Novel microorganism having high lipolytic ability at low temperature

Abstract

In an aspect, the present disclosure provides a novel microorganism having an ester degrading ability. In an aspect, the present disclosure provides a combination of novel microorganisms having an oil degrading ability. In an embodiment, the microorganism of the present invention includes a yeast belonging to the genus Yarrowia. In an embodiment, the microorganism of the present invention includes Yarrowia lipolytica. In an embodiment, the present disclosure provides a combination of a Burkholderia bacterium with a Yarrowia yeast. In an embodiment, provided is an oil degrading agent that comprises the microorganism of the present disclosure.

Description

Technical Field

[0001] The present disclosure relates to microorganisms having ester (e.g., fats and oils) and / or fatty acid degradation ability and their use. More specifically, it relates to Yarrowia yeast (e.g., Yarrowia lipolytica) that degrades fats and oils that are conventionally difficult to degrade, such as fats and oils containing trans fatty acids. The present disclosure also relates to Yarrowia yeast that improves the oil degradation ability of Burkholderia bacteria. Furthermore, the present disclosure relates to combinations of microorganisms having oil degradation ability and their use. More specifically, it relates to combinations of microorganisms having improved oil degradation ability compared to at least one microorganism alone in the combination. The present disclosure also relates to enhancing the expression of lipase by a combination of microorganisms. More specifically, it relates to a combination of Burkholderia bacteria and Yarrowia yeast.

Background Art

[0002] The wastewater from food factories and oil factories contains a large amount of oil. This oil causes various biological treatment function degradations, such as a decrease in the treatment ability by activated sludge, incomplete solid-liquid separation due to a decrease in sedimentation performance, membrane fouling in the membrane separation activated sludge process (MBR), and inhibition of methane fermentation in anaerobic digestion. Therefore, as a pre-stage of biological treatment of high-oil-content wastewater, oil is removed by, for example, a pressurized flotation separator. In addition, since the kitchen wastewater of the food service industry also contains a large amount of oil, a grease trap for removing oil is installed. Both the pressurized flotation separator and the grease trap have problems such as being a source of malodor and pests, the cost of recovering and transporting the separated oil and industrial waste treatment, and the labor and cost for management and cleaning. As a means to solve such problems, oil degradation technology using microorganisms has been studied, and a plurality of related microbial preparations are also commercially available. However, it is extremely difficult to lower the oil concentration to the desired level by microbial degradation within the set retention time. Therefore, currently, pressurized flotation separators and conventional grease traps are mostly used.

[0003] In addition, even in the fermentation treatment of food waste, when there is a large amount of oil, there are problems such as fermentation inhibition and high oil content in the drainage of the destruction type treatment machine. In addition, since the oily sludge separated and recovered by the pressure floatation separation device or the grease trap becomes industrial waste, its treatment requires a large cost. Therefore, decomposing these oils with microorganisms has been considered, but in fact, like the above-mentioned wastewater treatment, the decomposition ability of microorganisms has limitations.

[0004] In the removal of oil by microorganisms, as described above, the decomposition rate becomes a problem, but in particular, the decrease in activity due to the low temperature in winter often makes it difficult to apply microorganisms. Especially at low temperatures in winter, the decomposition rate of oils and fats by microorganisms is extremely slow, and it is considered impossible to perform wastewater treatment or waste treatment with specific microorganisms.

Prior Art Documents

Non-Patent Documents

[0005]

Non-Patent Document 1

Summary of the Invention

Means for Solving the Problems

[0006] As a result of intensive research, the inventors have found a microorganism belonging to the genus Yarrowia, which is a novel microorganism having a unique esterase activity. Aspects have also been found in which this microorganism can decompose and assimilate oils and fats containing trans fatty acids and / or trans fatty acids. Aspects have also been found in which this microorganism can decompose and assimilate oils and fats and / or fatty acids at low temperatures. Aspects have also been found in which this microorganism can widely decompose and assimilate fatty acids ranging from short-chain to long-chain and esters and oils and fats containing them. Further, the inventors have found Yarrowia yeast that improves the oil-decomposing ability of Burkholderia bacteria. Furthermore, the inventors have found a combination of Burkholderia bacteria that strongly decompose oils and fats and fatty acids and Yarrowia yeast. The present disclosure also relates to applications of the combinations of the microorganisms of the present disclosure, such as oil treatment. The present disclosure provides applications of the microorganisms of the present disclosure, novel combinations of microorganisms having oil-decomposing ability, and an oil-decomposing method using such combinations.

[0007] Accordingly, the present disclosure provides the following. (Item A1) Yarrowia yeast having the ability to decompose trans fatty acids. (Item A2) Yarrowia yeast having the ability to assimilate trans fatty acids. (Item A3) Yarrowia yeast having the ability to decompose oils and fats containing trans fatty acids. (Item A4) Yarrowia yeast having the ability to assimilate oils and fats containing trans fatty acids. (Item A5) Yarrowia yeast having the ability to decompose esters (e.g., oils and fats) and / or fatty acids at 15°C. (Item A6) Yarrowia yeast having the ability to assimilate esters (e.g., oils and fats) and / or fatty acids at 15°C. (Item A7) Yarrowia yeast according to any one of the above items, wherein the ability to assimilate or decompose is retained at 15°C. (Item A8) Yarrowia yeast having the ability to decompose esters containing short-chain to medium-chain fatty acids (C2 to C12). (Item A9) Yarrowia yeast having the ability to decompose oils and fats containing short-chain to long-chain fatty acids (C2 or higher). (Item A10) Yarrowia yeast having a higher decomposition activity for 4-nitrophenyl esters of short-chain to medium-chain fatty acids (C2 to C12) than for 4-nitrophenyl esters of long-chain fatty acids (C13 or higher), and having the ability to decompose triglycerides of long-chain fatty acids (C13 or higher). (Item A11) Yarrowia yeast according to any one of the above items, having two or more characteristics specified in Item A above. (Item A12) Yeast according to any one of the above items, which is Yarrowia lipolytica. (Item A13) Yeast according to any one of the above items, which is the Yarrowia yeast KH-2 strain identified by the accession number NITE BP-02732 or a derivative strain thereof, and the derivative strain has the characteristics of the Yarrowia yeast described in any one or more of Item A above. (Item A14) An oil decomposing agent containing Yarrowia yeast according to any one of the above items. (Item A15) An oil decomposing agent according to any one of the above items, further containing an additional oil treatment component. (Item A16) (a) For decomposing trans fatty acids, (b) For decomposing oils and fats containing trans fatty acids, (c) For decomposing esters (e.g., oils and fats) and / or fatty acids at 15°C, (d) For decomposing esters containing short-chain to medium-chain fatty acids (C2 to C12), and (e) For decomposing oils and fats containing short-chain to long-chain fatty acids (C2 or higher) A composition comprising the Yarrowia yeast according to any one of the above items or the oil-degrading agent according to any one of the above items for at least one selected from the group consisting of. (Item A17) A kit for ester (e.g., oil and fat) decomposition, comprising the Yarrowia yeast according to any one of the above items or the oil-degrading agent according to any one of the above items, or the composition according to any one of the above items, and a further oil treatment component. (Item A18) A method for ester (e.g., oil and fat) decomposition and removal, which includes allowing the Yarrowia yeast according to any one of the above items, or the oil-degrading agent according to any one of the above items, or the composition according to any one of the above items to act on a treatment target. (Item A19) The method according to any one of the above items, wherein the treatment target includes trans fatty acids or oils and fats containing trans fatty acids. (Item A20) (a) A step of decomposing trans fatty acids, (b) A step of decomposing oils and fats containing trans fatty acids, (c) A step of decomposing esters (e.g., oils and fats) and / or fatty acids at 15°C, (d) A step of decomposing esters containing short-chain to medium-chain fatty acids (C2 - C12), and (e) A step of decomposing oils and fats containing short-chain to long-chain fatty acids (C2 or more), The method according to any one of the above items, including at least one step selected from the group consisting of. (Item B1) A composition for treating oils and fats with a combination of Yarrowia yeast producing lipase and Burkholderia bacteria producing lipase, which contains Burkholderia bacteria. (Item B2) A composition for treating oils and fats with a combination of Yarrowia yeast producing lipase and Burkholderia bacteria producing lipase, which contains Yarrowia yeast. (Item B3) A combination for oil and fat treatment, comprising a combination of Burkholderia bacteria and Yarrowia yeast, wherein both the Burkholderia bacteria and the Yarrowia yeast produce lipase. (Item B4) The composition or combination according to any one of the above items, wherein the Yarrowia yeast includes Yarrowia lipolytica. (Item B5) The composition or combination according to any one of the above items, wherein the Burkholderia bacteria include Burkholderia bacteria. (Item B6) The composition or combination according to any one of the above items, wherein the Burkholderia bacteria include Burkholderia arboris, Burkholderia ambifaria, or Burkholderia cepacia complex. (Item B7) The composition or combination according to any one of the above items, wherein the combination of the Burkholderia bacteria and the Yarrowia yeast has a lipolytic ability higher than the lipolytic ability calculated from the lipolytic ability values of each single culture. (Item B8) The composition or combination according to any one of the above items, wherein the cell number of the Burkholderia bacteria: the cell number of the Yarrowia yeast is 1:20 to 20:1. (Item B9) The composition or combination according to any one of the above items, wherein at least one of the Burkholderia bacteria and the Yarrowia yeast has the ability to decompose fatty acids at 15°C. (Item B10) The Burkholderia bacteria is the composition or combination according to any one of the above items, which is Burkholderia arboris strain KH-1 (the strain identified by accession number NITE BP-02731), Burkholderia ambifaria strain KH-1AL1 (the strain identified by accession number NITE BP-02977), Burkholderia cepacia complex strain KH-1AL2 (the strain identified by accession number NITE BP-02978) or Burkholderia cepacia complex strain KH-1AL3 (the strain identified by accession number NITE BP-02979), or an induced strain thereof. (Item B11) The Yarrowia yeast is the composition or combination according to any one of the above items, which is Yarrowia lipolytica strain KH-2 (the microbial strain identified by accession number NITE BP-02732), Yarrowia lipolytica strain KH-2AL1 (the microbial strain identified by accession number NITE BP-03091), or Yarrowia lipolytica strain KH-2AL3 (the microbial strain identified by accession number NITE BP-03092), or an induced strain thereof. (Item B12) The composition or combination according to any one of the above items, which is an oil decomposing agent. (Item B13) The oil decomposing agent according to any one of the above items, which contains further oil treatment components. (Item B14) An oil decomposition and removal method, which includes making the composition or combination according to any one of the above items, or the oil decomposing agent according to any one of the above items act on the object to be treated. (Item B15) A composition for improving lipase production of Yarrowia yeast producing lipase, which composition contains Burkholderia bacteria. (Item B16) A composition for improving lipase production of Burkholderia bacteria producing lipase, which composition contains Yarrowia yeast. (Item B17) A composition for enhancing the ability of Yarrowia yeast producing lipase to process oils and fats, which composition contains Burkholderia bacteria. (Item B18) A composition for enhancing the ability of Burkholderia bacteria producing lipase to process oils and fats, which composition contains Yarrowia yeast. (Item B19) A method for improving lipase production of at least one of the Burkholderia bacteria and the Yarrowia yeast, which method includes a step of mixing and culturing the Burkholderia bacteria and the Yarrowia yeast. (Item B20) Use of Burkholderia bacteria for processing oils and fats in combination with Yarrowia yeast producing lipase and Burkholderia bacteria producing lipase. (Item B21) Use of Yarrowia yeast for processing oils and fats in combination with Yarrowia yeast producing lipase and Burkholderia bacteria producing lipase. (Item B22) Use of a combination of Burkholderia bacteria and Yarrowia yeast for processing oils and fats, wherein both the Burkholderia bacteria and the Yarrowia yeast produce lipase. (Item B23) The use according to any one of the above items, wherein the Yarrowia yeast includes Yarrowia lipolytica. (Item B24) The use according to any one of the above items, wherein the Burkholderia bacteria include Burkholderia bacteria. (Item B25) The Burkholderia bacterium includes Burkholderia arboris, Burkholderia ambifaria, or Burkholderia cepacia complex, and is used in any one of the above items. (Item B26) The combination of the Burkholderia bacterium and Yarrowia yeast has a lipolytic ability higher than the lipolytic ability calculated from the values of the lipolytic abilities of their respective monocultures, and is used in any one of the above items. (Item B27) The cell number of the Burkholderia bacterium: the cell number of the Yarrowia yeast is 1:20 to 20:1, and is used in any one of the above items. (Item B28) At least one of the Burkholderia bacterium and the Yarrowia yeast has the ability to decompose fatty acids at 15°C, and is used in any one of the above items. (Item B29) The Burkholderia bacterium is Burkholderia sp. strain KH-1 (a strain identified by accession number NITE BP-02731), KH-1AL1 strain (a strain identified by accession number NITE BP-02977), KH-1AL2 strain (a strain identified by accession number NITE BP-02978), or KH-1AL3 strain (a strain identified by accession number NITE BP-0297), or a derivative thereof, and is used in any one of the above items. (Item B30) The Yarrowia yeast is Yarrowia lipolytica strain KH-2 (a microbial strain identified by accession number NITE BP-02732), Yarrowia lipolytica KH-2AL1 strain (a microbial strain identified by accession number NITE BP-03091), or Yarrowia lipolytica KH-2AL3 strain (a microbial strain identified by accession number NITE BP-03092), or a derivative thereof, and is used in any one of the above items. (Item B31) The use according to any one of the above items, combining further oil treatment components. (Item B32) The use according to any one of the above items, including causing the combination of said Burkholderia bacterium and Yarrowia yeast to act on a treatment target. (Item B33) Use of a Burkholderia bacterium for improving the lipase production of a Yarrowia yeast that produces lipase. (Item B34) Use of a Yarrowia yeast for improving the lipase production of a Burkholderia bacterium that produces lipase. (Item B35) Use of a Burkholderia bacterium for enhancing the ability of a Yarrowia yeast that produces lipase to treat oils and fats. (Item B36) Use of a Yarrowia yeast for enhancing the ability of a Burkholderia bacterium that produces lipase to treat oils and fats. (Item B37) Use of said Burkholderia bacterium and said Yarrowia yeast for improving the lipase production of at least one of the Burkholderia bacterium and the Yarrowia yeast. (Item B38) A method for treating oils and fats with a combination of a Yarrowia yeast that produces lipase and a Burkholderia bacterium that produces lipase, including a step of contacting the Burkholderia bacterium. (Item B39) A method for treating oils and fats with a combination of a Yarrowia yeast that produces lipase and a Burkholderia bacterium that produces lipase, including a step of contacting the Yarrowia yeast. (Item B40) A method for treating oils and fats with a combination of a Yarrowia yeast that produces lipase and a Burkholderia bacterium that produces lipase, including a step of contacting the combination of the Burkholderia bacterium and the Yarrowia yeast. (Item B41) The method according to any one of the above items, wherein the Yarrowia yeast includes Yarrowia lipolytica. (Item B42) The method according to any one of the above items, wherein the Burkholderia bacterium includes Burkholderia bacteria. (Item B43) The method according to any one of the above items, wherein the Burkholderia bacterium includes Burkholderia arboris, Burkholderia ambifaria, or Burkholderia cepacia complex. (Item B44) The method according to any one of the above items, wherein the combination of the Burkholderia bacterium and the Yarrowia yeast has a lipolytic ability higher than the lipolytic ability calculated from the values of the lipolytic abilities of each single culture. (Item B45) The method according to any one of the above items, wherein the cell number of the Burkholderia bacterium: the cell number of the Yarrowia yeast is 1:20 to 20:1. (Item B46) The method according to any one of the above items, wherein at least one of the Burkholderia bacterium and the Yarrowia yeast has the ability to decompose fatty acids at 15°C. (Item B47) The method according to any one of the above items, wherein the Burkholderia bacterium is Burkholderia sp. strain KH-1 (strain identified by accession number NITE BP-02731), KH-1AL1 strain (strain identified by accession number NITE BP-02977), KH-1AL2 strain (strain identified by accession number NITE BP-02978), or KH-1AL3 strain (strain identified by accession number NITE BP-02979), or a derivative thereof. (Item B48) The method according to any one of the above items, wherein the Yarrowia yeast is the Yarrowia lipolytica KH-2 strain (a microbial strain identified by the accession number NITE BP-02732), the Yarrowia lipolytica KH-2AL1 strain (a microbial strain identified by the accession number NITE BP-03091), or the Yarrowia lipolytica KH-2AL3 strain (a microbial strain identified by the accession number NITE BP-03092), or a derivative strain thereof. (Item B49) The method according to any one of the above items, which uses an additional oil treatment component. (Item B50) The method according to any one of the above items, which is an oil decomposition and removal method. (Item B51) A method for improving the lipase production of Yarrowia yeast that produces lipase, which includes a step of introducing it to Burkholderia bacteria. (Item B52) A method for improving the lipase production of Burkholderia bacteria that produces lipase, which includes a step of introducing it to Yarrowia yeast. (Item B53) A method for enhancing the ability of Yarrowia yeast that produces lipase to process oils and fats, which includes a step of introducing it to Burkholderia bacteria. (Item B54) A method for enhancing the ability of Burkholderia bacteria that produces lipase to process oils and fats, which includes a step of introducing it to Yarrowia yeast. (Item C1) Yarrowia yeast having the ability to decompose trans fatty acids. (Item C2) Yarrowia yeast having the ability to decompose oils and fats containing trans fatty acids. (Item C3) Yarrowia yeast having the ability to decompose esters and / or fatty acids at 15°C. (Item C4) Yarrowia yeast having the ability to decompose esters containing short-chain to medium-chain fatty acids. (Item C5) Yarrowia yeast having the ability to decompose oils and fats containing short-chain to long-chain fatty acids. (Item C6) Yarrowia yeast having high decomposing activity against 4-nitrophenyl esters of short-chain to medium-chain fatty acids (C2 - C12) compared to 4-nitrophenyl esters of long-chain fatty acids (C13 or more), and having the ability to decompose triglycerides of long-chain fatty acids (C13 or more). (Item C7) Yarrowia yeast having the ability to improve the lipase production of Burkholderia bacteria that produce lipase. (Item C8) Yarrowia yeast having the ability to impart an ability to decompose oils or fatty acids higher than the ability to decompose oils or fatty acids during the single culture of Burkholderia bacteria to the Burkholderia bacteria. (Item C9) The Yarrowia yeast according to any one of the above items, wherein the Burkholderia bacteria include Burkholderia bacteria. (Item C10) The Yarrowia yeast according to any one of the above items, wherein the Burkholderia bacteria include Burkholderia arboris, Burkholderia ambifaria, or Burkholderia cepacia complex. (Item C11) The characteristics of the Yarrowia yeast according to any one or more of Items C1 - 6, and The characteristics of the Yarrowia yeast according to any one or more of Items C7 - 10, The Yarrowia yeast having these. (Item C12) The Yarrowia yeast according to any one of the above items, which is Yarrowia lipolytica. (Item C13) The Yarrowia lipolytica KH-2 strain (a microbial strain identified by the deposit number NITE BP-02732), the Yarrowia lipolytica KH-2AL1 strain (a microbial strain identified by the deposit number NITE BP-03091), or the Yarrowia lipolytica KH-2AL3 strain (a microbial strain identified by the deposit number NITE BP-03092), or an induced strain thereof, wherein the induced strain has the characteristics of the Yarrowia yeast described in any one of the above items, the Yarrowia yeast described in any one of the above items. (Item C14) An oil-decomposing agent containing the Yarrowia yeast described in any one of the above items. (Item C15) The oil-decomposing agent described in any one of the above items, further containing an additional oil treatment component. (Item C16) (a) For decomposing trans fatty acids, (b) For decomposing oils and fats containing trans fatty acids, (c) For decomposing esters and / or fatty acids at 15°C, (d) For decomposing esters containing short-chain to medium-chain fatty acids (C2 - C12), and (e) For decomposing oils and fats containing short-chain to long-chain fatty acids (C2 or more) A composition containing the Yarrowia yeast described in any one of the above items, or an oil-decomposing agent, for at least one selected from the group consisting of (Item C17) A kit for ester decomposition, comprising the Yarrowia yeast or oil-decomposing agent described in any one of the above items, or the composition described in any one of the above items, and an additional oil treatment component. (Item C18) A method for ester decomposition removal, including allowing the Yarrowia yeast, oil-decomposing agent, or composition described in any one of the above items to act on a treatment target. (Item C19) The method described in any one of the above items, wherein the treatment target contains trans fatty acids or oils and fats containing trans fatty acids. (Item C20) (a) The step of decomposing trans fatty acids, (b) The step of decomposing oils and fats containing trans fatty acids, (c) The step of decomposing esters and / or fatty acids at 15°C, (d) The step of decomposing esters containing short-chain to medium-chain fatty acids (C2 - C12), and (e) The step of decomposing oils and fats containing short-chain to long-chain fatty acids (C2 or more), The method according to any one of the above items, comprising at least one step selected from the group consisting of. (Item C21) A composition for treating oils and fats or fatty acids with a combination of Yarrowia yeast producing lipase and Burkholderia bacteria producing lipase, which contains Burkholderia bacteria. (Item C22) A composition for treating oils and fats or fatty acids with a combination of Yarrowia yeast producing lipase and Burkholderia bacteria producing lipase, which contains Yarrowia yeast. (Item C23) A combination for treating oils and fats or fatty acids, which contains a combination of Burkholderia bacteria and Yarrowia yeast, and both the Burkholderia bacteria and the Yarrowia yeast produce lipase. (Item C24) The composition or combination according to any one of the above items, wherein the Yarrowia yeast includes Yarrowia lipolytica. (Item C25) The composition or combination according to any one of the above items, wherein the Burkholderia bacteria include bacteria of the genus Burkholderia. (Item C26) The Burkholderia bacterium is the composition or combination according to any one of the above items, including Burkholderia arboris, Burkholderia ambifaria, or Burkholderia cepacia complex. (Item C27) The composition or combination according to any one of the above items, wherein the combination of the Burkholderia bacterium and Yarrowia yeast has an oil or fatty acid degradation ability higher than the oil or fatty acid degradation ability calculated from the values of the oil or fatty acid degradation ability of each single culture. (Item C28) The composition or combination according to any one of the above items, wherein the cell number of the Burkholderia bacterium: the cell number of the Yarrowia yeast is 1:20 to 20:1. (Item C29) The composition or combination according to any one of the above items, wherein at least one of the Burkholderia bacterium and the Yarrowia yeast has the ability to decompose fatty acids at 15°C. (Item C30) The Burkholderia bacterium is the composition or combination according to any one of the above items, which is the Burkholderia sp. strain KH-1 (the strain identified by the accession number NITE BP-02731), KH-1AL1 strain (the strain identified by the accession number NITE BP-02977), KH-1AL2 strain (the strain identified by the accession number NITE BP-02978), or KH-1AL3 strain (the strain identified by the accession number NITE BP-02979), or an induced strain thereof. (Item C31) The Yarrowia yeast is the composition or combination according to any one of the above items, which is the Yarrowia lipolytica KH-2 strain (a microbial strain identified by the accession number NITE BP-02732), the Yarrowia lipolytica KH-2AL1 strain (a microbial strain identified by the accession number NITE BP-03091), or the Yarrowia lipolytica KH-2AL3 strain (a microbial strain identified by the accession number NITE BP-03092), or an induced strain thereof. (Item C32) The composition or combination according to any one of the above items, which is an oil decomposing agent. (Item C33) The oil decomposing agent according to any one of the above items, which contains an additional oil treatment component. (Item C34) An oil decomposition and removal method, which includes making the composition or combination, or the oil decomposing agent according to any one of the above items act on the object to be treated. (Item C35) A composition for improving the lipase production of Yarrowia yeast that produces lipase and contains Burkholderia bacteria. (Item C36) A composition for improving the lipase production of Burkholderia bacteria that produces lipase and contains Yarrowia yeast. (Item C37) A composition for enhancing the ability of Yarrowia yeast that produces lipase and contains Burkholderia bacteria to process oils, fats, or fatty acids. (Item C38) A composition for enhancing the ability of Burkholderia bacteria that produces lipase and contains Yarrowia yeast to process oils, fats, or fatty acids. (Item C39) A method for improving the lipase production of at least one of the Burkholderia bacteria and the Yarrowia yeast, which includes a step of mixing and culturing the Burkholderia bacteria and the Yarrowia yeast.

