JP2017136033A - Oil content-decomposing microorganism - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a microorganism that is effective in reducing an oil content in a grease trap, particularly, a microorganism that can purify drain water even in water quality environments in a wide range of salt concentrations.SOLUTION: The problems is solved by an oil content-decomposing microorganism identified with Sphingomonas sp. LM02-032 strain (accession number NITE P-02069).SELECTED DRAWING: None

Description

  The present invention relates to a novel oil-degradable microorganism belonging to the genus Sphingomonas. The present invention also relates to a wastewater treatment method and wastewater treatment agent using the microorganism.

  Wastewater (waste water) from kitchens and food factories usually contains garbage and cooking oil. Solids such as raw garbage can be easily removed from the waste water by providing a basket or the like at the drain outlet, but it is not easy to remove liquid substances such as cooking oil. Therefore, in facilities such as kitchens and food factories that discharge wastewater mixed with a large amount of oil, a grease trap is provided for collecting oil and separating and discarding the oil floating on the upper layer.

  However, the oil accumulated in the grease trap solidifies and remains as scum (lumps of oil) on the water surface of the grease trap, or it accumulates and adheres to the inner wall surface of the grease trap or inside the piping to block the piping. There is. At this time, the accumulated oil may be oxidized and spoiled and cause odors and pests. Also, if the accumulated oil is left unattended, the oil trap's ability to remove oil will be reduced, causing oil to flow into sewage and rivers. For this reason, when oil is accumulated in the grease trap, it is necessary to request a specialized supplier to remove the oil by vacuum treatment or high-pressure washing treatment, which increases costs.

  Therefore, a method for efficiently reducing oil content in a grease trap, in particular, a method using microorganisms that decompose and assimilate the oil content has been studied. For example, in Patent Document 1, Bacillus subtilis BN1001 (Bacillus subtilis BN1001) is used as a microorganism that can be used in applications such as reducing n-hexane extract in oil-containing wastewater or decomposing scum that accumulates in drainage tanks such as kitchens. Is described. Patent Document 2 describes an invention relating to a culture solution of Bacillus megaterium that can be used in an oil trap.

JP-A-3-236771 Special table 2004-528852 gazette

  However, with conventional microorganisms, it may be difficult to sufficiently reduce the oil content contained in the wastewater in the grease trap. In particular, the water quality such as the salinity of the waste water in the grease trap can vary greatly depending on the food residue discharged. For this reason, microorganisms used in grease traps have a low salt concentration (for example, wastewater containing 0.005% (w / v) sodium chloride) to a high salt concentration (for example, 4% (w / v)). In other words, the wastewater containing sodium chloride is required to be able to purify the wastewater even in a wide variety of salt water environments. However, conventionally known microorganisms have not had such characteristics sufficiently.

  Therefore, this invention is made | formed in view of the said situation, and makes it a subject to provide the microorganisms excellent in the reduction effect of the oil content in a grease trap. In particular, it is an object of the present invention to provide a microorganism capable of purifying wastewater even in a water quality environment having a wide range of salt concentrations (for example, wastewater containing 0.005 to 4% (w / v) sodium chloride).

  The present inventors have intensively studied to solve the above problems. As a result, the present inventors have found that the above problems can be solved by an oil-degrading microorganism belonging to the genus Sphingomonas and exhibiting a predetermined mycological property, and the present invention has been completed.

  ADVANTAGE OF THE INVENTION According to this invention, the microorganisms excellent in the reduction effect of the oil content in a grease trap are provided. In particular, according to the present invention, a microorganism capable of purifying wastewater even in a water quality environment having a wide range of salt concentrations (for example, wastewater containing 0.005 to 4% (w / v) sodium chloride) is provided.

FIG. 1 schematically shows the mechanism of wastewater treatment by a grease trap.

  Embodiments of the present invention will be described below. In addition, this invention is not limited only to the following embodiment. In addition, the dimensional ratios in the drawings are exaggerated for convenience of explanation, and may be different from the actual ratios.

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

[Oil-degrading microorganisms]
In one aspect of the present invention, an oil-degrading microorganism belonging to the genus Sphingomonas and exhibiting the following mycological properties is provided. In one embodiment of the present invention, there is provided an oil-degrading microorganism belonging to the genus Sphingomonas, having the following mycological properties and having a 16S rDNA base sequence represented by SEQ ID NO: 1. In the present specification, the term “oil-decomposing microorganism” refers to microorganisms such as bacteria and fungi whose oil content reduction rate measured by the method described later is 10% by weight or more at least one of low salinity and high salinity. Say. Preferably, in the oil-degrading microorganism, the oil decrease rate measured by the method described later is 15% by weight or more in at least one of the low salinity concentration and the high salinity concentration.

(screening)
As a specific example of the oil-degrading microorganism of the genus Sphingomonas as described above, a strain isolated by the following screening method is illustrated and described in more detail.

1. Screening method The components of medium A (agar-free) shown in Table 4 below were dissolved in deionized water, and the pH was adjusted to 5 with hydrochloric acid. Next, an oil component (soybean oil: rapeseed oil = 1: 1 (w / w)) is added to the prepared solution so that the final concentration becomes 1% (w / v), and high-temperature and high-pressure sterilization is performed for accumulation culture. Used as medium.

