JP4566207B2 - Oil-degrading microorganism and method for treating oil-containing wastewater using the same - Google Patents

Description

The present invention relates to a novel oil-degrading microorganism capable of efficiently degrading animal and vegetable oils and fats contained in waste water discharged from kitchens, the oil-degrading enzymes produced by the microorganisms, and the oil-degrading microorganisms Alternatively, the present invention relates to a method for treating fat-containing wastewater using an oil-degrading enzyme produced by the microorganism.
More specifically, a novel Pseudomonas sp. WU-LM1, which has an excellent decomposing activity especially on animal fats such as lard and has an excellent decomposing activity even at low temperatures, The present invention relates to an oil-degrading enzyme secreted and produced, and a method for treating fat-containing wastewater using the oil-degrading microorganism or the oil-degrading enzyme produced by the microorganism.

Waste oil and environmental problems;
A large amount of fats and oils are used in establishments having commercial kitchens such as restaurants, restaurants, hotels, lunch centers, and hospitals, and such large amounts of fats and oils are discarded as waste oil. These waste oils are usually difficult to be decomposed and cause environmental pollution, and the oil and fat adhering to and accumulating on the drainage pipes causes bad odor and clogging of pipes.
Wastewater mixed with waste oil such as fats and oils has long been treated by methods such as ocean dumping and incineration. However, ocean dumping of wastewater has become a social problem due to pollution and river and ocean pollution, and at present, it is prohibited by law to dump wastewater containing waste oil such as fats and oils. On the other hand, incineration treatment of waste oil not only causes air pollution, but also has a problem from the viewpoint of emission of carbon dioxide, which is one of the major causes of global warming.

Waste water purification and grease wrapping;
Against this backdrop, according to the Ministry of Construction Notification No. 1597 “Standard for Installation of Waste Water Traps and Interceptors” in 1951, restaurants, school lunches, hospitals, employee cafeterias, nursing homes, food processing factories, etc. It was obliged to install a grease trap, a fat and oil interceptor, to discharge waste water containing waste oil such as min.
The grease trap is a device that temporarily collects waste water and residues from the kitchen, and so to speak, is a catcher that collects “oil” and “trash”. The collected garbage, oil and fat, and precipitated dirt (scum) are separated and removed with a wire mesh, ladle, etc. Cleaning the basket once a day, removing grease, residues, etc. once a month The inside of the trap needs to be cleaned once every several months.
Waste water containing fat and oil discarded from the kitchen flows into the grease trap, where the fat and oil is separated and accumulated, and further passes through the septic tank and the secondary tank, and the water quality is below the defined standard (standard value of water that can flow down the road) And then discharged into sewage through a septic tank.
The grease trap has an entrance through which wastewater flows and a wastewater outlet that discharges wastewater in the greasetrap to a drain pipe or septic tank, etc., and the inside of the tank is divided into three or four tanks, thus extending the residence time of the wastewater. By this, it is a water storage tank which has a structure where waste oil, such as fats and oils in wastewater, is trapped in the grease trap. The first layer is provided with a wire mesh basket to separate large garbage. The second layer is responsible for separation of fats and oils and dirt, where the oils and fats float near the upper water surface, and small dust and dirt scum float in water or settle in the lower part. A water distribution pipe is provided below the third layer, and water purified to some extent not containing oil and fat / precipitate is discharged into the sewer. The fat and oil floating in the grease trap and the sediment are treated by sucking the fat and scum in the tank by a pump and separating it into oil and water by a separator and then discarding or incineration as industrial waste.
In this way, the grease trap itself retains the physical function of separating and collecting waste oil such as fats and oils from the wastewater, but the function of biologically or chemically decomposing waste oils such as fats and oils is not. I don't have it.
However, with this device, if the wastewater contains fat or oil that exceeds the processing capacity, the processing capacity cannot catch up, and it adheres to and decays in the scum in the drainage pipe, causing malodors and pests. Not only does this cause the clogging of the exhaust pipe, but the function of the device itself stops, causing oil to spill into rivers and the like, causing even greater pollution problems.
Therefore, various ideas have been proposed and put into practical use in order to improve the performance of the grease strap. For example, it is as follows.
(1) A system that uses special equipment such as a filter or a tool (2) A method that uses ozone (3) A system that decomposes fats and oils using enzymes (4) Oils and fats that use aerobic oil-degrading microorganisms (5) Method of using an aeration pump to promote the growth of aerobic microorganisms In addition, aeration is the growth of anaerobic microorganisms that release malodors such as ammonia and hydrogen sulfide during the process of oil and fat decomposition. It is a treatment for growing oil-degrading microorganisms that are aerobic bacteria that suppress and require oxygen.

Use of microorganisms;
Examples of the microorganism used in the biological wastewater treatment method include Cryptococcus, Acinetobacter, Bacillus, Burkholderia, Staphylococcus, Many strains belonging to the genus Pseudomonas and Aspergillus have been reported.
Among them, Pseudomonas genus bacteria are known as bacteria that inhabit a wide range of environments and are involved in the degradation of various organic substances, but some of them also produce oil-degrading enzymes. The oil-degrading enzyme producing strains include Pseudomonas aeruginosa, Pseudomonas pseudoalgenes (Pseudomonas)
pseudoalkaligenes), Pseudomonas
alkaligenes), Pseudomonas fluorescens, Pseudomonas cepacia, Pseudomonas
fragi) and the like.