[0008] It is contemplated that the present disclosure may provide one or more of the above-described features in combinations other than those explicitly stated. Still further embodiments and advantages of the present disclosure will be recognized by those skilled in the art upon reading and understanding the following detailed description, if necessary. [Effects of the Invention]

[0009] The microorganism or combination of microorganisms disclosed herein, and the composition or combination that provides the same, can achieve rapid decomposition of oils and / or fatty acids, and are therefore applicable to a wide range of situations, such as the purification of environmental pollution caused by oil, waste treatment and composting, including food waste treatment, composting, and wastewater treatment. They can also handle a wide range of oil concentrations, and because they can decompose trans fatty acids and oils and fats containing trans fatty acids, they can be used to treat oil-containing substances, such as wastewater discharged from food factories.

[0010] The present disclosure can also solve the problem of oils that are difficult to decompose, i.e., oil species. The microorganisms and compositions containing the microorganisms disclosed herein can decompose trans fatty acids and the oils and fats containing them that are produced during the oil hydrogenation process, and are particularly effective in treating margarine, fat spreads, shortening, and other products that contain large amounts of such fatty acid-containing oils and fats, which could not be treated with conventional microorganisms. In particular, the microorganisms and compositions containing the microorganisms disclosed herein provide the effect of achieving trans fatty acid decomposition at a practical level, which can be used as yeast (microorganisms) that play a key role in wastewater treatment and waste disposal. [Brief explanation of the drawings]

[0011]

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Mode for Carrying Out the Invention

[0012] The following will describe the present disclosure while showing the best mode. Throughout this specification, it should be understood that the singular expressions include the concepts of their plural forms unless otherwise specified. Therefore, the singular articles (e.g., "a", "an", "the" in English, etc.) should be understood to include the concepts of their plural forms unless otherwise specified. Also, the terms used in this specification should be understood to be used in the ordinary meanings used in the relevant field unless otherwise specified. Therefore, unless otherwise defined, all technical terms and scientific and technological terms used in this specification have the same meanings as commonly understood by those skilled in the art to which the present disclosure pertains. In case of contradiction, this specification (including the definitions) shall prevail.

[0013] The definitions of the terms specifically used in this specification and / or the basic technical content will be appropriately described below. In this specification, the strain names such as KH-1 strain may sometimes omit the indication of "strain", but those skilled in the art should appropriately understand that there is an indication of strain according to the context.

[0014] (Definitions, etc.) In this specification, "esterase" refers to a hydrolase that decomposes an ester into an acid and an alcohol by a chemical reaction with water. In this specification, typically, esterase refers to a hydrolase that decomposes a fatty acid ester into a fatty acid and an alcohol by a chemical reaction with water.

[0015] In this specification, "lipase" is a kind of esterase and refers to an enzyme that reversibly catalyzes the reaction of hydrolyzing neutral fat (glycerol ester) into fatty acid and glycerol. For example, as lipase, there is triglyceride lipase classified as EC 3.1.1.3 by the enzyme number (EC number).

[0016] As used herein, the term "symbiotic system" refers to a combination of multiple types of microorganisms existing in the same environment or system. Also, "symbiotic culturing" refers to causing a combination of multiple types of microorganisms to exist in the same environment or system. For example, the symbiotic system can be used in any of the following senses: a combination of multiple types of microorganisms mixed and existing in a certain composition; a combination of multiple types of microorganisms carried on a carrier and existing in a certain composition; and a combination of multiple types of microorganisms formed as a result of separately introducing each microorganism species into a certain environment. In one embodiment, each microorganism species in the symbiotic system may be in a state where they can contact each other (including fusion and encapsulation). In one embodiment, the symbiotic system may be in a state where another microorganism species in the symbiotic system can utilize an environment generated by the action (e.g., release, decomposition) of at least one microorganism species in the symbiotic system. In this case, the respective microorganism species may or may not be able to contact each other (e.g., each microorganism is arranged upstream and downstream in the system). Examples of the environment generated by the action of at least one microorganism species in the symbiotic system include an environment in which a certain component has decreased, an environment in which a certain component (a component generated by decomposition by a certain microorganism species, a secondary component generated by the action (e.g., reaction) of this component, etc.) has increased, and an environment in which a certain factor (such as pH) has changed. In particular, a typical example of a symbiotic system is a case where a product generated by the decomposition of a chemical substance by one or more of the microorganisms constituting the symbiotic system becomes a growth substrate such as a carbon source for other microorganisms. Also, in a mixed system of microorganisms, microorganisms having the same ability to decompose and assimilate the same growth substrate generally tend to be in a competitive relationship with respect to the substrate, so it is considered that a symbiotic system is difficult to establish. As used herein, "symbiotic culturable" refers to the ability of a combination of multiple types of microorganisms to form a symbiotic system.

[0017] As used herein, the "lipolytic ability calculated from the values of the lipolytic ability of each individual culture" refers to a numerical value obtained by calculating based on the values obtained for the lipolytic ability of each microbial strain constituting a combination of microorganisms (for example, Burkholderia bacteria and Yarrowia yeast), taking into account the contribution degree in each mixture. Typically, it includes, but is not limited to, the lipolytic ability value calculated from the sum of the values obtained by multiplying the lipolytic ability values obtained based on the results of culturing under the same conditions (such as medium composition, time, temperature, etc.) for each by a simple ratio. In this case, it can be expressed as the "sum of the lipolytic abilities calculated from the values of the lipolytic ability of each individual culture". Additionally, a value considering the contribution degree based on the initial ratio at the time of mixing may also be used. In this case, it can be expressed as the "lipolytic ability calculated from the initial values of the lipolytic ability of each individual culture". For example, for a combination with a total cell concentration of 1×10 5 cells / mL consisting of one type of Burkholderia bacteria and one type of Yarrowia yeast, the "lipolytic ability calculated from the values of the lipolytic ability of each individual culture" is [(the lipolytic ability when this Burkholderia bacteria at 1×10 5 cells / mL is cultured alone)×(the mixing ratio of this Burkholderia bacteria)+(the lipolytic ability when this Yarrowia yeast at 1×10 5 cells / mL is cultured alone)×(the mixing ratio of this Yarrowia yeast)]. The cell concentration during the individual culture serving as the basis for the calculation is not particularly limited and can be appropriately selected by those skilled in the art and can be calculated by multiplying by an appropriate coefficient (which may be 1). Also, as another example, it may be calculated from the value of the lipolytic ability based on the initial inoculated microbial amount (optionally considering the growth rate of the microorganisms). The "fatty acid decomposition ability calculated from the values of the fatty acid decomposition ability of each individual culture" and the "sum of the fatty acid decomposition abilities calculated from the values of the fatty acid decomposition ability of each individual culture" are also understood in a similar context.

[0018] As used herein, "oil and fat" refers to oily substances, and oil and fat includes ester group-containing compounds formed by dehydration condensation of a compound containing a hydroxyl group and a fatty acid. Typically, the compound containing a hydroxyl group is glycerin, but others include polyglycerin and the like. Similar to the meaning commonly used in this technical field, in this specification, an ester group-containing compound formed by dehydration condensation of glycerin and a fatty acid is called "glyceride". When the compound containing a hydroxyl group has a plurality of hydroxyl groups, if at least one of the hydroxyl groups forms an ester by dehydration condensation with a fatty acid, it corresponds to the ester group-containing compound in this specification. In this specification, oil and fat may include cis-fatty acid-containing oil and fat, trans-fatty acid-containing oil and fat, or both.

[0019] Oil and fat is contained in, for example, the kitchen wastewater of the food service industry and the wastewater of food factories. Grease traps and pressurized flotation separation devices, which are treatment facilities for removing this by solid-liquid separation, are sources of bad odors and pests, and have many problems such as the labor and cost involved in the recovery, transportation, and cleaning of the separated oil, and the cost of the flocculant required for this. The combination of microorganisms of the present disclosure and the composition or combination providing the same can be used to eliminate the oil in the grease trap or in the factory wastewater treatment facility.

[0020] The present disclosure provides microbial preparations for grease traps and factory wastewater. In particular, when applied to factory wastewater, it is possible to reduce or even replace the operating rate of the pressurized flotation separation device. The kitchen wastewater of the food service industry not only contains high-concentration oil and fat of usually 1 g / L or more, and up to 10 g / L or more when high, but also the residence time of the wastewater in many grease traps is extremely short, about 10 minutes, but the microorganisms of the present disclosure can be used even in such an environment.

[0021] Oils and fats are also abundant in food waste, livestock waste, sludge from wastewater treatment plants, and the like. Microorganisms are often used to treat such solid waste, but if the oil content is high, the treatment becomes difficult or the oil remains. The combination of microorganisms and the composition or combination thereof provided by the present disclosure are also applicable to the decomposition treatment of oil in such waste.

[0022] As used herein, "oil" includes oils and fats and fatty acids.

[0023] As used herein, "fatty acid" is a compound having 2 to 100 carbon atoms and at least one carboxyl group. Typically, the carbon chain in a fatty acid is straight, but it may be branched or contain a ring. Typically, a fatty acid contains one carboxyl group, but may contain multiple carboxyl groups. The carbon chain in a fatty acid may contain a C=C double bond, and "trans fatty acid" is used in the meaning commonly used in the art and means an unsaturated fatty acid having a trans-type double bond.

[0024] As used herein, "trans fatty acid-containing oil and fat" refers to a compound formed by the dehydration condensation of a trans fatty acid and a compound containing a hydroxyl group. Trans fatty acids include elaidic acid, vaccenic acid, etc., but when mentioned in this specification, there is no particular limitation on the type of trans fatty acid. The ratio of trans fatty acids present in the trans fatty acid-containing oil and fat is not particularly limited. The "trans type" and "cis type" of double bonds are used in the meaning commonly used in the art and have the following structure in which four substituents (R1, R2, R3, and R4) are bonded to two carbon atoms forming a double bond

Chemical formula

[0025] As used herein, the "short-chain to medium-chain fatty acid-containing ester" refers to an ester of a fatty acid containing one or more of short-chain fatty acids or medium-chain fatty acids. "Short-chain fatty acid", "medium-chain fatty acid" and "long-chain fatty acid" are used in the meanings commonly used in the art, and mean fatty acids having 2 to 6 carbon atoms, 7 to 12 carbon atoms and 13 or more carbon atoms, respectively. Short-chain fatty acids include acetic acid (2 carbon atoms), butyric acid (4 carbon atoms), caproic acid (6 carbon atoms), etc. Medium-chain fatty acids include caprylic acid (8 carbon atoms), capric acid (10 carbon atoms), lauric acid (12 carbon atoms), etc. Long-chain fatty acids include myristic acid (14 carbon atoms), palmitic acid (16 carbon atoms), palmitelaidic acid (16 carbon atoms), stearic acid (18 carbon atoms), oleic acid (18 carbon atoms), elaidic acid (18 carbon atoms), linoleic acid (18 carbon atoms), vaccenic acid (18 carbon atoms), linolenic acid (18 carbon atoms), etc.

[0026] As used herein, the "normal hexane value" is the amount of non-volatile substances extracted by normal hexane, and refers to an index indicating the amount of oil in water (oils and fats, their hydrolysis products, etc.). The normal hexane value can be determined, for example, in accordance with JIS K 0102. It can also be determined using a simple measurement reagent kit by measuring the polynipam extract.

[0027] As used herein, "assimilation" means utilization as a nutrient source, and the substance (such as oil and fat) that has become the object of assimilation will be decomposed and as a result disappear or decrease.

[0028] As used herein, when referring to "decomposition" with respect to esters (such as oil and fat) and / or fatty acids, it means that the target ester (such as oil and fat) and / or fatty acid becomes smaller molecules. For example, it means being separated into glycerol and (free) fatty acids, and the conversion of fatty acids into fatty acids with fewer carbon atoms or the conversion into carbon dioxide and water is also called decomposition.

[0029] As used herein, "the ability to decompose esters" or "esterase activity" refers to the activity of hydrolyzing esters into alcohols and acids (such as free fatty acids). For example, "the ability to decompose esters" or "esterase activity" can be measured by contacting a microorganism, a combination of microorganisms, or their culture supernatant with an ester of 4-nitrophenol and a fatty acid and measuring the amount of 4-nitrophenol produced by the hydrolysis reaction, or it can be measured in the same way as the measurement of the ability to decompose the oil and fat described in this specification. It is also understood that more specific abilities among "the ability to decompose esters containing short-chain to medium-chain fatty acids" and "the ability to decompose oil and fat containing short-chain to long-chain fatty acids" of "the ability to decompose esters" or "esterase activity" can be measured in the same way.

[0030] As used herein, "(a microorganism, etc.) having lipase activity" means having the activity of hydrolyzing the oil and fat produced by the dehydration condensation of glycerol and fatty acids into glycerol and free fatty acids, and such lipase activity is also referred to as triglyceride lipase activity in this specification. For example, whether it has lipase activity can be confirmed by the decrease in the oil and fat (such as animal and vegetable oils such as canola oil, triolein, etc.) contained in the medium to which the microorganism is added.

[0031] As used herein, the term that a microorganism "produces lipase" means that the microorganism produces lipase extracellularly or intracellularly. In particular, when lipase is produced extracellularly, it is said that the lipase is secreted or secreted and produced. However, in that case, the secreted lipase may be released from the cells of the bacterial cells into the external environment, or may remain on the cell surface due to some interaction with the cell surface. However, for example, the production of lipase by a microorganism can be measured and identified by the following decomposition tests. It is sufficient that the decomposition ability is shown in any of the following tests, and it is not necessary that decomposition is recognized in all tests. · A test to confirm whether the microorganism has the ability to assimilate oil and fat at a predetermined temperature (for example, 15°C, 28°C). · A test to confirm whether a clear zone is observed around the colonies formed on an agar medium containing oil and fat at a predetermined temperature (for example, 15°C, 28°C). · A test in which oil and fat are used as a carbon source and cultured at a predetermined temperature (for example, 15°C, 28°C), and the amount of decrease in the normal hexane value in the culture supernatant is measured. · A test in which oil and fat are used as a carbon source and cultured at a predetermined temperature (for example, 15°C, 28°C), and the temporal changes in the amounts of oil and fat and free fatty acids in the culture supernatant are measured by thin-layer chromatography. If the amount of oil and fat decreases over time, it has the ability to decompose. Alternatively, if the amount of free fatty acids once increases, it can be said that it has the ability to decompose. · A test in which oil and fat are used as a carbon source and cultured at a predetermined temperature (for example, 15°C, 28°C), and the concentration of free fatty acids in the culture supernatant is measured by instrumental analysis such as gas chromatography, gas chromatography-mass spectrometry, high-performance liquid chromatography, etc. If the concentration of free fatty acids once increases, it can be said that it has the ability to decompose. · A test in which test water containing oil and fat as the main organic substance (for example, 70% by weight or more in all organic substances) is prepared, and the biochemical oxygen demand (BOD) is measured. · Analyze the proteins in the culture supernatant or cultured cells by electrophoresis or mass spectrometry to confirm the presence of lipase. · Confirm the expression of the lipase gene of the microorganism by quantifying or detecting its mRNA. · Analyze the "lipase activity" of microorganisms, combinations of microorganisms, or their culture supernatants according to the method for measuring the above-mentioned "esterase activity". Individual more detailed measurement methods are provided herein, and those skilled in the art can perform these measurements using any other equipment and conditions.

[0032] As used herein, the "ability to decompose esters at 15°C" means that (such as microorganisms) have the activity to hydrolyze esters into alcohols and acids at low temperatures. The ability to decompose fats and oils at 15°C can be measured by contacting the microorganisms of the present disclosure (including derivative strains of KH-2 strain) with an ester of 4-nitrophenol and an acid (such as free fatty acid) at 15°C and measuring the amount of 4-nitrophenol produced by the hydrolysis reaction, or it can be measured in the same manner as the measurement of the ability to decompose fats and oils at 15°C described herein.

[0033] As used herein, the "ability to decompose fats and oils at 15°C" means that (such as microorganisms) have the activity to hydrolyze fats and oils into glycerol and free fatty acids at low temperatures. The ability to decompose fats and oils at 15°C can be measured and identified by the following tests. It is only necessary to show the decomposition ability in any of the following tests, and it is not necessary that decomposition is recognized in all tests. · A test to confirm whether there is the ability to assimilate fats and oils at 15°C. · A test to confirm whether a clear zone is observed around the colonies formed on an agar medium containing fats and oils at 15°C. · A test in which fats and oils are provided as a carbon source and cultured at 15°C, and the amount of decrease in the normal hexane value in the culture supernatant is measured. · A test in which fats and oils are provided as a carbon source and cultured at 15°C, and the time change in the amounts of fats and oils and free fatty acids in the culture supernatant is measured by thin-layer chromatography. If the amount of fats and oils decreases over time, it has the decomposition ability. Alternatively, if the amount of free fatty acids once increases, it can be said that it has the ability to decompose. · A test in which oil and fat is provided as a carbon source and cultured at 15°C, and the concentration of free fatty acids in the culture supernatant is measured by instrumental analysis such as gas chromatography, gas chromatography-mass spectrometry, high-performance liquid chromatography, etc. If the concentration of free fatty acids once increases, it can be said that there is the ability to decompose. · A test in which test water containing oil and fat as the main organic substance (for example, 70% by weight or more in all organic substances) is prepared and the biochemical oxygen demand (BOD) is measured. Individual more detailed measurement methods are provided in this specification, and those skilled in the art can perform these measurements using any other equipment and conditions.

[0034] In this specification, the "ability to assimilate trans-fatty acid-containing oil and fat" refers to the activity to assimilate trans-fatty acid-containing oil and fat. In this specification, "assimilate trans-fatty acid-containing oil and fat" is used in the meaning commonly used in this technical field, and means that a microorganism takes in trans-fatty acid-containing oil and fat as a nutrient source such as a carbon source. When "assimilating", in addition to hydrolyzing into glycerol and free fatty acids, it also includes changing to a part of other substances. The ability to assimilate trans-fatty acid-containing oil and fat can be measured and identified by the following tests. It is sufficient that the assimilation ability is shown in any of the following tests, and it is not necessarily required that assimilation is recognized in all tests. · A test to confirm whether it is possible to grow in a medium containing trans-fatty acid-containing oil and fat as the sole carbon source. · A test to confirm whether colonies can be formed in a medium containing trans-fatty acid-containing oil and fat as the sole carbon source. · A test to measure the decrease in the normal hexane value in the culture supernatant with growth. · A test to measure the total amount of all fatty acids (the sum of fatty acids in oil and fat and free fatty acids) in the culture supernatant with growth after converting them into methyl esters and then measuring the total amount by gas chromatography. · A test to measure the amounts of oil and fat and free fatty acids in the culture supernatant with growth by thin-layer chromatography. Individual more detailed measurement methods are provided herein, and those skilled in the art can perform these measurements using any other equipment and conditions.

[0035] As used herein, the "ability to decompose oils and fats containing trans fatty acids" refers to the activity of hydrolyzing oils and fats containing trans fatty acids into glycerol and free fatty acids. The ability to decompose oils and fats containing trans fatty acids can be measured and identified by the following tests. It is sufficient that the decomposition ability is shown in any of the following tests, and it is not necessary that decomposition is recognized in all tests. · A test to confirm whether there is an ability to assimilate oils and fats containing trans fatty acids. · A test to measure the amounts of oils and fats and free fatty acids in the culture supernatant by thin layer chromatography. · A test to culture by providing oils and fats containing trans fatty acids as a carbon source and measure the decrease in the normal hexane value in the culture supernatant. · A test to convert the free fatty acids contained in the culture supernatant and the fatty acids in the oils and fats into methyl esters and then measure their concentrations by gas chromatography. · A test to measure the concentration of free fatty acids in the culture supernatant by gas chromatography-mass spectrometry. · A test to measure the concentration of free fatty acids in the culture supernatant by high performance liquid chromatography. · A test to confirm whether a clear zone is observed around the colonies formed on an agar medium containing oils and fats containing trans fatty acids. · A test to prepare a test water containing oils and fats containing trans fatty acids as the main organic matter (for example, 70% by weight or more of the total organic matter) and measure the biochemical oxygen demand (BOD). Individual more detailed measurement methods are provided herein, and those skilled in the art can perform these measurements using any other equipment and conditions.

[0036] As used herein, the "ability to assimilate trans fatty acids" refers to the ability to assimilate trans fatty acids (e.g., elaidic acid, palmitelaidic acid, and / or vaccenic acid). The ability to assimilate trans fatty acids can be measured and identified by the following tests. It is sufficient that the assimilation ability is shown in any of the following tests, and it is not necessary that assimilation is recognized in all tests. · A test to confirm whether it can grow in a medium containing trans fatty acids as the sole carbon source. · A test to confirm whether it can form colonies in a medium containing trans fatty acids as the sole carbon source. · A test in which trans fatty acids are given as a carbon source and cultured, and the decrease in the normal hexane value in the culture supernatant is measured as growth progresses. · A test to measure the concentration of trans fatty acids in the culture supernatant by gas chromatography as growth progresses. · A test to measure the concentration of trans fatty acids in the culture supernatant by gas chromatography-mass spectrometry as growth progresses. · A test to measure the concentration of trans fatty acids in the culture supernatant by high performance liquid chromatography as growth progresses. · A test to measure the amount of trans fatty acids in the culture supernatant by thin layer chromatography as growth progresses. Individual more detailed measurement methods are provided herein, and those skilled in the art can perform these measurements using any other equipment and conditions.

[0037] As used herein, the "ability to decompose trans fatty acids" refers to the ability to decompose trans fatty acids (e.g., elaidic acid, palmitelaidic acid, and / or vaccenic acid). The ability to decompose trans fatty acids can be measured and identified by the following tests. It is sufficient that the decomposition ability is shown in any of the following tests, and it is not necessary that decomposition is recognized in all tests. · A test to confirm whether it has the ability to assimilate trans fatty acids. · A test to measure the amount of trans fatty acids in the culture supernatant by thin layer chromatography. ·A test in which the culture is carried out with trans fatty acids as a carbon source and the decrease in the normal hexane value in the supernatant is measured. ·A test in which the concentration of trans fatty acids in the culture supernatant is measured by gas chromatography. ·A test in which the concentration of trans fatty acids in the culture supernatant is measured by gas chromatography-mass spectrometry. ·A test in which the concentration of trans fatty acids in the culture supernatant is measured by high performance liquid chromatography. ·A test to confirm whether a clear zone is observed around the colonies formed on an agar medium containing trans fatty acids. ·A test in which test water containing trans fatty acids as the main organic substance (for example, 70% by weight or more of all organic substances) is prepared and the biochemical oxygen demand (BOD) is measured. Individual more detailed measurement methods are provided in this specification, and those skilled in the art can perform these measurements using any other equipment and conditions.

[0038] In this specification, the "ability to assimilate fats and oils" refers to the activity of assimilating fats and oils. In this specification, "assimilating fats and oils" is used in the meaning commonly used in the technical field, which means that microorganisms take in fats and oils or their decomposition products as nutrient sources such as carbon sources. When "assimilating", in addition to hydrolyzing into glycerol and free fatty acids, it also includes changing to a part of other substances. The ability to assimilate fats and oils can be measured and identified by the following tests. It is only necessary that the assimilation ability be shown in any of the following tests, and it is not necessarily required that assimilation be recognized in all tests. ·A test to confirm whether it is possible to grow in a medium containing fats and oils as the sole carbon source. ·A test to confirm whether colonies can be formed in a medium containing fats and oils as the sole carbon source. ·A test in which the decrease in the normal hexane value in the culture supernatant is measured as growth progresses. ·A test in which all fatty acids (the sum of fatty acids in fats and oils and free fatty acids) in the culture supernatant as growth progresses are converted to methyl esters and the total amount is measured by gas chromatography. · A test for measuring the amounts of oils and fats and free fatty acids in the culture supernatant by thin layer chromatography as the cells grow. More detailed measurement methods for each are provided herein, and those skilled in the art can perform these measurements using any other equipment and conditions.

[0039] As used herein, the "ability to decompose oils and fats" refers to the activity of hydrolyzing oils and fats into glycerol and free fatty acids. The ability to decompose oils and fats can be measured and identified by the following tests. It is only necessary that the decomposition ability be shown in any of the following tests; it is not necessarily required that decomposition be recognized in all tests. · A test for confirming whether the cells have the ability to assimilate oils and fats. · A test for measuring the amounts of oils and fats and free fatty acids in the culture supernatant by thin layer chromatography. · A test for culturing cells with oils and fats as the carbon source and measuring the amount of decrease in the normal hexane value in the culture supernatant. · A test for converting the free fatty acids contained in the culture supernatant and the fatty acids in the oils and fats into methyl esters and then measuring their concentrations by gas chromatography. · A test for measuring the concentration of free fatty acids in the culture supernatant by gas chromatography - mass spectrometry. · A test for measuring the concentration of free fatty acids in the culture supernatant by high performance liquid chromatography. · A test for confirming whether a clear zone is observed around the colonies formed on an agar medium containing oils and fats. · A test for preparing a test water containing fatty acid - containing oils and fats as the main organic substance (for example, 70% by weight or more of the total organic substances) and measuring the biochemical oxygen demand (BOD). More detailed measurement methods for each are provided herein, and those skilled in the art can perform these measurements using any other equipment and conditions.

