JP2011125825A - Lypolytic bacteria and lipolytic agent - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide lypolytic bacteria with high propagation rate and lypolytic rate, when optimum lypolytic conditions (temperature, pH, etc.) are out of the scope of optimum growth conditions (temperatures, pH, etc.) of BOD degrading bacteria. <P>SOLUTION: The lipolytic bacteria belongs to Pseudmonas group and indicates the following mycologic characteristics, for example: growth + at 37°C, growth + at 45°C, growth - under an anaerobic condition, nitrate reduction activity +, indole production activity -, arginine dihydrolase activity +, urease activity -, gelatin hydrolysis activity +, β-galactosidase activity -, cytochrome oxidase activity +, dextrose assimilation nature +, L-arabinose assimilation nature, D-mannose assimilation nature -, D-mannitol assimilation nature -, N-acetyl-D-Glucosamine assimilation nature +, maltose assimilation nature -, and potassium gluconate assimilation nature +, where "+" is positivity and "-" is negative. <P>COPYRIGHT: (C)2011,JPO&INPIT

Description

  The present invention relates to an oil-degrading bacterium and an oil-degrading agent.

  As a wastewater treatment method using microorganisms, an activated sludge method using an aerobic microorganism (aerobic treatment method) and a methane fermentation method using an anaerobic microorganism (anaerobic treatment method) are generally known. The aerobic treatment method requires energy for aeration, whereas the anaerobic treatment method consumes less energy and can also obtain biogas mainly composed of methane. For this reason, it is said that the anaerobic processing method is more advantageous in terms of energy efficiency than the aerobic processing method.

  Various types of upflow anaerobic treatment tanks have been developed for treating high-load wastewater containing a high concentration of soluble organic matter by methane fermentation. For example, a processing tank using a UASB (Upflow Anaerobic Sludge Blanket) method or an improved EGSB (Expanded Granular Sludge Bed) method is known. These upward-flow anaerobic treatment tanks can store granular sludge called granular sludge in the tank, and the organic matter is made efficient by flowing the treated water upward while in contact with the granular sludge. Is broken down.

  By the way, when treating organic wastewater containing a large amount of fats and oils, such as wastewater from food factories that handle fats and oils in the manufacturing process, with the granular sludge, the fats and oils adhere to the granule sludge, and thereby the contact efficiency between the wastewater and the granular sludge. The organic matter is likely to be insufficiently processed due to the decrease. As means for solving such a problem, in Patent Document 1, prior to introducing the water to be treated into the upward flow type anaerobic treatment tank, oil is separated from the water to be treated, and this is decomposed by lipase producing bacteria. An anaerobic treatment apparatus equipped with an oil and fat decomposition tank is described.

  Moreover, Pseudomonas genus microbe which has oil and fat degradability is disclosed (patent document 2 and patent document 3).

JP 2005-270862 A JP 2008-220225 A JP-A-6-153922

  However, conventionally known oil-degrading bacteria are soluble organic matter-degrading bacteria (hereinafter referred to as BOD-degrading bacteria) whose optimum oil-degrading conditions (temperature, pH, etc.) have a faster growth rate than oil-degrading bacteria. It falls within the range of optimal growth conditions (temperature, pH, etc.). For this reason, BOD-degrading bacteria are dominant in the sludge of the fat and oil decomposition tank, and there is a problem that a large amount of excess sludge is generated due to decomposition of soluble organic matter (hereinafter referred to as BOD component) in addition to a decrease in the decomposition efficiency of fat and oil. In addition, BOD degrading bacteria become dominant in the sludge of the oil decomposing tank, and there is also a problem that the oil decomposing bacteria are not present.

  Accordingly, the present invention provides an oil and fat having a sufficient growth rate and an oil-and-oil decomposition rate because the optimum oil-and-oil decomposition conditions (temperature and pH, etc.) do not fall within the optimum growth conditions (temperature, pH, etc.) of BOD-degrading bacteria. The purpose is to provide degrading bacteria. Another object of the present invention is to provide an oil decomposing agent containing the above oil decomposing bacteria.

