TWI756538B - oil-decomposing microorganisms - Google Patents

Abstract

The invention provides a microorganism with good decomposing effect on mineral oil. The present invention relates to a microorganism belonging to Acinetobacter soli and having good decomposing property for mineral oil.

Description

oil-decomposing microorganisms

The present invention relates to a novel microorganism belonging to Acinetobacter soli and having excellent oil-decomposing properties, especially mineral oil-decomposing properties. The present invention more relates to a method for decomposing oil, particularly mineral oil, and oil decomposers (especially mineral oil decomposers) using the microorganism.

Inversely proportional to the development of the industry, the pollution of the natural environment such as the sea, rivers, and soil continues to progress. The natural environment is a living place for many living things including human beings, and in recent years, concerns about pollutants have become more severe. In particular, mineral oils such as petroleum have increased in recent years due to oil spills caused by oil tankers hitting rocks and soil contamination at gasoline filling stations, and the demand for safe removal has increased. Although there are chemical and physical methods as methods for removing mineral oil, bioremediation techniques for purifying environmental pollution by decomposing pollutants by the action of microorganisms and the like are attracting attention. Biological remediation using microorganisms has applicability to a variety of pollutants, and theoretically requires less energy. Generally speaking, purification costs are likely to be reduced, and it is presumed to be one of the main technologies in the future. For example, Japanese Patent Laid-Open No. 2006-296382 reports that Acinetobacter baumannii KIM30135 strain can decompose mineral oils such as engine oil even in the absence of carbon sources other than oil.

However, the microorganisms described in Japanese Patent Application Laid-Open No. 2006-296382 have a problem that the mineral oil decomposability is insufficient.

Therefore, this invention was made in view of the above-mentioned facts, and an object is to provide the microorganism which is excellent in oil (especially mineral oil) decomposing property.

The inventors of the present invention have made intensive studies in order to solve the above-mentioned problems. As a result, the inventors found that the above-mentioned problems can be solved by an oil-decomposing microorganism belonging to Acinetobacter soli and exhibiting specific microbiological properties, and the present invention was completed.

Hereinafter, embodiments of the present invention will be described. In addition, the present invention is not limited to the following embodiments.

In the present specification, the range is expressed as "X to Y", which means that X and Y are included, and "X or more and Y or less". In addition, unless otherwise stated, the measurement of operation, physical properties, etc. is carried out under the conditions of room temperature (20 to 25° C.)/relative humidity of 40 to 50% RH.

>Oil-decomposing microorganisms> One aspect of the present invention is to provide an oil-decomposing microorganism (particularly, a mineral oil-decomposing microorganism) belonging to Acinetobacter soli and exhibiting the following microbiological properties. One aspect of the present invention is to provide an oil-decomposing microorganism (particularly, a mineral oil-decomposing microorganism) belonging to Acinetobacter soli and having the 16S rDNA nucleotide sequence shown in SEQ ID NO: 1, exhibiting the following microbiological properties. The microorganisms relevant to the present invention are excellent in oil (especially mineral oil) decomposing properties. In this specification, oil-decomposing microorganisms belonging to Acinetobacter soli and exhibiting the following microbiological properties are sometimes simply referred to as "oil-decomposing microorganisms related to the present invention" or "microorganisms related to the present invention". .

+：陽性、 -：陰性。[Table 1]

+: Positive, -: Negative.

+：陽性、 -：陰性。[Table 2]

+: Positive, -: Negative.

+：陽性、 -：陰性、 w：微弱反應。[table 3]

+: Positive, -: Negative, w: Weak reaction.

As a specific example of the above-mentioned oil-decomposing microorganism belonging to Acinetobacter soli, a strain isolated by the following screening method will be described in detail. In addition, the 1-1357 strain related to the present invention was isolated from the soil of the coast of Shimane Prefecture, Japan by the following screening method.

[filter] An appropriate amount of soil, waste liquid, sewage from waste oil treatment facilities, or samples collected from river water in the coastal area of Shimane Prefecture, Japan was added to a liquid medium for primary screening prepared by the following method, and cultured at 30°C for 1 week. 100 μL of the cultured medium was further inoculated into 140 mL of the liquid medium for primary screening, and cultured at 30° C. for another week. In addition, in this specification, the liquid medium for primary screening produced by the following method is also abbreviated as "liquid medium for primary screening".

The liquid medium for primary screening was prepared by dissolving components other than mineral oil in pure water so as to have the composition shown in Table 4 below, so that the final concentration of mineral oil was 0.5w/v% (0.5g/100mL). It is added in the form of high temperature and high pressure sterilization and then prepared. And, mineral oil is obtained by mixing diesel engine oil, gasoline engine oil, engine oil, gear oil, and cutting oil in an equal weight ratio (that is, the mixing ratio of diesel engine oil: gasoline engine oil: engine oil: gear oil: cutting oil (weight ratio)=1:1:1:1:1) mixed and prepared. Here, the trade name of Fuji Kosan Co., Ltd.: CF Eco Diesel is used as diesel engine oil, and the trade name of Sanyou Chemical Industry Co., Ltd.: 4-stroke gasoline engine oil for general purpose engine is used as gasoline engine oil. Trade name from AZ CO., LTD.: Motor oil, trade name from Trusco Nakayama Corporation: Industrial gear oil ISO VG220 as gear oil, and trade name from AZ CO., LTD.: Tap & Drill Oil cutting Oil as cutting oil. In addition, the pH of the liquid medium for primary screening was not adjusted.

[Table 4] Table 4: Liquid medium for primary screening (without pH adjustment).

100 mL of the 104 ^- fold diluted culture solution after the primary screening was spread on the agar medium for secondary screening prepared by the following method, and cultured at 30°C for 1 week. After culturing, the strains confirmed to grow on agar were isolated. In addition, in the present specification, the agar medium for secondary screening produced by the following method may also be referred to as "agar medium for secondary screening".

The agar culture medium for secondary screening is obtained by dissolving mineral oil and components other than agar in pure water so that the final concentration of mineral oil is 0.5 w/v% (0.5 g /100mL) and agarose so that the final concentration of the agar is 2.0w/v% (2.0g/100mL), add mineral oil and agar, sterilize at high temperature and autoclave, divide the package appropriately, and then solidify and prepare. In addition, in the following Table 5, as mineral oil, the same mineral oil used for the liquid medium for primary screening was used. In addition, the pH of the agar medium for secondary screening was not adjusted.

[Table 5] Table 5: Agar medium for secondary screening (without pH adjustment).