More specifically, the following reports have been made on Pseudomonas bacteria related to fat and oil decomposition technology.
(A) A cell belonging to the genus Pseudomonas having a high ability to decompose fats and oils, capable of growing in the range of 15 ° C to 55 ° C, and having the ability to degrade animal fats, vegetable fats and free fatty acids. Highly functional oil-degrading bacteria (see Patent Document 1).
(B) Pseudomonas sp. AQ-T5 strain, which produces a membrane-bound oil-degrading enzyme that is not secreted extracellularly and whose 16S ribosomal RNA gene has a partial base sequence set forth in SEQ ID NO: 1 in the Sequence Listing. FERMP-17554) or a mutant thereof (see Patent Document 2).
(C) Pseudomonas genus microbe which can decompose | disassemble mineral oil (refer patent document 3).
(D) Pseudomonas denitrificans strain capable of growing at a temperature in the range of 4 to 40 ° C., characterized by having an oil-fat decomposing ability and a denitrifying ability Oil-degrading bacteria (see Patent Document 4).
(E) A cell belonging to the genus Pseudomonas having a high ability to decompose fats and oils, capable of growing in the range of 15 ° C to 55 ° C, and having the ability to degrade animal fats, vegetable fats and free fatty acids. Highly functional oil-degrading bacteria (see Patent Document 5)
(F) A microorganism belonging to the genus Pseudomonas that can decompose fats and oils at 10 to 37 ° C. (see Patent Document 6).
(G) In the method for treating oil-containing wastewater, a Pseudomonas cepacia LM2-1 strain, a mutant thereof having oil-and-fat resolving power, or a culture thereof is added to the treatment tank. How to treat oil-containing wastewater (see Patent Document 7).
(H) A method using a Pseudomonas sp. NO. AES053 strain (Microtechnological Bacteria No. 13230) that exhibits a degrading performance against a surfactant as a microorganism to be comprehensively immobilized on a floating filter medium (patent) Reference 8).
(I) A novel microorganism Pseudomonas putida SH-2992 (FERM P-13564) having the ability to decompose toluene (see Patent Document 9).
(J) Deposited as Pseudomonas sp. Strain SK0401 (No. 13231) and Acinetobactor sp. Strain SK0402A (No. 13232). Microorganisms that have a degradability for edible fats and oils (see Patent Document 10).
(K) Pseudomonas sp. S10-071 strain (see Patent Document 11) belonging to the genus Pseudomonas and having high lipase production ability.
These oil-decomposable microorganisms are used by being sprayed as powder or instilled as liquid in waste oil.
However, the technology described in the literature using these microorganisms is to decompose oils and fats in wastewater by using microorganisms alone, but its resolution does not necessarily satisfy the requirements of users. In particular, the resolution in winter and cold regions and the resolution against lard were still insufficient, and there was room for further improvement.
In addition, the AQ-T5 strain or its mutant disclosed in (b) above is different from the microorganism of the present invention in that it produces a membrane-bound type lipolytic enzyme.

Use of oil-degrading enzymes (lipases);
As described above, in addition to the physicochemical method, a method of treating fat and oil-containing wastewater with an enzyme produced by a microorganism is also known. In addition, it is also known to hydrolyze fats and oils in wastewater by adding fat-degrading enzymes (lipases) to the fat-containing wastewater. For example, the use of a lipase produced by Pseudomonas cepacia S16-019B strain (Microtechnological Laboratories No. 12574) has been reported (JP-A-5-123170).
As the lipase as such an oil-degrading enzyme, lipases derived from microorganisms as well as those derived from animals or plants are known. Among these, examples of the lipase derived from microorganisms include, for example, Rhizopus genus, Aspergillus genus, Mucor genus, Pseudomonas genus, Geotrichum genus, Penicillium genus, Candida (Candida ( Candida) and other origins (see Patent Document 12).
However, the resolution of these lipases, as in the case of the above-mentioned microorganisms, does not necessarily satisfy the user's requirements. In particular, the resolution in winter or cold regions and the resolution against lard are still insufficient. There was room for further improvement.

Problems that remain
Wastewater discharged from many business establishments that have commercial kitchens such as restaurants, cafeterias, hotels, lunch centers, hospitals, etc. has its own characteristics, and in order to efficiently treat these wastewaters, it takes time. Microbial treatment corresponding to wastewater is necessary. In addition, regarding microbial preparations, countermeasures are also required because microorganisms are killed or their ability to grow is reduced by hot water waste water or detergents with strong sterilizing power.
In addition, many microorganisms and enzymes with normal oil / fat degradability at normal temperatures are known, but they have sufficient resolution even under low temperature conditions in winter or in cold regions, and are difficult to decompose because they tend to solidify. No microorganism or enzyme has been known that has satisfactory resolution for lard.
Incidentally, the known microbial preparation 314 also has satisfactory fat and oil resolution for normal animal and plant oils at normal temperatures, but in terms of resolution under low-temperature conditions such as in winter or in cold regions, and resolution against lard. It is not always satisfactory. The difficulty of decomposition of lard by microorganisms and the difficulty of decomposition of fats and oils at low temperatures are as follows.

Difficulty of lard decomposition;
Lard is used in a variety of dishes, and the amount of lard contained in wastewater is never negligible. However, lard is not only difficult to be decomposed by microorganisms due to its large molecular weight, but also presents as a solid in the grease trap due to its low melting point and does not dissolve or suspend in water, making it more difficult for microorganisms to decompose It is said. As described above, various reports have been made on oil-degradable microorganisms and lipases produced by these microorganisms, but few reports have been made on microorganisms and lipases that can satisfactorily degrade lard.
For example, in “Heisei 14 Research Report Vol.52” of Ishikawa Prefectural Industrial Test Station, the difficulty of lard degradation by microorganisms is described as follows (see Non-Patent Document 1).
“In this study, we devised two types of biological oil treatment devices (bioreactors) and tried to microbially decompose three types of commercially available oil and waste oil. As a result, the microorganisms were olived in an aerated device at 27 ° C for 72 hours. The oil 3000ppm decomposed 92%, the engine oil 3000ppm decomposed 97% under the same conditions, and the waste oil discharged from the plating plant was decomposed about 65%, but the lard was hardly decomposed. "

The problem of oil degradation activity in low temperature environment;
The grease trap has to exhibit a sufficient effect even in the winter or in a cold region. However, many known microorganisms have decreased activity in the winter or in a cold region, and have not been able to maintain sufficient decomposition performance. In particular, lard solidified under such low temperature conditions and was difficult to decompose.
Thus, there is room for further improvement in terms of lard resolution and resolution in a low temperature environment.