The present invention is an oil-degrading bacterium belonging to the genus Pseudomonas and exhibiting the following mycological properties.
Cell morphology Neisseria gonorrhoeae cell diameter 0.6-0.7 × 1.0-1.5 μm
Presence or absence of spores −
Gram staining −
Motility +
Growth at 37 ° C +
Growth at 45 ° C +
Growth under anaerobic conditions −
Catalase +
Oxidase +
Acid / gas production from glucose-/-
Glucose oxidation / fermentation +/-
Nitrate reduction activity +
Indole production activity −
Glucose acidification activity −
Arginine dihydrolase activity +
Urease activity −
Esculin hydrolysis activity −
Gelatin hydrolysis activity +
β-galactosidase activity −
Cytochrome oxidase activity +
Glucose utilization ＋
L-arabinose assimilation-
D-Mannose assimilation-
D-mannitol assimilation-
N-acetyl-D-glucosamine assimilation +
Maltose utilization-
Potassium gluconate assimilation +
n-Capric acid assimilation +
Adipic acid utilization ＋
dl-Assimilation of malic acid +
Sodium citrate assimilation ＋
Utilization of phenyl acetate-
In addition, “+” indicates positive and “−” indicates negative.

  It is preferable that the fat-and-oil degrading bacteria form an opaque green colony having a peripheral wavy circular shape with a diameter of 2 to 3 mm, a smooth surface, and a flat shape when cultured on an agar medium at 30 ° C. for 24 hours.

  In addition, the oil-degrading bacterium preferably has a 16S rDNA base sequence represented by SEQ ID NO: 1.

  As the above-mentioned oil-degrading bacteria, the number of the NITE P at the National Institute of Technology and Evaluation Microorganisms (National Biotechnology Headquarters, postal number 292-0818; -847 and Pseudomonas aeruginosa SS-219 strain entrusted on November 25, 2009.

  The present invention also provides an oil decomposing agent comprising the above-described oil decomposing bacteria.

  Since the above oil-degrading bacteria can be efficiently grown and decomposed under alkaline conditions and high-temperature conditions, the fats and oils can be decomposed while suppressing the growth of BOD-degrading bacteria. Moreover, since the said fat-and-oil decomposing bacteria have a fat-and-oil decomposition rate quick compared with the conventional fat-and-oil decomposing bacteria, it can utilize suitably for the process of the waste_water | drain containing high concentration fats and oils.

  Since the oil-and-oil decomposing bacteria of the present invention can make the oil-and-oil decomposing conditions high alkali and high temperature, the growth of BOD-decomposing bacteria can be suppressed and the oil-and-oil degrading bacteria can be dominant or dominant. Moreover, since the fat-and-oil decomposing bacteria of this invention have a quick growth rate and fat-and-oil decomposition rate, the process of the waste_water | drain containing high concentration fats and oils is possible. Furthermore, since it is difficult for the BOD component to be decomposed during the oil and fat decomposition process, it is possible to obtain a high-concentration oil and fat decomposition product, and to obtain a fat and oil decomposition wastewater that maintains a high BOD component concentration. This is advantageous when the fat and oil decomposition wastewater is anaerobically treated at a later stage.

It is a photograph which shows the TLC (thin layer chromatography) analysis result of fat and oil decomposition product. In FIG. 1, TG, DG, MG, and FA represent triglyceride, diglyceride, monoglyceride, and fatty acid, respectively, and OA, O, S, W, and C represent oleic acid, olive oil, and mixed fat (salad oil / lard / Beef tallow, mass ratio 1: 1: 1), water layer after extraction, chloroform layer after extraction. It is a photograph which shows the colony form of Pseudomonas aeruginosa SS-219 strain (NITE P-847). It is a graph which shows the fat and oil decomposition rate with respect to (a) pH fluctuation of a culture solution or (b) fluctuation | variation of culture temperature. It is a photograph which shows a mode that NITE P-847 (Pseudomonas aeruginosa SS-219 strain) was grown on the fat emulsified plate. It is a photograph showing NITE P-847 (Pseudomonas aeruginosa SS-219 strain) cultured in a test tube. (A) Photographs of culture days 1 (A) and 3 (B), (b) culture days 12 (C).

  Hereinafter, the present invention will be described in detail based on embodiments.