Next, 0.05 g of mineral oil was added to 5 mL of the liquid medium for tertiary screening prepared by the following method, and the sterilized test solution (pH 6.0) was prepared (mineral oil concentration: 1% (w/v)). Each isolated strain obtained by the above secondary screening was inoculated into the test solution prepared by the above method using an inoculation rod, and was shaken and cultured at 30° C. for 24 hours (rotation frequency: 140 rpm). In addition, in this specification, the liquid medium for tertiary screening prepared by the following method is also abbreviated as "liquid medium for tertiary screening". In addition, the test liquid prepared above is also abbreviated as "test liquid" in this specification.

The liquid medium for tertiary screening was prepared by dissolving each component in pure water, adjusting to pH 6.0 with hydrochloric acid, and autoclaving the liquid medium so as to have the composition shown in Table 6 below. In addition, in the following Table 6, as the mineral oil, the same mineral oil used for the liquid medium for primary screening was used.

[Table 6] Table 6: Liquid medium (pH=6.0) for three screenings.

After culturing, n-hexane extract was prepared according to JIS K0102:2016 revision (industrial drainage test method). The n-hexane extract was regarded as the remaining amount of mineral oil, and the amount of The mineral oil reduction rate was obtained by the above equation (1). As a result, a strain with a high reduction rate of mineral oil can be isolated.

× 100。[Formula 1] Mathematical formula (1): Mineral oil reduction rate (% by weight)=

×100.

As a result, the bacterial strain with the highest reduction rate of mineral oil was isolated from the soil outside Shimane Prefecture. About this isolated strain (isolated microorganism), bacterial species identification (identification) was performed as follows.

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

1.16S rDNA base sequence analysis The 16S rDNA nucleotide sequence of the isolated microorganism was analyzed according to the following procedure guidelines (operation from PCR amplification to cycle sequencing). In addition, the base sequence is determined by visually confirming the raw data (electropherogram) from the sequencer, and performing calibration.

[Chemical 1] (16S rDNA base sequence analysis procedure guidelines) DNA extraction: achromopeptidase (achromopeptidase, Wako Pure Chemical Industries, Japan) PCR amplification: PrimeSTAR HS DNA Polymerase (Takara Bio, Japan) Cycle sequencing: BigDye Terminator v3.1 Cycle Sequencing Kit (provided by Biosystems, USA) Using primers ¹⁾ : PCR amplification: 9F, 1510R Sequencing: 9F, 515F, 785F, 536R, 802R, 1510R Sequencing: ABI PRISM 3130 ×l Genetic Analyzer System ( Provided by Biosystems, USA) Base sequence determination: ChromasPro 1.7 (Technelysium, AUS) BLAST sequence similarity search ²⁾ : Software TechnoSuruga Laboratory Microbial Species Identification System (TechnoSuruga Laboratory, Japan) Database DB-BA12.0 (TechnoSuruga Laboratory, Japan) , Japan) International Base Sequence Database (DDBJ/ENA(EMBL)/GenBank) Simple Molecular System Analysis: Inference of Family Trees: Neighbor Binding Method ³⁾ Base Substitution Model: Kimura-2-parameter ⁴⁾ Reliability of Trees Degree evaluation: boot-pulling and re-extraction method ⁵⁾ (1,000 repetitions) 1) Nakagawa Yasuyoshi , Kawasaki Hiroko (2001). Gene analysis method 16S rRNA gene base sequence determination method. Classification and identification of actinomycetes. pp. 88-117 Japan Actinomycetes Society ed. Tokyo: Japan Society Affairs Center 2) Altschul SF, Madden TF, Schäffer AA, Zhang J., Zhang Z., Miller W., & Lipman DJ, (1997). Gapped BLAST and PSI -BLAST: a new generation of protein database search programs. Nucleic Acids Res 25, 3389-3402. 3) Saitou N. & Nei M. (1987) . The neighbor-joining method: a new method for reconstruc ting phylogenetic trees. Mol Biol Evol 4 , 406-425 4) Kimura M. (1980). A simple method for estimating evolutionary rates of base substitutions through comparative studies of nucleotide sequences. J Mol Evol 16, 111-120. 5) Felsenstein J. (1985). Confidence limits on phylogenies: an approach using the bootstrap. Evolution 39, 783-791.

The 16S rDNA nucleotide sequence of the isolated microorganism identified above is shown in the following SEQ ID NO: 1.

[Chemical 2] SEQ ID NO: 1 (base sequence of 16S rDNA)

The nucleotide sequence of the 16S rDNA of the isolated microorganism was compared with that of Acinetobacter The nucleotide sequence of the 16S rDNA of the reference strain B1 ^T (Accession No. EU290155) of soli) showed a sequence similarity of 100% (see Table 7 below). In addition, according to the results of the BLAST sequence similarity search of the International Base Sequence Database (DDBJ/ENA (EMBL)/GenBank), the base sequence of the 16S rDNA of the isolated microorganism was compared with that of the 16S rDNA of Acinetobacter soli. The nucleotide sequence also showed a high sequence similarity of more than 99.8% sequence similarity (refer to Table 8 below). Table 7 shows the results of the sequence similarity search of the DNA database DB-BA12.0 for microbial species identification. In addition, Table 8 shows the results of the sequence similarity search with respect to the International Base Sequence Database (DDBJ/ENA (EMBL)/GenBank). In addition, in the following table, the so-called "BSL" refers to the biosafety level (biosafety level), and the biosafety level (BSL) is based on the "BSL level of pathogenic bacteria" (level 1: There is no possibility of causing human disease, or the possibility of causing veterinary important diseases in animals (including opportunistic infections), level 2: although pathogenic to humans or animals, but to laboratory staff, regional settlements, livestock , the environment, etc., will not become a major disaster person, although there is a possibility of serious infection when exposed in the laboratory, but there are effective treatment methods and prevention methods, and the possibility of transmission is low, level 3: Although when Causes serious illness when infecting humans, but has a low probability of spreading to other individuals). In the table below, opportunistic pathogens are designated "BSL1*". In addition, gray-scale dots represent sequence data provided for easy molecular system analysis.

[Table 7] Result of BLAST sequence similarity search for DB-BA12.0 of the isolated microorganism (sequence similarity to the upper 30-nucleotide sequence)

[Table 8] Result of BLAST sequence similarity search on the international base sequence database of isolated microorganisms (sequence similarity to the upper 30 base sequence)

In addition, based on the nucleotide sequence obtained by the sequence similarity search with respect to the DNA database DB-BA12.0 (Techno Suruga Laboratory Co., Ltd.) for identification of microorganisms, the results of analyzing the molecular family tree showed that the isolated microorganisms were included Within the cluster formed by the genus Acinetobacter, it is in the same molecular phylogeny position as the reference strain B1 ^T (Accession No. EU290155) of Acinetobacter soli (see below). the genealogy tree). In the following pedigree tree, the upper left line represents the scale bar, the numbers on the branches of the pedigrees represent the bootstrap value, the T at the end of the strain name represents the reference strain (Type Strain) of the species, and the BSL represents the reference strain (Type Strain) of the species. Biosafety protection level (marked BSL1* (opportunistic pathogens) or above).