  Samples taken from soil, grease trap drainage (wastewater), seawater, sewage, river water, hot spring water, mud, etc. were added in 0.1 g each to a test tube into which 5 mL of the culture medium for integrated culture was dispensed. The medium supplemented with the sample was cultured in an incubator with shaking at 110 rpm at 30 ° C. for 1 week. 100 μL of the culture solution was collected, added to 5 mL of the culture medium for accumulation culture separately prepared, and cultured for 1 week under the same conditions three times (cultured for a total of 4 weeks) to obtain the accumulation culture solution.

  Separately, the components of medium A shown in Table 5 below were dissolved in deionized water, and the pH was adjusted to 5 with hydrochloric acid.

  The medium A and the oil (soybean oil: rapeseed oil = 1: 1 (w: w)) were separately sterilized at high temperature and high pressure. 20 mL of sterilized A medium was dispensed into a petri dish and solidified. Thereafter, 100 μL of sterilized oil was smeared on the solidified medium A to obtain a medium for separation culture. 100 μL of the above enriched culture solution was smeared on a culture medium for separation culture and cultured in an incubator at 30 ° C. for 1 week.

  After culturing, single colonies were collected with a platinum loop and 5 mL of LB medium (1% (w / v) polypeptone (manufactured by Nippon Pharmaceutical), 5% (w / v) yeast extract (manufactured by Oriental Yeast), 1% (W / v) adjusted to pH 6.0 with sodium chloride and hydrochloric acid) and cultured for 24 hours in a 30 ° C. incubator with shaking at 140 rpm. After culture, 100 μL of the culture solution was added to LB agar medium (2% (w / v) agar, 1% (w / v) polypeptone (manufactured by Nippon Pharmaceutical), 5% (w / v) yeast extract (manufactured by Oriental Yeast) ) 1% (w / v) sodium chloride, pH adjusted to 6.0 with hydrochloric acid) and cultured in an incubator at 30 ° C. for 24 to 72 hours for purification.

  Next, the purified colony was inoculated with 1 platinum ear in 5 mL of LB medium and cultured in a 30 ° C. incubator for 24 hours with shaking at 140 rpm to obtain a preculture solution.

  In order to use it in the oil decomposition test, a TG medium was prepared with the following composition, and the pH was adjusted to 5.0 with hydrochloric acid.

  0.05 g of oil (rapeseed oil: soybean oil: beef tallow = 2: 2: 1 (w / w / w)) was added to the TG medium (5 mL) prepared by the above method and sterilized at high temperature and high pressure, Different oil decomposition test solutions were prepared (oil 1% (w / v)).

To each of the oil decomposition test solutions, 100 μL of a preculture solution (amount of bacterial cells: 2 × 10 ⁶ CFU) was added and cultured in a 30 ° C. incubator for 24 hours while shaking at 140 rpm.

  After the culture, a normal hexane extract was prepared according to JIS K0102: 2013 revision (industrial wastewater test method). Using the normal hexane extract as the remaining amount of oil, the oil content reduction rate was calculated from the oil content (0.05 g) added during the preparation of the test solution and the remaining amount of oil (the amount of normal hexane extract (g)) according to the following formula 1. Asked.

  As a result, a strain exhibiting a high oil content reduction rate was isolated from both 1m deep and low salinity oil decomposition test liquid and high salinity oil decomposition test liquid from mud 1m depth of Onna village Saeida fishing port in Okinawa Prefecture.

2. 2. Classification of strains 2-1. 16S rDNA base sequence The isolated strain was subjected to aerobic culture at 30 ° C. for 24 hours using an LB agar medium (Becton Dickinson), and used for classification of the following strains.

  About the isolated strain, the 16S rDNA base sequence was analyzed by ABI PRISM 3130 xl Genetic Analyzer System (Applied Biosystems). The determined 16S rDNA base sequence of the isolated microorganism is shown in SEQ ID NO: 1 below.

  As a result of BLAST homology search against DNA database DB-BA10.0 (Techno Surga Lab) for microorganism identification, the 16S rDNA base sequence of the isolated microorganism is highly homologous to the 16S rDNA base sequence of the genus Sphingomonas. (Homology rate). Isolated microorganisms are especially S. cerevisiae. It showed the highest homology of 99.9% with respect to the pseudosanguinis strain G1-2. Also in the homology search for GenBank / DDBJ / EMBL, the 16S rDNA base sequence of the isolated microorganism showed high homology to the 16S rDNA base sequence of the genus Sphingomonas. Table 7 shows the results of the homology search for the microorganism identification DNA database DB-BA10.0, and Table 8 shows the results of the homology search for the international nucleotide sequence database.

2-2. Mycological properties The mycological properties of the strains isolated by the above screening are shown below. Morphological observation was performed using an optical microscope (BX50F4, Olympus). Faber G “Nissui” (Nissui Pharmaceutical) was used for Gram staining. Tests for catalase, oxidase, acid / gas production from glucose, oxidation / fermentation from glucose are described by Barrow et al. (Barrow, (GI) and Feltham, (RKA): Cowan and Steel's Manual for the Identification of Medical Bacteria. 3rd edition. 1993, Cambridge University Press).