  The oil-degrading bacterium of the present invention has the above-mentioned mycological properties as described above. The above oil-degrading bacteria can exhibit high oil-degrading activity under high alkali and high-temperature conditions, for example, a culture temperature of 30 to 40 ° C. and a culture solution pH of 8.5 to 9.5. Moreover, the growth rate and the fat and oil decomposition rate are high, and edible fats and oils such as salad oil, lard, and beef tallow can be decomposed by 82.8 to 90.9% in 24 hours. In order to achieve such a degree of fat degradation by conventional fat-degrading bacteria, it usually takes 48 hours or more, and it can be seen that the oil-degrading speed of the present invention is high.

  Moreover, it is preferable to use Pseudomonas aeruginosa SS-219 strain (NITE P-847) as the oil-degrading bacterium according to the present embodiment. Pseudomonas aeruginosa SS-219 strain is a specific example of an oil-degrading bacterium having all the above-mentioned mycological properties. Although it is not Alcaligene genus or Achromobacter genus known as an alkalophilic bacterium, it can grow under highly alkaline conditions and has a high fat degradation rate.

  The fats and oils to be decomposed by the oil-degrading bacteria according to the present invention are mainly fats and oils derived from animals and plants, but are not limited to them, artificial fats and oils obtained by methods including various synthetic chemical methods, The derivatives are also included. Specifically, it can be edible fats and oils, industrial fats and oils, etc. contained in waste water, and in particular, edible fats and oils such as salad oil, corn oil, soybean oil, milk fat, butter, lard, beef tallow and the like.

  The oil-degrading bacterium according to the present invention can be isolated by culturing a group of bacteria collected from various environments in an alkaline medium containing oil and fat, and screening using the oil and fat resolution as an index.

  A specific method is, for example, as follows. An alkaline medium (for example, pH 8 to 10) containing oil or mixed fat is added to the test tube, and a small amount of sample collected from various environments (for example, soil, sand, mud, seawater, lakes, etc.) is added. Cover the test tube with aluminum foil, etc., and after shaking culture in a shaking incubator under appropriate conditions (for example, 37 ° C., 170 times / min, 24 hours), select a sample in which fat and oil decomposition has occurred. .

  Analysis of a sample in which fat and oil decomposition has occurred after shaking culture can be made by visual determination of whether or not there is residual fat or oil. For example, the determination of residual fat or oil by n-hexane extraction based on JIS standards or It can also be carried out by the method described in a reference (Journal of Bioscience and Bioengineering, 2007, 103 (4), pages 325-330).

  The oil-degrading bacteria can be isolated from the sample that has confirmed that the oil-degradation has occurred by pure separation. Pure isolation can be performed by methods well known to those skilled in the art. For example, a pure isolated colony can be formed by inoculating and culturing on an agar medium, and the colony is picked up. By doing so, the target oil-degrading bacteria can be isolated.

  In addition, after selecting a sample in which fat and oil decomposition has occurred, a part of the sample is again inoculated into a test tube to which an alkaline medium (for example, pH 8 to 10) containing fresh fat or mixed fat is added, and again. Shaking culture can also be performed. A sample that has undergone fat and oil degradation after this shaking culture is selected. The pure separation described above can be performed on the selected sample, or this shaking culture cycle can be repeated again. By increasing the shaking culture cycle, the target oil-degrading bacteria can be accumulated.

  The identification, properties and properties of the oil-degrading bacteria isolated from the sample collected from the environment as described above can be analyzed using various known identification test methods or commercially available identification kits. In addition, oil-degrading bacteria can be identified by sequencing the 16S rDNA base sequence and performing homology search and molecular phylogenetic analysis.

  The oil and fat decomposing agent of the present invention can be obtained by formulating the oil and fat decomposing bacterium of the present invention. Specifically, for example, “Applied Microbiology Revised Edition, Sawao Murao, Motoo Arai, Bafukan, 1993”, “Introduction to Biotechnology, Eizo Sada et al., Kodansha, 1996”, “Industrial Enzyme, Takayuki Kamijima It can be made into a fixed preparation, a liquefied preparation, and a powdery preparation by the method described in “Mr. Maruzen, 1995”.