[Chem.3] Simple molecular family tree based on 16S rDNA base sequences of isolated microorganisms

From the above, it is inferred that the above-mentioned isolated microorganisms belong to Acinetobacter soli.

2. The nature of microbiology The microbiological properties of the strains (isolated microorganisms) isolated by the above screening are shown below. Morphological observation was performed using an optical microscope (BX50F4, Olympus, Japan). Gram staining was performed using Favor-G "Nissui" (Nissui Pharmaceutical, Japan). Tests for catalase reaction, oxidase reaction, acid/gas production from glucose, oxidation/fermentation (O/F) of glucose, 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).

+：陽性、 -：陰性。[Table 9]

+: Positive, -: Negative.

From the results of the above Table 9, the strains (isolated microorganisms) isolated by the above-mentioned screening were Gram-negative coccobacilli with no motility, glucose showed oxidation, catalase reaction showed positive, and oxidase reaction showed negative . These characteristics are considered to be consistent with those of Acinetobacter.

Next, using API (registered trademark) 20NE and API (registered trademark)-ZYM (both bioMerieux, France), the following items were tested according to the manufacturer's procedure guidelines. The results are shown in Table 10.

*生化試驗、**微生物利用性試驗。[Table 10]

*Biochemical test, **Microbial availability test.

In addition, based on the technical cooperation matters between Techno Suruga Laboratory Co., Ltd. and NCIMB Ltd. in the United Kingdom and the known technology, the following items are tested. The results are shown in Table 11.

+：陽性、-：陰性、 w：微弱反應。[Table 11]

+: Positive, -: Negative, w: Weak reaction.

One aspect of the present invention provides an oil-decomposing microorganism (particularly, a mineral oil-decomposing microorganism) belonging to Acinetobacter soli and exhibiting the above-mentioned microbiological properties. In addition, one embodiment of the present invention provides an oil-decomposing microorganism (particularly, a mineral oil-decomposing microorganism) belonging to Acinetobacter soli, exhibiting the above-mentioned microbiological properties, and having the 16S rDNA base sequence shown in SEQ ID NO: 1.

From the results of the above Table 10, the strains (isolated microorganisms) isolated by the above screening did not reduce nitrate, did not produce indole, did not show arginine dihydrolase activity, and showed urease activity, although for glucose, gluconic acid Potassium and n-decanoic acid showed microbial availability, but L-arabinose and D-mannose did not show microbial availability. In addition, from the results of Table 11 above, the strains (isolated microorganisms) isolated by the above screening showed alkaline phosphatase, esterase (C4) (esterase (substrate: 2-naphthyl butyrate)), esterase lipase Activity of enzyme (C8) (esterase lipase (substrate: 2-naphthyl caprylate)) and lipase (C14) (lipase (substrate: 2-naphthyl myristate)). Furthermore, the strains (isolated microorganisms) isolated by the above screening were confirmed for their growth ability at 10°C and at 41°C. Its characteristics are almost the same as those suggested to be assigned to Acinetobacter soli in the above "1.16S rDNA nucleotide sequence analysis", but the characteristics of alkaline phosphatase and acid phosphatase activity shown are similar to those of Acinetobacter soli (Acinetobacter soli). Characteristics of Acinetobacter Soli) (Kim D., Baik KS, Kim MS, Park SC, Kim SS, Rhee MS, Kwak YS & Seong CN (2008), Acinetobacter Soli Sp. nov., isolated from forest soil. J. Microbiol. , 2008, 46, 396-401) different.

Therefore, since it was understood that the isolated strain (isolated microorganism) had different properties from the previously known Acinetobacter soli, it was judged to be a novel microorganism, and the present strain was named as Acinetobacter soli 1- 1357 (hereinafter referred to as "1-1357 strains"). In addition, on March 13, 2018, this 1-1357 strain was deposited in the Patent Microorganism Depositary (NPMD) (NITE Patent Microorganisms Depositary, NPMD) (Japan National 〒 292-0818 Kazusakamatari 2, Kisarazu City, Chiba Prefecture) on March 13, 2018. -5-8 Room 122), its deposit number is NITE BP-02665. That is, one aspect of the present invention relates to an oil-decomposing microorganism (in particular, a mineral oil-decomposing microorganism), specifically Acinetobacter soli strain 1-1357 (Accession No. NITE BP-02665).

Oil-decomposing microorganisms (especially strains 1-1357) exhibiting the above-mentioned microbiological properties have excellent decomposing power of mineral oil (hydrocarbon) and have water quality with a wide range of mineral oil concentrations including unsaturated hydrocarbons, polycyclic aromatics, etc. Characteristics of purifying drainage in the environment. Therefore, oil-decomposing microorganisms (especially strains 1-1357) exhibiting the above-mentioned microbiological properties are added to industrial waste water containing mineral oil, a hazardous substance removal facility of a facility for treating industrial waste water containing mineral oil, and installed in Degreasing wells and gasoline wells in maintenance factories, parking lots, car washes, gasoline gas stations, etc., as well as seas, rivers, and soils where mineral oil leaks, can effectively decompose mineral oil and prevent and suppress pollution of the natural environment. In addition, the oil-decomposing microorganisms (especially strain 1-1357) exhibiting the above-mentioned microbiological properties are microorganisms originally existing in nature. Therefore, even if the microorganisms related to the present invention are added, there is little or no adverse effect on the natural environment. Therefore, according to the microorganisms related to the present invention, mineral oil can be safely removed. Furthermore, since there is no need to additionally recover the microorganisms related to the present invention after the mineral oil is decomposed, there is also an advantage that purification costs are reduced.

Here, the mineral oil may be unused or after use. Although not limited to the following, specific examples of such mineral oils include diesel engine oil, gasoline engine oil, engine oil, gear oil, cutting oil, and the like. Oil-decomposing microorganisms (especially, mineral oil-decomposing microorganisms such as strain 1-1357) exhibiting the above-mentioned microbiological properties show high oil-decomposing energy (especially mineral oil-decomposing energy) for such a wide range of mineral oils. In this specification, "oil" refers to lubricating oil that reduces friction caused when metals come into contact with each other, and corresponds to machine oil, turbine oil, and spindle oil depending on the part and application of the machine used. (spindle oil), generator oil (dynamo oil), cylinder oil (cylinder oil), bearing oil (bearing oil), refrigerating machine oil (refrigerating machine oil), hydraulic oil (hydraulic fluid), gear oil (gear oil), compressor Oil (compressor oil), sliding face oil, etc. In this manual, "gear oil" is mainly used for lubricating and cooling the engines and gears of automobiles and motorcycles. Gear oils may contain antioxidants, anti-wear agents, detergent dispersants, tackifiers, defoamers, and pour point depressants in order to improve the intended properties. etc. additives. In addition, in this specification, "cutting oil" refers to a cutting oil that plays a role of lubricating, cooling, and removing cutting in processes such as cutting, turning, milling, and tapping.

