture solution, recovered as a solid content from the culture solution, or dried is added to wastewater or the like and brought into contact with the oil contained therein. The oil-degrading microorganisms immobilized on the carrier may be added to the wastewater or the like as described above.However, a carrier on which the oil-degrading microorganisms are immobilized is placed in a trap, and the wastewater or the like is placed on the microorganism-immobilized carrier. By allowing the solution to pass, the oil-decomposing microorganisms can be brought into contact with wastewater or the like. By disposing the oil-decomposing microorganisms immobilized on the carrier in the trap, it is possible to prevent the oil-decomposed microorganisms from flowing out together with the drainage and the like to reduce the number of bacteria.

  When using oil-decomposing microorganisms collected as a solid content from a culture solution, any method known to those skilled in the art can be used for the method of recovery. For example, the culture solution of the oil-degrading microorganism cultured by the above-described method can be obtained by solid-liquid separation by centrifugation, filtration, or the like, and collecting the solid content. If this solid content is dried (for example, freeze-dried), a dried oil-degrading microorganism can be obtained.

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

  The method for immobilizing the oil-decomposing microorganism on the carrier is not particularly limited, and a general microorganism immobilizing method may be used in the same manner or appropriately modified. For example, an immobilization method by pouring a culture solution of a microorganism into a carrier, an immobilization method by placing the support under reduced pressure using an aspirator, and a culture solution of a microorganism by pouring the culture solution into a carrier, and a medium in which the culture solution of the microorganism is sterilized. And a method in which the mixture is poured into a mixture of the mixture and a carrier, cultured with shaking, and the carrier removed from the mixture is naturally dried.

In the method according to the present invention, when an oil-decomposing microorganism is added to and brought into contact with wastewater or the like, the amount of the added microorganism can be arbitrarily set. The amount of bacteria added to the wastewater or the like is not particularly limited, but is, for example, 1 × 10 ⁴ to 1 × 10 ¹² CFU, preferably 1 × 10 ⁵ per 1 g of oil contained in the wastewater or the like. ^１1 × 10 ¹¹ CFU. Alternatively, the amount is, for example, 0.1 mg to 5 g (dry cell weight), preferably 1 mg to 1.5 g (dry cell weight), and more preferably 10 mg to 1 g of oil contained in wastewater. 150 mg (dry cell weight). Alternatively, the amount may be, for example, 1 × 10 ⁶ to 1 × 10 ¹² CFU / L, more preferably 1 × 10 ⁷ to 1 × 10 ¹¹ CFU / L, with respect to the waste water in the trap. Good. Alternatively, the amount is, for example, 10 mg to 15 g (dry cell weight) / L, preferably 0.1 g to 1.5 g (dry cell weight) / L for wastewater. When two or more microorganisms are used in combination, the amount of the microorganism added means the total amount. Pre-cultured microorganisms may be used as the microorganisms to be added to the wastewater or the like. By pre-culturing, the amount of bacteria to be inoculated can be easily adjusted.

  When draining waste water or the like to the external environment, oil-decomposing microorganisms not fixed to the carrier are discharged to the outside together with the waste water. For this reason, in the present invention, it is preferable to periodically add oil-decomposing microorganisms to wastewater or the like. The interval of addition is not particularly limited, but it is preferable to add, for example, once every 3 hours, once every 24 hours, or once every 2 to 3 days. The method of addition is not particularly limited, and when wastewater or the like continuously flows into the system, it may be added in a mixed manner with the wastewater or the like, or may be directly added to the wastewater or the like in the system. .

  As mentioned above, the microorganisms according to the invention show a high resolution for a wide range of concentrations of mineral oil. For this reason, the content of oil in drainage or the like is not particularly limited. The oil contained in the wastewater or the like is not limited to one kind, and may be two or more kinds.

  In the method of the present invention, in addition to the oil-decomposing microorganisms of the present invention, other components may be added to wastewater or the like from the viewpoint of more efficiently reducing oil content. Other components include, for example, other microorganisms that can coexist with the oil-decomposing microorganism according to the present invention, lipase, a pH adjuster, a mineral oil adsorbent, a surfactant, and the like.