[0040] In this specification, the "ability to assimilate fatty acids" refers to the ability to assimilate fatty acids. The ability to assimilate fatty acids can be measured and identified by the following tests. It is sufficient that the assimilation ability is shown in any of the following tests, and it is not necessary that assimilation is recognized in all tests. · A test to confirm whether it is possible to grow in a medium containing fatty acids as the sole carbon source. · A test to confirm whether colonies can be formed in a medium containing fatty acids as the sole carbon source. · A test in which fatty acids are given as a carbon source and cultured, and the decrease in the normal hexane value in the culture supernatant is measured as growth progresses. · A test in which the concentration of fatty acids in the culture supernatant is measured by gas chromatography as growth progresses. · A test in which the concentration of fatty acids in the culture supernatant is measured by gas chromatography-mass spectrometry as growth progresses. · A test in which the concentration of fatty acids in the culture supernatant is measured by high performance liquid chromatography as growth progresses. · A test in which the amount of fatty acids in the culture supernatant is measured by thin layer chromatography as growth progresses. Individual more detailed measurement methods are provided in this specification, and those skilled in the art can perform these measurements using any other equipment and conditions.

[0041] In this specification, the "ability to decompose fatty acids" refers to the ability to decompose fatty acids. The ability to decompose fatty acids can be measured and identified by the following tests. It is sufficient that the decomposition ability is shown in any of the following tests, and it is not necessary that decomposition is recognized in all tests. · A test to confirm whether there is an ability to assimilate fatty acids. · A test in which the amount of fatty acids in the culture supernatant is measured by thin layer chromatography. · A test in which fatty acids are given as a carbon source and cultured, and the decrease in the normal hexane value in the supernatant is measured. · A test in which the concentration of fatty acids in the culture supernatant is measured by gas chromatography. · A test in which the concentration of fatty acids in the culture supernatant is measured by gas chromatography-mass spectrometry. · A test for measuring the concentration of fatty acids in the culture supernatant by high-performance liquid chromatography. · A test for checking whether a clear zone is observed around the colonies formed on an agar medium containing fatty acids. · A test for preparing a test water containing fatty acids as the main organic substance (for example, 70% by weight or more of all organic substances) and measuring the biochemical oxygen demand (BOD). Individual more detailed measurement methods are provided herein, and those skilled in the art can perform these measurements using any other equipment and conditions.

[0042] In this specification, the "oil treatment component" means a component that aids in the assimilation and decomposition of oils and fats and / or fatty acids. Specifically, it includes components that promote the dispersion of oils and fats and / or fatty acids, such as surfactants, components that decompose oils and fats into fatty acids and glycerol, those that decompose fatty acids, those that decompose glycerol, and those that adsorb oil and remove it from the object to be treated.

[0043] As used herein, the "oil-degrading agent" refers to a preparation that contains at least one microorganism of the microorganisms of the present disclosure or a combination of the microorganisms of the present disclosure as an active ingredient and is capable of decomposing oils and / or fatty acids alone or in a combination of the microorganisms of the present disclosure. A preparation capable of decomposing oils and / or fatty acids in a combination of the microorganisms of the present disclosure may be a composition containing only one microorganism (for example, either Burkholderia bacteria or Yarrowia yeast) among the combinations of the microorganisms of the present disclosure, which by itself does not exhibit the desired oil and / or fatty acid decomposing ability. However, when it is used to form a combination of the microorganisms of the present disclosure and exhibits the desired oil and / or fatty acid decomposing ability, this composition may be an oil-degrading agent. In the present disclosure, the oil-degrading agent may be used in combination with an oil treatment component. In this case, the timing of the combined use of the oil-degrading agent and the oil treatment component may be simultaneous or either one may be used first. Furthermore, the oil-degrading agent may further contain components (such as a carbon source and a nitrogen source) that enhance the activity of the microorganism strain or esterase (for example, lipase) derived from the microorganism strain used, a surfactant, a drying protectant, a component for maintaining the microorganism for a long period, a preservative, an excipient, a strengthening agent, an antioxidant, and the like.

[0044] As used herein, "derivative strain", "similar strain" or "mutant strain" preferably, but not intending to be limiting, includes a gene (e.g., 16S rDNA or 26S rDNA) containing a region substantially homologous to the DNA of the target microorganism. Such strains, in various embodiments, when aligned by computer homology programs known in the art and compared to the sequence of the entire genome of the original strain, have a genome sequence that is at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or 99% identical. This refers to a microorganism modified by gene mutations, substitutions, deletions and / or additions, and the derivative strain thereof may or may not exhibit the biological functions of the original microorganism to the same extent. In one embodiment, such derivative strains, similar strains or mutant strains provided in the present disclosure are microorganisms whose biological functions (e.g., oil degradation ability, ability to enhance lipase production or oil degradation ability of other microorganisms) are equal to or greater than those of the original microorganism. For example, gene mutations can be introduced using any known mutagen, UV, plasma, etc. In one embodiment, the "derivative strain", "similar strain" or "mutant strain" is a strain belonging to the same genus and / or species as the original strain. For example, the biological functions of such microorganisms can be examined by appropriate and available in vitro assays described herein or known in the art. As used herein, the "similarity" of a gene or nucleotide sequence refers to the degree of similarity between two or more gene sequences to each other, indicating a high degree of similarity to other sequences in addition to identity. "Similarity" is a numerical value that takes into account similar bases in addition to identity, where similar bases refer to cases where there is partial identity in degenerate bases (e.g., R = A + G, M = A + C, W = A + T, S = C + G, Y = C + T, K = G + T, H = A + T + C, B = G + T + C, D = G + A + T, V = A + C + G, N = A + C + G + T).

[0045] As used herein, "protein", "polypeptide", "oligopeptide", and "peptide" are used interchangeably herein and refer to polymers of amino acids of any length. Such polymers may be linear, branched, or cyclic. The amino acids may be natural, non-natural, or modified amino acids. The term may also include assemblies of multiple polypeptide chains complexed together. The term also includes amino acid polymers that have been modified either naturally or artificially. Such modifications include, for example, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification (e.g., conjugation with a labeling component). This definition also includes, for example, polypeptides that contain one or more analogs of amino acids (e.g., including non-natural amino acids, etc.), peptidomimetic compounds (e.g., peptoids), and other modifications known in the art. As used herein, "amino acid" is a general term for organic compounds having an amino group and a carboxyl group. When a protein or enzyme according to an embodiment of the present disclosure "contains a specific amino acid sequence", any amino acid in the amino acid sequence may be chemically modified. Also, any amino acid in the amino acid sequence may form a salt or a solvate. Also, any amino acid in the amino acid sequence may be of the L-form or the D-form. Even in such cases, it can be said that the protein according to the embodiment of the present disclosure "contains the above-mentioned specific amino acid sequence". Known chemical modifications that amino acids in a protein undergo in vivo include, for example, N-terminal modifications (e.g., acetylation, myristoylation, etc.), C-terminal modifications (e.g., amidation, glycosylphosphatidylinositol addition, etc.), or side-chain modifications (e.g., phosphorylation, sugar chain addition, etc.). The amino acids may be natural or non-natural as long as the object of the present disclosure is satisfied.

[0046] As used herein, "polynucleotide", "oligonucleotide" and "nucleic acid" are used interchangeably and refer to polymers of nucleotides of any length. This term also includes "oligonucleotide derivatives" or "polynucleotide derivatives". "Base sequence" or "nucleic acid sequence" means the order of consecutive nucleobases in a "polynucleotide", "oligonucleotide" or "nucleic acid". "Oligonucleotide derivative" or "polynucleotide derivative" refers to an oligonucleotide or polynucleotide that contains derivatives of nucleotides or in which the bonds between nucleotides are different from normal, and are used interchangeably. Such oligonucleotides specifically include, for example, 2'-O-methyl-ribonucleotides, oligonucleotide derivatives in which the phosphodiester bond in the oligonucleotide is converted to a phosphorothioate bond, oligonucleotide derivatives in which the phosphodiester bond in the oligonucleotide is converted to an N3'-P5' phosphoramidate bond, oligonucleotide derivatives in which the ribose and phosphodiester bond in the oligonucleotide are converted to a peptide nucleic acid bond, oligonucleotide derivatives in which uracil in the oligonucleotide is replaced by C-5 propynyluracil, oligonucleotide derivatives in which uracil in the oligonucleotide is replaced by C-5 thiazoleuracil, oligonucleotide derivatives in which cytosine in the oligonucleotide is replaced by C-5 propynylcytosine, oligonucleotide derivatives in which cytosine in the oligonucleotide is replaced by phenoxazine-modified cytosine, oligonucleotide derivatives in which ribose in DNA is replaced by 2'-O-propylribose, and oligonucleotide derivatives in which ribose in the oligonucleotide is replaced by 2'-methoxyethoxyribose. Unless otherwise indicated, a particular base sequence is also intended to include its conservatively modified variants (e.g., degenerate codon substitutions) and complementary sequences, as well as the explicitly shown sequence.Specifically, a degenerate codon variant can be achieved by creating an array in which the third position of one or more selected (or all) codons is substituted with a mixed base and / or a deoxyinosine residue (Batzer et al., Nucleic Acid Res. 19:5081 (1991); Ohtsuka et al., J. Biol. Chem. 260: 2605-2608 (1985); Rossolini et al., Mol. Cell. Probes 8:91-98 (1994)). As used herein, "nucleic acid" is also used interchangeably with gene, cDNA, mRNA, oligonucleotide, and polynucleotide. As used herein, "nucleotide" may be natural or non-natural.

[0047] As used herein, "gene" refers to a factor that defines a genetic trait, and "gene" may refer to "polynucleotide", "oligonucleotide", and "nucleic acid".

[0048] As used herein, the "homology" of a gene refers to the degree of identity between two or more gene sequences with respect to each other. Generally, having "homology" means having a high degree of identity or similarity. Therefore, the higher the homology between two genes, the higher the identity or similarity of their sequences. Whether two genes have homology can be examined by direct comparison of the sequences or, in the case of nucleic acids, by a hybridization method under stringent conditions. When directly comparing two gene sequences, when the DNA sequences between the gene sequences are typically at least 50% identical, preferably at least 70% identical, more preferably at least 80%, 90%, 95%, 96%, 97%, 98% or 99% identical, those genes have homology.

[0049] Amino acids can be referred to herein by either their generally known three-letter symbols or the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Nucleotides can likewise be referred to by their generally recognized one-letter codes. In this specification, comparisons of amino acid sequences and nucleotide sequences for similarity, identity, and homology are calculated using default parameters with the BLAST sequence analysis tool. A search for identity can be performed, for example, using NCBI's BLAST 2.7.1 (released on October 19, 2017). The "identity" value in this specification usually refers to the value obtained when aligned under default conditions using the above BLAST. However, if a higher value is obtained by changing the parameters, the highest value shall be taken as the identity value. When identity is evaluated in multiple regions, the highest value among them shall be taken as the identity value. "Similarity" is a numerical value that takes into account similar amino acids in addition to identity.

[0050] In one embodiment of the present disclosure, the numerical value of "70% or more" for identity or the like may be, for example, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100% or more, or may be within the range of any two values of those starting values. The above "identity" is calculated according to a known method as described above, as the ratio of the number of identical amino acids or bases in an amino acid or base sequence between two or more. Specifically explained, before calculating the ratio, the amino acid or base sequences of the amino acid or base sequence groups to be compared are aligned, and a gap is introduced into a part of the amino acid or base sequence if necessary to maximize the ratio of the same amino acid or base. Methods for alignment, methods for calculating the ratio, comparison methods, and computer programs related thereto are well known in the art (for example, BLAST described above). In this specification, "identity" and "similarity" can be represented by values measured by NCBI's BLAST unless otherwise specified. When comparing amino acid or base sequences with BLAST, Blastp can be used with default settings. The measurement results are quantified as Positives or Identities. In this case, when "similarity" is used instead of "identity", it is a numerical value that also takes into account those that correspond to the definition of "similar" "amino acids" or "bases" described in this specification. In this specification, polynucleotides, oligonucleotides, nucleic acids, peptides, or proteins having homology, identity, and / or similarity between their sequences may be referred to as "variants" of each other.

[0051] As used herein, the term "polynucleotide that hybridizes under stringent conditions" refers to well-known conditions commonly used in the art. Such polynucleotides can be obtained by using, as a probe, a polynucleotide selected from the polynucleotides of the present disclosure, and employing methods such as colony hybridization, plaque hybridization, or Southern blot hybridization. Specifically, it means a polynucleotide that can be identified by performing hybridization at 65°C in the presence of 0.7 to 1.0 M NaCl using a filter immobilized with DNA derived from colonies or plaques, and then washing the filter at 65°C using an SSC (saline-sodium citrate) solution at a concentration of 0.1 to 2 times (the composition of a 1×SSC solution is 150 mM sodium chloride and 15 mM sodium citrate). "Stringent conditions" can employ, for example, the following conditions: (1) using low ionic strength and high temperature for washing (e.g., at 50°C, 0.015 M sodium chloride / 0.0015 M sodium citrate / 0.1% sodium dodecyl sulfate), (2) using a denaturing agent such as formamide during hybridization (e.g., at 42°C, 50% (v / v) formamide and 0.1% bovine serum albumin / 0.1% ficoll / 0.1% polyvinylpyrrolidone / 50 mM sodium phosphate buffer at pH 6.5, and 750 mM sodium chloride, 75 mM sodium citrate), or (3) incubating overnight at 37°C in a solution containing 20% formamide, 5×SSC, 50 mM sodium phosphate (pH 7.6), 5×Denhardt's solution, 10% dextran sulfate, and 20 mg / ml of denatured sheared salmon sperm DNA, and then washing the filter with 1×SSC at about 37 - 50°C. Note that the formamide concentration may be 50% or higher. The washing time may be 5, 15, 30, 60, or 120 minutes, or longer.Multiple factors such as temperature and salt concentration can affect the stringency of the hybridization reaction. For details, refer to Ausubel et al., Current Protocols in Molecular Biology, Wiley Interscience Publishers, (1995). Examples of "highly stringent conditions" are 0.0015 M sodium chloride, 0.0015 M sodium citrate, 65 - 68 °C, or 0.015 M sodium chloride, 0.0015 M sodium citrate, and 50% formamide, 42 °C. Hybridization can be carried out according to the methods described in experimental manuals such as Molecular Cloning 2nd ed., Current Protocols in Molecular Biology, Supplement 1 - 38, DNA Cloning 1: Core Techniques, A Practical Approach, Second Edition, Oxford University Press (1995). Here, sequences that hybridize under stringent conditions preferably exclude sequences containing only the A sequence or only the T sequence. Moderately stringent conditions can be easily determined by those skilled in the art based on, for example, the length of the DNA, as shown in Sambrook et al., Molecular Cloning: A Laboratory Manual, 3rd Edition, Vol. 1, 7.42 - 7.45 Cold Spring Harbor Laboratory Press, 2001, and for nitrocellulose filters, include the use of a pre - wash solution of 5×SSC, 0.5% SDS, 1.0 mM EDTA (pH 8.0), hybridization conditions of approximately 40 - 50 °C in approximately 50% formamide, 2×SSC - 6×SSC (or other similar hybridization solutions such as Stark's solution in approximately 50% formamide at approximately 42 °C), and washing conditions of approximately 60 °C, 0.5×SSC, 0.1% SDS.Accordingly, the polypeptides used in the present disclosure include polypeptides encoded by nucleic acid molecules that hybridize to nucleic acid molecules encoding the polypeptides specifically described in the present disclosure under highly or moderately stringent conditions.

[0052] As used herein, the term "biological function," when referring to a microorganism, refers to a specific function that the microorganism may possess, including, for example, but not limited to, the decomposition of esters (e.g., oils and fats) (e.g., decomposition at low temperatures), the production of lipase, the decomposition of oil, the ability to enhance the lipase production of other microorganisms, the ability to enhance the oil decomposition activity of other microorganisms, etc. As used herein, a biological function may be exerted by the corresponding "biological activity." As used herein, the term "biological activity" refers to the activity that a microorganism may possess in a certain environment, including activities that exhibit various functions (e.g., the oil decomposition activity at 15°C). Such biological activities can be measured by techniques well known in the art. Accordingly, "activity" refers to various measurable indicators that affect a response (i.e., have a measurable effect in response to some exposure or stimulus), and may include, for example, the amount of a protein upstream or downstream after some stimulation or event of the microorganisms of the present disclosure or other similar measures of function.

[0053] As used herein, the "amount" of an analyte in a sample generally refers to an absolute value that reflects the mass of the analyte that can be detected in the volume of the sample. However, the amount also contemplates a relative amount compared to another amount of analyte. For example, the amount of an analyte in a sample may be an amount greater than the control level or normal level of the analyte normally present in the sample.

[0054] The term "about" refers to plus or minus 10% of the indicated value.

[0055] As used herein, "kit" refers to a unit that is usually divided into two or more compartments and provided with parts to be provided (for example, compositions containing the microorganisms of the present disclosure, additional components, buffers, instructions, etc.). When the purpose is to provide a composition that should not be provided in a mixed state for reasons such as stability and is preferably mixed and used immediately before use, this kit form is preferred. Such a kit preferably includes instructions or manuals that describe how to use the provided parts (for example, compositions containing microorganisms, additional components), or how to process them. When a kit is used herein, the kit usually includes instructions that describe how to use the microorganisms and compositions of the present disclosure.

[0056] As used herein, "instructions" are those described for explaining to the user how to use the microorganisms of the present disclosure or combinations of the microorganisms of the present disclosure. These instructions describe the language that instructs how to use the microorganisms of the present disclosure or combinations of the microorganisms of the present disclosure. These instructions are prepared in accordance with the format specified by the regulatory authorities of the country where the method of the present disclosure is implemented (for example, the Ministry of Health, Labour and Welfare or the Ministry of Agriculture, Forestry and Fisheries in Japan, the Food and Drug Administration (FDA), the United States Department of Agriculture (USDA), etc. in the United States), and it is specified that approval has been obtained from the regulatory authorities. The instructions can be provided in a paper medium, but are not limited thereto, and can also be provided in forms such as an electronic medium (for example, a homepage provided on the Internet, an e-mail).

[0057] (Preferred Embodiment) Preferred embodiments of the present disclosure are described below. It is understood that the embodiments provided below are for better understanding of the present disclosure, and the scope of the present disclosure should not be limited to the following description. Therefore, it is obvious that those skilled in the art can make appropriate modifications within the scope of the present disclosure with reference to the descriptions in this specification. It is also understood that the following embodiments of the present disclosure can be used alone or in combination.

[0058] (Microorganisms with new capabilities) In one aspect, the present disclosure provides microorganisms having newly discovered capabilities regarding the degradation of oils and fats and / or fatty acids. In particular, the microorganisms of the present disclosure have lipase activity against triglycerides containing long-chain fatty acids (fatty acids containing 13 or more carbon atoms, for example, fatty acids containing 14 to 22 carbon atoms), and have higher hydrolytic activity against 4-nitrophenyl esters of short-chain to medium-chain fatty acids (fatty acids containing 2 to 12 carbon atoms) than 4-nitrophenyl esters of long-chain fatty acids, have the ability to degrade short- to long-chain fatty acid-containing oils and fats (fatty acids containing 2 or more carbon atoms), have the ability to degrade short- to medium-chain fatty acid-containing esters (fatty acids containing 2 to 12 carbon atoms), have the ability to assimilate esters (for example, oils and fats) and / or fatty acids at 15°C, have the ability to degrade esters (for example, oils and fats) and / or fatty acids at 15°C, have the ability to assimilate trans-fatty acid-containing oils and fats, have the ability to degrade trans-fatty acid-containing oils and fats, have the ability to assimilate trans-fatty acids (for example, elaidic acid, palmitelaidic acid and / or vaccenic acid), and / or have the ability to degrade trans-fatty acids (for example, elaidic acid, palmitelaidic acid and / or vaccenic acid), and have the ability to improve the lipase production of Burkholderia bacteria, and have the ability to improve the fatty acid and / or oil and fat degradation ability of Burkholderia bacteria. In one aspect, the present disclosure provides new microorganisms with the ability to degrade esters (for example, oils and fats). The microorganisms of the present disclosure may have broad hydrolytic activity against short- to long-chain fatty acid-containing oils and fats. In one aspect, the present disclosure provides new microorganisms that have the ability to improve the lipase production and / or oil (including fatty acids and oils and fats) degradation ability of Burkholderia bacteria when combined with Burkholderia bacteria.

[0059] In one embodiment, the microorganism of the present disclosure is a yeast of the genus Yarrowia. Microorganisms of the genus Yarrowia are fungi, and during a certain period of their life cycle, their vegetative bodies are unicellular and have cell walls. Morphologically, they are circular or elliptical with few characteristics. The genus Yarrowia includes species such as bubula, deformans, lipolytica, porcina, and yakushimensis. In one embodiment, the microorganism of the present disclosure is Yarrowia lipolytica. The inventor identified a new strain of microorganism found by examining the trans-fatty acid degradation and assimilation ability and the low-temperature ester (e.g., oil and fat) degradation ability as Yarrowia lipolytica, and deposited it with the Patent Microorganisms Depositary, National Institute of Technology and Evaluation. It was received on June 4, 2018, and a deposit certificate was issued on June 12, 2018. The deposit number is NITE BP-02732. Further strains of Yarrowia lipolytica (KH-2AL1 strain and KH-2AL3 strain) were further identified and deposited with the Patent Microorganisms Depositary, National Institute of Technology and Evaluation. They were received on December 23, 2019, and a deposit certificate was issued on January 16, 2020. The deposit numbers are NITE BP-03091 and NITE BP-03092, respectively. In one embodiment, the microorganism of the present disclosure is the Yarrowia yeast KH-2 strain (the strain specified by the deposit number NITE BP-02732), the KH-2AL1 strain (the strain specified by the deposit number NITE BP-03091), or the KH-2AL3 strain (the strain specified by the deposit number NITE BP-03092), or an induced strain thereof. In a further aspect of the present disclosure, the Yarrowia yeast of the present disclosure can be a yeast of the genus Candida, Zygoascus, Ogataea, Pichia, or Aciculoconidium.Examples of yeasts of the genus Candida include deformans, oslonensis, galli, phangngensis, hollandica, alimentaria, hispaniensis, and incommunis. Examples of the genus Zygoascus include steatolyticus var. steatolyticus, and examples of the genus (Aciculoconidium) include aculeatum.

[0060] In one embodiment, the microorganism of the present disclosure is a derivative strain of Yarrowia yeast strain KH-2 (strain identified by accession number NITE BP-02732), KH-2AL1 (strain identified by accession number NITE BP-03091), or KH-2AL3 (strain identified by accession number NITE BP-03092). Here, the derivative strain does not necessarily have to be a strain obtained based on Yarrowia yeast strain KH-2, KH-2AL1, or KH-2AL3, and refers to a microorganism that exhibits the biological functions of Yarrowia yeast strain KH-2, KH-2AL1, or KH-2AL3, although not necessarily to the same extent. In one embodiment, the microorganism that is a derivative strain of the present disclosure has lipase activity, the ability to assimilate (decompose) esters (e.g., oils and fats containing trans fatty acids) at low temperatures (e.g., 25°C or lower, 20°C or lower, 15°C or lower, 10°C or lower, 5°C or lower, etc.), the ability to assimilate (decompose) trans fatty acids (e.g., elaidic acid, palmitelaidic acid, and / or vaccenic acid), the ability to improve the lipase production of Burkholderia bacteria, and the ability to improve the fatty acid and / or oil and fat decomposition ability of Burkholderia bacteria, similar to Yarrowia yeast strain KH-2, KH-2AL1, or KH-2AL3, although the degree of its biological functions may be different from that of KH-2 strain, KH-2AL1 strain, or KH-2AL3. In one embodiment, the microorganism that is a derivative strain of the present disclosure is a yeast of the genus Yarrowia, and more specifically, it may be Yarrowia lipolytica.