JP2003-116526A JP 2001-178451 A JP 2005-313159 A JP-A-2005-117773 JP2003-116526A JP 2000-270845 A JP 11-47789 A JP 7-2222988 JP-A-6-277045 JP-A-6-153922 JP-A-5-304949 JP 2004-113238 A Ishikawa Prefectural Industrial Experiment Station “FY2014 Research Report Vol.52”

Accordingly, one of the objects of the present invention is not only having sufficient resolution for a wide range of animal and vegetable oils and oils under normal conditions, but also having effective resolution especially for lard, and It is to provide a novel oil-degrading microorganism having activity even under low temperature conditions in winter and cold regions, and a novel enzyme (lipase) having a similar resolution secreted by these microorganisms. Another object of the present invention is to provide a method for decomposing oil and fat in wastewater using these oil-degrading microorganisms and / or an enzyme (lipase) having a similar resolution secreted by the microorganisms.

As a result of diligent research to solve the above-mentioned problems, the present inventors succeeded in separating a novel microorganism having lard resolution and excellent fat-and-oil decomposition activity even at low temperatures from kitchen wastewater. Was completed.
More specifically, the present invention is as follows.
1. Pseudomonas sp. (Pseudomonas sp.) WU-LM1 strain (reception number; NITE) having a partial base sequence described in SEQ ID NO: 1 in the 16S ribosomal RNA gene having a lipolytic enzyme secretory ability and lard resolution AP-324) and variants thereof having the ability to secrete oleolytic enzymes and lard resolution.
2. An oil-degrading enzyme secreted extracellularly by a Pseudomonas sp. WU-LM1 strain or a variant thereof according to 1 above.
3. Pseudomonas sp. (Pseudomonas sp.) WU-LM1 strain or a variant thereof and / or a fat-containing wastewater treated with the fat-degrading enzyme described in 2 above Processing method.
4). 4. The method for treating oil-containing wastewater according to 3 above, wherein the oil-containing wastewater is wastewater containing lard.
5). Furthermore, surfactant is used together, The processing method of the fat-and-oil containing wastewater of said 3 or 4 characterized by the above-mentioned.
6). Pseudomonas sp. (Pseudomonas sp.) WU-LM1 strain or its variant is diluted 10 to 20,000 times and used, The processing method of fat-and-oil containing wastewater in any one of said 3 thru | or 5 characterized by the above-mentioned.

According to the present invention, not only exhibits a sufficient fat and oil decomposition effect on a wide range of animal and vegetable fats and oils under normal conditions, but also effectively decomposes particularly against lard, and in winter and cold. The oil and fat decomposition effect can be exhibited even under low temperature conditions. Therefore, it can be effectively used in restaurants that frequently use lard, restaurants in cold regions, and the like.

As described above, one form of the present invention is as follows. “Pseudomonas having an oil-degrading enzyme secretory ability and lard resolution, wherein the 16S ribosomal RNA gene has a partial base sequence set forth in SEQ ID NO: 1 in the Sequence Listing. sp.) WU-LM1 strain (reception number; NITE AP-324) and its variants having the same ability to secrete oil-degrading enzymes and lard resolution as the WU-LM1 strain. In addition, WU-LM1 stock was deposited at the receiving organization independent administrative institution National Institute of Technology and Evaluation Patent Microorganism Deposit Center (NPMD). The receipt number is NITE AP-324 and the receipt date is February 28, 2007.
Here, the “oil-degrading enzyme secretion ability” means having the ability to secrete an oil-degrading enzyme, ie, lipase, outside the cell. Further, “having lard resolution” specifically means having the ability to reduce lard resolution after 3 days to 40% or less of the initial concentration in an environment of 15 ° C. and 30 ° C. The “mutant” means a mutant having the same properties as the WU-LM1 strain, that is, “oil-degrading enzyme secretion ability and lard resolution” equivalent to the WU-LM1 strain. Also, “equivalent” means matching within a range of 10%. When these microorganisms are used, they may be liquid or dry solids. Here, “resolution” and “decomposition activity” are synonymous.

Another form of the present invention is “Pseudomonas sp. As described in 1 above”.
sp.) An oil-degrading enzyme secreted extracellularly by the WU-LM1 strain or a variant thereof. Is.
The “oil-degrading enzyme” is an oil-degrading enzyme contained in the supernatant obtained by culturing the microorganism of the present invention. In wastewater treatment, these supernatants may be used as they are, or may be used after being concentrated or purified. In use, it may be liquid or dry solid content. The “oil-degrading enzyme” is not necessarily limited to one enzyme, and may be a so-called “oil-degrading enzyme composition” containing a plurality of lipases. Usually, it is an “oil-degrading enzyme composition” containing a plurality of lipases. Further, a preferable “oil-degrading enzyme” herein has a lipase activity of 100 (unit / l) or more after 3 days at 30 ° C.