  The oil-degrading bacterium and the oil-degrading agent of the present invention are not limited thereto, but can be used for treatment of organic wastewater containing a large amount of fats and oils, such as wastewater from food factories that handle fats and oils in the production process. The wastewater treatment method is not particularly limited, and in the conventional wastewater treatment using oil-decomposing microorganisms, the oil-decomposing bacteria or oil-decomposing agent of the present invention can be used instead of the oil-decomposing microorganisms. Moreover, when using the oil-and-oil decomposing bacteria or oil-and-oil degrading agent of this invention, it is preferable to set the temperature of the waste_water | drain to process to 30-40 degreeC, and to set the pH of the waste_water | drain to process to 8.5-9.5. As a result, the oil-degrading bacterium of the present invention is not only a condition showing high oil-and-oil degradability, but also the growth of BOD-degrading bacteria can be suppressed, so that the amount of sludge generation can be reduced and the oil-degrading efficiency can be further increased. Can do.

  In addition, the oil-degrading bacterium and the oil-degrading agent of the present invention are combined with various types of upward flow anaerobic treatment tanks for treating high-load wastewater containing a high concentration of soluble organic matter by methane fermentation. And more preferably used. As the upward flow type anaerobic treatment tank, for example, a treatment tank using a UASB (Upflow Anaerobic Sludge Blanket) method or an improved EGSB (Expanded Granular Sludge Bed) method can be used. Wastewater and granules in the post-treatment step by an upward flow type anaerobic treatment tank by removing the fats and oils by treating organic wastewater containing a lot of fats and oils with the oil-degrading bacteria and fat-degrading agent of the present invention in the pretreatment step Reduction in contact efficiency with sludge can be prevented, and wastewater can be treated efficiently. In addition, since the decomposition of the BOD component is unlikely to occur in the pretreatment process, the wastewater supplied to the posttreatment process contains a high-concentration fat and oil decomposition product as the BOD component. Therefore, by performing anaerobic treatment in the post-treatment step, a high concentration of BOD component is converted into high energy, and a sludge generation amount is small, so that high treatment efficiency can be achieved.

  Hereinafter, the present invention will be described more specifically with reference to examples, but the present invention is not limited thereto.

[Search for alkali-resistant oil-degrading bacteria]
The soil collected in Kanagawa, Fukushima, and Ehime was used as a sample, suspended in sterilized physiological saline, and then subjected to ultrasonic treatment with an ultrasonic cleaner for 5 minutes. After the treatment, it is allowed to stand for about 5 minutes, and an alkaline artificial sewage medium 1 (Table 1) or an alkaline artificial sewage medium to which a mixed fat (salad oil / lard / beef tallow, mass ratio 1: 1: 1) is added so that the supernatant becomes 3000 ppm. 2 (Table 2) An 18φ × 180 mm test tube containing 5 ml each was inoculated to 1% (v / v).

  The test tube was covered with aluminum foil, and cultured with shaking in a shaking incubator at 37 ° C. and 170 times / minute. About the sample in which the growth of microorganisms was recognized, inoculate fresh alkaline artificial sewage culture medium 1 or alkaline artificial sewage culture medium 2 to which the above-mentioned mixed fats and oils were added so as to be 3000 ppm to 1% (v / v). In the same manner, shaking culture was performed. By repeating this, the target microorganism was accumulated. A flask test was performed on a sample that had grown when it was repeated three times or more and in which oil decomposition was observed.

  The flask test was performed according to the following procedure. 100 ml of alkaline artificial sewage medium 1 or alkaline artificial sewage medium 2 added with the above mixed fats and oils to 3000 ppm in a 500 ml baffle flask, inoculated with a test tube culture solution to 1% (v / v), 37 ° C. And shaking culture at 120 rpm for 24 hours. After this was treated with an autoclave (121 ° C., 20 minutes), the remaining fats and oils were quantified by an n-hexane extraction method according to JIS K0101, and the fats and oils decomposition rate was calculated from the difference from the control without inoculating bacteria. .