[0061] The microorganisms of the present disclosure (including derivatives of strain KH-2, strain KH-2AL1, or strain KH-2AL3) can be isolated on an inorganic salt agar medium containing ester (e.g., oil and fat) as the sole carbon source and adjusted to a pH of 6 to 8. Also, in one embodiment, the microorganisms of the present disclosure (including derivatives of strain KH-2, strain KH-2AL1, or strain KH-2AL3) can be discriminated by confirming that a clear zone (halo) is formed around the colonies grown on an agar medium containing ester (e.g., oil and fat) dispersed therein. In one embodiment, the microorganisms of the present disclosure (including derivatives of strain KH-2, strain KH-2AL1, or strain KH-2AL3) can form colonies and grow on an agar medium supplemented with 10 g / L of canola oil as a carbon source at 15°C or 28°C, preferably 15°C. Although not intended to be limiting, it may be useful for the microorganisms of the present disclosure to have a minimum ability to assimilate (decompose) ester (e.g., oil and fat) or resistance to ester (e.g., oil and fat).

[0062] In one embodiment, the microorganism of the present disclosure (including derivatives of KH-2 strain, KH-2AL1 strain or KH-2AL3 strain) has esterase activity. In one embodiment, the microorganism of the present disclosure (including derivatives of KH-2 strain, KH-2AL1 strain or KH-2AL3 strain) has lipase activity against triglycerides containing long-chain fatty acids (fatty acids containing 13 or more carbon atoms, for example, fatty acids containing 14 to 22 carbon atoms). In one embodiment, the microorganism of the present disclosure (including derivatives of KH-2 strain, KH-2AL1 strain or KH-2AL3 strain) has esterase activity against esters (for example, triglycerides) containing short- to medium-chain fatty acids (fatty acids containing 2 to 12 carbon atoms). In one embodiment, the microorganism of the present disclosure (including derivatives of KH-2 strain, KH-2AL1 strain or KH-2AL3 strain) has higher hydrolytic activity against 4-nitrophenyl esters of short- to medium-chain fatty acids (fatty acids containing 2 to 12 carbon atoms) than 4-nitrophenyl esters of long-chain fatty acids. In one embodiment, the microorganism of the present disclosure (including derivatives of KH-2 strain, KH-2AL1 strain or KH-2AL3 strain) has the ability to decompose or assimilate esters (for example, oils and fats) and / or fatty acids at 15°C. In one embodiment, the microorganism of the present disclosure (including derivatives of KH-2 strain, KH-2AL1 strain or KH-2AL3 strain) retains the ability to assimilate or decompose trans fatty acids (for example, elaidic acid, palmitelaidic acid and / or vaccenic acid) or trans fatty acid-containing oils and fats at 15°C.

[0063] In one embodiment, the microorganism of the present disclosure (including derivatives of KH-2 strain, KH-2AL1 strain or KH-2AL3 strain) is added to an inorganic salt medium without added canola oil at an initial cell optical density OD 660After inoculation to achieve a value of 0.05, it was cultured at 28°C for 24 hours, at 28°C for 48 hours, or at 15°C for 48 hours, and then centrifuged to remove the cells. When 1 ml of the supernatant, 1 ml of a substrate solution prepared by dissolving 0.05 mol of a fatty acid 4-nitrophenyl ester in 12 ml of a 3% (v / v) Triton® X-100 aqueous solution, and 1 ml of 150 mM GTA buffer (pH 7.0) were mixed and the absorbance at 410 nm was monitored for 1 minute while stirring, it had a hydrolysis activity 1-fold, 1.5-fold, 2-fold, 5-fold, 10-fold, 20-fold, 50-fold, 100-fold, 200-fold, or 500-fold higher than that of the 4-nitrophenyl ester of long-chain fatty acids (fatty acids containing 13 or more carbon atoms) with respect to the 4-nitrophenyl ester of short-chain to medium-chain fatty acids (fatty acids containing 2 to 12 carbon atoms).

[0064] In one embodiment, the microorganism of the present disclosure (including derivatives of KH-2 strain, KH-2AL1 strain, or KH-2AL3 strain) has the ability to assimilate trans fatty acids (e.g., elaidic acid, palmitelaidic acid, and / or vaccenic acid) or oils and fats containing trans fatty acids. In one embodiment, the microorganism of the present disclosure (including derivatives of KH-2 strain, KH-2AL1 strain, or KH-2AL3 strain) has the ability to decompose trans fatty acids (e.g., elaidic acid, palmitelaidic acid, and / or vaccenic acid). In one embodiment, the microorganism of the present disclosure (including derivatives of KH-2 strain, KH-2AL1 strain, or KH-2AL3 strain) has the ability to decompose oils and fats containing trans fatty acids.

[0065] In one embodiment, the microorganism of the present disclosure (including derivatives of KH-2 strain, KH-2AL1 strain, or KH-2AL3 strain) has lipase activity to decompose triolein. In one embodiment, the microorganism of the present disclosure (including derivatives of KH-2 strain, KH-2AL1 strain, or KH-2AL3 strain) has lipase activity to decompose trielaidin.

[0066] In one embodiment, the microorganism of the present disclosure (including derivatives of KH-2 strain, KH-2AL1 strain or KH-2AL3 strain) has the ability to decompose or assimilate various fatty acids (oleic acid, elaidic acid, palmitelaidic acid, vaccenic acid) including trans-fatty acids at low temperatures.

[0067] In one embodiment, the microorganism of the present disclosure (including derivatives of KH-2 strain, KH-2AL1 strain or KH-2AL3 strain) may have the ability to assimilate and / or decompose oils and fats containing trans-fatty acids (e.g., elaidic acid) in a medium at pH 7.0 and 15 °C or 28 °C containing oils and fats containing trans-fatty acids.

[0068] In one embodiment, the microorganism of the present disclosure (including derivatives of KH-2 strain, KH-2AL1 strain or KH-2AL3 strain) is inoculated into an inorganic salt medium containing Triton (registered trademark) X-100 and elaidic acid at concentrations of 0.25 wt% and 0.2 wt% respectively, with an optical density at OD 660 = 0.5 or 0.8, and when cultured at pH 7.0 and 28 °C, the elaidic acid concentration in the supernatant after 24 hours or 46 hours (e.g., in an inorganic salt medium containing Triton (registered trademark) X-100 and elaidic acid at concentrations of 0.25 wt% and 0.2 wt% respectively, with an optical density at OD 660 = 0.5, the elaidic acid concentration in the supernatant after 24 hours when cultured at pH 7.0 and 28 °C, or in an inorganic salt medium containing Triton (registered trademark) X-100 and elaidic acid at concentrations of 0.25 wt% and 0.2 wt% respectively, with an optical density at OD 660Inoculate the microorganism at a cell optical density such that it becomes 0.8, and when cultured at pH 7.0 and 28°C, the elaidic acid concentration in the supernatant after 46 hours) is less than 1500 mg / L, less than 1200 mg / L, less than 1000 mg / L, less than 900 mg / L, less than 800 mg / L, less than 700 mg / L, less than 600 mg / L, less than 500 mg / L, less than 400 mg / L, less than 300 mg / L, less than 200 mg / L, less than 150 mg / L, less than 100 mg / L, less than 70 mg / L, less than 50 mg / L, less than 20 mg / L, less than 10 mg / L, or less than 5 mg / L, and has the ability to decompose trans fatty acids. The microorganism of the present disclosure (including derivatives of the KH-2 strain, KH-2AL1 strain, or KH-2AL3 strain) preferably has the ability to reduce the elaidic acid concentration in the supernatant to less than 800 mg / L, less than 700 mg / L, less than 600 mg / L, or less than 500 mg / L, particularly less than 700 mg / L, when determined under these conditions. Such a microorganism having the ability to decompose trans fatty acids can be beneficially used in various applications of the present disclosure.

[0069] In one embodiment, the microorganism of the present disclosure (including derivatives of the KH-2 strain, KH-2AL1 strain, or KH-2AL3 strain) has an OD in an inorganic salt medium containing Triton (registered trademark) X-100 and elaidic acid at concentrations of 0.25 wt% and 0.2 wt%, respectively. 660Inoculate the microorganism at a cell optical density such that it becomes 0.8, and when cultured at pH 7.0 and 15°C, the elaidic acid concentration in the supernatant after 48 hours or 90 hours is less than 1500 mg / L, less than 1200 mg / L, less than 1000 mg / L, less than 900 mg / L, less than 800 mg / L, less than 700 mg / L, less than 600 mg / L, less than 500 mg / L, less than 400 mg / L, less than 300 mg / L, less than 200 mg / L, less than 150 mg / L, less than 100 mg / L, less than 70 mg / L, less than 50 mg / L, less than 20 mg / L, less than 10 mg / L, or less than 5 mg / L, having the ability to decompose trans fatty acids. The microorganism of the present disclosure (including derivatives of KH-2 strain, KH-2AL1 strain or KH-2AL3 strain) preferably has the ability to reduce the elaidic acid concentration in the supernatant to less than 1000 mg / L, less than 700 mg / L, less than 500 mg / L, less than 400 mg / L, less than 300 mg / L, less than 200 mg / L or less than 100 mg / L, particularly less than 500 mg / L when judged under this condition. Such a microorganism having the ability to decompose trans fatty acids can be beneficially used in various applications of the present disclosure.

[0070] In one embodiment, the microorganism of the present disclosure (including derivatives of KH-2 strain, KH-2AL1 strain or KH-2AL3 strain) has an OD in an inorganic salt medium containing Triton (registered trademark) X-100 and trielaidin at concentrations of 0.25 wt% and 0.1 wt% respectively 660Inoculate the microorganism at a cell optical density such that it becomes 0.8, and when cultured at pH 7.0 and 28°C, the residual oil concentration in the supernatant after 5 days is less than 800 mg / L, less than 700 mg / L, less than 600 mg / L, less than 500 mg / L, less than 400 mg / L, less than 350 mg / L, less than 300 mg / L, less than 250 mg / L, less than 200 mg / L, less than 150 mg / L, less than 100 mg / L, less than 70 mg / L, less than 50 mg / L, less than 20 mg / L, or less than 10 mg / L, and it has the ability to decompose trans-fatty acid-containing oils and fats. The microorganism of the present disclosure (including derivatives of strain KH-2, strain KH-2AL1, or strain KH-2AL3) preferably has the ability to reduce the residual oil concentration in the supernatant to less than 400 mg / L, less than 350 mg / L, less than 300 mg / L, less than 250 mg / L, less than 200 mg / L, less than 150 mg / L, or less than 100 mg / L, particularly less than 350 mg / L when judged under these conditions. Such a microorganism having the ability to decompose trans-fatty acid-containing oils and fats can be beneficially used in various applications of the present disclosure.

[0071] In one embodiment, the microorganism of the present disclosure (including derivatives of strain KH-2, strain KH-2AL1, or strain KH-2AL3) is inoculated into an inorganic salt medium containing 1% (v / v) canola oil at a cell optical density such that the final concentration is OD 660 = 0.05, and when cultured at pH 7.0 and 15°C, the residual oil content 24 hours after the start of culture is less than 95%, less than 90%, less than 85%, less than 80%, less than 75%, less than 70%, less than 65%, less than 60%, less than 55%, less than 50%, less than 45%, less than 40%, less than 35%, less than 30%, less than 25%, less than 20%, less than 15%, less than 10%, less than 7%, less than 5%, less than 2%, or less than 1% of that at the start of culture, and it has the ability to decompose oils and fats. The microorganism of the present disclosure (including derivatives of strain KH-2, strain KH-2AL1, or strain KH-2AL3) preferably has the ability to decompose oils and fats such that the residual oil content in the 15°C culture is less than 65%, less than 60%, less than 55%, less than 50%, less than 45%, or less than 40%, particularly less than 45% when judged under these conditions. Such a microorganism having the ability to decompose oils and fats at low temperatures can be beneficially used in various applications of the present disclosure.

[0072] In one embodiment, the microorganism of the present disclosure (including derivatives of KH-2 strain, KH-2AL1 strain or KH-2AL3 strain) is inoculated into an inorganic salt medium containing 1% (v / v) canola oil at a cell optical density such that the final concentration is OD 660 = 0.05, and when cultured at pH 7.0 and 28°C, the residual oil content 24 hours after the start of culture is less than 50%, less than 45%, less than 40%, less than 35%, less than 30%, less than 25%, less than 20%, less than 17%, less than 15%, less than 12%, less than 10%, less than 7%, less than 5%, less than 4%, less than 3%, less than 2%, or less than 1% of that at the start of culture. The microorganism of the present disclosure (including derivatives of KH-2 strain, KH-2AL1 strain or KH-2AL3 strain) preferably has an oil-decomposing ability such that the residual oil content in the 28°C culture is less than 20%, less than 17%, less than 15%, less than 12%, or less than 10%, particularly less than 10% when judged under these conditions. Microorganisms having such a high-speed oil-decomposing ability can be beneficially used in various applications of the present disclosure.

[0073] In one embodiment, the microorganism of the present disclosure (including derivatives of KH-2 strain, KH-2AL1 strain or KH-2AL3 strain) is inoculated into an inorganic salt medium containing 1% (v / v) canola oil at a cell optical density such that the final concentration is OD 660When inoculating the microorganism at a cell optical density such that it becomes 0.05 and culturing it at pH 7.0, 15°C, and 28°C respectively, the oil content equivalent to the normal hexane value in the supernatant after 24 hours of culturing at 15°C is 1000% or less, 800% or less, 600% or less, 400% or less, 200% or less, 150% or less, 100% or less, 80% or less, 60% or less, 40% or less, 20% or less, 10% or less, or 5% or less compared to the oil content equivalent to the normal hexane value in the supernatant after 24 hours of culturing at 28°C. It has an oil-decomposing ability. The microorganism of the present disclosure (including derivatives of strain KH-2, strain KH-2AL1, or strain KH-2AL3) preferably has an oil-decomposing ability such that when judged under this condition, the oil residue rate in culturing at 15°C is 800% or less, 700% or less, 600% or less, 500% or less, 400% or less, particularly 500% or less compared to culturing at 28°C. Such a microorganism with low-temperature oil-decomposing ability can be beneficially used in various applications of the present disclosure.

[0074] In one embodiment, the microorganism of the present disclosure (including derivatives of strain KH-2, strain KH-2AL1, or strain KH-2AL3) is inoculated into an inorganic salt medium containing 1% (v / v) canola oil at a cell optical density such that the final concentration is OD 660 =0.05, and when starting culturing at pH 7.0, 15°C, and 28°C respectively, the total fatty acid decomposition rate in culturing at 15°C is 1000% or more, 800% or more, 600% or more, 400% or more, 200%, or more, 150% or more, 100% or more, 80% or more, 60% or more, 50% or more, 40% or more, 30% or more, 20% or more, 10% or more, or 5% or more compared to culturing at 28°C. It has an oil-decomposing ability. The microorganism of the present disclosure (including derivatives of strain KH-2, strain KH-2AL1, or strain KH-2AL3) preferably has an oil-decomposing ability such that when judged under this condition, the total fatty acid decomposition rate in culturing at 15°C is 50% or more, 40% or more, 30% or more, 20% or more, or 10% or more, particularly 30% or more compared to culturing at 28°C. Such a microorganism with low-temperature oil-decomposing ability can be beneficially used in various applications of the present disclosure.

[0075] In one embodiment, the Yarrowia yeast of the present disclosure has the ability to improve the lipase production of Burkholderia bacteria that produce lipase. In one embodiment, the Yarrowia yeast of the present disclosure has the ability to confer on the Burkholderia bacteria a lipolytic ability higher than that during the single culture of the Burkholderia bacteria. In one embodiment, the Yarrowia yeast of the present disclosure has the ability to confer on the Burkholderia bacteria a fatty acid degradation ability higher than that during the single culture of the Burkholderia bacteria. In one embodiment, the Burkholderia bacteria are any of the Burkholderia bacteria in the combination of microorganisms of the present disclosure. Any feature of the combination of microorganisms of the present disclosure described below in this specification is also intended to be a description of the ability that the Yarrowia yeast of the present disclosure confers on the Burkholderia bacteria of the present disclosure. For example, when Burkholderia bacteria and Yarrowia yeast are inoculated in an inorganic salt medium supplemented with 1% (v / v) canola oil at a cell concentration such that the total cell number is 5×10 5 cells / mL at a mixing ratio of 1:1 (based on the number of cells) and cultured at pH 7.0 and 28°C for 18 hours, compared to the lipolytic ability calculated from the values of the lipolytic ability of each single culture based on gas chromatography analysis, the disclosure regarding the combination of microbial strains of Burkholderia bacteria and Yarrowia yeast that is 100% or more is also a disclosure regarding the ability of Yarrowia yeast to confer the same lipolytic ability on Burkholderia bacteria under the same culture conditions and at the same mixing ratio. Similarly, the disclosure regarding the characteristics of the combination of microorganisms of the present disclosure is respectively equivalent to a description of the ability that the Yarrowia yeast of the present disclosure confers on the Burkholderia bacteria of the present disclosure. In one embodiment, the Yarrowia yeast of the present disclosure has both one or more abilities (described above) based on the Yarrowia yeast alone described herein, and the ability to confer on the Burkholderia bacteria in the combination of microorganisms of the present disclosure (described below). The Yarrowia yeast having the abilities of these different aspects is particularly unexpected. Examples of the Yarrowia yeast having the abilities of these different aspects include, but are not limited to, strains such as KH-2 strain, KH-2AL1 strain, KH-2AL3 strain, etc.

[0076] (Combination of oil-degrading microorganisms) In one aspect, the present disclosure provides a combination of Burkholderia bacteria and Yarrowia yeast that degrade fats, oils and / or fatty acids (also referred to herein as "the combination of microorganisms of the present disclosure"). As used herein, the term "Burkholderia bacteria" refers to bacteria including microorganisms of the order Burkholderiales, the details of which will be described later in this specification. As used herein, the term "Yarrowia yeast" refers to yeast including microorganisms of the genus Yarrowia, the details of which are described elsewhere in this specification. Among various combinations of microorganisms, the combination of Burkholderia bacteria and Yarrowia yeast may be a combination that can achieve particularly good oil-degrading ability. As shown in the examples, an improvement in oil-degrading ability was observed with the combination of Burkholderia bacteria and Yarrowia yeast. Without wishing to be bound by theory, this is presumably because the substances resulting from the degradation of oil by Yarrowia yeast improved the oil-degrading ability of Burkholderia bacteria as inducers. Therefore, microorganisms having similar oil-degrading ability can be preferably used as Yarrowia yeast, and strains having similar sensitivity can be preferably used as Burkholderia bacteria. Similarly, since it is expected that the substances resulting from the degradation of oil by Burkholderia bacteria improved the oil-degrading ability of Yarrowia yeast as inducers, microorganisms having similar oil-degrading ability can be preferably used as Burkholderia bacteria, and strains having similar sensitivity can be preferably used as Yarrowia yeast.

[0077] In one embodiment, at least one of the Burkholderia bacteria and Yarrowia yeast in the microbial combination of the present disclosure has the ability to produce lipase. In one embodiment, both the Burkholderia bacteria and Yarrowia yeast in the microbial combination of the present disclosure have the ability to produce lipase. The inventors unexpectedly found that a combination of Burkholderia bacteria and Yarrowia yeast, each of which produces lipase alone, exhibits unexpectedly high lipolytic activity, despite the expectation that they would compete with each other and suppress each other's abilities (resulting in a decrease in overall oil degradation ability) due to having similar properties. In one embodiment, the Burkholderia bacteria and Yarrowia yeast in the microbial combination of the present disclosure are symbiotic. In one embodiment, the Yarrowia yeast in the microbial combination of the present disclosure can improve the lipase expression and / or production of the Burkholderia bacteria. In one embodiment, the Burkholderia bacteria in the microbial combination of the present disclosure can improve the lipase expression and / or production of the Yarrowia yeast. The microorganisms in the microbial combination of the present disclosure can be produced by culturing by any suitable method.

[0078] In one aspect, the microbial combination of the present disclosure provides a lipolytic ability that exceeds the lipolytic ability of each microorganism used in this combination (e.g., the ability to decompose oils and fats and / or fatty acids at 28°C, the ability to decompose oils and fats and / or fatty acids at 15°C, etc.). In one aspect, the microbial combination of the present disclosure provides a lipolytic ability that exceeds the sum of the lipolytic abilities of each microorganism used in this combination (e.g., the ability to decompose oil at 28°C, the ability to decompose oil at 15°C, etc.). In particular, as long as any of the lipolytic abilities exceeds the lipolytic ability calculated from the value of the lipolytic ability of each single culture, the microbial combination can be usefully used even if another ability decreases. In one embodiment, the microbial combination of the present disclosure has the ability to decompose oils and fats containing cis-fatty acids, trans-fatty acids, or both.

[0079] In one aspect, the microbial combinations of the present disclosure provide improved lipase expression and / or production of Burkholderia bacteria and / or Yarrowia yeast used in this combination.

[0080] In one embodiment, the ratio of the cell number of Burkholderia bacteria to the cell number of Yarrowia yeast in the microbial combination of the present disclosure is from about 1000:1 to 1:100, from about 1000:1 to 1:50, from about 1000:1 to 1:20, from about 1000:1 to 1:10, from about 1000:1 to 1:5, from about 1000:1 to 1:2, from about 1000:1 to 1:1, from about 1000:1 to 2:1, from about 1000:1 to 5:1, from about 500:1 to 1:100, from about 500:1 to 1:50, from about 500:1 to 1:20, from about 500:1 to 1:10, from about 500:1 to 1:5, from about 500:1 to 1:2, from about 500:1 to 1:1, from about 500:1 to 2:1, from about 500:1 to 5:1, from about 200:1 to 1:100, from about 200:1 to 1:50, from about 200:1 to 1:20, from about 200:1 to 1:10, from about 200:1 to 1:5, from about 200:1 to 1:2, from about 200:1 to 1:1, from about 200:1 to 2:1, from about 200:1 to 5:1, from about 100:1 to 1:100, from about 100:1 to 1:50, from about 100:1 to 1:20, from about 100:1 to 1:10, from about 100:1 to 1:5, from about 100:1 to 1:2, from about 100:1 to 1:1, from about 100:1 to 2:1, from about 100:1 to 5:1, from about 50:1 to 1:100, from about 50:1 to 1:50, from about 50:1 to 1:20, from about 50:1 to 1:10, from about 50:1 to 1:5, from about 50:1 to 1:2, from about 50:1 to 1:1, from about 50:1 to 2:1, from about 50:1 to 5:1, from about 20:1 to 1:100, from about 20:1 to 1:50, from about 20:1 to 1:20, from about 20:1 to 1:10, from about 20:1 to 1:5, from about 20:1 to 1:2, from about 20:1 to 1:1, from about 20:1 to 2:1, from about 10:1 to 1:10, from about 9:1 to 1:9, from about 8:1 to 1:8, from about 1000:1, from about 100:1, from about 50:1, from about 20:1, from about 10:1, from about 9:1, from about 8:1, from about 7:1, from about 6:1, from about 5:1, from about 2:1, from about 1:1, from about 1:2, from about 1:5, from about 1:6, from about 1:7, from about 1:8, from about 1:9, from about 1:10, from about 1:100.

[0081] The ratio of the number of cells and the cell concentration can be determined and / or prepared using any suitable method. As one example, regarding the relationship between the cell number concentration of a microorganism and the optical density (OD 660 ) of the bacterial cells, by creating a calibration curve in advance, it is possible to determine the cell number concentration and prepare a microbial cell suspension with the desired cell number concentration based on the optical density. For example, the KH-1 strain with OD 660 = 0.01 is approximately 1×10 6 cells / mL, and the KH-2 strain with OD 660 = 0.01 can be converted to approximately 1×10 5 cells / mL.

[0082] In one embodiment, the dry weight ratio of Burkholderia bacteria to Yarrowia yeast in the combination of microorganisms of the present disclosure is about 100:1 to 1:100, about 100:1 to 1:50, about 100:1 to 1:20, about 100:1 to 1:10, about 100:1 to 1:5, about 100:1 to 1:2, about 100:1 to 1:1, about 100:1 to 2:1, about 100:1 to 5:1, about 50:1 to 1:100, about 50:1 to 1:50, about 50:1 to 1:20, about 50:1 to 1:10, about 50:1 to 1:5, about 50:1 to 1:2, about 50:1 to 1:1, about 50:1 to 2:1, about 50:1 to 5:1, about 20:1 to 1:100, about 20:1 to 1:50, about 20:1 to 1:20, about 20:1 to 1:10, about 20:1 to 1:5, about 20:1 to 1:2, about 20:1 to 1:1, about 20:1 to 2:1, about 20:1 to 5:1, about 10:1 to 1:100, about 10:1 to 1:50, about 10:1 to 1:20, about 10:1 to 1:10, about 10:1 to 1:5, about 10:1 to 1:2, about 10:1 to 1:1, about 10:1 to 2:1, about 10:1 to 5:1, about 5:1 to 1:100, about 5:1 to 1:50, about 5:1 to 1:20, about 5:1 to 1:10, about 5:1 to 1:5, about 5:1 to 1:2, about 5:1 to 1:1, about 5:1 to 2:1, about 2:1 to 1:100, about 2:1 to 1:50, about 2:1 to 1:20, about 2:1 to 1:10, about 2:1 to 1:5, about 2:1 to 1:2, about 2:1 to 1:1, about 100:1, about 10:1, about 5:1, about 2:1, about 1:1, about 1:2, about 1:5, about 1:10, about 1:20, about 1:50, about 1:100.