Another aspect of the present invention is “Pseudomonas sp. As described in 1 above”.
sp.) A method for treating fat-containing wastewater, comprising treating fat-containing wastewater using the WU-LM1 strain or a variant thereof and / or the oil-degrading enzyme described in 2 above. Is.
Specifically, it relates to the treatment of oil-containing wastewater in grease traps. The WU-LM1 strain and its mutant may be used alone or in combination. In addition, the “oil-degrading enzyme” may be used alone or in combination with “WU-LM1 strain or a variant thereof”. The advantage of microbial administration is that frequent use is not required because the oleaginous microorganism itself grows spontaneously in the grease trap.
For the WU-LM1 strain and its mutants, the powdered microorganisms may be put directly into the grease trap, these microorganisms may be solidified and suspended in the grease trap, or water. You may melt | dissolve in and put into a bottle, and you may throw in from a bottle. You may melt | dissolve in water and may supply automatically with an infusion apparatus. Further, these microorganisms may be supported on a floating lightweight porous material (for example, pumice, glass foam, ceramic ball, nonwoven fabric sheet) and floated in the grease strap. The timing of adding these microorganisms to the grease trap is preferably after closing the store where drainage is stopped, but may be during opening.
As the enzyme, the culture supernatant of the microorganism may be used as it is, or it may be used after being concentrated or purified. It may be liquid or dry solid content. As with the case of microorganisms, the addition timing is preferably after closing, but may be during opening. The advantage of enzyme administration is that microorganisms may be killed by the presence of a surfactant or hot water, whereas that is not the case with enzymes.

Moreover, another form of this invention is "the processing method of fat-and-oil containing wastewater of said 3 whose fat-and-oil containing wastewater is wastewater containing lard." Lard contains a relatively large amount of saturated fatty acids such as palmitic acid, oleic acid and stearic acid, and has a high boiling point, so it is solid at room temperature. In this way, lard is composed of a saturated fatty acid that is chemically stable compared to an unsaturated fatty acid, and therefore cannot be sufficiently degraded by ordinary microorganisms. Further, since it is a solid, it is not dissolved or dispersed in water, and it is difficult to decompose by microorganisms in this respect. However, since the microorganisms and / or enzymes of the present invention have excellent resolution against lard, it is possible to effectively cope with wastewater containing lard.

Moreover, another form of this invention is "the processing method of fat-and-oil containing wastewater of said 3 or 4 characterized by using surfactant further together." Surfactants themselves do not have fat-and-oil decomposability, but the fat and oil components in grease traps, such as lard, are dissolved or dispersed in the liquid to increase the chance of contact with microorganisms and enzymes, and the fats and oils are decomposed. Facilitate. The surfactant that can be used is not particularly limited as long as it does not kill microorganisms.
Examples of surfactants include trideceth-3, a linear primary C12-14 alcohol ethylene oxide 3 mol adduct, a linear primary C12-14 alcohol ethylene oxide 7 mol adduct, sodium lauryl sulfate, lauryl ammonium sulfate , Dodecylbenzene sulfonic acid, ammonium lauryl sulfate, sodium xylene sulfonate, sodium lauryl sulfate, cocamide diethanolamine, lauramine oxide, α sulfomethyl C12-18 sodium ester and α sulfo C12-18 fatty acid disodium salt, sodium dodecylbenzene sulfonate, Alkylpolyglycosides, nonylphenoxypoly (ethyleneoxy) ethanol, branched, nonylphenoxypoly (ethyleneoxy) ethanol, branched, alkoxylated linear alcohol , Mixtures of linear primary C12-14 alcohol ethoxylates, linear alkyl sulfates, alkanolamides, octylphenoxypolyethoxyethanol adsorbed on magnesium carbonate, sodium dodecylbenzenesulfonate and isopropyl alcohol, poe (6 ) Tridecyl alcohol, poly (oxy-1,2-ethanediyl), α (nonylphenyl) -ωhydroxy and the like. A nonionic surfactant is preferable, Tween (registered trademark) is particularly preferable, and Tween 60 or Tween 80 is particularly preferable. Although there will be no restriction | limiting in particular if a compounding quantity is an quantity which does not kill microorganisms, Preferably, it is 0.01-10 weight times with respect to microorganisms or an enzyme.

Another form of the present invention is “Pseudomonas sp.”
sp.) WU-LM1 strain (reception number; NITE AP-324) or a variant thereof diluted 100 to 20,000 times and used, containing fats and oils according to any one of 3 to 5 above Wastewater treatment method. Is. A preferable dilution ratio is 500 to 15,000 times.