  The following items were tested using API (registered trademark) 20NE and API (registered trademark) -ZYM (both Sysmex Biomelu) and according to the manufacturer's protocol. The result is shown. Regarding the evaluation of acid production using cellobiose, maltose, D-mannose, melibiose, and rhamnose, the following items were tested based on “New Bacteriological Culture Course” written by Toshikazu Sakazaki (published by Modern Publishing).

The test results, "YABUUCHI E. and KOSAKO Y.: Genus I. Sphingomonas Yabuuchi, Yano, Oyaizu, Hashimoto, Ezaki and Yamamoto 1990b, 321 VP (Effective publication: Yabuuchi, Yano, Oyaizu, Hashimoto, Ezaki and Yamamoto 1990a, 116 Emend.Takeuchi, Hamana and Hiraishi 2001, 1414; emed.Yabuchi, Kosako, Fujiwara, Naka, Matsunaga, Ogura and Kobayashi, 2002'14. The Systemic Bacteriology, 2nd edn.vol.Two, The Proteobacteria, Part C The Alpha-, Beta-, Delta, and Eplonproteobacteria, ER. Kampfer P. et al., Described in Sphingomonasseudosanguinis sp. Nov., Isolated from the water reservoir of an air humidifier.Int.J. They were compared with the bacteria.

  In one aspect of the present invention, an oil-degrading microorganism belonging to the genus Sphingomonas and exhibiting the above mycological properties is provided. In one embodiment of the present invention, there is provided an oil-degrading microorganism belonging to the genus Sphingomonas, exhibiting the above bacteriological properties, and having the 16S rDNA base sequence represented by SEQ ID NO: 1.

3. Various properties The isolated strain was a gram-negative bacillus having motility, the colony color was yellow on the LB agar medium, glucose was not oxidized, catalase reaction was positive, and oxidase reaction was negative. These properties were consistent with those of the known genus Sphingomonas. In addition, the isolated strain does not reduce nitrate (reduction of potassium nitrate to potassium nitrite, reduction of potassium nitrate to N ₂ gas), does not show arginine dihydrolase activity, hydrolyzes esculin, glucose, D -Assimilated mannose, maltose and dl-malic acid and not assimilated L-arabinose, D-mannitol and sodium citrate. Furthermore, the isolated strain exhibits alkaline phosphatase activity, acid phosphatase activity and β-galactosidase activity, no β-glucuronidase and α-glucosidase activity, oxidizes cellobiose and D-mannose, maltose, melibiose and rhamnose Did not oxidize. Although these properties are in common with those of the genus Sphingomonas, differences were also observed. In particular, L-arabinose and sodium citrate were not assimilated, α-glucosidase activity was not exhibited, and cellobiose and D-mannose were oxidized, and the properties were different from known microorganisms of the genus Sphingomonas.

  Therefore, it was determined that the isolated strain was a novel microorganism because it was revealed that it had different properties from the conventionally known microorganisms of the genus Sphingomonas, and this strain was determined to be a sphingomonas sp. .) LM02-032 strain (hereinafter, also simply referred to as “LM02-032 strain”). In addition, this LM02-032 strain was incorporated in the Patent Microorganism Depositary Center of the National Institute for Product Evaluation and Technology (Kazusa Kamashichi 2-5-8, Kisarazu City, Chiba Prefecture, Japan 292-0818) on June 19, 2015. Deposited under the deposit number NITE P-02069. That is, one aspect of the present invention relates to an oil-degrading microorganism identified by Sphingomonas sp. LM02-032 strain (Accession No. NITE P-02069).

The sphingomonas oil-degrading microorganisms exhibiting the above mycological properties have salt tolerance as described above. Therefore, when an oil-decomposing microorganism belonging to the genus Sphingomonas exhibiting the above mycological properties is applied to a grease trap, it contains a wide range of salt concentrations (for example, 0.005 to 4% (w / v) sodium chloride). The wastewater can be purified even in the water quality environment. In this specification, “having salt tolerance” means low salt concentration TG medium (0.005% (w / v) NaCl) and high salt concentration TG medium (4% (w / v) NaCl). The oil reduction rate is 10% by weight or more. Preferably, the oil reduction rate in the low salt concentration TG medium and the high salt concentration TG medium is 30% by weight or more, more preferably 45%. % Or more, more preferably 50% by weight or more (upper limit 100% by weight). In one embodiment, the oil-degrading microorganism has an oil content reduction rate measured by the following method of 10 wt% or more, preferably 30 wt% or more, within a NaCl concentration range of 0.005 to 4% (w / v). More preferably, it is 45 weight% or more, More preferably, it is 50 weight% or more (upper limit 100 weight%). “Oil content reduction rate” is 5 mL of TG medium containing 1% (w / v) oil (rapeseed oil: soybean oil: beef tallow = 2: 2: 1 (w / w / w)) as described above. From a normal hexane extract measured according to JIS K0102: 2013 revision, using a reaction solution obtained by adding 100 μL of a preculture solution (OD ₆₀₀ = 7.8) to the reaction mixture at 30 ° C. and 140 rpm for 24 hours. , The value calculated by Equation 1 above.