  Screening was performed on 25 samples using alkaline artificial sewage medium 1 and 49 samples using alkaline artificial sewage medium 2. As a result of screening using the alkaline artificial sewage medium 2, two types of samples (samples No. 192 and No. 219) showing a high oil-fat decomposition rate of 78 to 88% / d were obtained. Sample No. 192 is derived from a sample obtained from the soil near the rail in the Yokosuka Works, Sumitomo Heavy Industries, Ltd. 219 was derived from a sample obtained from bonfire fire soil (ash) in Ehime Prefecture.

  As a result of pure separation of the microorganisms contained in these samples, both samples were in a single fungus state by enrichment culture. Master plates and glycerol stocks were made from the isolated colonies. Each bacterium picked up from the master plate was cultured on an agar plate (30 ° C., 24 hours), and one plate for identification analysis was prepared for each bacterium.

[Measurement of oil decomposability]
Sample No. 192 and No. The reproducibility of the oil and fat decomposability of the strain isolated from 219 was confirmed. In n-hexane extraction, it was thought that the extraction accuracy of the remaining fats and oils was low due to the formation of an intermediate layer and an emulsion during extraction. Therefore, extraction with 40 ml of chloroform / methanol (3: 1) and centrifugation (5,000 rpm, 30 min) After that, the mixture was gently transferred to a separatory funnel, and the chloroform (lower) layer was placed in a new centrifuge tube and centrifuged again (10,000 rpm, 10 min). After centrifugation, the mixture was gently transferred to a separatory funnel, the chloroform (lower) layer was taken in a flask, dehydrated with anhydrous magnesium sulfate, filtered, and evaporated to measure the residual oil. As a result, it was found that both strains were highly reproducible and showed high fat and oil degradability (Table 3).

[TLC analysis of fat and oil decomposition process]
In order to grasp the fat and oil decomposition process, TLC analysis of the extracted residual oil was performed. The result is shown in FIG. No. The 192 strain (left panel) is, in order from the left lane, oleic acid (OA), olive oil (triolein; O), mixed fat (salad oil / lard / beef tallow, mass ratio 1: 1: 1; S), The aqueous layer (W) and the chloroform layer (C) containing the extracted residual oil were loaded. Similarly, no. 219 strains (right panel) were extracted in the order from the left lane, oleic acid (OA), olive oil (triolein; O), mixed fat (salad oil / lard / beef tallow, mass ratio 1: 1: 1; S), remaining extracted A chloroform layer (C) containing oil and an aqueous layer (W) after extraction were loaded. No. 192 strain and No. In any of the 219 strains, no fat or oil was detected from the aqueous layer (W), and it was found that the remaining fat or oil was completely extracted into the chloroform layer (C). No. Compared to 219 strains, It was found that the 192 strain had a higher amount of diglyceride (DG), monoglyceride (MG), and fatty acid (FA). This is no. This suggests that degradation of triglyceride (TG) by lipase and fatty acid metabolism (β oxidation) are rate-determining during the oil and fat decomposition process of 192 strain. On the other hand, no. In 219 strain, DG and MG are hardly observed in the extracted oil, and since the amount of FA is small, it is presumed that the degradation of glycerides is rapid.

  Hereinafter, sample no. Further detailed analysis was performed on a strain (No. 219 strain) purely isolated from 219.

[Taxonomy identification of oil-degrading bacteria]
Based on the nucleotide sequence analysis of 16S rDNA, no. 219 strains were identified. No. DNA extraction from strain 219 was performed using InstaGene Matrix (BioRad, CA, USA). Full length 16S rDNA was amplified by PCR reaction. PrimeSTAR HS DNA Polymerase (manufactured by Takara Bio Inc.) was used for the PCR reaction. Using the obtained PCR product as a template, cycle sequencing was performed using BigDye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems, CA, USA). The sequence was analyzed by ABI PRISM 3130x1 Genetic Analyzer System (Applied Biosystems, CA, USA), and the nucleotide sequence was determined using ChromasPro1.4 (Technyllium Pty Ltd., Tewantin, AUS) software. The determined No. 219 strain 16S rDNA nucleotide sequences were subjected to homology search and simple molecular phylogenetic analysis using Apollon 2.0 (Techno Suruga Labs) software and Apollon DB-BA ver4.0 (Techno Suruga Labs) database. did. As a result, no. The 16S rDNA base sequence of the 219 strain showed a high homology of 99.9% with the 16S rDNA base sequence of the Pseudomonas aeruginosa ATCC 10145 strain (Table 4). In addition, as a result of homology search performed on the international base sequence database (GenBank / DDBJ / EMBL), No. The 16S rDNA nucleotide sequence of the 219 strain is described in P.A. Although it showed 100% homology with 16S rDNA nucleotide sequences of aeruginosa MCCB123 strain, MML2212 strain, NK2.1B-1 strain, etc., 16S rDNA derived from the reference strain was not searched.