The oil-decomposing microorganisms (especially strain 1-1357) related to the present invention belonging to Acinetobacter soli exhibiting the above-mentioned microbiological properties show high decomposing energy for a wide range of concentrations of mineral oil. Specifically, the oil-decomposing microorganism of the present invention has a mineral oil reduction rate of more than 50% by weight, preferably 60% by weight, in at least one mineral oil concentration in the range of 1,000 to 20,000 ppm measured by the method described later. Above, more preferably 70% by weight or more, particularly preferably more than 80% by weight (upper limit: 100% by weight). Preferably, the oil-decomposing microorganism related to the present invention has a mineral oil reduction rate of 50% by weight or more, more preferably 60% by weight or more (upper limit: 100 weight%). In addition, in this specification, "mineral oil reduction rate in the range of 1,000 to 20,000 ppm mineral oil concentration measured by the following method" is also simply referred to as "mineral oil reduction rate". That is, in a preferred form of the present invention, the oil-decomposing microorganism according to the present invention has a mineral oil reduction rate of 60% by weight or more ( It is more preferably 70% by weight or more, particularly preferably more than 80% by weight) (upper limit: 100% by weight). Further, a preferred form of the present invention is the oil-decomposing microorganism according to the present invention, wherein the mineral oil reduction rate is 50% by weight or more (more preferably 60% by weight) in the total mineral oil concentration in the range of 1,000 to 20,000 ppm measured by the following method. % by weight or more) (upper limit: 100% by weight). In general, when oil-decomposing microorganisms are cultured with oil for a long time, the oil is decomposed slowly but the oil is decomposed (the reduction rate of mineral oil increases). However, for example, when microorganisms are added to a hazardous substance removal facility or the like, they are sequentially discharged from the hazardous substance removal facility. Usually, microorganisms are supplied to the hazardous substance removal facility about every 1 to 3 days. On the other hand, the oil-decomposing microorganisms related to the present invention show a high reduction rate of mineral oil in a short period of so-called 24 hours, as is clear from the following method, and can decompose a considerable amount of mineral oil in a harmful substance removal facility, Good in application. In addition, in this specification, "mineral oil reduction rate" is the value measured by the following method.

[Evaluation of Mineral Oil Reduction Effect (Measurement of Mineral Oil Reduction Rate)] In this specification, the mineral oil reduction effect was evaluated based on the mineral oil reduction rate by the following method. That is, mineral oil was added to the aseptically treated medium (5 mL) for oil decomposition evaluation as the above-mentioned liquid medium for three screenings so that the concentration of mineral oil was 1,000 to 20,000 ppm, to prepare a test solution (pH 6.5. 0)(Oil content: 0.1~2%(w/v)(1g/L~20g/L)). For this test solution, the microorganisms to be cultured are inoculated on a plate medium (eg, agar medium for secondary screening), and cultured with shaking (140 rpm) at an arbitrary temperature zone (eg, 30° C.) for 24 hours. With the inoculation rod, the amount of bacteria inoculated is controlled to be about one inoculation rod. As the microorganisms inoculated in the test solution, those pre-cultured in a liquid medium for tertiary screening or the like can also be used. By performing pre-culture, the amount of bacteria to be inoculated can be easily adjusted. When using precultured microorganisms, 1 mL of the test solution was inoculated so as to be 1.5×10 ⁶ CFU/ml. The culturing temperature may be set according to the temperature zone where the oil content of the cells is decomposed and the utilization rate of microorganisms is high, but it is, for example, 10°C or more and less than 45°C, preferably 15 to 35°C.

After culturing, n-hexane extract was prepared according to JIS K0102:2016 revision (industrial drainage test method). The n-hexane extract is regarded as the remaining amount of mineral oil, the amount of mineral oil (g) and the remaining amount (g) of mineral oil added during the preparation of the test solution (the amount of n-hexane extract (g)), through the following Mathematical formula (1) calculates the mineral oil reduction rate.

× 100。[Numerical 2] Mathematical formula (1): Mineral oil reduction rate (% by weight)=

×100.

[Cultivation of Microorganisms] Any method for culturing oil-decomposing microorganisms belonging to Acinetobacter soli (hereinafter simply referred to as "microorganisms") according to the present invention may be used as long as the microorganisms can grow and proliferate. For example, the medium used for culturing the microorganism of the present invention may be either a solid or a liquid medium, as long as it contains a carbon source, an appropriate amount of nitrogen source, inorganic salts, and other nutrients available to the microorganism to be used. The medium may be either a synthetic medium or a natural medium. Typically, the medium contains carbon sources, nitrogen sources, and inorganics.

The carbon source that can be used for culturing the microorganism of the present invention is not particularly limited as long as it is a carbon source that can be used by the strain to be used. Specifically, considering the microbial availability of microorganisms, glucose, fructose, cellobiose, raffinose, xylose, galactose, sorbose ( sorbose), glucosamine, ribose, rhamnose, sucrose, trehalose, α-methyl-D-glucoside, salicin, melibiose, lactose , melezitose, inulin, erythritol, ribitol, xylitol, sorbitol, glucitol, galactitol, inositol (inositol), sorbitol (sorbitol), amygdalin (amygdalin), starch, starch hydrolyzate, sugar, molasses (treacle), waste molasses (molasses) and other sugars, wheat, rice and other natural products, glycerin, methanol Alcohols such as ethanol, acetic acid, phenyl acetate, lactic acid, succinic acid, gluconic acid, capric acid, adipic acid, pyruvic acid, citric acid, malic acid and other organic acids and their salts, hexadecanoic acid, etc. alkane (hexadecane) and other hydrocarbons. The above-mentioned carbon sources are appropriately selected in consideration of the availability of microorganisms according to the microorganisms to be cultivated. For example, when using the 1-1357 strain, among the above carbon sources, glucose, gluconic acid, capric acid, adipic acid, malic acid, citric acid, phenyl acetate and their salts (eg, sodium salt, potassium salt), etc. are used. More preferably, glucose is used. In addition, one type or two or more types of the above-mentioned carbon sources can be selected and used. The above-mentioned carbon source is appropriately selected in consideration of the availability of microorganisms according to the microorganisms to be cultured.