  Other microorganisms that can coexist with the oil-decomposing microorganism according to the present invention include, for example, genus Yarrowia, genus Candida, genus Pichia, genus Hansenula, genus Saccharomyces, The genus Kluyveromyces, the genus Trichosporon, the genus Bacillus, the genus Lactobacillus, the genus Aspergillus, the genus Rhodococcoscus, Rhodococcoscoscus genus, Rhodococcoscoscus genus, Rhodococcoscoscus genus, Rhodococcoscoscus genus, Rhodococcoscoscus genus, Rhodococcoscoscus genus, Rhodococcoscus genus, Rhodococcoscus genus, Rhodococcoscus genus, Rhodococcoscus ssp. ) Genus, Enterobacter genus, Burkholderi Genus (Burkholderia), genus Pseudomonas, genus Penicillium, genus Staphylococcus, genus Rhizopus, genus Rhizobium, genus Rhizobium, Aciensaeb genus Aciensaeb The genus (Fusarium), the genus Serratia, the genus Tetrasphaera, the genus Humicola, and the genus Stenotrophomonas can be exemplified. These microorganisms may be obtained from culture collections such as ATCC, NBRC, DSMZ. Among these microorganisms, it is preferable to use an oil-degrading microorganism of the genus Yarrowia, Enterobacter, or Sphingomonas due to the high resolution of the oil component.

  Examples of oil-degrading microorganisms of the genus Yarrowia include Yarrowia lipolytica ATCC 48436, Yarrowia lipolytica NBRC 1548, Yarrowia lipolytica LM02-011 (Accession number NITE P-01813), Yarrowia lipolytica NBRC 0746, and Yarrowia lipopoly BRC 120 such as Yarrowia N. Examples include Yarrowia sp. Such as Yarrowia lipolytica and Yarrowia YH-01, but Yarrowia lipolytica is more preferable, and Yarrowia lipolytica LM02-01 is independent and Yarrowia lipolytica LM02-01 is independent. National Institute of Technology and Evaluation Patent Microorganisms Depositary Center ( To 292-0818 Japan Chiba Prefecture Kisarazu Kazusa legs bowed in 2-5-8), has been deposited as accession number NITE P-01813 in the March 6 date 2014) is more preferred.

  As the oil-degrading microorganisms of the genus Enterobacter, Enterobacter sp. LM02-030 strain (LM02-030 strain is a patent microorganism deposit center of the National Institute of Technology and Evaluation, National Institute of Technology and Evaluation (Chiba, Japan 292-0818). 2-5-8, Kazusa-Kamashita, Kisarazu-shi, Japan) and deposited on May 12, 2015 under the accession number NITE P-02048).

  Examples of oil-degrading microorganisms of the genus Sphingomonas include, for example, Sphingomonas sp. 2629-3b described in JP-A-2006-166874 and Sphingomonas sp. LM02-032 strain described in JP-A-2017-136033 (LM02). -032 strain was registered with the National Institute of Technology and Evaluation, Patent Microorganisms Depositary Center (2-5-8, Kazusa-Kamashita, Kisarazu-shi, Chiba, Japan 292-0818 Japan) on accession number NITE P on June 19, 2015. Sphingomonas sp. (Deposited as 02069).

  An oil-degrading enzyme such as lipase or phospholipase may be added to wastewater or the like to assist the decomposition of mineral oil by the oil-decomposing microorganism used in the method according to the present invention. Examples of the lipolytic enzyme include, for example, Pseudomonas, Aspergillus, Bacillus, Penicillium, Rhizopus, Rhizomucor, Rhizomucor and Rhizomucor. Genus, Paecilomyces, Rhizoctonia, Absidia, Achromobacter, Aeromonas, Alternaria, Aurenia, Aurenia and Aureobia, Aureobia di aureba Genus Beauveria, Chromobacter ) Genus, Coprinus, Fusarium, Geotricum, Humicola, Hyphozyma, Lactobacillus, Metallidium, Polymorphism Rhodosporium, Trichoderma, Yarrowia, Candida, Pichia, Hansenula, Saccharomyces, Saccharomyces, Vero And / or obtained from the genus Trichosporon Door can be.

  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)", Newase (registered trademark) F ( Sumiteam (registered trademark) NLS, Sumiteam (registered trademark) RLS (Nippon Chemical Industry Co., Ltd.); Lilipase (registered trademark) A-10D, Lilipase (registered trademark) AF-5, PLA2 Nagase (Nagase) Chemtex Co., Ltd.); Entilon AKG-2000, Entilon LP, Entilon LPG (Rakuto Kasei Kogyo Co., Ltd.) Company); Lipolase (registered trademark) 100T, Lipolase (registered trademark) 100L, Palatase 20000L, Lipex (registered trademark) 100T, Lipex (registered trademark) 100L, Lipozyme (registered trademark) RMIM, Lipozyme (registered trademark) TLIM, Novozime (registered) Trade name) 435FG (manufactured by Novozymes); picantase A, picantase R800 (manufactured by DS Japan). These may be used in combination of two or more.