  The sphingomonas oil-degrading microorganism according to the present invention can efficiently purify wastewater under a wide range of salt concentrations. Specifically, the oil-decomposing microorganism according to the present invention has an oil reduction rate of 0.7 to 1. in the high salinity TG medium when the oil reduction rate in the low salt concentration TG medium is 1. 4 is preferable from the viewpoint of wastewater treatment, and more preferably 0.8 to 1.25.

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

  The carbon source that can be used in the culture of the microorganism of the present invention is not particularly limited as long as the carbon source can be assimilated by the strain used. Specifically, in consideration of microbial assimilation, glucose, fructose, cellobiose, raffinose, xylose, maltose, galactose, sorbose, glucosamine, ribose, rhamnose, sucrose, trehalose, α-methyl-D-glucoside, salicin , Melibiose, lactose, melezitose, inulin, erythritol, glucitol, mannose, mannitol, galactitol, N-acetyl-D-glucosamine, sorbitol, amidagulin, starch, starch hydrolysate, sugars such as white sugar, molasses, molasses, wheat , Natural products such as rice, alcohols such as glycerol, methanol and ethanol, organic acids such as acetic acid, lactic acid, succinic acid, gluconic acid, pyrpinic acid, citric acid and malic acid and salts thereof, and hydrocarbons such as hexadecane And the like. The carbon source is appropriately selected in view of assimilation by the microorganism to be cultured. For example, when using LM02-032 strain, it is preferable to use glucose, maltose, mannose, malic acid and the like among the above carbon sources. Moreover, the said carbon source can be used 1 type or 2 types or more selected.

  Examples of nitrogen sources that can be used in the culture of the microorganism of the present invention include meat extract, fish extract, peptone, polypeptone, yeast extract, malt extract, soybean hydrolysate, soybean powder, casein, milk casein, casamino acid, glycine, Various amino acids such as glutamic acid and aspartic acid, organic nitrogen sources such as corn steep liquor, other animal, plant, and microorganism hydrolysates; ammonium salts such as ammonia, ammonium nitrate, ammonium sulfate, and ammonium chloride; nitrates such as sodium nitrate; Examples thereof include nitrites such as sodium nitrate and inorganic nitrogen sources such as urea. The nitrogen source is appropriately selected in view of assimilation by the microorganism to be cultured. For example, when using LM02-032 strain, it is preferable to use meat extract (for example, derived from bovine), fish extract, polypeptone, yeast extract and the like among the nitrogen sources. Further, one or more of the nitrogen sources can be selected and used.

  Examples of inorganic substances that can be used in the present invention include magnesium, manganese, calcium, sodium, potassium, copper, iron, zinc, and other halides such as phosphates, hydrochlorides, sulfates, acetates, carbonates, chlorides, and the like. Is mentioned. The said inorganic substance is suitably selected according to the microorganisms to culture. The inorganic substance contained in the medium is, for example, 0.01 to 4% (w / v). In addition, one or more of the above inorganic materials can be selected and used. Moreover, you may add surfactant etc. in a culture medium as needed.

  In order for microorganisms to efficiently decompose oil or to maintain the oil content of microorganisms, it is preferable to add oil to the medium. Examples of the oil component include edible fats and oils, industrial fats and oils, and fatty acids described below. The amount of oil added is not particularly limited, and can be appropriately selected in consideration of the oil resolution by the microorganism to be cultured. Specifically, it is preferable to add oil at a concentration of 1 to 30 g, more preferably 5 to 15 g, in 1 L of the medium. With such an added amount, the microorganism can maintain a high oil content resolution. The oil component may be added alone or in the form of a mixture of two or more.

  Microorganisms can be cultured by ordinary methods. For example, depending on the type of microorganism, the cells are cultured under aerobic conditions or anaerobic conditions. In the former case, the culture of microorganisms is performed by shaking or aeration stirring. Alternatively, the microorganisms may be cultured continuously or in batches. The culture conditions are appropriately selected depending on the composition of the medium and the culture method, and are not particularly limited as long as the microorganisms of the present invention can grow, and can be appropriately selected according to the type of microorganism to be cultured. Usually, culture | cultivation temperature becomes like this. Preferably it is 15-45 degreeC, More preferably, it is 25-35 degreeC. The pH of the medium suitable for culture is preferably 3 to 11, more preferably 3.5 to 10.5. The culture time is not particularly limited, and varies depending on the type of microorganism to be cultured, the amount of medium, culture conditions, and the like. Usually, the culture time is preferably 16 to 48 hours, more preferably 20 to 30 hours.

[Wastewater treatment method]
In one embodiment of the present invention, there is provided a wastewater treatment method including a step of bringing the oil-decomposing microorganisms into contact with wastewater containing oil. Hereinafter, the waste water treatment method according to the present embodiment will be described in more detail with reference to FIG. The embodiment of the present invention is not limited to FIG.