In addition, as a nitrogen source that can be used in the culture of the oil-decomposing microorganism according to the present invention, meat extract, fish meat extract, peptone, polypeptone, trypsin, yeast extract, wheat Sprout extract, soybean hydrolyzate, soybean powder, casein, milk casein, various amino acids such as casamino acid, glycine, glutamic acid, aspartic acid, corn Organic nitrogen sources for corn steep liquor, hydrolysates of other animals, plants, microorganisms, etc.; ammonium salts of ammonia, ammonium nitrate, ammonium sulfate, ammonium chloride, etc., nitrates such as sodium nitrate, sodium nitrite, etc. Inorganic nitrogen sources such as nitrite, urea, etc. The above-mentioned nitrogen source is appropriately selected in consideration of the availability of microorganisms according to the microorganisms to be cultured. For example, when strain 1-1357 is used, among the above nitrogen sources, trypsin, yeast extract, ammonium chloride and the like are preferably used. In addition, one type or two or more types of the above-mentioned nitrogen sources can be selectively used.

Examples of inorganic substances usable in the present invention include magnesium, manganese, calcium, sodium, potassium, copper, iron, zinc, and the like, phosphates, hydrochlorides, sulfates, acetates, carbonates, chlorides, and the like. Halides, etc. The above-mentioned inorganic substances are appropriately selected according to the microorganisms to be cultured. In addition, one kind or two or more kinds of the above-mentioned inorganic substances can be selected and used. In addition, a surfactant and the like may be added to the medium as necessary.

To make the microorganisms related to the present invention decompose and utilize oil (especially mineral oil) more effectively, or to maintain the oil decomposition and microorganism utilization (especially mineral oil decomposition, microorganism utilization) of the microorganisms related to the present invention, It is better to add oil (especially mineral oil) to the medium. Examples of the oil include mineral oils (hydrocarbons) such as the above-mentioned diesel engine oil, gasoline engine oil, motor oil, gear oil, and cutting oil; edible oils and fats, industrial oils and fats, and fatty acids. The amount of oil to be added is not particularly limited, and is appropriately selected in consideration of the ability to decompose oil, etc., depending on the microorganism to be cultured. Specifically, the oil is preferably added at a concentration of 0.5 to 50 g, more preferably 1 to 30 g, and particularly preferably 5 to 15 g, in 1 L of the medium. With this amount of addition, microorganisms can maintain a high oil-decomposing ability. Especially when using mineral oil, add mineral oil (diesel engine oil: gasoline engine oil: motor oil: gear oil: cutting oil = 1:1:1:1:1 (w/w/w/w/w)) in the medium It is preferable to add it at 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) per liter. With this amount of addition, microorganisms can maintain high mineral oil decomposition and microbial utilization. In addition, the oil (mineral oil) may be added alone or in the form of a mixture of two or more.

Cultivation of microorganisms can be carried out by a general method. For example, depending on the type of microorganisms, the culture is carried out under aerobic conditions or anaerobic conditions. In the former case, the culture of microorganisms can be performed by shaking or aeration stirring. Furthermore, the microorganisms can be cultured either continuously or in batches. The culturing conditions are appropriately selected depending on the composition of the medium and the culturing method, and are not particularly limited as long as the microorganisms of the present invention can proliferate, and are appropriately selected according to the type of microorganisms to be cultured. Usually, the culture temperature is preferably 10°C or more and less than 45°C, more preferably 15 to 35°C. In addition, it is preferable that the pH of the appropriate medium is 5 to 8, more preferably 5.5 to 7.5, during the culture. The culture time is also not particularly limited, and varies depending on the type of microorganism to be cultured, the amount of the medium, the culture conditions, and the like. Usually, the culturing time is preferably 16 to 48 hours, more preferably 20 to 30 hours.

>How to break down oil> One aspect of the present invention is to provide a method for decomposing oil, comprising the step of contacting the oil-decomposing microorganism of the present invention with oil, or a method for treating waste water, comprising causing the oil-decomposing microorganism to be associated with the oil-decomposing microorganism of the present invention in the drainage water containing oil. Contact steps. A preferred form of the present invention is to provide a method for decomposing mineral oil, which includes the step of contacting the oil decomposing microorganism of the present invention with mineral oil. Furthermore, a preferred aspect of the present invention is to provide a method for treating waste water including a step of contacting the waste water containing mineral oil with the oil-decomposing microorganism of the present invention. A particularly preferred form of the present invention is a method for decomposing mineral oil, comprising the step of contacting Acinetobacter soli strain 1-1357 (Accession No. NITE BP-02665) with mineral oil. Furthermore, a particularly preferred embodiment of the present invention is a method for treating waste water, comprising the step of contacting waste water containing mineral oil with Acinetobacter soli strain 1-1357 (Accession No. NITE BP-02665). Here, the objects that the oil-decomposing microorganisms of the present invention come into contact with are not particularly limited as long as they contain oil. In addition to the above-mentioned mineral oil itself, there are also industrial waste water containing oil (especially mineral oil), which is used for treatment Drainage of hazardous substance removal facilities (drainage treatment facilities) of industrial drainage facilities containing oil (especially mineral oil), oil removal wells, gasoline wells installed in maintenance factories, parking lots, car washes, gasoline filling stations, etc. Seas, rivers, soils, etc., from which oil (especially mineral oil) leaks. The oil-decomposing microorganisms (especially strain 1-1357) related to the present invention have high decomposition energy for oil, especially mineral oil (hydrocarbon), and can effectively remove oil in a water quality environment with a wide range of oil concentrations. Therefore, according to the method of the present invention, by adding the oil-decomposing microorganisms (especially strains 1-1357) related to the present invention in drainage, drainage treatment facilities, oil removal wells, gasoline wells, and seas where oil (oil) leaks , rivers, soil, etc., can effectively decompose oil (especially mineral oil), can effectively prevent and inhibit the pollution of the natural environment. In addition, the oil-decomposing microorganisms (especially strain 1-1357) related to the present invention are microorganisms originally existing in nature. Therefore, the method of the present invention has little or no adverse effect on the natural environment (oil can be safely removed). Furthermore, since there is no need to additionally recover the microorganisms relevant to the present invention after the oil (especially mineral oil) is decomposed, the method of the present invention can reduce the purification cost and is also economically advantageous.

Here, the method of contacting the oil-decomposing microorganism of the present invention with oil (oil) (especially mineral oil) is not particularly limited as long as the oil-decomposing microorganism of the present invention can be brought into contact with the oil. For example, the oil-decomposing microorganisms of the present invention can be added directly to desired locations (eg, mineral oil, mineral oil-containing drainage, drainage treatment facilities, oil removal traps, gasoline traps, and seas, rivers, and Soil, etc.; hereinafter, collectively referred to as "drainage, etc."). Alternatively, a carrier or the like in which the oil-decomposing microorganisms related to the present invention are immobilized may be provided in a channel (pipeline) or a storage tank, and the microorganisms may be contacted by oil flowing therethrough.