Next, terms used in this specification will be described.
In the present invention, the term “lipid” means an oily substance derived from animals and plants containing a saturated or unsaturated fatty acid and its glycerin ester. In addition, when they are edible oils, they also include modified oils modified by cooking or the like. Generally, it means oil that is liquid at normal temperature and fat that is solid at normal temperature. Although it does not specifically limit, Preferably it is animal and vegetable oil used for edible and cooking. Specific examples include olive oil, soybean oil, rapeseed oil, beef tallow, lard and the like. In addition, components include saturated fatty acids and unsaturated fatty acids, and specific examples include oleic acid, linoleic acid, docosenoic acid, palmitic acid, stearic acid, lauric acid, oleic acid, myristic acid and the like. it can.
“Saturated fatty acids” include palmitic acid, stearic acid, lauric acid, myristic acid and the like, and are often contained in animal fats such as beef tallow and lard and palm oil. Saturated fatty acids are usually solid because of their high melting points, and are chemically stable because they are saturated, and are more difficult to decompose than unsaturated fatty acids.
Among unsaturated fatty acids, “monounsaturated fatty acid” means an unsaturated fatty acid having one double bond, and specific examples include olive oil and rapeseed oil.
“Polyunsaturated fatty acid” means an unsaturated fatty acid having two or more double bonds. Specifically, α-linolenic acid (eg perilla oil), docosahexaenoic acid, eicosapentaenoic acid, linoleic acid, etc. Can be mentioned.
“Animal and vegetable oil” means vegetable oils such as olive oil, safflower oil, soybean oil, and animal oils such as het (beef tallow) and lard (pig tallow). Vegetable oil contains a large amount of oleic acid and linoleic acid and is usually liquid. Animal oils contain a relatively large amount of palmitic acid, oleic acid, and stearic acid, and are usually solid. Oils and fats of aquatic animals such as whales and dolphins are liquid because they contain a lot of unsaturated fatty acids (eicopentaenoic acid, docosahexaenoic acid, etc.).
“Waste oil” or “oil-and-fat-containing wastewater” to “oil-and-fat-containing wastewater” means wastewater or wastewater containing these oils and fats as waste.
The meanings of “oil-degrading enzyme secretory ability” and “Lard resolution” are as described above.
“Secreted extracellularly” means that the produced enzyme is completely released outside the cell and can be separated from the cell. Usually, it is released into the culture medium. Therefore, the microorganism of the present invention can be clearly distinguished from a membrane-bound enzyme as reported in JP-A No. 2001-178451 in this sense.
“Treatment of fat / oil-containing wastewater” means “oil / fat” in the above-mentioned “oil / fat-containing wastewater” is an oil-degrading microorganism comprising the WU-LM1 strain and / or an oil-degrading enzyme (lipase) produced by the microorganism. It means to decompose. Thereby, the fats and oils in fat-containing wastewater can be reduced, thereby reducing or eliminating contamination and bad odor.
“Oil-degradable Pseudomonas sp. WU-LM1 strain (reception number: NITE AP-324)” in which the 16S ribosomal RNA gene has a partial base sequence described in SEQ ID NO: 1 of the Sequence Listing is a deposit number It is an oil-degradable Pseudomonas sp. (Pseudomonas sp.) WU-LM1 strain in which the 16S ribosomal RNA gene which is 06-161 has the partial base sequence described in SEQ ID NO: 1 in the sequence listing, and more specifically is contained in Pseudomonas , A new strain belonging to P. Koreesis.
The processing method of the fat-and-oil containing waste water in a grease lap is not specifically limited, What is necessary is just to perform according to a well-known method. For example, an appropriate amount of the microorganism or enzyme of the present invention may be added to the grease trap at night when business is terminated and the inflow of wastewater stops. At this time, the timing of addition may be controlled using a timer. When using microorganisms, it is preferable to send air by an aeration pump. When an enzyme is used, the supernatant obtained when the microorganism is cultured may be used as it is, or it may be concentrated or purified. The form of the microorganism and the enzyme may be a powder, or may be a liquid containing the microorganism or the enzyme. Moreover, you may use this microorganism and an enzyme together.

Hereinafter, a specific test example will be described. However, it goes without saying that the present invention is not limited to these specific examples.

1. Test material and measurement method;
(1) Used fats and oils;
The relationship between the main fats and oils used in this test and their main components is as shown in Table 1 below.

(2) liquid medium;
Medium A shown in Table 2 below was used as the liquid medium, and Medium B shown in Table 2 below was used as the plate medium for selection. As the substrate, various animal and vegetable oils (olive oil, rapeseed oil, soybean oil, beef tallow, lard) were used.

(3) Simulated drainage;
The composition of the simulated wastewater used in this experiment is shown in Table 3 below.

(4) Measuring method of lipase activity The lipase activity was analyzed using Lipase Kit S (Dainippon Pharmaceutical Co., Ltd.). The amount of enzyme that liberates 1 μmol SH group per minute at 30 ° C. when dimercaprol tributyrate was used as a substrate was defined as 1 unit.

(5) Method for measuring fat / oil decomposition activity using simulated wastewater Here, lipid resolution using simulated wastewater is measured by placing 200 ml of simulated wastewater in a 500 ml container, The prepared 314 microbial preparation was added, cultured with shaking at 150 rpm and 30 ° C. for 3 days, and the amount of residual fat was measured by hexane extraction according to JISK-0102.
Pre-culture conditions are LB medium added with beef tallow 2% (w / v), 30 ° C., 240 rpm, and the culture time is 1 day.

2. Oil and fat degradation test of microorganism preparation 314 at 30 ° C. and 15 ° C .;
(1) Resolution test of known microorganism preparation 314 at 30 ° C .;
A known 314 preparation was diluted 3,000 times, and the degradation activity on oils and fats (olive oil, soybean oil, rapeseed oil, beef tallow and lard) was measured at 30 ° C. The results are shown in Table 4. The concentration of olive oil, soybean oil, rapeseed oil and beef tallow was reduced from 500 mg / l to 80 to 125 mg / l after 8 hours. The rapeseed oil had a slightly slower oil and fat decomposition rate than olive oil and soybean oil. This is probably because the main constituent fatty acid of rapeseed oil is docosenoic acid and has a long carbon number of 22. However, Lard was 410 mg / l after 8 hours, and its decomposition effect was not sufficient. It is unclear why beef tallow is broken down and lard is not broken down.

(2) Table 4 shows the results of the test conducted in the same manner as in the above (1) except that the temperature of the known microorganism preparation 314 at 15 ° C was 15 ° C. The oil / fat concentrations after 8 hours were 380 mg / l for olive oil, 420 mg / l for soybean oil, 470 mg / l for rapeseed oil, and 500 mg / l for beef tallow and lard. The degradation rate of various fats and oils at 15 ° C. was lower than that at 30 ° C., and the microbial preparation 314 was not satisfactory for use in cold regions or in winter.

  As is clear from Table 4 above, the microbial preparation 314 had a degrading activity against olive oil, soybean oil, rapeseed oil and beef tallow at 30 ° C., but did not show satisfactory resolution against lard. Further, at 15 ° C., no effect could be expected from the viewpoint of practical use.

3. Search for oil-degradable microorganisms;
In order to efficiently treat oil-containing wastewater under low-temperature conditions, we searched for microorganisms with high oil-decomposing activity even at low temperatures. Specifically, we searched for new strains from wastewater samples from a large restaurant kitchen. Medium A was used as the liquid medium, and Medium B was used as the selection plate medium. The composition of Medium A and Medium B is as described above. As the substrate, various animal and vegetable oils (olive oil, rapeseed oil, soybean oil, beef tallow, lard) were used.
As a result, we succeeded in newly separating four oil-degrading microorganism strains (A strain, B strain, C strain and D strain) from the wastewater sample. Among these, the A strain and the B strain showed particularly high lipase activity and fat-and-oil decomposition activity. All strains produced an oil-degrading enzyme (extracellular enzyme) in the culture supernatant.
These strains A and B were identified, and further, SDS-PAGE analysis of extracellular enzyme, measurement of extracellular enzyme amount and lipase activity were performed as follows.