  FIG. 1 schematically shows a mechanism of waste water treatment (waste water treatment) by the grease trap 10. In the method according to the present invention, the oil-decomposing microorganisms may be added in advance to the waste water before being discharged into the grease trap 10, but typically, it is added to the waste water in the waste water treatment tank 1. However, the wastewater treatment method according to the present invention is not particularly limited as long as the oil-decomposing microorganism according to the present invention and the oil-containing wastewater can be brought into contact with each other. For example, a carrier on which the oil-decomposing microorganisms according to the present invention are immobilized may be installed in a drainage channel or a drainage storage tank and contacted with oil-containing wastewater.

  The installation form of the grease trap 10 is not particularly limited, such as an embedded type and a movable type. In the case of the buried type, for example, in a kitchen or a food processing plant, the grease trap 10 is buried so that the wastewater that has flowed into the drainage channel is poured into the residue receiver 3. In the case of the movable type, for example, the grease trap 10 is installed so that the residue receiver 3 is positioned below the drainage groove of the sink.

  In FIG. 1, drainage flows in the direction of the arrow. The drainage to the grease trap 10 may be a batch type or a continuous type. Oil-containing wastewater flows into the wastewater treatment tank 1 through the residue receiver 3. At this time, all or a part of the residue such as garbage is collected in the residue receiver 3, but most of the oil passes through the residue receiver 3 and flows into the waste water treatment tank 1. The oil 6 flowing into the waste water treatment tank 1 floats toward the water surface 5 and collects in a space partitioned by the partition plates 2a and 2c. Therefore, when the wastewater treatment agent containing oil-decomposable microorganisms is not added to the wastewater, the oil 6 gradually aggregates in the space partitioned by the partition plates 2a and 2c to form scum.

  When the oil-decomposable microorganism according to the present invention is applied to the grease trap 10, the wastewater containing the oil and the oil-degradable microorganism are disposed in the wastewater treatment tank 1 (mainly in the space partitioned by the partition plates 2a and 2c). Will come into contact. Since the oil-decomposing microorganism according to the present invention has high oil-decomposing activity and has assimilability, it can suppress aggregation of oil 6 and effectively prevent scum from being formed. In particular, the oil-degrading microorganism according to the present invention efficiently reduces oil content in water quality with a wide range of salt concentrations (for example, wastewater containing 0.005 to 4% (w / v) sodium chloride). For this reason, the oil-decomposing microorganism according to the present invention has a high salinity drainage discharged, and when the salinity of the drainage in the grease trap becomes high (for example, 4% (w / v) sodium chloride is added. The wastewater purification effect is excellent. This prevents the oil from flowing out to the external environment through the trap pipe 4 without depending on the salinity concentration of the waste water, and is also advantageous from the viewpoint of environmental conservation.

  The oil-degrading microorganisms used in the method according to the present invention are various in a state suspended in a culture solution, a state recovered from a culture solution as a solid content, a dried state, a state immobilized on a carrier, and the like. Can be contacted with drainage in any form. The oil-decomposing microorganisms suspended in the culture solution, recovered as a solid content from the culture solution, or dried are added to, for example, waste water and brought into contact with the waste water. The oil-degrading microorganisms immobilized on the carrier may be added to the waste water, but the carrier on which the oil-degrading microorganisms are immobilized is placed in a grease trap and the microorganism-immobilized carrier is allowed to pass waste water. Thus, the oil-decomposing microorganism and the waste water can be brought into contact with each other. By installing the oil-decomposing microorganisms immobilized on the carrier in the grease trap, it is possible to prevent the oil-decomposing microorganisms from flowing out together with the waste water and reducing the number of bacteria.

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

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

  Further, the method for immobilizing the oil-decomposing microorganisms on the carrier is not particularly limited, and a general method for immobilizing microorganisms is used in the same manner or appropriately modified. For example, an immobilization method by pouring a microorganism culture solution into a carrier, an immobilization method by pouring the carrier under a reduced pressure using an aspirator, and a microorganism culture solution into the carrier, and a medium in which the microorganism culture solution is sterilized For example, a method of pouring into a mixture of a carrier and a carrier, culturing with shaking, and naturally drying the carrier taken out of the mixture.

In the method according to the present invention, when an oil-decomposing microorganism is added to and contacted with wastewater, the amount of bacteria to be added can be arbitrarily set. The amount of bacteria added to the wastewater is not particularly limited, but is, for example, 1 × 10 ⁴ to 1 × 10 ¹² CFU, preferably 1 × 10 ⁵ to 1 × 10, per 1 g of oil contained in the wastewater. ¹¹ CFU. Alternatively, for example, 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 (1 g of oil contained in the waste water). Dry cell weight). Alternatively, the amount may be, for example, 1 × 10 ⁶ to 1 × 10 ¹² CFU / L, more preferably 1 × 10 ⁷ to 1 × 10 ¹¹ CFU / L with respect to the wastewater in the grease trap. . Or it is 10 mg-15 g (dry cell weight) / L with respect to the waste_water | drain in a grease trap, for example, Preferably it is 0.1g-1.5g (dry cell weight) / L. In addition, when using combining 2 or more types of microorganisms, the total amount is meant. In addition, you may use the pre-cultured microorganisms added to waste water. By pre-culturing, the amount of bacteria to be inoculated can be easily adjusted.