The oil-decomposing microorganisms used in the method according to the present invention can be in various forms such as suspended in the culture solution, recovered as a solid from the culture solution, dried, and immobilized on a carrier. Drainage, etc. The oil-decomposing microorganisms suspended in the culture solution, recovered as a solid content from the culture solution, or dried are added to drain water or the like, and are brought into contact with the oil contained therein. The oil-decomposing microorganisms immobilized on the carrier may be added to the drainage etc. as described above, or the carrier immobilized with the oil-decomposing microorganisms may be placed in a trap, and the drainage or the like may be passed through the carrier immobilized with the microorganisms. The oil-decomposing microorganisms come into contact with drainage, etc. By arranging the oil-decomposing microorganisms immobilized on the carrier in the trap, it is possible to prevent the oil-decomposing microorganisms from flowing out together with the above-mentioned drainage and the like, thereby reducing the number of bacteria.

When oil-decomposing microorganisms are used which are recovered as a solid from the culture solution, the recovery method may employ any means known in the art. For example, the solid content can be recovered by subjecting the culture solution of the oil-decomposing microorganisms cultivated by the above method to centrifugation, filtration, or the like to separate the solid and the liquid. If the solid content is dried (eg, freeze-dried), oil-decomposing microorganisms in a dry state can be obtained.

When oil-decomposing microorganisms immobilized on a carrier are used, the carrier for immobilizing oil-decomposing microorganisms is not particularly limited as long as the microorganisms can be immobilized, and the same carriers as those generally used for immobilizing microorganisms can be used. , or appropriately modified. For example, a coating on a colloidal substance such as alginic acid, polyvinyl alcohol, gellan gum, agarose, cellulose, dextran and the like can be used Buried fixation method, adsorption fixation method on the surface of glass, activated carbon, polystyrene, polyethylene, polypropylene, wood, silica gel, etc.

In addition, the method of immobilizing the oil-decomposing microorganism on the carrier is not particularly limited, but the same method as the immobilization method of a general microorganism, or one modified appropriately can be used. For example, an immobilization method in which a culture solution of microorganisms is poured into a carrier, an immobilization method in which a culture solution of microorganisms is poured into a support by making the support under reduced pressure using an aspirator, and A method of pouring a microbial culture solution into a mixture of a sterilized medium and a carrier, shaking culture, and naturally drying the carrier taken out from the mixture, etc.

In the method according to the present invention, the amount of bacteria to be added can be arbitrarily set when the oil-decomposing microorganisms are added and brought into contact with waste water or the like. The amount of bacteria added to the drainage etc. is not particularly limited, but it is, for example, 1×10 ⁴ to 1×10 ¹² CFU, preferably 1×10 ⁵ to 1×, relative to 1 g of the oil content contained in the drainage etc. 10 ¹¹ CFU. Or, relative to 1 g of oil contained in the drainage etc., for example, it is 0.1 mg to 5 g (dry cell weight), preferably 1 mg to 1.5 g (dry cell weight), more preferably 10 mg to 150 mg ( dry cell weight). In addition, with respect to the drainage etc. in the well, for example, the amount is set to be 1×10 ⁶ to 1×10 ¹² CFU/L, more preferably 1×10 ⁷ to 1×10 ¹¹ CFU/L. Alternatively, it is 10 mg to 15 g (dry cell weight)/L, preferably 0.1 g to 1.5 g (dry cell weight)/L, relative to drainage or the like. In addition, when using a combination of two or more types of microorganisms, the added amount of the above-mentioned microorganisms refers to the total amount thereof. In addition, as the microorganisms added to drainage or the like, precultured ones can also be used. By pre-cultivation, the amount of bacteria to be inoculated can be easily adjusted.

When the drainage or the like is discharged to the external environment, the oil-decomposing microorganisms that are not immobilized on the carrier are discharged out of the system along with the drainage or the like. Therefore, in the present invention, it is preferable to periodically add oil-decomposing microorganisms to drainage or the like. The addition interval is not particularly limited, but for example, it is preferably added at an interval of once/3 hours, once/24 hours, or once every 2 to 3 days. The method of adding is not particularly limited, but when the drainage or the like continuously flows into the system, it may be added to the drainage or the like, or it may be directly added to the drainage or the like in the system.

As described above, the microorganisms related to the present invention exhibit high decomposition energy for a wide range of concentrations of mineral oil. Therefore, the content of oil in drain water and the like is not particularly limited. The oil contained in drain water etc. is not limited to 1 type, and may be 2 or more types.

In the method of the present invention, in addition to the oil-decomposing microorganisms according to the present invention, other components may be added to drainage and the like from the viewpoint of more effectively reducing oil content. Other components include, for example, other microorganisms capable of coexisting with the oil-decomposing microorganisms of the present invention, lipases, phospholipases, pH adjusters, mineral oil adsorbents, surfactants, and the like.

Examples of other microorganisms that can coexist with the oil-decomposing microorganisms of the present invention include, for example, Yarrowia, Candida, Pichia, and Hansenula Genus Hansenula, Saccharomyces, Kluyveromyces, Trichosporon, Bacillus, Lactobacillus, Aspergillus ), Rhodococcus, Enterococcus, Sphingomonas, Enterobacter, Burkholderia, Pseudomonas Genus, Penicillium, Staphylococcus, Rhizopus, Rhizobium, Acinetobacter, Alcaligenes, Fusarium genus, Serratia, Tetrasphaera, Humicola, Stenotrophomonas and the like. Such microorganisms can be obtained from species conservation centers of ATCC, NBRC, DSMZ, etc. Among these microorganisms, oil-decomposing microorganisms of the genus Yarrowia, Enterobacter, or Sphingomonas are preferably used in terms of the level of oil-decomposing energy.

Examples of oil-decomposing microorganisms of the genus Yarrowia include Yarrowia lipolytica ATCC48436, Yarrowia lipolytica NBRC1548, Yarrowia lipolytica LM02-011 (Accession No. NITE P-01813), Yarrowia lipolytica such as Yarrowia lipolytica NBRC0746, Yarrowia lipolytica such as Yarrowia lipolytica NBRC1209, Yarrowia sp. Yarrowia lipolytica (Yarrowia lipolytica) is better, and Yarrowia lipolytica LM02-011 (LM02-011 strain was deposited on March 6, 2014 as the deposit number NITE P-01813 in the independent administrative agency product evaluation The technical base institution Patent Microorganism Deposit Center (〒292-0818 Kazusakamatari 2-5-8, Kisarazu City, Chiba Prefecture, Japan)) is further preferable.

Examples of the oil-decomposing microorganisms of the genus Enterobacter include Enterobacter sp. LM02-030 strain (the LM02-030 strain was deposited with the Incorporated Administrative Agency on May 12, 2015 as accession number NITE P-02048). Product Evaluation Technology Base Mechanism Patent Microorganism Deposit Center (〒292-0818 Kazusakamatari 2-5-8, Kisarazu City, Chiba Prefecture, Japan)), etc.