(1) Measurement of fat and oil decomposing ability of novel oil-degrading microorganisms;
In accordance with the method described above, 200 ml of the simulated waste water was placed in a 500 ml container, then inoculated with 1% preculture solution, inoculated, and incubated at 150 rpm at 30 ° C. for 1 day. The residual fat amount was measured by hexane extraction based on the above.

(2) Measuring method of lipase activity;
This was carried out according to the above-mentioned “Method for measuring lipase activity”.

(3) Identification and activity evaluation of novel oil-degrading microorganisms;
The strains were identified by 168 rRNA gene analysis for four new oil-degrading microorganism strains (A strain, B strain, C strain and D strain). The control is strain OFD1 taken from microbial preparation 314.
The evaluation results were as shown in Table 5 below. Details of the identification and characteristics of the A strain will be described later.

Results A and B strains showed high homology with Pseudomonas sp. The C strain is Pseudomonas aeruginosa and the D strain is Serratia.
High homology with liquefaciens and Serratia protemaculams.
Pseudomonas aeruginosa and Serratia are reported as lipase producing bacteria, but both are opportunistic infections. Therefore, C and D strains are not suitable for wastewater treatment of grease traps.
In addition, the A strain and the B strain are considered to be effective in wastewater treatment of grease traps because they have both higher oil decomposition activity and lipase activity than Bacillus sp. WU-OFD1, which is an isolate from the microbial preparation 314. Therefore, the A strain and the B strain were selected as candidate strains. In addition, the A strain was named “WU-LM1”.

(4) SDS-PAGE analysis of extracellular enzymes of novel oil-degrading microorganisms SDS-PAGE analysis of culture supernatants (extracellular enzymes) of oil-degrading microorganisms was performed on these four strains.
The molecular weight of lipase is generally reported as 25 to 65 kDa. Since any of the strains A to D shows a band in this molecular weight range, it is considered that lipase is secreted as an extracellular enzyme. However, since a plurality of bands were seen instead of a single band, it is considered that a plurality of extracellular enzymes may be involved in fat and oil degradation.

(5) Measurement of growth and degradation activity of novel oil-degrading microorganisms A and B strains that showed higher oil-degrading activity against olive oil and beef tallow than OFD1 strain, their growth (according to OD660) and degradation activity over time And growth was measured. For comparison, the same measurement was performed for OFD1 strain. The results are shown in Tables 6 and 7. All are the measurement results after 3 days at 30 ° C.

As is clear from Tables 6 and 7, the A strain and the B strain are not only excellent in fat and oil resolution but also excellent in viability.

(6) Measurement of the amount of extracellular enzyme produced by a novel oil-degrading microorganism and its lipase activity Extracellular enzymes produced by strains A and B which showed higher oil-degrading activity than olive oil and beef tallow than OFD1 strain The amount of enzyme and the lipase activity were measured. For comparison, the same measurement was performed for OFD1 strain. The results are shown in Tables 8 and 9. All are the measurement results after 3 days at 30 ° C.

Results A strain and B strain showed high fat-and-oil decomposition activity. In particular, it was revealed that the A strain showed higher fat decomposing activity than the OFD1 strain, and also had higher fat decomposing activity against animal fats such as beef tallow.
Furthermore, both the A strain and the B strain showed higher lipase activity (oil resolving power) than the OFD1 strain in both cases of olive oil degradation and beef fat degradation. In particular, the A strain showed a very high lipase activity. Moreover, from the result of the protein amount of the extracellular enzyme, it can be seen that the A strain is particularly high as the lipase activity per unit protein amount.
From these facts, it can be said that the A strain is a strain having high fat decomposing activity and is very promising in the fat decomposing treatment.

(7) Identification of a novel WU-LM1 strain (A strain);
The WU-LM1 strain (A strain) was further classified and analyzed for mycological characteristics as follows.

(I) culture conditions;
A strain obtained by aerobic culture for 24 hours on an LB agar medium (1% tryptone, 0.5% dry yeast extract, 1% NaCl) at a culture temperature of 30 ° C. was used as a test cell.

(Ii) 16S rDNA homology search;
The operations from extraction to cycle sequence were performed based on the following protocol.
・ DNA extraction InstaGene Matrix (BIO RAD, CA, USA)
・ PCR PrimeSTAR HS DNA Polymerase (Takara Bio, Shiga)
Cycle sequence BigDye Terminator v3.1 Cycle Sequencing Kit
(Applied Biosystems, CA, USA)
・ Primers used 9F, 339F, 785F, 1099F, 536R, 802R, 1242R, 1510R
・ Sequence ABI PRISM 3100 Genetic Analyzer System
(Applied Biosystems, CA, USA)
・ Analysis software Auto Assembler (Applied Biosystems, CA, USA)
DNASIS Pro (Hitachi Software Engineering, Tokyo)
・ Homology search Bacteria reference strain database (Techno Suruga, Shizuoka)
International nucleotide sequence data pace (GenBank / DDBJ / EMBL)

(Iii) Bacteria first stage test;
Based on morphological observation with an optical microscope and the method of BARROW ¹⁾ and others, tests were conducted on catalase reaction, oxidase reaction, acid / gas production from glucose, and oxidation / fermentation (O / F) of glucose.
・ Gram dyeing Faber G "Nissui" (Nissui Pharmaceutical, Tokyo)
・ Microscope Optical microscope BX50F4 (Olympus, Tokyo)
The results were as follows.