[0083] In one embodiment, when the combination of microorganisms of the present disclosure is cultured at a predetermined temperature (for example, 15 °C or 28 °C) in an inorganic salt medium supplemented with 1% (v / v) canola oil, it has a lipolytic ability higher than the lipolytic ability calculated from the lipolytic ability values of each single culture. In one embodiment, when the combination of microorganisms of the present disclosure is cultured at a predetermined temperature in an inorganic salt medium supplemented with 1% (v / v) canola oil, it has a lipolytic ability higher than that of either Burkholderia bacteria alone or Yarrowia yeast alone in this combination with the same number of cells as the total number of cells in this combination. In one embodiment, when the combination of microorganisms of the present disclosure is cultured at a predetermined temperature (for example, 28 °C) in an inorganic salt medium supplemented with 1% (v / v) oleic acid, it has a fatty acid-degrading ability higher than the fatty acid-degrading ability calculated from the fatty acid-degrading ability values of each single culture. In one embodiment, when the combination of microorganisms of the present disclosure is cultured at a predetermined temperature in an inorganic salt medium supplemented with 1% (v / v) oleic acid, it has a fatty acid-degrading ability higher than that of either Burkholderia bacteria alone or Yarrowia yeast alone in this combination with the same number of cells as the total number of cells in this combination. This lipolytic and / or fatty acid-degrading ability can also be tested by thin-layer chromatography described herein. For example, the culture supernatant can be collected over time, and it can also be determined by comparing the time until the spots corresponding to the oil and / or the spots corresponding to the fatty acid disappear.

[0084] In one embodiment, Burkholderia bacteria and Yarrowia yeast are combined to a total of 5×10 in an inorganic salt medium supplemented with 1% (v / v) canola oil. 5When inoculated at a cell concentration of cells / mL and cultured at pH 7.0 and 15°C, the total fatty acids determined by gas chromatography analysis of the culture supernatant 48 hours after the start of culture are less than 95%, less than 90%, less than 85%, less than 80%, less than 75%, less than 70%, less than 65%, less than 60%, less than 55%, less than 50%, less than 45%, less than 40%, less than 35%, less than 30%, less than 25%, less than 20%, less than 15%, less than 10%, less than 7%, less than 5%, less than 2%, or less than 1% of that at the start of culture. A combination of a Burkholderia bacterium and a Yarrowia yeast microbial strain and its mixing ratio are used.

[0085] In one embodiment, a combination of a Burkholderia bacterium and a Yarrowia yeast and its mixing ratio are used such that when the Burkholderia bacterium and the Yarrowia yeast are co-inoculated at a cell concentration of 5×10 5 cells / mL into an inorganic salt medium supplemented with 1% (v / v) canola oil and cultured at pH 7.0 and 15°C for 48 hours, the lipolytic ability calculated from the lipolytic ability values of each single culture based on gas chromatography analysis is 100% or more, 101% or more, 102% or more, 103% or more, 104% or more, 105% or more, 106% or more, 107% or more, 108% or more, 109% or more, 110% or more, 115% or more, 120% or more, or 125% or more compared to that of each single culture.

[0086] In one embodiment, a combination of a Burkholderia bacterium and a Yarrowia yeast and its mixing ratio are used such that when the Burkholderia bacterium and the Yarrowia yeast are inoculated at a total cell concentration of 5×10 5 cells / mL into an inorganic salt medium supplemented with 1% (v / v) canola oil and cultured at pH 7.0 and 28°C, the total fatty acids determined by gas chromatography analysis of the culture supernatant 18 hours after the start of culture are less than 95%, less than 90%, less than 85%, less than 80%, less than 75%, less than 70%, less than 65%, less than 60%, less than 55%, less than 50%, less than 45%, less than 40%, less than 35%, less than 30%, less than 25%, less than 20%, less than 15%, less than 10%, less than 7%, less than 5%, less than 2%, or less than 1% of that at the start of culture.

[0087] In one embodiment, Burkholderia bacteria and Yarrowia yeast are inoculated into an inorganic salt medium supplemented with 1% (v / v) canola oil at a cell concentration such that the total cell count is 5×10 5 cells / mL, and when cultured at pH 7.0 and 28°C for 18 hours, the lipolytic ability calculated from the values of the lipolytic ability of each single culture based on gas chromatography analysis is 100% or more, 101% or more, 102% or more, 103% or more, 104% or more, 105% or more, 106% or more, 107% or more, 108% or more, 109% or more, 110% or more, 115% or more, 120% or more, or 125% or more compared to that. A combination of microbial strains of Burkholderia bacteria and Yarrowia yeast and their mixing ratio are used.

[0088] In one embodiment, Burkholderia bacteria and Yarrowia yeast are inoculated into an inorganic salt medium supplemented with 1% (v / v) oleic acid at a total cell concentration of 5×10 5 cells / mL, and when cultured at pH 7.0 and 15°C, the total fatty acids determined by gas chromatography analysis of the culture supernatant 48 hours after the start of culture are less than 95%, less than 90%, less than 85%, less than 80%, less than 75%, less than 70%, less than 65%, less than 60%, less than 55%, less than 50%, less than 45%, less than 40%, less than 35%, less than 30%, less than 25%, less than 20%, less than 15%, less than 10%, less than 7%, less than 5%, less than 2%, or less than 1% of those at the start of culture. A combination of microbial strains of Burkholderia bacteria and Yarrowia yeast and their mixing ratio are used.

[0089] In one embodiment, Burkholderia bacteria and Yarrowia yeast are inoculated into an inorganic salt medium supplemented with 1% (v / v) oleic acid at a total cell count of 5×10 5When co-inoculating at a cell concentration of cells / mL and culturing at pH 7.0 and 15°C for 48 hours, compared with the fatty acid resolution calculated from the values of the fatty acid resolution of each single culture based on gas chromatography analysis, a combination of Burkholderia bacteria and Yarrowia yeast microbial strains and their mixing ratios that are 100% or more, 105% or more, 110% or more, 120% or more, 130% or more, 140% or more, 150% or more, 160% or more, 170% or more, 180% or more, 190% or more, or 200% or more are used.

[0090] In one embodiment, the combination of microorganisms of the present disclosure is a combination that improves the lipase expression and / or production of Burkholderia bacteria in the combination of microorganisms of the present disclosure. In one embodiment, the lipase of Burkholderia bacteria includes a first lipase (nucleotide sequence: SEQ ID NO: 1, amino acid sequence: SEQ ID NO: 2, or variants thereof, etc.) and / or a second lipase (nucleotide sequence: SEQ ID NO: 3, amino acid sequence: SEQ ID NO: 4, or variants thereof, etc.). In one embodiment, the improvement in lipase expression and / or production can be determined by comparing the lipase gene expression (such as RNA amount, production protein amount, secreted protein amount, etc.) when using the combination of microorganisms of the present disclosure with the results when compared with the single culture of Burkholderia bacteria.

[0091] In one embodiment, the combination of microorganisms of the present disclosure is inoculated with each microorganism at an optical density of the cell mass such that the measured value of OD 660 is Burkholderia bacteria:Yarrowia yeast = 0.018:0.02 in an inorganic salt medium supplemented with 1% (v / v) canola oil, and when cultured at pH 7.0 and 15°C, total RNA is extracted from the cells 71 hours after the start of culture and quantitative PCR analysis is performed for a specific Burkholderia bacteria lipase, the expression level is 6 times or more, 7 times or more, or 8 times or more compared to the case of inoculating Burkholderia bacteria alone at an optical density of the cell mass such that the measured value of OD 660 is 0.02.

[0092] In one embodiment, the combination of microorganisms of the present disclosure is a combination that improves the lipase expression and / or production of Yarrowia yeast in the combination of microorganisms of the present disclosure. In one embodiment, the lipase of Yarrowia yeast includes a first lipase (nucleotide sequence: SEQ ID NO: 5, amino acid sequence: SEQ ID NO: 6, or variants thereof, etc.) and / or a second lipase (nucleotide sequence: SEQ ID NO: 7, amino acid sequence: SEQ ID NO: 8, or variants thereof, etc.). In one embodiment, the improvement in lipase expression and / or production can be determined by comparing the lipase gene expression (such as RNA amount, amount of produced protein, amount of secreted protein, etc.) when using the combination of microorganisms of the present disclosure with the result when compared with the single culture of Yarrowia yeast.

[0093] In one embodiment, when the combination of microorganisms of the present disclosure is inoculated with 2.5×10 5 cells / mL of Burkholderia bacteria and 2.5×10 5 cells / mL of Yarrowia yeast into an inorganic salt medium supplemented with 1% (v / v) canola oil and cultured at pH 7.0 and 15 °C, and total RNA is extracted from the cells 48 hours after the start of culture and quantitative PCR analysis is performed for a specific Yarrowia yeast lipase, the expression level is 5×10 5 times or more, 1.2 times or more, 1.5 times or more, 1.7 times or more, 2 times or more, 5 times or more, 10 times or more, or 20 times or more compared to the case of inoculating only 5×10

[0094] In one aspect, the present disclosure provides a composition for improving lipase production of Burkholderia bacteria that produce lipase, including Yarrowia yeast. Alternatively, the present disclosure provides Yarrowia yeast for improving lipase production of Burkholderia bacteria that produce lipase. In another aspect, the present disclosure provides a composition for improving lipase production of Yarrowia yeast that produce lipase, including Burkholderia bacteria. Alternatively, the present disclosure provides Burkholderia bacteria for improving lipase production of Yarrowia yeast that produce lipase. As described herein, in the present disclosure, a combined effect of two microorganisms has been observed, which, from another perspective, is based on the discovery that Yarrowia yeast may have a new function, ability or use of improving lipase production of Burkholderia bacteria that produce lipase, or based on the discovery that Burkholderia bacteria may have a new function, ability or use of improving lipase production of Yarrowia yeast that produce lipase. Therefore, in these aspects, it is understood that the matters regarding the combination of the microorganisms of the present disclosure described herein are equally applicable in these new uses.

[0095] In another aspect, the present disclosure provides a composition for enhancing the ability of Burkholderia bacteria producing lipase to process oils and fats and / or fatty acids, comprising Yarrowia yeast. Alternatively, the present disclosure provides Yarrowia yeast for enhancing the ability of Burkholderia bacteria producing lipase to process oils and fats and / or fatty acids. In yet another aspect, the present disclosure provides a composition for enhancing the ability of Yarrowia yeast producing lipase to process oils and fats and / or fatty acids, comprising Burkholderia bacteria. Alternatively, the present disclosure provides Burkholderia bacteria for enhancing the ability of Yarrowia yeast producing lipase to process oils and fats and / or fatty acids. In the present disclosure, a combined effect of two microorganisms has been observed, which, from another perspective, is an invention based on the finding that there may be a new function, ability or use of Yarrowia yeast to enhance the ability to process oils and fats and / or fatty acids of Burkholderia bacteria producing lipase, or an invention based on the finding that there may be a new function, ability or use of Burkholderia bacteria to enhance the ability to process oils and fats and / or fatty acids of Yarrowia yeast producing lipase. Therefore, in these aspects, it is understood that the matters regarding the combination of the microorganisms of the present disclosure described herein are equally applicable in these new uses.

[0096] While not wishing to be bound by theory, in one preferred embodiment, it is preferred to provide a composition for improving the lipase production of Burkholderia bacteria producing lipase, including Yarrowia yeast, and / or a composition for enhancing the ability to process oils and fats and / or fatty acids of Burkholderia bacteria producing lipase, including Yarrowia yeast. The reason is that by using a small amount of Yarrowia yeast, the ability of Burkholderia bacteria, which is relatively easy to prepare, can achieve an oil and fat and / or fatty acid decomposition effect equal to or greater than that of the individual bacteria. While not wishing to be bound by theory, for example, according to Ngamdee W et al., BMC Microbiol. 2015 Mar 3;15:56. "Competition between Burkholderia pseudomallei and B.thailandensis" and Mitch R et al., Appl Microbiol Biotechnol. 2015; 99(22):9723-9743. "Irradiation of Yarrowia lipolytica NRRL YB-567 creating novel strains with enhanced ammonia and oil production on protein and carbohydrate substrates", the doubling time of Burkholderia bacteria is about 39 minutes, and the doubling time of Yarrowia yeast is about 1.5 hours, so Burkholderia bacteria have a faster growth rate. The inventor has found that the combination of the microorganisms of the present disclosure may have an oil and fat and / or fatty acid decomposition ability exceeding that calculated from the values of the oil and fat and / or fatty acid decomposition ability of each single culture. Based on this finding, by using the combination of the microorganisms of the present disclosure, the oil and fat and / or fatty acid decomposition ability of Burkholderia bacteria with a fast growth rate is improved, and the overall oil and fat and / or fatty acid decomposition is accelerated, enabling efficient and stable use of the microorganisms.

[0097] In one embodiment, the Burkholderia bacteria in the combination of microorganisms of the present disclosure are bacteria of the order Burkholderiales. In one embodiment, the Burkholderia bacteria in the combination of microorganisms of the present disclosure are bacteria of the family Burkholderiaceae. In one embodiment, the Burkholderia bacteria in the combination of microorganisms of the present disclosure are bacteria of the genus Burkholderia. The genus Burkholderia is a Gram-negative, non-spore-forming, aerobic, rod-shaped bacterium with polar flagella and is the type genus of the family Burkholderiaceae. In one embodiment, the Burkholderia bacteria in the combination of microorganisms of the present disclosure are Burkholderia arboris, Burkholderia ambifaria or Burkholderia cepacia, preferably Burkholderia arboris or Burkholderia ambifaria. In one embodiment, the Burkholderia bacteria in the combination of microorganisms of the present disclosure are microorganisms belonging to the Burkholderia cepacia complex. The Burkholderia cepacia complex is a classification of genetically very closely related Burkholderia microorganisms, including ambifaria, anthina, arboris, cenocepacia, cepacia, contaminans, diffusa, dolosa, lata, latens, metallica, multivorans, pseudomultivorans, puraquae, pyrrocinia, seminalis, stabilis, stagnalis, territorii, ubonensis, and vietnamiensis (Martina P et al., Int J Syst Evol Microbiol. 2018 Jan;68(1):14-20.).In another embodiment, the Burkholderia bacteria in the combination of microorganisms of the present disclosure may be metallica, seminalis, anthina, ambifaria, diffusa, ubonensis, multivorans, latens, cenocepacia, vietnamiensis, pyrrocinia, stabilis, glumae, gladioli, plantarii, oklahomaensis, thailandensis, mallei, pseudomallei or phytofirmans. In a further aspect of the present disclosure, the Burkholderia bacteria in the combination of microorganisms of the present disclosure may be bacteria of the genus Ralstonia or Pseudomonas. The inventors identified a new microbial strain (strain KH-1) as Burkholderia arboris by determining the nucleotide sequence of 16S ribosomal DNA and phylogenetic analysis, and deposited it with the Patent Microorganisms Depositary, National Institute of Technology and Evaluation, Japan, which was received on June 4, 2018, and a deposit certificate was issued on June 12, 2018. The deposit number is NITE BP-02731. In addition, strains of Burkholderia bacteria (strains KH-1AL1, KH-1AL2 and KH-1AL3) were further identified and deposited with the Patent Microorganisms Depositary, National Institute of Technology and Evaluation, Japan, which was received on June 26, 2019, and a deposit certificate was issued on July 8, 2019. The deposit numbers are NITE BP-02977, NITE BP-02978, and NITE BP-02979, respectively. In one embodiment, the Burkholderia bacteria in the combination of microorganisms of the present disclosure are the Burkholderia bacterium KH-1 strain (the strain specified by the deposit number NITE BP-02731), the KH-1AL1 strain (the strain specified by the deposit number NITE BP-02977), the KH-1AL2 strain (the strain specified by the deposit number NITE BP-02978) or the KH-1AL3 strain (the strain specified by the deposit number NITE BP-02979), or an induced strain thereof.

[0098] In one embodiment, the Burkholderia bacteria in the combination of microorganisms of the present disclosure are derivative strains of Burkholderia bacterium KH-1 strain (the strain identified by accession number NITE BP-02731), KH-1AL1 strain (the strain identified by accession number NITE BP-02977), KH-1AL2 strain (the strain identified by accession number NITE BP-02978), or KH-1AL3 strain (the strain identified by accession number NITE BP-02979). Here, the derivative strain does not necessarily have to be a strain obtained from the Burkholderia bacterium KH-1 strain, KH-1AL1 strain, KH-1AL2 strain, or KH-1AL3 strain, and it is a microorganism that exhibits the biological functions of these Burkholderia strains, not necessarily to the same extent, preferably a microorganism having biological activity to the same degree or higher. In one embodiment, the Burkholderia bacteria in the combination of microorganisms of the present disclosure, similar to the Burkholderia bacterium KH-1 strain, KH-1AL1 strain, KH-1AL2 strain, or KH-1AL3 strain, exhibit at least one biological function selected from the group consisting of the ability to produce lipase and the ability to assimilate (decompose) oils and fats and / or fatty acids at low temperatures (for example, 25°C or lower, 20°C or lower, 15°C or lower, 10°C or lower, 5°C or lower, etc.), but the degree of its biological function may be different from that of the KH-1 strain, KH-1AL1 strain, KH-1AL2 strain, or KH-1AL3 strain. In one embodiment, the Burkholderia bacteria that are derivative strains in the combination of microorganisms of the present disclosure are bacteria of the family Burkholderiaceae, more specifically, bacteria of the genus Burkholderia, and even more specifically, are microorganisms belonging to Burkholderia arboris, Burkholderia ambifaria, or Burkholderia cepacia complex.

[0099] The Burkholderia bacteria in the microbial combination of the present disclosure contain oil and fat as the sole carbon source and can be isolated on an inorganic salt agar medium with the pH adjusted to 6-8. In one embodiment, the Burkholderia bacteria in the microbial combination of the present disclosure produce lipase. Also, in one embodiment, the Burkholderia bacteria in the microbial combination of the present disclosure can be discriminated by confirming that a clear zone (halo) is formed around the colonies formed on the agar medium.

[0100] In one embodiment, the Burkholderia bacteria in the microbial combination of the present disclosure have the ability to decompose oil and fat and / or fatty acids at 15°C. In one embodiment, the ability of the Burkholderia bacteria in the microbial combination of the present disclosure to produce lipase and / or the ability to assimilate or decompose fatty acids or oil and fat is retained at 15°C.

[0101] In one embodiment, the Burkholderia bacteria in the microbial combination of the present disclosure can secrete biosurfactant when cultured in a medium containing oil and fat or fatty acids.

[0102] In one embodiment, the Burkholderia bacteria in the microbial combination of the present disclosure were inoculated with microorganisms at a cell concentration such that the cell concentration was 3×10 6 cells / mL or 2×10 6 cells / mL into an inorganic salt medium containing 10 g / L of canola oil, and when cultured at pH 7.0 and 15°C under an air reflux of 200 ml / min, the oil content equivalent to the normal hexane value in the supernatant after 24 or 48 hours was less than 9 g / L, less than 8 g / L, less than 7 g / L, less than 6 g / L, less than 5 g / L, less than 4 g / L, less than 3 g / L, less than 2 g / L, less than 1 g / L, less than 0.7 g / L, less than 0.5 g / L, less than 0.2 g / L, less than 0.1 g / L, less than 0.07 g / L, less than 0.05 g / L, less than 0.02 g / L, or less than 0.01 g / L, having an oil and fat decomposing ability. In particular, the Burkholderia bacteria in the microbial combination of the present disclosure, when judged under this condition, have 2×10 6When inoculated at a cell concentration of cells / mL and cultured for 48 hours, it preferably has an oil-decomposing ability to reduce the oil content equivalent to the normal hexane value to less than about 6 g / L, and 3×10 6 When inoculated at a cell concentration of cells / mL and cultured for 24 hours, it particularly preferably has an oil-decomposing ability to reduce the oil content equivalent to the normal hexane value to less than 6 g / L. Microorganisms having such a low-temperature oil-decomposing ability can be beneficially used in various applications of the present disclosure.

[0103] In one embodiment, the Burkholderia bacterium in the combination of microorganisms of the present disclosure is added to an inorganic salt medium containing 10 g / L of canola oil at a final concentration of 3×10 6 When the microorganisms are inoculated at a cell concentration such that the cell concentration becomes cells / mL and cultured at pH 7.0, 15°C, and 28°C under an air reflux of 200 ml / min, respectively, the oil content equivalent to the normal hexane value in the supernatant after 24 hours of culturing at 15°C is 1000% or less, 800% or less, 600% or less, 400% or less, 200% or less, 150% or less, 100% or less, 80% or less, 60% or less, 40% or less, 20% or less, 10% or less, or 5% or less compared to the oil content equivalent to the normal hexane value in the supernatant after 24 hours of culturing at 28°C. The Burkholderia bacterium in the combination of microorganisms of the present disclosure preferably has an oil-decomposing ability such that the oil residue rate in culturing at 15°C is 800% or less, 700% or less, 600% or less, 500% or less, 400% or less, particularly 700% or less, when judged under this condition. Microorganisms having such a low-temperature oil-decomposing ability can be beneficially used in various applications of the present disclosure.

[0104] In one embodiment, the Burkholderia bacterium in the combination of microorganisms of the present disclosure is added to an inorganic salt medium containing 10 g / L of canola oil at a final concentration of 3×10 6When inoculating microorganisms at a cell concentration of cells / mL and starting culturing at pH 7.0, 15°C, and 28°C respectively under an air reflux of 200 ml / min, the total fatty acid decomposition rate in the 15°C culture is 1000% or more, 800% or more, 600% or more, 400% or more, 200% or more, 150% or more, 100% or more, 80% or more, 60% or more, 50% or more, 40% or more, 30% or more, 20% or more, 10% or more, or 5% or more compared to the 28°C culture, having an oil decomposition ability. Burkholderia bacteria in the combination of microorganisms of the present disclosure, when judged under this condition, preferably have an oil decomposition ability such that the total fatty acid decomposition rate in the 15°C culture is 50% or more, 40% or more, 30% or more, 20% or more, or 10% or more, particularly 30% or more compared to the 28°C culture. Microorganisms having such a low-temperature oil decomposition ability can be beneficially used in various applications of the present disclosure.

[0105] In one embodiment, Yarrowia yeast in the combination of microorganisms of the present disclosure can be any yeast of the genus Yarrowia described herein.

[0106] At least one microorganism in the combination of microorganisms of the present disclosure can contain oil as the sole carbon source and can be isolated on an inorganic salt agar medium with the pH adjusted to 6 - 8. Also, in one embodiment, at least one microorganism in the combination of microorganisms of the present disclosure can be distinguishable by confirming that a clear zone (halo) is formed around the colony formed on the agar medium. In one embodiment, it is preferable that one microorganism in the combination of microorganisms of the present disclosure can form colonies and grow on an agar medium supplemented with 10 g / L of canola oil as a carbon source at 15°C or 28°C, preferably 15°C. It is more preferable that both Burkholderia bacteria and Yarrowia yeast in the combination of microorganisms of the present disclosure can form colonies and grow under the same conditions. In the combination of microorganisms of the present disclosure, it may be useful that both Burkholderia bacteria and Yarrowia yeast have a minimum oil assimilation (decomposition) ability.

[0107] In one embodiment, the ability of microorganisms to decompose and assimilate oils and fats and fatty acids can be evaluated by quantifying the fatty acids contained in the oils and fats remaining in the medium and the free fatty acids generated by decomposition using gas chromatography. To show the specific quantification procedure, first, 3 mL of the culture supernatant is acidified with hydrochloric acid, and an equal volume of ethyl acetate is added. After stirring for 5 minutes, it is centrifuged, and 1 mL of the ethyl acetate layer is transferred to another container and the solvent is evaporated and concentrated. It is dissolved in 1 ml of chloroform, 4 mL of a methanolysis solution (methanol:sulfuric acid = 17:3) is added, and it is heated at 100 °C for 2 hours to methyl-esterify the fatty acids and free fatty acids in the oils and fats. Then, a solution of chloroform:pure water = 1:1 is added and stirred, and the chloroform layer is mixed with methyl octanoate at a ratio of 1:1 and analyzed by gas chromatography to quantify the methyl esters of all fatty acids. The amounts of reagents, the types of extraction solvents, etc. can be changed as appropriate. For example, the following alternative procedures are possible. 1 mL of the culture supernatant is acidified with hydrochloric acid, and 2 mL of chloroform is added. After stirring for 2 minutes, it is centrifuged, and 1 mL of the chloroform layer is transferred to another container and the solvent is evaporated and concentrated. 2 mL of a methanolysis solution (methanol:sulfuric acid = 17:3) is added and heated at 100 °C for 2 hours to methyl-esterify the fatty acids and free fatty acids in the oils and fats. Then, 2 mL of chloroform and 1 mL of pure water are added and stirred, and the chloroform layer is analyzed by gas chromatography to quantify the methyl esters of all fatty acids.