Examples of oil-decomposing microorganisms of the genus Sphingomonas include, for example, Sphingomonas 2629-3b described in JP-A-2006-166874 , and those described in JP-A-2017-136033 . Sphingomonas sp. LM02-032 strain (the LM02-032 strain was deposited in the Patent Microorganism Deposit Center of the National Product Evaluation Technology Base Agency (〒292-0818 Japan) on June 19, 2015 as deposit number NITE P-02069 Sphingomonas sp., etc., such as Kazusakamatari 2-5-8), Kisarazu City, Chiba Prefecture.

Since the decomposition of mineral oil is assisted by the oil-decomposing microorganisms used in the method according to the present invention, oil-decomposing enzymes such as lipase and phospholipase may be added to waste water and the like. As the oil-decomposing enzyme, for example, Pseudomonas (Pseudomonas), Aspergillus (Aspergillus), Bacillus (Bacillus), Penicillium (Penicillium), Rhizopus (Rhizopus), Rhizomyces Rhizomucor, Mucor, Paecilomyces, Rhizoctonia, Absidia, Achromobacter, Aeromonas Genus, Alternaria, Aureobasidium, Beauveria, Chromobacter, Coprinus, Fusarium, Geotrichum Genus Geotricum, Humicola, Hyphozyma, Lactobacillus, Metarhizium, Rhodosporidium, Trichoderma Genus, Yarrowia, Candida, Pichia, Hansenula, Saccharomyces, Kluyveromyces (Kluyveromyces) and/or Trichosporon.

As commercially available oil-decomposing enzymes, Lipases MY, Lipases OF, Lipases PL, Lipases QLM (Mingsu Industrial Co., Ltd.); Lipases A "Amano (registered trademark)" 6, Lipases DF "Amano (registered trademark)" )" 15, Lipases G "Amano (registered trademark)" 50, Lipases AY "Amano (registered trademark)" 30SD, Lipases R "Amano (registered trademark)", Lipases MER "Amano (registered trademark)", Newlase (registered trademark) ) F (Amano Enzyme inc); Sumiteam (registered trademark) NLS, Sumiteam (registered trademark) RLS (Shin Nippon Chemical Industry Co., Ltd.); Riripaze (registered trademark) A-10D, Riripaze (registered trademark) AF-5, PLA2 Nagase (Nagase Chemtex Corporation); Enchiron AKG-2000, Enchiron LP, Enchiron LPG (Nakdong Chemical Industry Co., Ltd.); 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, Novozyme (registered trademark) 435FG (manufactured by Novozymes); Pikantaze A, Pikantaze R800 (manufactured by DSM Japan Co. Ltd), and the like. These two or more types can also be used in combination.

The amount of the oil-decomposing enzyme is not particularly limited as long as the enzyme can react with the oil, but 10 to 2,000 U is preferably used relative to 1 g of the oil contained in the drainage or the like. More preferably, it is 50~1,500U, and still more preferably, it is 100~1,000U. Moreover, with respect to the capacity of the well, it is preferably 1,000 to 100,000 U/L, more preferably 2,000 to 80,000 U/L. In addition, the activity unit (U) of the oil-decomposing enzyme is the amount of the enzyme that liberates 1 μmol of fatty acid in 1 minute under the conditions of 37° C. and pH 7.

In the method according to the present invention, inorganic polymers such as shirasu balloon, diatomaceous earth, pearlite, polyurethane, polymer, etc. described in Japanese Patent Laid-Open No. 2012-206084 may also be added. Oil adsorbents such as ethylene and organic polymers such as melamine resins are added to the drainage.

In the present invention, in order to prevent the aggregation of oil and the formation of scum, sodium dodecyl sulfate (SDS), dodecylbenzenesulfonic acid (NaDDBS), ammonium lauryl sulfate, sodium caseinate, etc. anionic surfactants; cationic surfactants of aliphatic amine salts, 4-level ammonium salts, etc.; fatty acid esters (monoglycerol fatty acid esters, diglycerol fatty acid esters, sorbitan fatty acid esters, propylene glycol fatty acid esters) , sucrose fatty acid ester), polyethylene glycol, polyethylene glycol-3-octyl phenyl ether (Triton X-100), lecithin and other non-ionic surfactants; betaine, amine oxide, saponin Surfactants such as amphoteric surfactants, etc., are added to the drainage.

In the method of the present invention, wastewater containing oil (especially mineral oil) may be continuously introduced into the system having the oil-decomposing microorganisms according to the present invention, and the treated wastewater or the like may be continuously discharged (continuous treatment). ), or a form (batch treatment) in which drainage water containing oil (especially mineral oil) is introduced, and all treated wastewater is discharged at one time.

In addition, 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 drainage or the like, can be arbitrarily set. In addition, the pH when the oil-decomposing microorganisms are brought into contact with the mineral oil, that is, the pH as drainage or the like, can be arbitrarily set. Generally, the temperature is, for example, 10°C or more and less than 45°C, preferably 15 to 35°C. The pH is, for example, 5 to 8, preferably 5.5 to 7.5. Furthermore, if necessary, aeration is performed in the drainage by aeration or the like.

>Oil decomposers> One aspect of the present invention is to provide an oil-decomposing agent (drainage treatment agent) containing the oil-decomposing microorganism according to the present invention. One embodiment of the present invention provides a mineral oil decomposing agent (drainage treatment agent) containing the oil decomposing microorganism of the present invention. A preferred aspect of the present invention is to provide a mineral oil decomposing agent (drainage treatment agent) containing Acinetobacter soli strain 1-1357 (Accession No. NITE BP-02665). The oil-decomposing microorganisms related to the present invention are excellent in oil (especially mineral oil) reducing effect (high oil decomposition rate (especially mineral oil reduction rate)). In addition, the oil-decomposing microorganisms according to the present invention can purify waste water and the like (remove oil in waste water) even in a water quality environment with a wide range of oil concentrations (eg, 1,000 to 20,000 ppm). Therefore, the drainage treatment agent containing the oil-decomposing microorganisms related to the present invention is used for oil (especially mineral oil), drainage containing oil (especially mineral oil), and oil (especially mineral oil) leakage. Seas, rivers and soils, as well as their treatment equipment (eg, hazardous substance removal facilities, oil removal traps, gasoline traps), can effectively decompose (effectively purify drainage, etc.) oil (especially mineral oil). In addition, the description of the above-mentioned oil-decomposing microorganisms and the wastewater treatment method can be applied to the present embodiment with changes as necessary.