(Iv) Bacteria second stage test;
API 20NE (bioMerieux. Lyon,
France) kit was used.
In addition, additional tests were conducted on the production of fluorescent dyes. The results are as follows.

(V) Conclusion;
As a result of homology search against the bacterial reference strain database using BRAST ²⁾ , the 16S rDNA base sequence of the A strain (WU-LM1 strain) showed a high homology with the 16S rDNA derived from Pseudomonas, and the homology rate was 99.4. % P. Koreensis ³⁾ Highest homology to 16S rDNA of strain Ps 9-14.
As a result of homology search against GenBank / DDBJ / EMBL, 16S rDNA of A strain (WU-LM1 strain) showed high homology to 16S rDNA derived from Pseudomonas, but 16S rDNA derived from the reference strain was searched. There wasn't. In addition, as a result of simple molecular phylogenetic analysis using 16S rDNA of A strain (WU-LM1 strain) and 16S rDNA of the top 30 homology search against the bacterial reference strain database, strain A (WU-LM1 strain) was obtained. ) Formed a phylogenetic branch with 16S rDNA of P. Koreesis.
From the above, it is considered that the A strain (WU-LM1 strain) is included in Pseudomonas and may belong to P. Koreesis.
However, both 16S rDNAs do not completely match, and although the A strain (WU-LM1 strain) is closely related to P. Koreesis, it may be a different strain as a species. Therefore, from the result of this 16S rDNA nucleotide sequence analysis, the A strain (WU-LM1 strain) was presumed to be Pseudomonas sp. Closely related to P. Koreesis.
As a result of the bacterial first stage test, the A strain (WU-LM1 strain) was a gram-negative gonococci having motility and oxidized glucose, and both the catalase reaction and the oxidase reaction were positive. As a result of the bacterial second stage test using the API kit, the A strain (WU-LM1 strain) did not reduce nitrate, showed arginine dihydrolase activity, and assimilated glucose, arabinose and the like. Furthermore, as a result of the additional test, the A strain (WU-LM1 strain) produced a fluorescent dye on King's B agar and grew at 4 ° C, but did not grow at 41 ° C. Moreover, it did not grow on a medium containing 5% NaCI, did not produce levan, showed no lecithinase activity, and showed lipase activity. These properties are thought to have many similarities to P. Koreesis, which was suggested to be related in 16S rDNA nucleotide sequence analysis. However, it was different from P. Koreesis in that it did not grow at 5% NaCI.
From the above, A strain (WU-LM1 strain) is Pseudomonas
It is considered to be most closely related to P. Koreesis among known species. However, the results of 16S rDNA nucleotide sequence analysis and physiological and biochemical property tests suggested that the A strain (WU-LM1 strain) was slightly different from P. Koreesis.
Therefore, from the results of the bacterial premium test this time, it is considered appropriate that the A strain (WU-LM1 strain) is Pseudomonas sp. Closely related to P. Koreesis.
The WU-LM1 strain (A strain) was deposited on February 28, 2007 at the Patent Microorganism Deposit Center, National Institute of Technology and Evaluation. The receipt number is NITE AP-324.
16S rDNA is as shown in SEQ ID NO: 1.
1); BARROW, (G.1.)
and FELTHANM, (RKA): Cowal and Steel's Manual for the Identification of
Medical Bacteria. 3 ^rd edition. 1993, Cambridge University Press.
2); ALTSCHUL,
(SF), MADDEN, (TF), SCHAFFER, (AA), ZHANG, (1.), ZHANG Z., MILLER, (w.),
and LIPMAN, (DJ): Gapped BLAST and PS1-BLAST: a new
generation of protein database search programs, Nucleic Acids. 1997. 25,
3389-3402.
3) ； KWON (SW),
KIM (JS), PARK (IC), YOON (SH), PARK (DH), LIM (CK), and GO (SJ):
Pseudomonas koreesis sp. Nov., Pseudomonas umsongensis sp. Nov. and Pseudomonas
jinjuensis sp. Nov., novel species from farm soid in Korea. Int. J. Syst. Evol.
Microbiol., 2003, 53, 21-27.

4). Oil and fat decomposition activity of WU-LM1 strain (A strain) under various temperature conditions Next, fats and oils for various fats and oils at 15 ° C and 30 ° C of WU-LM1 strain (A strain) obtained according to the above examples. Degradation activity was measured. As a control, microbial preparation 314 was used.

(1) Method for Measuring Oil Decomposition Activity Using Simulated Wastewater Using the aforementioned “simulated wastewater”, the method was carried out according to the above-mentioned “Method for measuring oil and fat decomposition activity using simulated wastewater”.

(2) Measuring method of lipase activity It carried out according to the above-mentioned "Method for measuring lipase activity".

(3) Various fats and oils described in Table 1 were used as the fats and oils used.

4-1 Measurement of degradation activity on various oils and fats at 1.30 ° C. Degradation activity on various oils and fats of Pseudomonas sp. WU-LM1 at 30 ° C. considered to be the most standard temperature during grease trap operation and WU-LM1 The lipase activity derived from the strain was measured. In addition, the growth ability was also measured. As a control, microbial preparation 314 was used. The results are shown in Tables 12-14. All are the measurement results after 3 days at 30 ° C.

Results The WU-LM1 strain showed a lipase activity significantly higher than that of the microorganism preparation 314 (see Table 13), and also showed a high degradation activity against animal and vegetable oils and fats (see Table 12). In particular, it showed higher degradation activity than animal microorganisms 314 for animal fats and oils. Regarding the viability, the WU-LM1 strain showed nearly twice the viability of the microbial preparation 314.

4. Measurement of Degradation Activity on Various Oils at 4-2.15 ° C. The degradation activity and lipase activity on various fats and oils of Pseudomonas sp. WU-LM1 strain at 15 ° C. were measured assuming cold regions or winter. The same experiment was performed for the microbial preparation 314 and compared. The results are shown in Tables 15-17. All are the measurement results after 3 days at 30 ° C.