[0108] In one embodiment, the ability of the microorganism to decompose and assimilate oils and fats and fatty acids can be evaluated by analyzing the oils and fats remaining in the medium and the fatty acids, which are decomposition products, by thin-layer chromatography. To show the specific procedure, first, the oils and fats are extracted by adding an equal volume of chloroform to the culture supernatant. 5 μL of this extract is developed on a silica gel-coated plate using a developing solvent containing chloroform, acetone, and methanol at a volume ratio of 96:4:1, respectively. The ratio of the developing solvent and the like can be changed as appropriate. For example, even when chloroform, acetone, and methanol are at a volume ratio of 96:4:2, respectively, good results can be obtained. The plate is treated with molybdotungstic acid n-hydrate to develop the color of the oils and fats and / or fatty acids.

[0109] In one embodiment, the ability of the microorganism to decompose and assimilate esters (e.g., oils and fats) and fatty acids can be evaluated by examining the growth ability in a medium using each oil or fatty acid as the sole carbon source.

[0110] In one embodiment, the microorganisms in the combination of the microorganisms of the present disclosure have the ability to produce esterases (e.g., lipases).

[0111] In one embodiment, lipase activity can be determined by performing an enzymatic reaction using 4-nitrophenyl palmitate (4-NPP), which is an ester of palmitic acid and 4-nitrophenol, as a substrate, and measuring the amount of p-nitrophenol generated by hydrolysis of the ester by measuring the absorbance at 410 nm. First, 4-NPP (18.9 mg) is added to 3% (v / v) Triton® X-100 (12 ml) and dissolved at 70 °C to obtain a substrate solution. 1 mL of the substrate solution, 0.9 mL of ion-exchanged water, and 1 mL of 150 mM GTA buffer (prepared by adding NaOH or HCl to 150 mM 3,3-dimethylglutaric acid, 150 mM Tris, and 150 mM 2-amino-2-methyl-1,3-propanediol to pH 7.0) are placed in a cell and incubated at 28 °C for 5 minutes. 0.1 mL of the culture supernatant is added thereto, and the value at 410 nm is measured while stirring. Lipase activity is measured by defining the amount of enzyme that produces 1 μmol of 4-nitrophenol as 1 unit (U), and the units per 1 mL of the culture supernatant are calculated.

[0112] In one embodiment, the microorganisms in the combination of microorganisms of the present disclosure may be capable of growing and degrading oil under weakly acidic conditions (for example, pH of about 5.5 to 6.0).

[0113] In one embodiment, the growth ability of microorganisms can be examined by a method of measuring the absorbance (turbidity) at 660 nm as the optical density of the cells, a method of measuring colony-forming units (CFU), or the like. In the latter method, a certain amount of the stock solution and dilution of the culture solution are spread on an agar medium, and the colonies formed by static culture are counted.

[0114] Those skilled in the art can appropriately use the above measurement methods to test the induced strains of KH-1 strain, KH-1AL1 strain, KH-1AL2 strain, KH-1AL3 strain or KH-2 strain, KH-2AL1 strain, KH-2AL3 strain, and obtain induced strains having the above biological functions (and the degree thereof).

[0115] In one embodiment, the Burkholderia bacterium and / or Yarrowia yeast in the combination of microorganisms of the present disclosure can be selected based on the result that KH-1 strain + this Yarrowia yeast or this Burkholderia bacterium + KH-2 strain is equivalent to or superior to KH-1 strain alone or KH-2 strain alone.

[0116] In one embodiment, for example, KH-1 strain + Yarrowia yeast or Burkholderia bacterium + KH-2 strain is inoculated into an inorganic salt medium supplemented with 1% (v / v) canola oil at a total cell concentration of 5×10 5 cells / mL, cultured at pH 7.0 and 15°C for 48 hours, or when cultured at pH 7.0 and 28°C for 18 hours, and the total fatty acid measurement value determined by gas chromatography analysis of the culture supernatant is less than 200%, less than 180%, less than 160%, less than 140%, less than 120%, less than 100%, less than 70%, or less than 50% compared to the total fatty acid measurement value of KH-1 strain + KH-2 strain under the same conditions, then the Burkholderia bacterium and / or Yarrowia yeast can be preferably used.

[0117] In one embodiment, each microorganism is inoculated at an optical density of cells such that the OD 660 measurement value of KH-1 strain:Yarrowia yeast or Burkholderia bacterium:KH-2 strain = 0.018:0.02 into an inorganic salt medium supplemented with 1% (v / v) canola oil, total RNA is extracted from the cells cultured at pH 7.0 and 15°C for 71 hours, and when quantitative PCR analysis is performed for a specific Burkholderia bacterium lipase, the expression level is 50% or more, 70% or more, 100% or more, 120% or more, 140% or more, 160% or more, 180% or more, 200% or more compared to the expression level of Burkholderia bacterium lipase during the mixed culture of KH-1 strain + KH-2 strain under the same conditions, then the Burkholderia bacterium and / or Yarrowia yeast can be preferably used.

[0118] In one embodiment, KH-1 strain:Yarrowia yeast or Burkholderia bacterium:KH-2 strain = 2.5×10 5Cells / mL: 2.5×10 5 When each microorganism was inoculated to reach 2.5×10 cells / mL, total RNA was extracted from the cells cultured at pH 7.0 and 15°C for 48 hours, and when quantitative PCR analysis was performed for a specific Yarrowia lipase, the expression level was 50% or more, 70% or more, 100% or more, 120% or more, 140% or more, 160% or more, 180% or more, 200% or more compared to the expression level of Yarrowia lipase during the mixed culture of KH-1 strain + KH-2 strain under the same conditions, the Burkholderia bacterium and / or Yarrowia lipase can be preferably used.

[0119] (Composition or combination) In one aspect, the present disclosure provides a composition or combination comprising the microorganisms of the present disclosure. In one aspect, the present disclosure provides a composition or combination comprising the culture supernatant of the microorganisms of the present disclosure. The microorganisms of the present disclosure can be produced by culturing by any suitable method. In one embodiment, the composition or combination is an oil-degrading agent. In one embodiment, it is an oil-degrading agent for decomposing trans fatty acids (for example, elaidic acid, palmitelaidic acid and / or vaccenic acid), for decomposing oils and fats containing trans fatty acids, for decomposing esters (for example, oils and fats) and / or fatty acids at 15°C, for decomposing esters containing short-chain to medium-chain fatty acids (C2 - C12), and / or for decomposing oils and fats containing short-chain to long-chain fatty acids (C2 or more). In one embodiment, the composition is a fatty acid-degrading agent. By treating with the fatty acid-degrading agent of the present disclosure, a compound containing less carbon than the number of carbon atoms contained in the fatty acid can be produced. In one embodiment, the composition is a trans fatty acid (for example, elaidic acid, palmitelaidic acid and / or vaccenic acid) decomposing agent.

[0120] In one aspect, the present disclosure provides a composition or combination comprising at least one microorganism in the combination of microorganisms of the present disclosure. In this aspect, the combination of microorganisms of the present disclosure is provided in the composition (optionally in combination with microorganisms such that they form the combination of microorganisms of the present disclosure) or combination. At least one microorganism in the combination of microorganisms of the present disclosure can be produced by culturing by any suitable method. The combination of microorganisms of the present disclosure used in this composition or combination can be any of the combinations of microorganisms of the present disclosure described above.

[0121] In one embodiment, the composition or combination of the present disclosure is an oil-degrading agent. In one embodiment, the composition of the present disclosure is a composition for improving the lipase expression and / or production of one of Burkholderia bacteria and Yarrowia yeast in the combination of microorganisms of the present disclosure, and in one embodiment, the lipase is the first lipase of Burkholderia bacteria (base sequence: SEQ ID NO: 1, amino acid sequence: SEQ ID NO: 2, or variants thereof, etc.), the second lipase (base sequence: SEQ ID NO: 3, amino acid sequence: SEQ ID NO: 4, or variants thereof, etc.), the first lipase of Yarrowia yeast (base sequence: SEQ ID NO: 5, amino acid sequence: SEQ ID NO: 6, or variants thereof, etc.) and / or the second lipase (base sequence: SEQ ID NO: 7, amino acid sequence: SEQ ID NO: 8, or variants thereof, etc.). In one embodiment, it is an oil-degrading agent for decomposing oils and fats and / or fatty acids at a predetermined temperature (e.g., 15 °C, 28 °C), for decomposing oils and fats containing short-chain to medium-chain fatty acids (C2 - C12), and / or for decomposing oils and fats containing short-chain to long-chain fatty acids (C2 or more). The oil-degrading agent contains only one kind of microorganism (e.g., either Burkholderia bacteria or Yarrowia yeast) among the combinations of microorganisms of the present disclosure, and even if it does not exhibit the desired oil and fat and / or fatty acid degradation ability by itself, it should exhibit the desired oil and fat and / or fatty acid degradation ability when used to form the combination of microorganisms of the present disclosure. In one embodiment, the composition is a fatty acid-degrading agent. By treating with the fatty acid-degrading agent of the present disclosure, a compound containing less carbon than the number of carbon atoms contained in the fatty acid can be produced.

[0122] (Applicable object) In one embodiment, examples of the fats and oils to which the oil decomposing agent of the present disclosure is applied include vegetable fats and oils (such as cottonseed oil, rapeseed oil, soybean oil, corn oil, olive oil, safflower oil, rice bran oil, sesame oil, palm oil, coconut oil, peanut oil, etc.), animal fats and oils (such as lard, beef tallow, milk fat, etc.), fish oil, processed products of these fats and oils (such as margarine, shortening, butter, etc.), insulating oil, lubricating oil, etc., but are not limited thereto. The fats and oils may exist in the form of an emulsion or in a free state.

[0123] As a specific embodiment, examples of the fats and oils to which the oil decomposing agent of the present disclosure is applied include fats and oils containing trans fatty acids (such as elaidic acid, palmitelaidic acid and / or vaccenic acid), and such fats and oils include processed products (such as margarine, shortening, butter, etc.) of fats and oils produced by hydrogenation, etc., but are not limited thereto. By adding hydrogen, the number of double bonds of unsaturated fatty acids decreases and the proportion of saturated fatty acids increases, but this may generate trans fatty acids. It is said that margarine, fat spreads, shortening produced by hydrogenation, and Western confectionery such as bread, cakes, donuts using them as raw materials, and fried foods contain trans fatty acids. In the process of refining oils and fats taken from plants and fish, treatment is carried out at a high temperature to remove unpleasant odors. At this time, trans fatty acids are formed from the cis-type unsaturated fatty acids contained in the oil, so it is said that refined vegetable oils such as salad oil also contain trace amounts of trans fatty acids.

[0124] The target to which the oil decomposer or fatty acid decomposer of the present disclosure is applied is not particularly limited. For example, industrial wastewater, domestic wastewater, industrial waste, domestic waste (such as garbage), livestock waste, fish farms (and their wastewater), livestock houses (and their wastewater), slaughterhouses (and their wastewater), soil contaminated with oil, water contaminated with oil (such as the sea, ponds, rivers, drinking water for animals), the body surface of animals, water tanks (for aquaculture, ornamental, etc.), any oil-contaminated products (such as tableware, machine parts), grease traps installed in kitchens, drain pipes, fatbergs, insulating oil leaked from transformers or deteriorated insulating oil, etc. can be mentioned, but are not limited thereto. A "grease trap" is a device for separating and collecting oil in wastewater and typically consists of three tanks. The first tank is equipped with a basket to capture food scraps and leftover rice. In the second tank, oil and water are separated. The wastewater separated from the oil is sent to the third tank to remove sedimentable garbage, etc. The installation of grease traps is mandatory in commercial kitchens such as restaurants, hospitals, and hotels. When applying to a grease trap, a separate decomposition treatment tank may be provided, but it is also possible to directly input an oil decomposer or microorganisms into the grease trap for decomposition treatment within the grease trap.

[0125] Specifically, preparations, etc. can be introduced or added, or carriers on which one or more of the combinations of microorganisms of the present disclosure are immobilized can be installed in drainage paths, drainage storage tanks, inside grease traps, etc. It is also possible to separately provide a dedicated decomposition treatment tank outside the grease trap.

[0126] Since the combination of microorganisms of the present disclosure is also highly efficient in treatment at low temperatures, it can be a desirable embodiment for low-temperature treatment. For example, cases where treatment at less than 20°C (such as 15°C) is assumed, such as industrial wastewater, domestic wastewater, industrial waste, domestic waste (such as garbage), soil contaminated with oil, water contaminated with oil (such as the sea, ponds, rivers, drinking water for animals), etc., are also a preferred example of the objects to be treated by the combination of microorganisms of the present disclosure.

[0127] In one embodiment, examples of the wastewater include, but are not limited to, wastewater from restaurants, hospitals, hotels, etc., domestic wastewater, and industrial wastewater discharged from food processing plants, oil processing plants, etc.

[0128] (Usage form) Examples of the form of the combination, combination product or composition of the microorganisms of the present disclosure include, for example, a liquid state, a solid state, etc. Examples of the combination, combination product or composition of microorganisms in a liquid state include a culture solution of microorganisms, and after collecting the microorganisms from the culture solution by centrifugation or the like, those redispersed in water, a buffer solution or a culture solution, etc. Examples of the microorganisms or composition in a solid state include those dehydrated by centrifugation, press compression, etc., those in a paste state or mayonnaise state like an intermediate between solid and liquid, and dried bodies dried (for example, vacuum drying, freeze drying). Examples of the shape of the solid include powder, granule, tablet, etc. Further, the combination of the microorganisms of the present disclosure may be provided in a state where the microorganisms or the culture supernatant is immobilized on a carrier.

[0129] In one embodiment, the combination of the microorganisms of the present disclosure and the composition or combination product providing the same may be added to a liquid so that the total number of microorganisms is about 1×10 8 cells / mL, about 1×10 7 cells / mL, about 1×10 6 cells / mL, about 1×10 5 cells / mL, about 1×10 4 cells / mL, about 1×10 3 cells / mL, about 1×10 2 cells / mL, about 10 cells / mL or about 1 cell / mL.

[0130] (Applicable environment) The microbial combinations of the present disclosure and the compositions or combinations providing the same can be used in any suitable environment. In one embodiment, the microbial combinations of the present disclosure and the compositions or combinations providing the same can be used in an environment of 10 to 60 °C, 12 to 50 °C, 15 to 40 °C, 20 to 35 °C, less than 60 °C, less than 50 °C, less than 40 °C, less than 30 °C, less than 25 °C, less than 20 °C, less than 15 °C, about 60 °C, about 50 °C, about 40 °C, about 30 °C, about 25 °C, about 15 °C, or about 10 °C.

[0131] In one embodiment, the microbial combinations of the present disclosure and the compositions or combinations providing the same can be used in an environment with a pH of 4 to 12, pH 5 to 11, pH 6 to 10, pH 7 to 9, pH 5.5 to 8.5, about pH 4, about pH 5, about pH 6, about pH 7, about pH 8, about pH 9, about pH 10, or about pH 11.

[0132] In one embodiment, the microbial combinations of the present disclosure and the compositions or combinations providing the same can be used in an environment with a dissolved oxygen concentration (DO) of 0.05 mg / L or more, 0.1 mg / L or more, 0.5 mg / L or more, or 1 mg / L or more.

[0133] In one embodiment, the microbial combinations of the present disclosure and the compositions or combinations providing the same can be used in an environment with a normal hexane value of 100 to 40000 mg / L, 200 to 30000 mg / L, 300 to 30000 mg / L (for example, in wastewater). In solid wastes such as sludge slurries and food waste (which may contain water), there may be higher concentrations of oils and fats. However, in one embodiment, the microbial combinations of the present disclosure and the compositions or combinations providing the same can also be usefully applied to such solid wastes.

[0134] In one embodiment, the microorganism, composition or combination of the present disclosure can be added to a subject containing 50 wt% or more, 20 wt% or more, 10 wt% or more, 7 wt% or more, 5 wt% or more, 2 wt% or more, 1 wt% or more, 0.7 wt% or more, 0.5 wt% or more, 0.2 wt% or more, 0.1 wt% or more, 0.07 wt% or more, 0.05 wt% or more, 0.02 wt% or more, 0.01 wt% or more, 0.007 wt% or more, 0.005 wt% or more, 0.002 wt% or more or 0.001 wt% or more of trans fatty acids.

[0135] In one embodiment, the microorganism, composition or combination of the present disclosure can be added to a subject in which the proportion of trans fatty acids in the esters (e.g., fats and oils) contained is 50 wt% or more, 20 wt% or more, 10 wt% or more, 7 wt% or more, 5 wt% or more, 2 wt% or more, 1 wt% or more, 0.7 wt% or more, 0.5 wt% or more, 0.2 wt% or more, 0.1 wt% or more, 0.07 wt% or more, 0.05 wt% or more, 0.02 wt% or more, 0.01 wt% or more, 0.007 wt% or more, 0.005 wt% or more, 0.002 wt% or more or 0.001 wt% or more.

[0136] In one embodiment, the subject to which the microorganism, combination of microorganisms and the composition or combination providing the same of the present disclosure are added may contain nitrogen in a form available to the microorganism, preferably in the form of ammonium salts, nitrates, sulfates, or organic nitrogen compounds, more preferably ammonium sulfate, urea, amino acids, or peptides such as peptone, tryptone, and casamino acids. The amount of nitrogen present may be in the range of C / N = 2 to 50, preferably in the range of C / N = 2 to 30, more preferably in the range of C / N = 2 to 20. However, C / N is the weight ratio of normal hexane-derived carbon atoms to nitrogen atoms contained in the wastewater. In one embodiment, nitrogen may be further added so as to be within these ranges.

[0137] In one embodiment, for the object to which the microorganism, combination of microorganisms, and composition or combination thereof provided by the present disclosure are added, phosphorus (P) may be present in a form that can be utilized by the microorganism, preferably in the form of phosphate or nucleic acid, more preferably in the form of phosphate. The amount of phosphorus present may be such that N / P = 1 to 20 with respect to nitrogen. However, N / P is the weight ratio of nitrogen atoms to phosphorus atoms contained in the wastewater. In one embodiment, phosphorus may be further added so as to fall within these ranges.

[0138] In one embodiment, the microorganism, combination of microorganisms, and composition or combination thereof provided by the present disclosure may be used under conditions where salts, surfactants, light, electric current, stirring operation, aeration operation, or any combination thereof are present.

[0139] In one embodiment, the microorganism, combination of microorganisms, and composition or combination thereof provided by the present disclosure may be applied after removing substances (such as chlorine, antibiotics, etc.) that kill the microorganisms of the present disclosure and suppress growth.

[0140] In one embodiment, the microorganisms, combinations of microorganisms, and compositions or combinations thereof of the present disclosure may be used with a carrier capable of immobilizing the microorganisms. By using such a carrier, washout can be effectively avoided. The material of the carrier is not particularly limited as long as it can immobilize microorganisms. Examples include carbon fibers (PAN-based, pitch-based, phenolic resin-based, etc.), polyethylene resin, polypropylene resin, polyurethane resin, polystyrene resin, polyvinyl chloride resin, polyvinyl acetate resin, polyvinyl alcohol resin, polyethylene glycol resin, acrylic resin, gelatin, sodium alginate, carrageenan, dextrin, ceramics, silicon, metal, charcoal, activated carbon, minerals (zeolite, diatomaceous earth, etc.), and composites thereof. In order to increase the immobilization rate of the microorganisms and the action efficiency of the microorganisms, it is preferable to use a porous or fibrous carrier. Also, the microorganisms may be included in a gel-like carrier. Examples of the shape of the carrier include cubic, rectangular parallelepiped, cylindrical, spherical, disc-shaped, sheet-shaped, and membrane-shaped. For the microorganism immobilization technique, refer to, for example, "Wastewater Treatment by Microorganism Immobilization Method (edited by Ryuichi Sudo, Industrial Water Research Association)" and "Water Treatment by Microorganism Immobilization Method - Carrier Immobilization Method, Comprehensive Immobilization Method, Biological Activated Carbon Method (New Water Treatment Series (1)) (Kazuhiro Mochizuki, Katsutoshi Hori, Hideki Tatemoto (authors), N.T.S. Co., Ltd.)".

[0141] (Additional components) In one embodiment, the microorganisms, combinations of microorganisms, and compositions or combinations thereof of the present disclosure may be used in combination with additional components. In one embodiment, the additional components may be added to the composition or combination, or may be used separately from the microorganisms, combinations of microorganisms, or composition. When used separately, they may be provided as a kit.

[0142] In one embodiment, as additional components, components that enhance the activity of the microorganisms to be used (e.g., carbon source, nitrogen source), surfactants, cryoprotectants, components for maintaining the microorganisms for a long period, preservatives, excipients, enhancers, antioxidants, other microorganisms, etc. can be mentioned, but are not limited thereto, and any suitable components can be used.

[0143] In one embodiment, examples of other microorganisms include microorganisms that decompose (assimilate) glycerol, microorganisms that decompose (assimilate) proteins, amino acids, nucleic acids, or polysaccharides (e.g., cellulose), etc. Other microorganisms are preferably symbiotic with the microorganisms of the present disclosure.

[0144] As the microorganisms that decompose (assimilate) glycerol, for example, eubacteria, yeasts, filamentous fungi can be used. Preferably, yeasts of the genus Candida are used. A specific example of the yeast of the genus Candida is Candida cylindracea SL1B2 (deposited with the Patent Microorganisms Depositary, National Institute of Technology and Evaluation at the accession number NITE P-714). This strain is excellent in glycerol assimilation ability. By using in combination microorganisms that decompose (assimilate) glycerol, a decrease in the rate of oil decomposition due to the accumulation of glycerol can be prevented, and more efficient oil decomposition can be achieved.

[0145] (Method of using microorganisms) In one aspect, the present disclosure provides a method for decomposing and removing esters (e.g., fats and oils) and / or fatty acids, which includes causing the microorganisms, compositions or combinations of the present disclosure to act on the object to be treated. The object to be treated may contain trans fatty acids (e.g., elaidic acid, palmitelaidic acid and / or vaccenic acid) or fats and oils containing trans fatty acids. The object to be treated can be any object to be treated described herein to which the microorganisms, compositions or combinations of the present disclosure can be applied. The method for decomposing and removing esters (e.g., fats and oils) and / or fatty acids of the present disclosure can be carried out in any environment described herein to which the microorganisms, compositions or combinations of the present disclosure can be applied. In one embodiment, the method for decomposing and removing esters (e.g., fats and oils) and / or fatty acids of the present disclosure includes the steps of decomposing trans fatty acids (e.g., elaidic acid, palmitelaidic acid and / or vaccenic acid), decomposing fats and oils containing trans fatty acids, decomposing esters (e.g., fats and oils) and / or fatty acids at 15 °C, decomposing esters containing short-chain to medium-chain fatty acids (C2-C12), and / or decomposing fats and oils containing short-chain to long-chain fatty acids (C2 or more). In the method for decomposing and removing esters (e.g., fats and oils) and / or fatty acids of the present disclosure, any additional components described herein that can be used in combination with the microorganisms, compositions or combinations of the present disclosure can be used.

[0146] In one aspect, the present disclosure provides a method for decomposing and removing fats, oils and / or fatty acids, which includes acting on a composition or combination to be treated with the combinations of microorganisms of the present disclosure and the compositions or combinations providing the same. In one aspect, the present disclosure provides a method for improving the lipase production of at least one of Burkholderia bacteria and Yarrowia yeast in a combination of microorganisms of the present disclosure, the method including the step of culturing (for example, symbiotically culturing) by mixing the Burkholderia bacteria and the Yarrowia yeast in the combination of microorganisms of the present disclosure. The combination of microorganisms of the present disclosure may be applied as a combination, or may be applied such that the combination of microorganisms of the present disclosure results from applying each microorganism (or a composition containing the same), and in this specification, any embodiment is described as acting on, applying or introducing the combination of microorganisms of the present disclosure.