  The amount of the oil-degrading enzyme is not particularly limited as long as the enzyme can react with the oil, but it is preferably used in an amount of 10 to 2,000 U per 1 g of the oil contained in the wastewater or the like. More preferably, it is 50 to 1,500 U, and still more preferably 100 to 1,000 U. Alternatively, the amount may be preferably from 1,000 to 100,000 U / L, more preferably from 2,000 to 80,000 U / L, based on the capacity of the trap. The activity unit (U) of the oil-decomposing enzyme is the amount of the enzyme that releases 1 μmol of fatty acid per minute under the conditions of 37 ° C. and pH 7.

  In the method according to the present invention, a shirasu balloon described in JP2012-206084A, an inorganic polymer such as diatomaceous earth, perlite, polyurethane, polyethylene, an organic polymer such as a melanin resin, an oil adsorbent. It may be added to wastewater.

  In the present invention, an anionic surfactant such as sodium dodecyl sulfate (SDS), dodecylbenzenesulfonic acid (NaDDBS), ammonium lauryl sulfate, sodium caseinate, etc .; Cationic surfactants such as quaternary ammonium salts; fatty acid esters (monoglycerin fatty acid ester, diglycerin fatty acid ester, sorbitan fatty acid ester, propylene glycol fatty acid ester, sucrose fatty acid ester), polyethylene glycol, polyethylene glycol-t-octylphenyl Nonionic surfactants such as ether (Triton X-100) and lecithin; surfactants such as amphoteric surfactants such as betaine, amine oxide and saponin may be added to the wastewater.

  In the method of the present invention, wastewater containing oil (particularly, mineral oil) is continuously introduced into the system having the oil-decomposing microorganism according to the present invention, and wastewater after treatment is continuously discharged (continuous treatment). Alternatively, wastewater containing oil (particularly mineral oil) or the like may be introduced, treated collectively, and then treated wastewater may be collectively discharged (batch processing).

  In the method of the present invention, the temperature at which the oil-decomposing microorganisms are brought into contact with the oil, that is, the temperature of the wastewater or the like, can be arbitrarily set. Further, the pH at the time of bringing the oil-decomposing microorganisms into contact with the mineral oil, that is, the pH of the wastewater or the like can be arbitrarily set. Generally, the temperature is, for example, 10 ° C. or more and less than 45 ° C., and preferably 15 to 35 ° C. The pH is, for example, 5 to 8, preferably 5.5 to 7.5. Further, the drainage may be aerated by aeration or the like as necessary.

<Oil decomposer>
According to one aspect of the present invention, there is provided an oil decomposing agent (a wastewater treatment agent) containing the oil decomposing microorganism according to the present invention. In one embodiment of the present invention, there is provided a mineral oil decomposing agent (a wastewater treatment agent) containing the oil-decomposing microorganism according to the present invention. The oil-decomposing microorganism according to the present invention is excellent in the effect of reducing oil (particularly, mineral oil) (the oil-decomposition rate (particularly, the rate of reduction in mineral oil) is high). Further, the oil-decomposing microorganism according to the present invention can purify wastewater and the like (can remove oil in the wastewater) even in a water quality environment having a wide range of oil concentrations (for example, 1,000 to 10,000 ppm). Therefore, the wastewater treatment agent containing the oil-decomposing microorganism according to the present invention can be used as an oil (particularly mineral oil), a wastewater containing oil (particularly mineral oil), and the sea, river, and soil from which oil (particularly mineral oil) has leaked, and a treatment thereof. When used in equipment (for example, abatement facilities, oil traps and gasoline traps), oil (particularly mineral oil) can be effectively decomposed (effectively purifying wastewater and the like). The description of the oil-decomposing microorganisms and the wastewater treatment method described above can be modified as necessary and applied to the present embodiment.

  The oil decomposer (sewage treatment agent) may be in a dry form or in a liquid form, but a dry form such as powder, granules, pellets and tablets is preferred from the viewpoint of storage stability. As the oil-decomposing microorganism according to the present invention used in such an oil-decomposing agent in a dried form (a wastewater treatment agent), a cell powder obtained by drying a culture solution by spray drying or freeze-drying, or a carrier as described above is used. The cells may be immobilized cells, or may be formed into powder, granules, pellets, or tablets. Alternatively, the cells or culture solution may be encapsulated with hydroxypropylmethylcellulose, gelatin, or the like. The oil disintegrant (sewage treatment agent) may also contain excipients such as hydroxypropylcellulose, dextrin, lactose, starch and the like.

  The oil-decomposing microorganisms according to the present invention contained in the oil-decomposing agent (sewage treatment agent) may be dead or viable, but may be viable from the viewpoint of the sustained oil-decomposing activity. preferable.