  When draining wastewater to the outside environment, oil-degrading microorganisms that are not immobilized on the carrier are discharged with the wastewater to the outside of the grease trap. Therefore, in the present invention, oil-decomposing microorganisms are periodically added to the grease trap (drainage). It is preferable to do this. The addition interval is not particularly limited, but for example, it is preferably added once every 3 hours, once every 24 hours, or once every 2 to 3 days. The method of adding is not particularly limited, and when the wastewater flows continuously into the grease trap, it may be added to the wastewater or may be added directly to the wastewater in the grease trap. If microorganisms are added from a drain outlet such as a kitchen sink, the microorganisms can be introduced into the grease trap together with the wastewater discharged by washing.

  In the present specification, “oil” includes 50% by weight or more of acylglycerol such as triglyceride, diglyceride and monoglyceride (for example, 65% by weight or more, preferably 80% by weight or more, more preferably 90% by weight or more (upper limit) Edible or industrial oils and fats (including 100% by weight) glycerides. The fats and oils may contain lipids other than acylglycerols such as sterols and phospholipids.

  The fats and oils include various animal and vegetable fats and oils such as olive oil, canola oil, coconut oil, sesame oil, rice oil, rice bran oil, safflower oil, soybean oil, corn oil, rapeseed oil, palm oil, palm kernel oil, Sunflower oil, cottonseed oil, palm oil, peanut oil, cacao butter, beef tallow, lard, chicken oil, fish oil, whale oil, butter, margarine, fat spread, shortening, etc .; and linseed oil, jatropha oil, tall oil, hamana Industrial fats and oils such as oil, castor oil, jojoba oil and the like can be exemplified, but edible fats and oils that are frequently discharged in restaurants and the like where a grease trap is often installed are preferable.

  The fatty acid is not particularly limited, for example, butyric acid, hexanoic acid, heptanoic acid, octanoic acid, decanoic acid, lauric acid, tridecanoic acid, myristic acid, pentadecanoic acid, pentadecanoic acid, palmitic acid, heptadecanoic acid, Saturated fatty acids such as stearic acid, arachidic acid, behenic acid, lignoceric acid; decenoic acid, myristoleic acid, pentadecenoic acid, palmitoleic acid, heptadecenoic acid, oleic acid, icosenic acid, docosenoic acid, tetracosenoic acid, hexadecadienoic acid, hexa Decatrienoic acid, hexadecatetraenoic acid, linoleic acid, α-linolenic acid, γ-linolenic acid, octadecatetraenoic acid, icosadienoic acid, icosatrienoic acid, icosatetraenoic acid, arachidonic acid, icosapentaenoic acid, henicosapentaenoic acid, docosadienoic acid , And unsaturated fatty acids such as docosatetraenoic acid, docosapentaenoic acid, docosapentaenoic acid, and docosahexaenoic acid. Fatty acids may be produced by decomposing edible or industrial fats and oils.

  The oil content in the wastewater is not particularly limited. Two or more kinds of oil may be contained in the waste water.

  In the method of the present invention, in addition to the sphingomonas oil-decomposing microorganism according to the present invention, other components may be added to the waste water from the viewpoint of more efficiently reducing the oil content. Examples of other components include other microorganisms that can coexist with the sphingomonas oil-degrading microorganisms according to the present invention, lipases, pH adjusters, oil adsorbents, surfactants, and the like.

  Examples of other microorganisms that can coexist with the oil-degrading microorganisms of the genus Sphingomonas according to the present invention include, for example, the genus Yarrowia, the genus Candida, the genus Pichia, the genus Hansenula, and the saccharomyces ( Saccharomyces genus, Kluyveromyces genus, Trichosporon genus, Bacillus genus, Lactobacillus genus, Aspergillus genus ccco Sphingomonas genus, Enterobacte ), Burkholderia, Pseudomonas, Penicillium, Staphylococcus, Rhizopus, Rhizob, Rhizobet, Rhizobet, Rhizobet (Alcaligenes) genus, Fusarium genus, Serratia genus, Tetrasphaera genus, Humicola genus, and Stenotrophomonas genus can be exemplified. These microorganisms may be obtained from culture collections such as ATCC, NBRC, and DSMZ. Among these microorganisms, it is preferable to use an oil-decomposing microorganism of the genus Yarrowia, Enterobacter or Sphingomonas because of high resolution of the oil.

  The oil-degrading microorganisms of the genus Yarrowia include Yarrowia lipolytica ATCC 48436, Yarrowia lipolytica NBRC1548, Yarrowia lipolytica LM02-011 (accession number NITE P-01813), Yarrowia lipolytica NBRC0746, Yarrowia lipolytica 9 Yarrowia lipolytica), Yarrowia sp. Such as Yarrowia YH-01 can be exemplified, but Yarrowia lipolytica is more preferable, Yarrowia lipolytica LM02-011 (M02-011), M02-011 Japan Foundation for Product Evaluation Technology Patent Microorganism Deposit Center To Yubinbango292-0818 Japan Chiba Prefecture Kisarazu Kazusa legs bowed in 2-5-8), has been deposited as accession number NITE P-01813 in the March 6 date 2014) is more preferred.