[0147] In one embodiment, the method for decomposing and removing esters (for example, fats, oils) and / or fatty acids of the present disclosure includes the step of introducing the microorganisms of the present disclosure, combinations of microorganisms and the compositions or combinations providing the same into a lipolysis tank, and the introduction may be continuous or sequential. The HRT (hydraulic retention time) of the lipolysis tank is usually 12 hours or more, preferably 18 hours or more, more preferably 20 hours or more, and still more preferably 24 hours or more. For wastewater with a normal hexane content exceeding 10,000 mg / L, when an 80% or more reduction in the normal hexane value is expected, the HRT can usually be 18 hours or more, preferably 20 hours or more, and more preferably 24 hours or more. For wastewater with a normal hexane value of 3,000 mg / L or less, when an 80% or more reduction in the normal hexane value is expected, the HRT can usually be 8 hours or more, preferably 12 hours or more, and more preferably 18 hours or more.

[0148] The microbial concentration in the grease decomposition tank may depend on the ester (e.g., grease) and / or fatty acid concentration in the wastewater, and the higher the grease and / or fatty acid concentration, the higher the cell concentration can be maintained. When the grease decomposition tank foams, as a countermeasure, defoaming operations such as shortening the HRT, showering, and adding defoamers can be performed. However, since the defoamer can inhibit the growth of microorganisms, it is desirable to set the addition amount considering this matter.

[0149] For the normal hexane value of the effluent from the grease decomposition tank, in the case of low-concentration wastewater with a normal hexane value of the influent of about 300 mg / L or less, it is preferably 60 mg / L or less, more preferably 30 mg / L or less. In the case of medium-concentration wastewater with a normal hexane value of the influent of about 3000 mg / L, it is preferably 600 mg / L or less, more preferably 300 mg / L or less, still more preferably 150 mg / L or less, and most preferably 30 mg / L or less. In the case of high-concentration wastewater with a normal hexane value of the influent of about 10000 mg / L, it is preferably 1000 mg / L or less, more preferably 500 mg / L or less, still more preferably 100 mg / L or less, and most preferably 30 mg / L or less. In the case of ultra-high-concentration wastewater with a normal hexane value of the influent of about 30000 mg / L or more, it is preferably 3000 mg / L or less, more preferably 1000 mg / L or less, still more preferably 300 mg / L or less.

[0150] In one embodiment, by the method of the present disclosure, the normal hexane value of the wastewater containing grease and / or fatty acid can be reduced preferably by 80% or more, more preferably by 90% or more, still more preferably by 95% or more, and most preferably by 99% or more. As a result, in many wastewaters, it is also possible to lower the normal hexane value of the effluent from the grease decomposition tank to less than 30 mg / L, which is the discharge standard value to the sewer in many local governments. When this standard value is achieved, if only the normal hexane value is considered, even the subsequent main treatment such as activated sludge treatment may not be necessary.

[0151] In the effluent water from the oil decomposition tank, it is not necessary for the amount of the introduced microorganisms to increase. The amount of the introduced microorganisms in the effluent water is preferably 0.01 times or more, more preferably 0.1 times or more, still more preferably 0.5 times or more, and most preferably 1 time or more with respect to the introduced amount.

[0152] The method for decomposing and removing oil and fat and / or fatty acids of the present disclosure may include additional steps other than the above steps. Examples of such steps include a step of returning all or part of the effluent water from the oil decomposition tank to the oil decomposition tank again. However, in the method of the present disclosure, a sufficient oil and fat and / or fatty acid decomposition effect can be obtained without performing such a return treatment, so it is not essential to return all or part of the effluent water from the oil decomposition tank to the oil decomposition tank again.

[0153] (General technology) The molecular biological techniques, biochemical techniques, and microbiological techniques used in this specification are well-known and commonly used in the art. For example, Savli, H., Karadenizli, A., Kolayli, F., Gundes, S., Ozbek, U., and Vahaboglu, H. 2003. Expression stability of six housekeeping genes: A proposal for resistance gene quantification studies of Pseudomonas aeruginosa by real-time quantitative RT-PCR. J. Med. Microbiol. 52:403-408., Marie-Ange Teste, Manon Duquenne, Jean M Francois and Jean-Luc Parrou 2009. Validation of reference genes for quantitative expression analysis by real-time RT-PCR in Saccharomyces cerevisiae. BMC Molecular Biology 10:99, Seiji Ishii, Hiroshi Okumura, Chiyo Matsubara, Fumi Ninomiya, Hiroshi Yoshioka, 2004, "Simple method for measuring oil content in water using thermosensitive polymer", Vol. 46, No. 12, "Water Supply and Sewerage", etc. are described, and the relevant parts (which may be all) thereof are incorporated herein by reference.

[0154] (Note) As used herein, "or" is used when "at least one or more" of the items listed in the text can be adopted. The same applies to "or else". When it is specified herein that a value is "within the range" of "two values", the range includes the two values themselves.

[0155] References such as scientific literature, patents, and patent applications cited in this specification are incorporated herein by reference in their entirety to the same extent as if each were specifically described.

[0156] As described above, the present disclosure has been described by showing preferred embodiments for ease of understanding. Hereinafter, the present disclosure will be described based on examples. However, the above description and the following examples are provided for illustrative purposes only and not for the purpose of limiting the present disclosure. Therefore, the scope of the present invention is not limited to the embodiments or examples specifically described herein, but is limited only by the claims.

Examples

[0157] Examples are described below. When necessary, the handling of organisms used in the following examples complied with the standards defined in Nagoya University, regulatory authorities, and the Cartagena Act, if necessary. Specifically, the reagents used were the products described in the examples, but equivalents from other manufacturers (Sigma-Aldrich, Fujifilm, Wako Pure Chemical Industries, Nacalai, R&D Systems, USCN Life Science INC, Kanto Chemical, Funakoshi, Tokyo Chemical Industry, Merck, etc.) could also be substituted. Also, unless otherwise specified, the addition concentration of various oils, fats, fatty acids, etc. to the medium refers to the final concentration of the substance in the medium. For percentage notation, when the oil, fat, or fatty acid is in a liquid state, it represents volume / volume (v / v%), and when it is in a solid state, it represents weight / volume (w / v%).

[0158] (Example 1: Identification of microorganisms capable of assimilating and decomposing oils and fats containing trans fatty acids) A conserved population of yeast was cultured, and microorganisms were isolated therefrom. To examine whether the isolated microorganisms could be cultured under low-temperature conditions (15°C), each microorganism was streaked on an agar medium containing canola oil (Nisshin Canola Oil, Nisshin Oillio, Tokyo) as the sole carbon source and cultured at 15°C for 5 days. As a result, microorganisms capable of growing at low temperatures using oils and fats as a nutrient source were found.

[0159] In addition, the ability of each microorganism to decompose oil and fat at low temperature (15°C) was examined. The colonies of each microorganism were inoculated into 20 mL of an inorganic salt medium (3.5 g / L of Na2HPO4, 2.0 g / L of KH2PO4, 4.0 g / L of (NH4)2SO4, 0.34 g / L of MgCl2·6H2O, 2.8 mg / L of FeSO4·7H2O, 2.4 mg / L of MnSO4·5H2O, 2.4 mg / L of CoCl2·6H2O, 1.7 mg / L of CaCl2·2H2O, 0.2 mg / L of CuCl2·2H2O, 0.3 mg / L of ZnSO4·7H2O, and 0.25 mg / L of Na2MoO4) containing 10 g / L of canola oil as the sole carbon source using a cotton swab and cultured in a 100 mL Erlenmeyer flask. As a result, microorganisms that decompose, assimilate, and grow on oil and fat at low temperature were found.

[0160] Next, the ability of each microorganism to decompose and assimilate trans fatty acids was examined. Each microorganism was inoculated into 2 mL of an inorganic salt medium (the above composition) supplemented with elaidic acid at a final concentration of 0.2% as the sole carbon source so that the optical density OD 660 of the final cell suspension was 0.04. Cultivation was carried out at 28°C and 130 rpm for 24 hours in a 15 mL Harmon centrifuge tube (Eppendorf, Tokyo). As a result, microorganisms that can decompose, assimilate, and grow on elaidic acid, a trans fatty acid, were found.

[0161] Through these tests, it was found that a certain isolated microbial strain can be cultured in a low-temperature environment using oil and fat as a nutrient source, can assimilate (decompose) oil and fat at low temperature, and can decompose and assimilate oils and fats containing trans fatty acids and trans fatty acids. This novel strain was named KH-2 strain.

[0162] Subsequent analysis revealed that the KH-2 strain is Yarrowia lipolytica. Here, although the analysis was conducted focusing on the KH-2 strain, the ability of other strains to decompose oil and fat and / or fatty acids can also be identified by examining their oil and fat and / or fatty acid decomposition ability through similar tests.

[0163] (Example 2: Oil and Fat and Fatty Acid Assimilation and Decomposition Abilities of KH-2 Strain) Regarding the KH-2 strain, the bacteria were streaked on an agar medium (lipid or fatty acid final concentration 1%, Triton® X-100 final concentration 0.25%, polyvinyl alcohol final concentration 0.5%, inorganic salt agar medium (inorganic salt medium containing 1.5% agar) (pH 7.0)) containing triolein, oleic acid, or canola oil (described above) as the sole carbon source, and cultured at 28°C for 3 days. The composition of the inorganic salt medium is 3.5 g / L of Na2HPO4, 2.0 g / L of KH-2 strain PO4, 4.0 g / L of (NH4)2SO4, 0.34 g / L of MgCl2·6H2O, 2.8 mg / L of FeSO4·7H2O, 2.4 mg / L of MnSO4·5H2O, 2.4 mg / L of CoCl2·6H2O, 1.7 mg / L of CaCl2·2H2O, 0.2 mg / L of CuCl2·2H2O, 0.3 mg / L of ZnSO4·7H2O, and 0.25 mg / L of Na2MoO4. The results are shown in Figure 1. Colonies were formed on all agar media, indicating that the KH-2 strain can decompose, assimilate, and grow on canola oil, a typical oil-containing vegetable oil, triolein, a triglyceride lipid, and oleic acid, a typical example of free fatty acid as a decomposition product of these lipids.

[0164] Furthermore, regarding the KH-2 strain, the bacteria were streaked on an agar medium (elaidic acid final concentration 0.1%, Triton® X-100 final concentration 0.25%, inorganic salt agar medium (pH 7.0)) containing elaidic acid as the sole carbon source, and cultured at 15°C for 14 days. The results are shown in Figure 2. Colonies were formed on the agar medium, indicating that the KH-2 strain can decompose, assimilate, and grow on elaidic acid, a typical trans fatty acid.

[0165] (Example 3A: Trans Fatty Acid Degrading Ability of KH-2 Strain at 28°C) The trans fatty acid degrading abilities of the KH-2 strain and BioRemove 3200 (BR3200) (Novozymes, Denmark) at 28°C were compared. The cells were added to 20 mL of an inorganic salt medium (the above composition, pH 7.0) containing 0.2% elaidic acid and 0.25% Triton® X-100 until the optical density OD of the final cell mass 660= 0.5 (KH-2 strain of HITACHI U-2810 spectrophotometer (Hitachi, Ltd., Tokyo)) or 5 times the manufacturer's recommended concentration, 5×10 6 CFU / ml of BR3200 was inoculated and cultured at 28 °C with shaking at 130 rpm for 24 hours.

[0166] The oil and fat in the supernatant after cultivation was analyzed by thin layer chromatography. Specifically, an equal volume of chloroform was added to the culture supernatant and stirred. Then, 12 μl of the chloroform layer was applied to a silica gel plate and developed with a chloroform:acetone:methanol (96:4:2) solution. After development, a solution of molybdotungstic acid n-hydrate (2.4 g / 60 ml ethanol) was sprayed, and the fatty acids were visualized by heating at 110 °C for 12 minutes to compare the amount of fatty acids remaining in the medium (Figure 3A(a)).

[0167] Also, 0.5 mL of the 24-hour culture supernatant was analyzed using an oil content measurement reagent kit (Kyoritsu Chemical-Check Lab., Co., Ltd., Tokyo) (measurement reagent kit by the method for measuring polynippam extractable substances) according to the manufacturer's recommended protocol (Figure 3A(b)).

[0168] It was found that the KH-2 strain has strong trans fatty acid decomposition ability.

[0169] (Example 3B: Trans fatty acid decomposition ability of the KH-2 strain at 15 °C) The trans fatty acid decomposition ability of the KH-2 strain and BioRemove3200 (BR3200) (Novozymes, Denmark) at 15 °C was compared. The cells were added to 20 mL of inorganic salt medium (the above composition, pH 7.0) containing 0.2% elaidic acid and 0.25% Triton® X-100 until the optical density OD of the final cells 660 = 0.8 (HITACHI U-2810 spectrophotometer (Hitachi, Ltd., Tokyo)) of the KH-2 strain or 8 times the manufacturer's recommended concentration, 8×10 6 CFU / ml of BR3200 was inoculated and cultured at 15 °C with shaking at 130 rpm for 48 hours.

[0170] The fats and oils in the supernatant after cultivation were analyzed by thin-layer chromatography. Specifically, half the volume of chloroform was added to the culture supernatant and stirred. Then, 5 μl of the chloroform layer was applied to a silica gel plate and developed with a chloroform:acetone:methanol (96:4:1) solution. After development, a solution of molybdotungstic acid n-hydrate (2.4 g / 60 ml ethanol) was sprayed, and heating at 110 °C for 12 minutes was performed to visualize the fatty acids, and the amount of fatty acids remaining in the medium was compared (Fig. 3B(a)).

[0171] Also, 0.5 mL of the supernatant after 48-hour cultivation was analyzed using an oil content measurement reagent kit (Kyoritsu Chemical-Check Lab., Tokyo) (measurement reagent kit by the polynippam extraction substance measurement method) according to the manufacturer's recommended protocol (Fig. 3B(b)).

[0172] It was found that the KH-2 strain has strong trans-fatty acid decomposition ability even at low temperature (15 °C).

[0173] (Example 4A: Trans-fatty acid-containing oil and fat decomposition ability of the KH-2 strain) The decomposition ability of the KH-2 strain for trans-fatty acid-containing oils and fats at 15 °C and 28 °C was tested. The KH-2 strain with an optical density at the end of the cell OD 660 = 0.8 (HITACHI U-2810 spectrophotometer (Hitachi, Tokyo)) was inoculated into an inorganic salt medium (BS) containing 0.1% triolein and cultured at 15 °C or 28 °C with shaking at 130 rpm for 6 days. It was compared with a control sample without adding microorganisms.

[0174] The fats and oils in the supernatant after cultivation were analyzed by thin-layer chromatography. Specifically, an equal volume of chloroform was added to the culture supernatant and stirred. Then, 12 μl of the chloroform layer was applied to a silica gel plate and developed with a chloroform:acetone:methanol (96:4:2) solution. After development, a solution of molybdotungstic acid n-hydrate (2.4 g / 60 ml ethanol) was sprayed, and heating at 110 °C for 12 minutes was performed to visualize the fats and oils and free fatty acids, and the amount of fats and oils remaining in the medium and the amount of fatty acids, which are their decomposition products, were compared (Fig. 4). The KH-2 strain was found to have the ability to decompose oils and fats containing trans fatty acids at both normal temperature (28 °C) and low temperature (15 °C).

[0175] (Example 5: Lipid-decomposing ability of the KH-2 strain in actual wastewater) Using actual wastewater, the lipid-decomposing abilities of the KH-2 strain and BioRemove3200 (BR3200) (Novozymes, Denmark) were compared (Figure 5). To a wastewater sample containing a large amount of oils and fats containing trans fatty acids from a food factory using hydrogenated oils, nitrogen (ammonium sulfate) and phosphorus equivalent to the inorganic salt medium (described above) were added and cultured. The KH-2 strain was cultured in LB medium, washed twice with the inorganic salt medium, and then inoculated so that the optical density at 600 nm of the cell suspension OD 660 = 0.1 (HITACHI U-2810 spectrophotometer (Hitachi, Ltd., Tokyo)), and BR3200 was inoculated at 1 × 10 7 CFU / ml, which is 10 times the manufacturer's recommended concentration. A control sample without adding microorganisms was also compared. The samples were cultured at 28 °C, and samples were collected after 24 hours and 48 hours. The samples were analyzed using thin-layer chromatography (applied to a silica gel plate, developed with a chloroform:acetone:methanol (96:4:1) solution, and visualized with molybdotungstic acid n-hydrate) (Figure 5A) and an oil content measurement reagent kit (measurement of normal hexane value, described above) (Figure 5B). BR3200 showed slow decomposition progress even at an excessive amount, while the KH-2 strain showed excellent decomposition ability.

[0176] (Example 6: Lipid-decomposing ability of the KH-2 strain at 15 °C) The cells of the KH-2 strain were adjusted to an optical density at 600 nm of the cell suspension OD 660Inoculated so that it would be 0.05 (HITACHI U-2810 Spectrophotometer (Hitachi, Ltd., Tokyo)), and cultured with a fermenter at 15°C. Samples were collected at 0 hours, 24 hours, 48 hours, and 72 hours of culture. The cells were removed from the sampled culture broth by centrifugation, and in this supernatant, the oil content equivalent to the normal hexane value was measured with an oil content measurement reagent kit (normal hexane extraction, as described above) (Figure 6A, upper part). The total fatty acids (total amount of fatty acids in triglyceride and free fatty acids) were quantified by gas chromatography (Figure 6A, lower part), and analyzed by thin layer chromatography (applied to a silica gel plate, developed with a chloroform:acetone:methanol (96:4:1) solution, and visualized with molybdotungstic acid n-hydrate) (Figure 6B). Quantification by gas chromatography was performed as follows. 3 ml of the culture supernatant was acidified with hydrochloric acid, and an equal volume of ethyl acetate was added. After stirring for 5 minutes, centrifuged, and 1 ml of the ethyl acetate layer was transferred to an organic solvent-resistant tube and completely evaporated. Dissolved in 1 ml of chloroform, 4 ml of a methanolysis solution mixed with methanol:sulfuric acid = 17:3 was added, and heated at 100°C for 2 hours to methyl-esterify the total fatty acids. A solution mixed at a ratio of chloroform:pure water = 1:1 was added and stirred well, then the chloroform layer was mixed with 0.5% methyl octanoate (internal standard) at a ratio of 1:1, and analyzed by gas chromatography (GC-17A (Shimadzu Corporation, Kyoto)) equipped with an FID detector. From these results, it was confirmed that the KH-2 strain has high oil-decomposing and assimilating ability even at low temperatures.

[0177] (Example 7: Oil-decomposing ability of the KH-2 strain at 28°C) The cells of the KH-2 strain were added to an inorganic salt medium (pH 7) containing 1% canola oil (Nisshin Canola Oil, Nisshin Oillio) until the optical density OD of the final cells 660Inoculation was carried out so that it would be 0.05 (HITACHI U-2810 spectrophotometer (Hitachi, Ltd., Tokyo)), and it was cultured in a fermenter at 28°C. Samples were taken at 0 hours, 12 hours, 24 hours, and 30 hours of culture. The cells were removed from the sampled culture broth by centrifugation. In this supernatant, in the same manner as in Example 6, the oil content equivalent to the normal hexane value was measured (Figure 7A, upper part), the total fatty acids were quantified by gas chromatography (Figure 7A, lower part), and analysis was performed by thin-layer chromatography (Figure 7B). The KH-2 strain decomposed fats and oils extremely rapidly at 28°C. More than 70% of the 1% fats and oils initially contained in the medium were decomposed in 12 hours, and both fats and oils and fatty acids almost disappeared within 24 hours.

[0178] (Example 8: Fatty acid and fat and oil decomposition by additional strains) Samples were collected from a river near a food factory where fat and oil-containing wastewater flowed out, and microorganisms were isolated from them. Regarding the isolated microorganisms, it was examined whether they could produce lipase and decompose fats and oils under a low-temperature environment (15°C). As a result, microorganisms capable of decomposing fats and oils at low temperatures were found. These microbial strains were named KH-2AL1 strain and KH-2AL3 strain, respectively.

[0179] In order to further characterize the KH-2AL1 strain and the KH-2AL3 strain, gene sequence analysis of 26S rDNA was performed. Since the partial base sequence of 26S rDNA of the KH-2AL1 strain was 100% identical in homology to Yarrowia lipolytica, it was identified as Yarrowia lipolytica. Since the partial base sequence of 26S rDNA of the KH-2AL3 strain was 100% identical in homology to Yarrowia lipolytica, it was identified as Yarrowia lipolytica.

[0180] Regarding these strains, the trans-fatty acid decomposing ability, the ability to decompose oils and fats containing trans-fatty acids, and the fat and oil decomposing ability were also examined.

[0181] (Trans-fatty acid decomposition) Similar to Example 3, the ability of the KH-2AL1 strain and the KH-2AL3 strain to decompose trans fatty acids was also tested. The cells were inoculated into 10 mL of an inorganic salt medium (the above composition, pH 7.0) containing 0.2% elaidic acid and 0.25% Triton® X-100 to obtain a final cell optical density OD 660 = 0.8 (HITACHI U-2810 spectrophotometer (Hitachi, Ltd., Tokyo)) of the KH-2 strain, KH-2AL1 strain or KH-2AL3 strain, and cultured at 28°C for 46 hours or at 15°C for 90 hours while shaking at 130 rpm. Similar to Example 3, thin layer chromatography (28°C; Fig. 8A(a), 15°C; Fig. 8B(a)) and analysis using an oil content measurement reagent kit (28°C; Fig. 8A(b), 15°C; Fig. 8B(b)) were performed to examine the lipid decomposition in the culture supernatant.

[0182] The KH-2AL1 strain and the KH-2AL3 strain were found to have the same trans fatty acid decomposition ability as the KH-2 strain.

[0183] (Decomposition of oils and fats containing trans fatty acids) Similar to Example 4, the ability of the KH-2AL1 strain and the KH-2AL3 strain to decompose oils and fats containing trans fatty acids was also tested. The cells were inoculated into an inorganic salt medium (BS) containing 0.1% trielaidin and 0.25% Triton® X-100 to obtain a final cell optical density OD 660 = 0.8 (HITACHI U-2810 spectrophotometer (Hitachi, Ltd., Tokyo)) of the KH-2 strain, KH-2AL1 strain or KH-2AL3 strain, and cultured at 28°C for 5 days while shaking at 130 rpm. A comparison was made with a control sample without adding microorganisms. Similar to Example 3, the lipid decomposition in the culture supernatant was examined. The analysis results using an oil content measurement reagent kit are shown in Fig. 9.

[0184] The KH-2AL1 strain and the KH-2AL3 strain were found to have the same ability to decompose oils and fats containing trans fatty acids as the KH-2 strain.

[0185] (Decomposition of oils and fats) The cells of KH-2AL1 strain or KH-2AL3 strain were inoculated into an inorganic salt medium (pH 7) containing 1% canola oil (Nisshin Canola Oil, Nisshin Oillio) so that the optical density at the end of the cells OD 660 = 0.05 (HITACHI U-2810 spectrophotometer (Hitachi, Ltd., Tokyo)), and cultured in a fermenter at 15°C for 72 hours. Using the culture supernatant, analysis with an oil content measurement reagent kit (upper part of FIGS. 10A and 11A), total fatty acid analysis by gas chromatography (lower part of FIGS. 10A and 11A), and thin layer chromatography analysis (FIGS. 10B and 11B) were performed.

[0186] From these results, it was confirmed that the Yarrowia yeast of the present disclosure has high oil-decomposing and assimilating abilities even at low temperatures.

[0187] (Example 9: Assimilation ability of trans fatty acids and the oils and fats containing them of KH-2 strain at 15°C) The assimilation ability of trans fatty acids and the oils and fats containing them of KH-2 strain at 15°C was tested. An inorganic salt medium (pH 7.0) containing 0.2% elaidic acid or 0.2% trielaidin in terms of elaidic acid and 0.25% Triton® X-100 was inoculated with the cells of KH-2 strain with an optical density at the end of the cells OD 660 = 0.08 (HITACHI U-2810 spectrophotometer (Hitachi, Ltd., Tokyo)), and cultured at 15°C with shaking at 130 rpm for 5 days. It was compared with a control sample to which no microorganism was added. The results are shown in FIG. 12. In the sample inoculated with KH-2 strain, it was observed that the medium was turbid. From this, it was shown that KH-2 strain can grow in an environment using elaidic acid or trielaidin as the sole carbon source even at 15°C and has the ability to assimilate these compounds.