  No. Simplified molecular phylogenetic analysis was performed using 16S rDNA sequences of 219 strains and 16S rDNA sequences of the top 10 homology search strains for Apollon DB-BA ver4.0 (Table 5). In Table 5, the numbers located at the branches of the system branches indicate bootstrap values.

  As a result of simple molecular phylogenetic analysis, no. 219 strain 16S rDNA Aeruginosa 16S rDNA was clustered and showed the same molecular phylogenetic position (Table 5). Based on the above results, this strain was isolated from P. It is considered that the strain belongs to the species Aeruginosa. Therefore, this strain was named Pseudomonas aeruginosa SS-219 strain.

[Analysis of mycological characteristics of oil-degrading bacteria]
For Pseudomonas aeruginosa SS-219 strain, morphological observation by optical microscope (BX50F4, Olympus), Barrow et al. (Cowan and Steel's Manual for the Identity of Medicine. Based on the above, tests such as catalase reaction, oxidase reaction, Gram stainability, acid / gas production from glucose, oxidation / fermentation (O / F) of glucose, etc. were carried out. Faber G “Nissui” (manufactured by Nissui Pharmaceutical) was used for the Gram stain analysis. The results are shown in Table 6. FIG. 2 shows the colony morphology of Pseudomonas aeruginosa SS-219 strain.

  Further, various properties described in Table 7 were tested using an API20NE kit (Biomerieux, France). Determination was according to the manual attached to the kit. In addition, tests on matters described in Table 8 were performed.

  As a result, the Pseudomonas aeruginosa SS-219 strain was able to grow at a culture temperature of 45 ° C. (Table 6), and was different from the Pseudomonas family described in Patent Document 2 and Patent Document 3. Further, it was different from the Pseudomonas family described in Patent Document 2 in that L-arabinose and D-mannose were not assimilated (Table 7).

[Relationship between culture broth pH and oil degradation rate]
The influence of the culture solution pH on the oil degradation rate of Pseudomonas aeruginosa SS-219 strain was analyzed. A medium having the same composition as that of the alkaline artificial sewage medium 2 except for the pH adjuster (1M Tris-HCl, pH 9) was used. Based on this medium, medium of pH 3-8 uses hydrochloric acid or sodium hydroxide as a pH adjuster, medium of pH 9 uses 1M Tris-HCl (pH 9) as a pH adjuster, and medium of pH 10 serves as a pH adjuster. The pH was adjusted using 1 M glycine / sodium hydroxide buffer (pH 10). 100% of these culture mediums with mixed fats and oils (salad oil / lard / beef tallow, mass ratio 1: 1: 1) added to 3000 ppm are placed in a 500 ml baffle flask and pre-cultured for 24 hours in 1% (V / v) was inoculated and cultured with shaking at 120 rpm and 37 ° C. for 24 hours. After culturing, the mixture was treated with an autoclave (121 ° C., 20 minutes), extracted with 40 ml of chloroform / methanol (3: 1), centrifuged (5,000 rpm, 30 min), and then gently transferred to a separatory funnel. ) Layer was placed in a new centrifuge tube and centrifuged again (10,000 rpm, 10 min). After centrifugation, the mixture was gently transferred to a separating funnel, the chloroform (lower) layer was taken in a flask, dehydrated with anhydrous magnesium sulfate, filtered and evaporated to quantify the residual oil. The oil degradation rate was calculated from the difference from the control without inoculating the fungus.

  The results are shown in FIG. When the pH of the culture solution is 3, 4, 5, 6, 7, 8, 9, 10, the oil degradation rate after 24 hours culture of Pseudomonas aeruginosa SS-219 is 72, 58, 63, 80, respectively. 85, 80, 83%. A high oil degradation rate was exhibited at a medium pH of 8.5 to 9.5, and the optimum medium pH was around 9. In addition, even when the pH was 6 to 8 and pH 3, the oil decomposition rate was 70% or more.