The oil decomposer (drainage treatment agent) may be in either a dry form or a liquid form, but a dry form such as powder, granule, tablet, tablet or the like is preferable from the viewpoint of storage stability. As the oil-decomposing microorganism according to the present invention, which can be used in the oil-decomposing agent (drainage treatment agent) in such a dry form, the bacterial cell powder dried by spray-drying, freeze-drying or the like of the culture solution, or the above-mentioned The bacterial cells in the state of being immobilized on the carrier can also be shaped into powder, granule, tablet, or tablet. Alternatively, the cells and the culture solution may be encapsulated with hydroxypropyl methylcellulose, gelatin, or the like. The oil decomposer (drainage treatment agent) may further contain excipients such as hydroxypropyl cellulose, dextrin, lactose, and starch.

The oil-decomposing microorganisms according to the present invention contained in the oil-decomposing agent (drainage treatment agent) may be either dead bacteria or living bacteria, but from the viewpoint of sustainability of the oil-decomposing activity, living bacteria are preferred.

The amount of the oil-decomposing microorganisms according to the present invention contained in the oil-decomposing agent (drainage treatment agent) is, for example, 10 to 100% by weight in the solid content of the oil-decomposing agent (drainage treatment agent). In addition, the amount of the oil-decomposing microorganisms related to the present invention contained in the oil-decomposing agent (drainage treatment agent) is, for example, 1×10 ² to 1×10 ¹⁰ CFU/g with respect to the oil-decomposing agent (drainage treatment agent) amount. In addition, the oil decomposing agent (drainage treatment agent) may contain at least one selected from the group consisting of other microorganisms, oil decomposing enzymes, oil decomposing enzymes, and oil adsorbing microorganisms that can coexist with the oil decomposing microorganisms according to the present invention, as long as the objective effect of the present invention is achieved. Agents, and additives such as surfactant groups. [Example]

The effect of this invention is demonstrated using the following Example and a comparative example. However, the technical scope of the present invention is not limited to the following examples.

Example 1: Isolation of microorganisms An appropriate amount of a sample taken from the soil of the coast of Shimane Prefecture was added to a liquid medium for primary screening prepared in the same manner as above, and cultured at 30°C for one week. 100 μL of the cultured medium was further inoculated into 5 mL of the liquid medium for primary screening, and cultured at 30° C. for another week.

100 μL of the 104 ^- fold diluted culture solution after primary screening was spread on the agaric medium for secondary screening prepared in the same manner as above, and cultured at 30° C. for one week. After culturing, strains that could confirm growth on agar were 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 isolated strain obtained by the above secondary screening was inoculated into the test solution prepared by the above method with an inoculation rod, and was cultured with shaking at 30°C (rotation frequency: 140 rpm) for 24 hours.

After the cultivation, n-hexane extract was prepared according to JIS K0102:2016 (industrial drainage test method). Taking the n-hexane extract as the remaining amount of mineral oil, 0.05 g of mineral oil and the remaining amount of mineral oil (the amount of n-hexane extract (g)) added from the preparation of the test solution were calculated by the following mathematical formula ( 1) Obtain the mineral oil reduction rate. As a result, a strain with a high reduction rate of mineral oil was isolated.

× 100。[Numerical 3] Mathematical formula (1): Mineral oil reduction rate (% by weight)=

×100.

The isolated strain was named as Acinetobacter soli strain 1-1357, and was deposited in the Patent Microorganism Deposit Center, an independent administrative agency for product evaluation technology (deposit number NITE BP-02665).

Example 2: Evaluation of mineral oil decomposition energy (mineral oil reduction rate) To 5 mL of the liquid medium for tertiary screening prepared in the same manner as above, mineral oil was added so that the mineral oil concentration would be 1,000 to 20,000 ppm (1 to 20 g/L) to prepare a sterilized test solution. On agar medium for secondary screening, the cultured isolated strains were inoculated into the test solution prepared by the above-mentioned method using inoculation sticks, respectively, and cultured with shaking (140 rpm) at 30° C. for 24 hours.

In addition, the test solution prepared in the same manner as above was inoculated with the S30 strain of Acinetobacter baumannii as a comparison object in an inoculated stick, followed by shaking culture at 30°C (140 rpm) for 24 hours.

After culturing, n-hexane extract was prepared according to JIS K0102:2016 revision (industrial drainage test method). The n-hexane extract is regarded as the remaining amount of oil, the oil (g) added from the preparation of the test solution and the remaining amount of oil (the amount of n-hexane extract (g)), through the above mathematical formula (1) ) to obtain the mineral oil reduction rate (% by weight). The results are shown in Table 12 below. In addition, in the following Table 12, "Acinetobacter soli strain 1-1357" is described as "1-1357 strain", and "Acinetobacter baumannii S30 strain" is described as "S30 strain".

[Table 12]

As shown in Table 12, the 1-1357 strain related to the present invention showed a significantly higher reduction rate of mineral oil at all mineral oil concentrations (1,000-20,000 ppm) compared to the Acinetobacter baumannii S30 strain . Therefore, the oil-decomposing microorganisms according to the present invention can be expected to decompose mineral oil of a wide concentration (in a water quality environment containing mineral oil of a wide concentration, to purify waste water, etc.).

The present application is based on Japanese Patent Application No. 2018-094418 for which it applied on May 16, 2018, and the entirety of the disclosure is incorporated herein by reference.

without.

without.

Overseas storage information Japan, Patent Microorganism Deposit Center, Independent Administrative Incorporated Product Evaluation Technology Base Agency, March 13, 2018, NITE BP-02665

<110> CCI HOLDINGS INC.

<120> Oil-decomposing microorganisms

<130> 04176JP01

<160> 1

<170> PatentIn version 3.5

<210> 1

<211> 1494

<212> DNA

<213> Acinetobacter monocytogenes

<400> 1

without.

Claims (6)

A mineral oil decomposing microorganism belonging to Acinetobacter soli, the microorganism is Acinetobacter soli 1-1357 strain (Accession No. NITE BP-02665), showing the following microbiological properties,

+: positive, -: negative

+: positive, -: negative

+: positive, -: negative, w: weak reaction. The mineral oil-decomposing microorganism according to claim 1, which has the 16S rDNA nucleotide sequence shown in SEQ ID NO: 1. The mineral oil-decomposing microorganism according to claim 1 or claim 2, wherein the mineral oil reduction rate of at least one mineral oil concentration in the range of 1,000 to 20,000 ppm is 60% by weight or more. A method for decomposing oil, comprising the step of contacting the mineral oil decomposing microorganism described in any one of claims 1 to 3 with the oil. An oil-decomposing agent, comprising the mineral oil-decomposing microorganism described in any one of the 1st to 3rd claims in the scope of application. A use of a microbial preparation for mineral oil decomposition, comprising the mineral oil decomposing microorganism described in any one of items 1 to 3 of the patent application scope.