  Enterobacter sp. LM02-030 strain (LM02-030 strain is the National Institute of Technology and Evaluation, Japan Patent Evaluation Center for Microorganisms, Japan 292-0818, Chiba, Japan). In the prefecture, Kisarazu City, Kazusa Kamashichi 2-5-8), the deposit number is NITE P-02048 as of May 12, 2015).

  The oil-degrading microorganism belonging to the genus Sphingomonas is a microorganism different from the microorganism according to the present invention. For example, Sphingomonas sp. 2629-3b described in JP-A No. 2006-166874 is a Sphingomonas sp. sp.) and the like.

  An oil-degrading enzyme such as lipase or phospholipase may be added to the waste water in order to assist the decomposition of the oil by the sphingomonas oil-decomposing microorganism used in the method according to the present invention. Examples of the oil-degrading enzymes include the genus Pseudomonas, the genus Aspergillus, the genus Bacillus, the genus Penicillium, the genus Rhizopus, and the genus Rhizomucor. , The genus Paecilomyces, the genus Rhizoctonia, the genus Absidia, the genus Achromobacter, the genus Aeromonas, the genus Alternaria, i Beauveria), Cromobacter (Chromobac) er), Coprinus genus, Fusarium genus, Geotricum genus, Humicola genus, Hyphozyma genus, Lactobacillus genus, Metal ridium genus (Rhodosporidium), Trichoderma, Yarrowia, Candida, Pichia, Hansenula, Saccharomyces, and Saccharomyces And / or from the genus Trichosporon Rukoto can.

  Commercially available oil-degrading enzymes include lipase MY, lipase OF, lipase PL, lipase QLM (name sugar industry); lipase A “Amano (registered trademark)” 6, lipase DF “Amano (registered trademark)” 15, Lipase G "Amano (registered trademark)" 50, Lipase AY "Amano (registered trademark)" 30SD, Lipase R "Amano (registered trademark)", Lipase MER "Amano (registered trademark)", Neurase (registered trademark) F ( Amano Enzyme Co., Ltd.); Sumiteam (registered trademark) NLS, Sumiteam (registered trademark) RLS (Shin Nihon Chemical Industry Co., Ltd.); Lilipase (registered trademark) A-10D, Lilipase (registered trademark) AF-5, PLA2 Nagase (Nagase) Chemtex Co., Ltd.); Entilon AKG-2000, Entilon LP, Entilon LPG 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); picantase A, picantase R800 (manufactured by DSM Japan) and the like. Two or more of these may be used in combination.

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

  In the method according to the present invention, an oil adsorbent such as an inorganic polymer such as shirasu balloon, diatomaceous earth or pearlite, or an organic polymer such as polyurethane, polyethylene or melanin resin described in JP2012-206084A is used. It may be added to the waste water.

  In the present invention, in order to prevent oil aggregation and scum formation, anionic surfactants such as sodium dodecyl sulfate (SDS), dodecylbenzene sulfonic acid (NaDDBS), ammonium lauryl sulfate, sodium caseinate; aliphatic amine salts, Cationic surfactants such as quaternary ammonium salts; nonionic interfaces such as fatty acid esters (monoglycerin fatty acid ester, diglycerin fatty acid ester, sorbitan fatty acid ester, propylene glycol fatty acid ester, sucrose fatty acid ester), polyethylene glycol, lecithin, etc. Activators: Surfactants such as amphoteric surfactants such as betaine, amine oxide and saponin may be added to the waste water.

  The grease trap may be configured to continuously introduce the oil-containing wastewater and continuously discharge the treated wastewater, or after introducing the oil-containing wastewater and treating it all at once, the treated wastewater May be discharged in a batch.

  In the method of the present invention, the temperature at which the oil-decomposing microorganism and the oil are brought into contact with each other, that is, the temperature of the waste water in the grease trap can be arbitrarily set. Moreover, it can also set arbitrarily as pH at the time of making an oil-decomposing microorganism and oil contact, ie, the pH of the waste_water | drain in a grease trap. Generally, temperature is 10-60 degreeC, for example, 20-50 degreeC is preferable and 25-40 degreeC is more preferable. The pH is, for example, 3 to 10, preferably 4 to 9, and more preferably 5 to 8. Further, the waste water may be aerated by aeration or the like as necessary.

[Wastewater treatment agent]
In one Embodiment of this invention, the waste water treatment agent containing said oil-decomposing microorganism is provided. The sphingomonas oil-degrading microorganism according to the present invention has an excellent oil-reducing effect, particularly in a water quality environment having a wide range of salt concentrations (for example, wastewater containing 0.005 to 4% (w / v) sodium chloride). Also has the property of purifying waste water. Accordingly, the oil content can be effectively reduced by using the wastewater treatment agent containing the sphingomonas oil-degrading microorganisms according to the present invention in a wastewater treatment facility (drainage treatment facility) such as a grease trap. In addition, the description regarding the above oil-decomposing microorganism and the wastewater treatment method may be modified as necessary and applied to the present embodiment.