[Relationship between culture temperature and oil degradation rate]
The influence of the culture temperature on the fat and oil degradation rate of Pseudomonas aeruginosa SS-219 strain was analyzed. In a 500 ml baffle flask, 100 ml of alkaline artificial sewage medium 2 to which mixed fats and oils (salad oil / lard / beef tallow, mass ratio 1: 1: 1) were added so as to be 3000 ppm, and test tube culture solution preliminarily cultured for 24 hours was 1 % (V / v) was inoculated. The culture temperature was set to 20, 25, 30, 37, 40, and 45 ° C., respectively, and cultured with shaking at 120 rpm for 24 hours. Thereafter, the residual oil was quantified in the same manner as described above, and the oil decomposition rate was calculated.

  The results are shown in FIG. When the culture temperature is 20, 25, 30, 37, 40, and 45 ° C., the degradation rate of oil and fat after 24 hours of Pseudomonas aeruginosa SS-219 is 17, 65, 75, 83, 70, and 13%, respectively. there were. The oil and fat decomposition rate showed a high value at 30 to 40 ° C., and the optimum temperature was around 37 ° C.

[Other features]
Antibiotic susceptibility testing was performed using antibiotic-containing plates. As a result, the Pseudomonas aeruginosa SS-219 strain showed resistance to ampicillin and chloramphenicol.

  FIG. 4 shows a state in which the Pseudomonas aeruginosa SS-219 strain was grown on an oil emulsified plate. Clear halo was formed around the colony of Pseudomonas aeruginosa strain SS-219, suggesting that lipase was secreted outside the cells. The appearance of Pseudomonas aeruginosa strain SS-219 in vitro is shown in FIGS. 5 (a) and 5 (b). The Pseudomonas aeruginosa SS-219 strain produced a yellow fluorescent dye, and its color became darker with the increase in the number of culture days on the first day (A) and the third day (B). Furthermore, on the 12th day of culture (C), the fats and oils were almost completely decomposed. Moreover, since the turbidity of the culture solution was also significantly reduced, lysis is considered to have occurred.

Claims (5)

An oil-degrading bacterium belonging to the genus Pseudomonas and exhibiting the following mycological properties.
Cell morphology Neisseria gonorrhoeae cell diameter 0.6-0.7 × 1.0-1.5 μm
Presence or absence of spores −
Gram staining −
Motility +
Growth at 37 ° C +
Growth at 45 ° C +
Growth under anaerobic conditions −
Catalase +
Oxidase +
Acid / gas production from glucose-/-
Glucose oxidation / fermentation +/-
Nitrate reduction activity +
Indole production activity −
Glucose acidification activity −
Arginine dihydrolase activity +
Urease activity −
Esculin hydrolysis activity −
Gelatin hydrolysis activity +
β-galactosidase activity −
Cytochrome oxidase activity +
Glucose utilization ＋
L-arabinose assimilation-
D-Mannose assimilation-
D-mannitol assimilation-
N-acetyl-D-glucosamine assimilation +
Maltose utilization-
Potassium gluconate assimilation +
n-Capric acid assimilation +
Adipic acid utilization ＋
dl-Assimilation of malic acid +
Sodium citrate assimilation ＋
Utilization of phenyl acetate-
In addition, “+” indicates positive and “−” indicates negative.   The fat-and-oil degrading bacterium according to claim 1, which forms an opaque green colony having a peripheral corrugated circular shape having a diameter of 2 to 3 mm, a smooth surface, and a flat shape on an agar medium by culturing at 30 ° C for 24 hours.   The oil- and fat-degrading bacterium according to claim 1 or 2, which has the 16S rDNA base sequence represented by SEQ ID NO: 1.   An oil-degrading bacterium identified by the Patent Microorganism Deposit Center of the National Institute of Technology and Evaluation, Accession Number NITE P-847 (Pseudomonas aeruginosa SS-219).   The fat and oil decomposition agent containing the fat-and-oil decomposing bacteria as described in any one of Claims 1-4.