Results The WU-LM1 strain was able to grow and proliferate even under a low temperature condition of 15 ° C., and showed higher degradation activity and lipase activity than various microorganism preparations 314 against various oils and fats. From this, it can be seen that the WU-LM1 strain is extremely suitable for the oil and fat decomposition treatment at low temperatures.

5. Effect of addition of surfactant (Tween 80) With regard to the effect of the presence or absence of addition of surfactant (Tween 80) on the oil degradation activity, the degradation activity of olive oil and lard of WU-LM1 strain at 15 ° C and 30 ° C was measured. did.
As a result, it was confirmed that the addition of a surfactant (Tween 80) slightly increased the oil decomposing activity and the lipase activity.

6). Examination of dilution rate of WU-LM1 strain As a result of examination on the dilution rate and fat resolution of microbial preparations using WU-LM1 strain, good results were obtained when the dilution rate ranged from 10 to 20,000 times. It was. A particularly preferable range was 500 times to 10,000 times.

7). Surfactant (Tween 80) was added to the culture solution of Pseudomonas sp. WU-LM1 strain cultured in the field introduction test laboratory of WU-LM1 strain to prepare a microbial preparation. After adding 70 l (70,000 ml) of water to 100 ml of the microbial preparation and diluting with a plastic tank, the diluted solution was put into a medium-sized Japanese restaurant kitchen grease lap after the end of business hours. Then, the fats and oils decomposition process was performed, performing aeration using an aeration apparatus.

Results Before the introduction of the microbial preparation using the WU-LM1 strain, the amount of fat and oil in the grease trap wastewater was 1,500 to 2,500 mg / l, and a large amount of fat was visible, but the WU-LM1 strain was On the 4th day after the introduction of the used microbial preparation, the amount of fat and oil was greatly reduced to 150 mg / l, the grease trap was remarkably clarified, and it was demonstrated that the WU-LM1 strain was effective for the fat and oil decomposition treatment of the wastewater.

8). Oil degradation activity of enzymes produced from WU-LM1 strain
The culture supernatant 1-5% of WU-LM1 strain was added to 200 ml of simulated waste water, and the degradation activity against olive oil and lard at 30 ° C, 15 ° C and 45 ° C was measured.
8-1. Measurement result of degradation activity at 30 ° C. The degradation activity on olive oil and lard increased with the increase of the amount of WU-LM1 strain culture supernatant added, and 5% addition showed degradation activity on olive oil and lard. Was found to be approximately equivalent to the degradation activity of the WU-LM1 strain.
8-2. Measurement result of degradation activity at 15 ° C. The degradation activity on olive oil and lard increased with the increase of the amount of WU-LM1 strain culture supernatant added. Was found to be approximately equivalent to the degradation activity of the WU-LM1 strain.
8-3. Measurement result of degradation activity at 45 ° C. At 45 ° C., the oil degradation activity of WU-LM1 strain was low with respect to olive oil and lard, but with addition of WU-LM1 strain culture As the amount of the supernatant added increased, the degradation activity against olive oil and lard increased, and when 5% was added, the degradation activity against olive oil and lard was considerably high.
The WU-LM1 strain cannot grow at 45 ° C. and thus shows a low value for the oil decomposing activity. However, the extracellular enzyme of the WU-LM1 strain is considered to have a high oil decomposing activity even at 45 ° C.

As described above, the oil-degradable Pseudomonas sp.
sp.) WU-LM1 strain (reception number; NITE AP-324) is similar to that of existing fungi but produces enzymes outside the cells and does not grow on 5% NaCl. It can be said that it is a new type of strain due to its outstanding decomposition activity against lard compared to conventional products and its excellent oil and fat decomposition activity even at low temperatures.
This WU-LM1 strain has a particularly excellent resolution against lard, which has been difficult until now, and also has an excellent oil / fat resolution even in a low temperature region. Enzymes produced by the WU-LM1 strain outside the cells also have excellent oil decomposing activity.

The oil-degradable Pseudomonas sp. WU-LM1 strain (reception number; NITE AP-324) of the present invention and its variant having the same oil-degrading enzyme secretion ability and lard resolution as the WU-LM1 strain are: , Not only has an excellent oil decomposition effect on a wide range of animal and vegetable oils and oils under normal conditions, but also has an effective resolution especially against lard and low temperature conditions in winter and cold regions But it demonstrates its fat and oil decomposition effect. Therefore, it can be effectively used in restaurants that frequently use lard, restaurants in cold regions, and the like.

Claims (5)

Oil-degrading Pseudomonas sp. (Pseudomonas sp.) WU-LM1 strain (Accession number; NITE) having a partial base sequence described in SEQ ID NO: 1 in the Sequence Listing, which has an oil-degrading enzyme secretion ability and lard resolution P-324) or a variant thereof having an oil-degrading enzyme secretion ability and lard resolution equivalent to those of the WU-LM1 strain. Pseudomonas sp according to claim 1 (Pseudomonas sp.) WU- LM1 strain or variant and / or the Pseudomonas thereof
A method for treating fat-containing wastewater, comprising treating a fat-containing wastewater with a supernatant containing an oil-degrading enzyme or a concentrate thereof obtained by culturing the WU-LM1 strain or a mutant thereof .   The method for treating fat-containing wastewater according to claim 2, wherein the fat-containing wastewater is wastewater containing lard.   Furthermore, surfactant is used together, The processing method of the fat-and-oils containing wastewater of Claim 2 or 3 characterized by the above-mentioned. Pseudomonas sp. (Pseudomonas sp.) WU-LM1 strain or its variant is diluted 10 to 20,000 times and used, The processing method of the oil-containing wastewater in any one of Claim 2 thru | or 4 characterized by the above-mentioned .