The amount of the oil-decomposing microorganism according to the present invention contained in the oil-decomposing agent (sewage treatment agent) is, for example, 10 to 100% by weight in the solid content of the oil-decomposition agent (sewage treatment agent). Alternatively, the amount of the oil-decomposing microorganisms according to the present invention contained in the oil-decomposing agent (sewage treatment agent) is, for example, 1 × 10 ² to 1 × 10 ¹⁰ CFU / g with respect to the oil-decomposition agent (sewage treatment agent). Is the amount In addition, as long as the object effect of the present invention is achieved, other oil-degrading agents (oil-degrading enzymes, oil-adsorbing agents, and oil-degrading enzymes) can be used as long as the objective effects of the present invention are achieved. And one or more additives selected from the group consisting of surfactants.

  The effects of the present invention will be described using the following examples and comparative examples. However, the technical scope of the present invention is not limited only to the following examples.

Example 1: Isolation of microorganisms An appropriate amount of a sample collected from soil in Seki City, Gifu Prefecture, was added to a primary screening liquid medium prepared in the same manner as described above, and cultured at 30 ° C for 1 week. 100 μL of the culture solution after the culture was further inoculated into 5 mL of the primary screening liquid medium, and cultured again at 30 ° C. for one week.

10 ⁴ fold culture 100μL after primary screening were diluted and applied to the secondary screening agar fabricated in the same manner as described above, and cultured for one week at 30 ° C.. After the cultivation, a strain whose growth on agar was confirmed was isolated.

  Next, 0.05 g of mineral oil was added to 5 mL of the liquid medium for tertiary screening prepared in the same manner as above to prepare a sterilized test solution (mineral oil concentration: 1% (w / v)). Each of the isolated strains obtained in the secondary screening was inoculated with one loop of platinum loop into the test solution prepared by the above method, and cultured at 30 ° C. for 24 hours with shaking (rotation speed: 140 rpm).

  After culturing, a normal hexane extract was prepared according to JIS K0102: 2016 (industrial wastewater test method). Using the normal hexane extract as the residual amount of mineral oil, the mineral oil reduction rate was calculated from 0.05 g of mineral oil added at the time of preparing the test solution and the residual amount of mineral oil (the amount of normal hexane extract (g)) according to the following mathematical formula (1). I asked. As a result, a strain with a high mineral oil reduction rate was isolated.

  The isolated strain was named Acinetobacter guillouiae 1-2350 strain and deposited at the National Institute of Technology and Evaluation, Patent Microorganisms Depositary (Accession No. NITE BP-027334).

Example 2 Evaluation of Mineral Oil Degradability (Mineral Oil Decrease Rate) In 5 mL of the tertiary screening liquid medium prepared in the same manner as above, the mineral oil concentration was adjusted to 1,000 to 10,000 ppm (1 to 10 g / L). , Mineral oil was added to prepare a sterilized test solution. The isolated strain cultured on the agar medium for secondary screening was inoculated with one platinum loop using a platinum loop into the test solution prepared by the above method, and cultured at 30 ° C. for 24 hours with shaking (140 rpm).

  A platinum loop of Acinetobacter baumannii S30 strain was inoculated as a control to the test solution prepared in the same manner as above with a platinum loop, followed by shaking culture (140 rpm) at 30 ° C. for 24 hours.

  After the culture, a normal hexane extract was prepared according to JIS K0102: 2016 revision (industrial wastewater test method). Using the normal hexane extract as the residual oil content, the mineral oil reduction rate (the amount (g) of the normal hexane extract) and the oil content (g) added at the time of preparation of the test solution was calculated from the above equation (1). % By weight). The results are shown in Table 12 below. In Table 12, "Acinetobacter guillouiae 1-2350 strain" is described as "1-2350 strain" and "Acinetobacter baumannii (Acinetobacter baumannii) S30 strain" is described as "S30 strain", respectively. I do.

  As shown in Table 12, the 1-2350 strain according to the present invention exhibited a high mineral oil reduction rate at all mineral oil concentrations (1,000 to 10,000 ppm) as compared to Acinetobacter baumannii S30 strain. Show. Therefore, the oil-decomposing microorganism according to the present invention can be expected to be able to purify wastewater even in a water environment having a wide range of mineral oil concentrations.

Claims (6)

An oil-degrading microorganism belonging to Acinetobacter guillouiae and having the following mycological properties.
  The oil-decomposing microorganism according to claim 1, which has a 16S rDNA base sequence represented by SEQ ID NO: 1.   The oil-degrading microorganism according to claim 1 or 2, wherein the mineral oil reduction rate at at least one mineral oil concentration in the range of 1,000 to 10,000 ppm is more than 40% by weight.   The oil-decomposing microorganism according to any one of claims 1 to 3, which is specified by Acinetobacter guillouiae strain 1-2350 (accession number NITE BP-02734).   A method for decomposing oil, comprising a step of contacting the oil-decomposing microorganism according to any one of claims 1 to 4 with oil.   An oil-decomposing agent comprising the oil-decomposing microorganism according to any one of claims 1 to 4.