  The wastewater treatment agent may be in either a dry form or a liquid form, but a dry form such as powder, granule, pellet, tablet or the like is preferable from the viewpoint of storage stability. The microorganism according to the present invention used for such a wastewater treatment agent in a dry form includes a bacterial powder obtained by drying a culture solution by spray drying, freeze drying, or the like, or a bacterium in a state of being immobilized on a carrier as described above It may be a body, and may be formed into a powder, granule, pellet, or tablet. Alternatively, the bacterial cells and the culture solution may be encapsulated with hydroxypropylmethylcellulose, gelatin, or the like. The wastewater treatment agent may also contain excipients such as hydroxypropylcellulose, dextrin, lactose, starch and the like.

  The oil-degrading microorganism of the genus Sphingomonas according to the present invention contained in the wastewater treatment agent may be dead or live, but is preferably live from the viewpoint of the persistence of oil-degrading activity. .

The amount of the microorganism according to the present invention contained in the wastewater treatment agent is, for example, 10 to 100% by weight in the solid content of the wastewater treatment agent. Alternatively, the amount of the oil-decomposing microorganism of the genus Sphingomonas according to the present invention contained in the wastewater treatment agent is, for example, an amount that is 1 × 10 ² to 1 × 10 ¹⁰ CFU / g with respect to the whole wastewater treatment agent. . In addition, the wastewater treatment agent is, as long as the object and effect of the present invention are achieved, other microorganisms that can coexist with the above-described sphingomonas genus oil-decomposing microorganisms, oil-degrading enzymes, oil adsorbents, One or more additives selected from the group consisting of surfactants may be included.

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

  Mud (sample) collected from a depth of 1 m at Saeida Fishing Port, Onna Village, Okinawa Prefecture was cultured in a culture medium for accumulation culture by the above method to obtain an accumulation culture solution. The enrichment culture solution was smeared on a culture medium for separation culture and cultured, a single colony was collected, cultured in LB medium and purified.

  Purified microorganisms were treated with 5 mL of sterilized LB medium (1% (w / v) polypeptone (Nippon Pharmaceutical Co., Ltd.), 5% (w / v) yeast extract (Oriental Yeast Co., Ltd.), 1% (w / v) sodium chloride. The pH was adjusted to 6.0 with hydrochloric acid and 1 platinum ear was inoculated, and cultured for 24 hours while shaking at 140 rpm in a 30 ° C. incubator to obtain a precultured solution.

  In order to use it for the oil decomposition test, a TG medium was prepared with the following composition, and the pH was adjusted to 5.0 with hydrochloric acid. Polypeptone was purchased from Nippon Pharmaceutical Co., Ltd., and fish meat extract was purchased from Kyokuto Pharmaceutical Co., Ltd.

  0.05 g of oil (rapeseed oil: soybean oil: beef tallow = 2: 2: 1 (w / w / w)) is added to the TG medium (5 mL) prepared by the above method, and sterilized at high temperature and high pressure. Prepared (oil 1% (w / v)).

To the above test solution, 100 μL of a preculture solution (amount of bacterial cells: 1 × 10 ⁶ CFU) was added and cultured in a 30 ° C. incubator for 24 hours while shaking at 140 rpm.

  After the culture, a normal hexane extract was prepared according to JIS K0102: 2013 revision (industrial wastewater test method). Using the normal hexane extract as the remaining amount of oil, the oil content reduction rate was calculated from the oil content (0.05 g) added during the preparation of the test solution and the remaining amount of oil (the amount of normal hexane extract (g)) according to the following formula 1. Asked. As a result, a strain having a high oil content reduction rate was isolated in both the low salinity oil decomposition test solution and the high salinity oil decomposition test solution.

  The isolated strain was designated as Sphingomonas sp. LM02-032, and deposited with the Patent Microorganism Depositary, National Institute of Technology and Evaluation (Accession No. NITE P-02069).

  The oil reduction rate (% by weight) when LM02-032 strain and conventionally known microorganisms are used is shown in the table below. As known microorganisms, Bacillus subtilis BN1001 and bacterial preparation BI-CHEM TD800S (Novozymes, including Bacillus megaterium) were used.

  As shown in the above table, the microorganism of the genus Sphingomonas according to the present invention has a low salt concentration (drainage containing 0.005% (w / v) sodium chloride) to a high salt concentration (4% (w / v). It can be seen that the oil reduction rate is high in a wide range of water environments with a wide range of salt concentrations.

1 Wastewater treatment tank,
2a, 2b, 2c partition plate,
3 Receiving residue
4 Trap tube,
5 Water surface,
6 Oil,
10 Grease trap.

Claims (5)

An oil-degrading microorganism belonging to the genus Sphingomonas and exhibiting the following mycological properties.
  The oil-degrading microorganism according to claim 1, which has a 16S rDNA base sequence represented by SEQ ID NO: 1.   An oil-degrading microorganism specified by Sphingomonas sp. LM02-032 strain (Accession No. NITE P-02069).   A wastewater treatment method comprising a step of bringing the oil-decomposing microorganism according to any one of claims 1 to 3 into contact with wastewater containing oil.   A wastewater treatment agent comprising the oil-decomposing microorganism according to any one of claims 1 to 3.