[0188] (Example 10: Comparison of detergency between KH-2 strain and detergent) The ventilation fan filter with oil stains was treated with the culture supernatant of KH-2 strain (culture conditions: inorganic salt medium supplemented with 1% canola oil (the above composition), initial optical density of cells OD 660Inoculate to achieve a value of 0.01, culture at 28 °C for 70 hours. After culturing, centrifuge to remove the cells, and adjust the pH of the supernatant to 8.0 using an aqueous sodium hydroxide solution. Then, immerse and wash it at room temperature (25 °C) in an oil detergent (diluted 143-fold with water according to the instructions for the natural enzyme detergent Nicoeco Taisho (Nicoeco, Nagano)) and a general detergent (Family (registered trademark) (Kao, Tokyo), diluted 666-fold according to the instructions) (Figure 13). The washing times were 30 minutes for the culture supernatant of KH-2 strain, 2 hours for the oil detergent, and 4 hours for the general detergent. Even with 2-hour and 4-hour immersion washing in the general detergent and the oil detergent respectively, the oil stains could not be completely removed. However, with the KH-2 strain, after 30 minutes of immersion washing, the filter became as white as new.

[0189] (Example 11: Degradation of various esters) The substrate specificity of the ester degraded in the culture supernatant of KH-2 strain was examined. An inorganic salt medium with 1% canola oil added (the above composition), initial cell optical density OD 660 Inoculate the KH-2 strain to achieve a value of 0.05, then culture at 28 °C for 24 hours, at 28 °C for 48 hours, or at 15 °C for 48 hours. After culturing, centrifuge to remove the cells, and use the supernatant.

[0190] Mix 0.05 mol of any one of the 4-nitrophenyl esters (substrates) of 5 types of fatty acids (acetic acid (C2), butyric acid (C4), octanoic acid (C8), lauric acid (C12), palmitic acid (C16)) with 12 ml of 3% (v / v) Triton (registered trademark) X-100 aqueous solution, and melt at 70 °C to prepare a substrate solution. Mix 1 ml each of the substrate solution, 150 mM GTA buffer (pH 7.0), and each supernatant, and monitor the absorbance at 410 nm (corresponding to 4-nitrophenol generated by hydrolysis) for 1 minute while stirring.

[0191] The results are shown in the following table. Taking the one with the maximum degradation (4-nitrophenol release) among the 5 types of substrates as 100%, the percentage of the amount of 4-nitrophenol released by each substrate is shown.

Table 1

[0192] Generally, the lipase activity of microorganisms is measured by its hydrolysis activity using an ester (4-nitrophenyl ester) of 4-nitrophenol and a long-chain fatty acid (e.g., palmitic acid) as a model substrate. Since the hydrolysis product, 4-nitrophenol, exhibits a yellow color, it can be easily quantitatively evaluated by a colorimetric method. At this time, in order to correlate with the decomposition activity of animal and vegetable oils and fats (triglycerides), usually, an ester of a long-chain fatty acid (e.g., C16 or higher) constituting animal and vegetable oils and fats and 4-nitrophenol is used. However, it has been found that the KH-2 strain has low activity against an ester substrate of a long-chain fatty acid and 4-nitrophenol, and high activity against an ester substrate of a short-chain fatty acid (C6 or lower) or a medium-chain fatty acid (C7-12). That is, based on the experiments usually conducted by those skilled in the art (experiments for examining the decomposing ability of an ester of a long-chain fatty acid and 4-nitrophenol), a conclusion is drawn that the KH-2 strain does not have the ability to decompose triglycerides of long-chain fatty acids. However, as shown in the above examples, the present inventor has found that the KH-2 strain has a high activity of decomposing triglycerides of long-chain fatty acids, which can be said to be a completely unexpected finding. Further, from this result, it is predicted that the microorganisms of the present disclosure may have a broad lipase activity against oils and fats of short-chain to long-chain fatty acids.

[0193] (Example 12: Assimilation and decomposing ability of various trans fatty acids) The activity of the KH-2 strain to decompose palmitelaidic acid (16:1) and vaccenic acid (18:1), which are trans fatty acids, was compared with that of BioRemove3200 (BR3200) (Novozymes, Denmark).

[0194] KH-2 strain and BR3200 were each inoculated into 5 mL of an inorganic salt medium (the above composition, pH 7) prepared such that the final concentration of palmitic acid or vaccenic acid was 0.2% and the final concentration of Triton® X-100 was 0.25%. Using a HITACHI U-2810 spectrophotometer (Hitachi, Ltd., Tokyo), the KH-2 strain was inoculated so that the final concentration based on the optical density of the bacterial cells was OD 660 = 0.5, and BR3200 was inoculated so that the concentration was 5 × 10 6 CFU / ml, which is 5 times the manufacturer's recommended concentration. This was cultured at 15°C for 48 hours or 72 hours, or at 28°C for 24 hours while shaking at 130 rpm, and then the culture supernatant of each strain was obtained. A control sample without using microorganisms was also prepared.

[0195] Thereafter, the residual oil in the culture solution was analyzed by thin layer chromatography. Specifically, fatty acids were extracted with chloroform in an amount equal to half of the sample, 6 μl of the extract was applied to a silica gel plate, and developed with a chloroform:acetone:methanol (96:4:2) solution. After development, the fatty acids were visualized by a color reaction with molybdotungstic acid n-hydrate in the same manner as in Example 4, and the amount of fatty acids remaining in the medium was compared (Figs. 14 and 15).

[0196] The KH-2 strain was able to completely decompose palmitic acid and vaccenic acid within 24 hours at 28°C and within 48 to 72 hours at 15°C. On the other hand, BR3200 did not have the ability to decompose these fatty acids. Thus, the microorganisms of the present disclosure may be able to assimilate and decompose various trans fatty acids and oils and fats containing trans fatty acids.

[0197] (Example 13: Acquisition of related strains) Related strains can be obtained as follows. Add PBS to various sources of isolation such as yeast preservation libraries, soil, rivers, lake water, and activated sludge to prepare dilution series, and spread them on an inorganic salt agar medium using trielazine or elaidic acid as the sole carbon source. Alternatively, add 1-10% by weight of the above isolation source to an inorganic salt medium containing 0.3-1% by weight of shortening, and culture at 15°C or 28°C until emulsification and microbial growth are observed. After repeating this enrichment culture any number of times, a dilution series of the culture solution may be prepared and spread as described above. Colonies are obtained by statically culturing the inoculated agar medium at 15°C or 28°C. Among them, pick up those that form a clear zone around the colony and inoculate them into about 2 mL of inorganic salt agar medium (containing trielazine or trielaidic acid at the above concentration), and culture at 15°C or 28°C. After culture, analyze the degree of oil decomposition in the culture supernatant by thin layer chromatography to obtain microorganisms capable of oil decomposition at low temperatures or having the ability to decompose oils containing trans fatty acids.

[0198] (Example 14: Use in the device) Culture strain KH-2 in an inorganic salt medium containing 10 mL / L of canola oil to 2×10 9 cells / mL to obtain a culture stock solution. Dilute this 10-fold to obtain a microbial preparation (2×10 8 cells / mL). Refrigerate and store this in the microbial preservation tank of the automatic amplification input device as the inoculum. Automatically inoculate this inoculum into the inorganic salt medium in the culture amplification tank of the same device at a rate of 1 / 100 per day, and culture until the number of microorganisms increases 100-fold, i.e., until the cell concentration is the same as that of the microbial preparation. By introducing this into 1 / 1000 of the drainage volume of the oil decomposition treatment tank, the microbial concentration of the decomposing bacteria in the oil-treated water is set to 2×10 5 cells / mL, and decompose the drainage from a food factory that discharges drainage containing a large amount of oils containing trans fatty acids for 24 hours. The season at this time is winter, and the water temperature during the treatment fluctuates between 12 and 17°C. As a result, a significant reduction in the normal hexane value is observed compared to the control example without the addition of microorganisms.

[0199] (Example 15: Other embodiments) Inoculate strain KH-2 into the disposable food waste processor at 1×106 Inoculate to achieve a cell density of cells / mL, and treat at 25 to 35°C for 12 to 24 hours. Measure the normal hexane value in the wastewater discharged from the food waste treatment machine. A significant reduction in value is observed compared to the control example without the addition of strain KH-2.

[0200] Put the oily sludge recovered by flotation separation into a culture tank, and add an inorganic salt medium with a weight of 10 to 1000% of the input weight. Add strain KH-2 at 1×10 5 Inoculate to achieve a cell density of cells / mL, and incubate at 20 to 35°C for 12 to 240 hours while stirring and aerating. Then, investigate the decomposition and reduction amount of the oily sludge by measuring the oil content based on the normal hexane value of the treated liquid, or by measuring the weight of the residue mainly composed of the oil remaining after evaporating the water. As a result, significant decomposition of the oily sludge is observed compared to the control example without the addition of strain KH-2.

[0201] Put carriers such as charcoal, various plastics, and ceramic pieces into the grease trap, and automatically add an appropriate amount (for example, 1×10 5 cells / mL) of strain KH-2 every day after the operation of the cafeteria ends. Collect water immediately before the start of the operation every day and analyze the normal hexane value. After one week, a significant decrease in the normal hexane value is observed compared to the control example without the addition of strain KH-2, and effects such as a reduction in the adhesion and floating of oil are also observed in the appearance of the grease trap itself.

[0202] (Example 16: Combination of microorganisms with useful oil-decomposing ability) There are strains with oil-decomposing ability among Burkholderia bacteria and Yarrowia yeast. By combining these strains with another microorganism strain having oil-decomposing ability, a combination of microorganisms with excellent oil-decomposing ability can be obtained.

[0203] In the examples, the oil-decomposing ability by co-culture was tested using representative combinations of Burkholderia bacteria and several Yarrowia yeast. Excellent oil-decomposing ability was observed in the combination of Burkholderia bacteria and Yarrowia yeast. Details are described below.

[0204] Co-culture a combination of Burkholderia bacteria and Yarrowia yeast, both of which have lipase secretion ability and lipid decomposition ability, to test the lipid decomposition ability and obtain a combination with excellent lipid decomposition ability when co-cultured. The symbiotic system of Burkholderia bacteria and Yarrowia yeast was co-cultured in an inorganic salt medium (Na2HPO4 3.5 g / L, KH2PO4 2.0 g / L, (NH4)2SO4 4.0 g / L, MgCl2·6H2O 0.34 g / L, FeSO4·7H2O 2.8 mg / L, MnSO4·5H2O 2.4 mg / L, CoCl2·6H2O 2.4 mg / L, CaCl2·2H2O 1.7 mg / L, CuCl2·2H2O 0.2 mg / L, ZnSO4·7H2O 0.3 mg / L, and NaMoO4 0.25 mg / L) containing canola oil (Nisshin Canola Oil, Nisshin OilliO, Tokyo) at a final concentration of 1% (v / v). The culture supernatant was obtained and the decomposition of lipids was tested by thin layer chromatography analysis, gas chromatography analysis, etc. As a result, a symbiotic system of Burkholderia bacteria and Yarrowia yeast with excellent lipid decomposition ability was found. As microbial strains that can be usefully used in this symbiotic system, for example, KH-1 strain (Burkholderia arboris) and KH-2 strain (Yarrowia lipolytica) were found.

[0205] In the following examples, analysis was performed focusing on this representative combination, but other useful combinations can be found by examining the lipid decomposition ability by similar tests.

[0206] (Example 17: Further analysis of the lipid decomposition ability of the symbiotic system) The lipid decomposition ability of combinations of KH-1 strain and KH-2 strain at various temperatures with various mixing ratios was tested.

[0207] (Analysis at 15°C) KH-1 strain and KH-2 strain were added to an inorganic salt medium (the above composition, pH 7) containing canola oil (Nisshin Canola Oil, Nisshin OilliO, Tokyo) at a final concentration of 1% (v / v) so that the total cell concentration was 5×10 5They were added at ratios of KH-1 strain:KH-2 strain = 10:0, 9:1, 5:5, 1:9, and 0:10 (based on the number of cells) so as to be 1 cell / mL, and cultured at 15°C. Regarding the culture supernatant obtained at the time of culturing for 48 hours, total fatty acid analysis by gas chromatography was performed according to the following procedure.

[0208] Total fatty acid analysis by gas chromatography: 3 mL of the culture supernatant was acidified with hydrochloric acid, and an equal volume of ethyl acetate was added. After stirring for 5 minutes, centrifugation was performed, and 1.5 mL of the ethyl acetate layer was transferred to a tube resistant to organic solvents and completely evaporated. It was dissolved in 1 mL of chloroform, 4 mL of a methanolysis solution of methanol:sulfuric acid = 17:3 was added, and the total fatty acids were methyl-esterified by heating at 100°C for 2 hours. A solution mixed at a ratio of chloroform:pure water = 1:1 was added and stirred well, then the chloroform layer and 0.5% methyl octanoate (internal standard) were mixed at a ratio of 1:1, and analyzed by gas chromatography (GC-17A (Shimadzu Corporation, Kyoto)) equipped with an FID detector.

[0209] (Results) The results of the gas chromatography analysis are shown in Fig. 16. The symbiotic systems of the KH-1 strain and the KH-2 strain showed a lipolytic ability exceeding the lipolytic ability calculated from the values of the lipolytic ability of each single culture at various mixing ratios. As observed from the results of the KH-1 strain alone and the KH-2 strain alone, each of the KH-1 strain and the KH-2 strain produces lipase alone, and it is usually expected that microorganisms having such similar properties compete with each other and as a result suppress each other's abilities (the overall lipolytic ability decreases), but the inventors unexpectedly found an improvement in the lipolytic ability in the symbiotic system.

[0210] (Analysis at 28°C) The lipolytic ability of the combination of the KH-1 strain and the KH-2 strain was also tested at 28°C.

[0211] Inorganic salt medium (the above composition, pH 7) containing 1% (v / v) of canola oil (Nisshin Canola Oil, Nisshin OilliO, Tokyo) at the final concentration was inoculated with KH-1 strain and KH-2 strain such that the total cell concentration became 5×10 5 cells / mL, at ratios of KH-1 strain:KH-2 strain = 10:0, 9:1, 5:5, 1:9, and 0:10 (based on the number of cells), and cultured at 28°C. The culture supernatant obtained at the time of culturing for 18 hours was subjected to total fatty acid analysis by gas chromatography in the same manner as above.

[0212] (Results) The results of gas chromatography analysis are shown in Fig. 17. The symbiotic systems of KH-1 strain and KH-2 strain showed lipolytic ability exceeding that calculated from the lipolytic ability values of each single culture at various mixing ratios. Thus, the symbiotic systems of KH-1 strain and KH-2 strain have high lipolytic ability in various temperature ranges.

[0213] (Example 18: Analysis of fatty acid decomposition ability of symbiotic system) The fatty acid decomposition ability of combinations of KH-1 strain and KH-2 strain at various mixing ratios was tested. (Analysis at 28°C) The lipolytic ability of combinations of KH-1 strain and KH-2 strain was tested at 28°C.

[0214] Inorganic salt medium (the above composition, pH 7) containing 1% (v / v) of oleic acid at the final concentration was inoculated with KH-1 strain and KH-2 strain such that the total cell concentration became 5×10 5 cells / mL, at ratios of KH-1 strain:KH-2 strain = 10:0, 9:1, 5:5, 1:9, and 0:10 (based on the number of cells), and cultured at 28°C. The culture supernatant obtained at the time of culturing for 18 hours was subjected to total fatty acid analysis by gas chromatography in the same manner as above.

[0215] (Results) The results of gas chromatography analysis are shown in Fig. 18. The symbiotic systems of the KH-1 strain and the KH-2 strain showed fatty acid degradation ability exceeding that of each single culture at various mixing ratios. Thus, it was found that the symbiotic system of the KH-1 strain and the KH-2 strain is a very preferable combination having high fatty acid degradation ability.

[0216] (Example 19: Gene expression in symbiotic system) Changes in gene expression of each were examined in the symbiotic system of the KH-1 strain and the KH-2 strain.

[0217] (Analysis of KH-1 strain) First, gene expression of the KH-1 strain in the symbiotic system was analyzed.

[0218] The procedure was as follows. After culturing the KH-1 strain and the KH-2 strain in LB medium overnight, they were washed twice with PBS buffer to remove the medium components. 3 L of an inorganic salt medium (the above composition) containing canola oil (Nisshin Canola Oil, Nisshin Oillio, Tokyo) at a final concentration of 1% (v / v) was inoculated with the KH-1 strain alone having an OD 660 measurement value of 0.02, or each microorganism at a cell optical density such that the measurement value of OD 660 was KH-1 strain: KH-2 strain = 0.018: 0.02, and then cultured in a fermenter at 15°C. After culturing for 71 hours, total RNA was extracted using the Cica geneus RNA Prep Kit (For Tissue) (Kanto Chemical).

[0219] Using total RNA as a template, genomic DNA was removed and cDNA was synthesized using the PrimeScript TM RT reagent Kit with gDNA Eraser Perfect Real Time (Takara Bio Inc.). Thereafter, the cDNA stock solution was diluted 3-fold using the dilution solution attached to the kit. Using synthetic primers specific to the genes encoding the first lipase and the second lipase of the KH-1 strain, Applied Biosystems (registered trademark) StepOnePlus TM(Applied Biosystems) performed quantitative real-time RT-PCR. Here, the first lipase and the second lipase of the KH-1 strain refer to those whose representative amino acid sequences and base sequences are shown in SEQ ID NOs: 1 to 4. The PCR reaction was carried out in a 20 μl solution containing PowerUp TM SYBR® Green Master Mix (Thermo Fisher Scientific) (10 μl), each primer (final concentration 0.5 μM), and cDNA (1 μl). The PCR reaction was performed using the fast cycling mode. After performing 1 cycle of denaturation at 95°C for 2 minutes, a program of repeating 40 cycles of 3 seconds at 95°C and 30 seconds at 60°C was carried out.

[0220] The expression level was normalized by the expression level of RNA polymerase, sigma 70 (rpoD). After confirming that the melting curve was a single peak, the data was analyzed by the comparative Ct method (ΔΔCt method).

[0221] (Results) The results are shown in the left panel of Figure 19. In the symbiotic system of the KH-1 strain and the KH-2 strain, the expression of the first lipase and the second lipase of the KH-1 strain was unexpectedly improved compared to the KH-1 strain alone. This indicates that the presence of the KH-2 strain induces the enhancement of lipase expression in the KH-1 strain. Without wishing to be bound by theory, it is expected that the lipid degradation products generated by the KH-2 strain function as inducers to activate the expression of the lipase in the KH-1 strain. Therefore, it is expected that microorganisms having similar lipid degradation ability can be suitably used as Yarrowia yeast, and strains having similar sensitivity can be suitably used as Burkholderia bacteria.

[0222] (Analysis of the KH-2 strain) Similarly, the gene expression of the KH-2 strain in the symbiotic system was analyzed.

[0223] In the same manner as described above, after culturing the KH-1 strain and the KH-2 strain in LB medium overnight, they were washed twice with PBS buffer to remove the medium components. 3 L of an inorganic salt medium (the above composition) containing 1% (v / v) of canola oil (Nisshin Canola Oil, Nisshin Oillio, Tokyo) at a final concentration was inoculated with 5×10 5 cells / mL of the KH-2 strain alone, or 2.5×10 5 cells / mL of the KH-1 strain + 2.5×10 5 cells / mL of the KH-2 strain, and then cultured in a fermenter at 15°C for 48 hours.

[0224] In the same manner as the above KH-1 strain, total RNA was extracted, cDNA was synthesized, and quantitative real-time RT-PCR was performed using synthetic primers specific to the genes encoding the first lipase and the second lipase of the KH-2 strain. Here, the first lipase and the second lipase of the KH-2 strain refer to those whose representative amino acid sequences and nucleotide sequences are shown in SEQ ID NOs: 5 to 8. The PCR reaction was performed in the same manner as the above KH-1 strain.

[0225] The expression level was normalized by the expression level of alpha-1,2-mannosyltransferase (alg9). After confirming that the melting curve was a single peak, the data was analyzed by the comparative Ct method (ΔΔCt method).

[0226] (Results) The results are shown in the right panel of Figure 19. In the symbiotic system of the KH-1 strain and the KH-2 strain, the expression of the first lipase and the second lipase of the KH-2 strain was unexpectedly improved compared to the KH-2 strain alone. This indicates that the presence of the KH-1 strain also induces an enhancement of the lipase expression of the KH-2 strain, and it is expected that the lipid degradation products generated by the KH-1 strain function as inducers to activate the expression of the lipase of the KH-2 strain. Therefore, it is expected that microorganisms having similar lipid degradation ability as Burkholderia bacteria can be preferably used, and strains having similar sensitivity as Yarrowia yeast can be preferably used.

[0227] (Example 20: Lipase activity of symbiotic culture supernatant) Lipase activity in the culture supernatants of strains KH-1 and KH-2 was measured using a model substrate.

[0228] The KH-1 and KH-2 strains were cultured overnight in LB medium, then washed twice with PBS buffer to remove medium components. The OD was measured by adding 3 L of inorganic salt medium (composition as above) containing 1% (v / v) canola oil (Nissin Canola Oil, Nisshin Oillio, Tokyo). 660 The KH-1 strain alone, which had an OD of 0.02, or 660 Each microorganism was inoculated at an optical density such that the measured values of KH-1 strain:KH-2 strain were 0.018:0.02, and then cultured in a fermenter at 15°C. The supernatant was collected 48 and 71 hours after the start of culture. 4-Nitrophenyl palmitate or 4-nitrophenyl butyrate was added to a 3% (v / v) aqueous Triton® X-100 solution and heated at 70°C to prepare a substrate solution with a final concentration of 5 mM. 60 μL each of the substrate solution, 150 mM GTA buffer (pH 7.0), and the culture supernatant were mixed, and the absorbance at 410 nm (indicating free 4-nitrophenol) was monitored for 1 minute at room temperature.

[0229] The results are shown in Figure 20. The culture supernatant of the symbiotic system of KH-1 and KH-2 strains showed stronger lipase activity than the culture supernatant of KH-1 strain alone. Thus, it can be seen that the combination of microorganisms disclosed herein can decompose various oils and fats using the lipase produced.

[0230] Example 21: Identification of other strains Acquisition of different strains Samples were collected from a river near a food factory where oil-containing wastewater is discharged, and microorganisms were isolated from the samples. The isolated microorganisms were tested for lipase production in a low-temperature environment (15°C) and their ability to decompose oils and fats was examined. As a result, microorganisms capable of decomposing oils and fats at low temperatures were found. These microbial strains were named KH-1AL1, KH-1AL2, and KH-1AL3, respectively.

[0231] To further characterize the KH-1AL1 strain, KH-1AL2 strain, and KH-1AL3 strain, gene sequence analysis of 16S rDNA was performed. Since the partial nucleotide sequence of 16S rDNA of the KH-1AL1 strain was 100% identical in homology to Burkholderia ambifaria, it was identified as Burkholderia ambifaria. The partial nucleotide sequence of 16S rDNA of the KH-1AL2 strain was 99.9% identical in homology to Burkholderia contaminans and was classified into the same group as B. seminalis, B. territorii, and B. cepacia (homologies in the partial nucleotide sequences of 16S rDNA were 99.7%, 99.7%, and 99.8%, respectively) on the molecular phylogenetic tree. As a result, the KH-1AL2 strain was identified as a bacterium of the Burkholderia cepacia complex. The partial nucleotide sequence of 16S rDNA of the KH-1AL3 strain was 99.9% identical in homology to Burkholderia contaminans and was classified into the same group as B. seminalis, B. territorii, and B. cepacia (homologies in the partial nucleotide sequences of 16S rDNA were 99.7%, 99.7%, and 99.8%, respectively) on the molecular phylogenetic tree. As a result, the KH-1AL3 strain was identified as a bacterium of the Burkholderia cepacia complex. The Burkholderia cepacia complex is a classification of genetically very close Burkholderia - genus microorganisms, including ambifaria, anthina, arboris, cenocepacia, cepacia, contaminans, diffusa, dolosa, lata, latens, metallica, multivorans, pseudomultivorans, puraquae, pyrrocinia, seminalis, stabilis, stagnalis, territorii, ubonensis, and vietnamiensis (Martina P et al., Int J Syst Evol Microbiol. 2018 Jan;68(1):14 - 20.). As a result of the analysis, since various bacteria belonging to the Burkholderia cepacia complex showed high oil and / or fatty acid degradation ability, the bacteria belonging to the Burkholderia cepacia complex are expected to be particularly useful.

[0232] (Example 22: Symbiotic system using other combinations) Similar to Example 17, the alternative strains identified in Examples 8 and 21 were added, and a comparison was made of mixed cultures in which the KH-1 strain, KH-1AL1 strain, and KH-1AL3 strain were each replaced with one-tenth of the cell number of the KH-2 strain, KH-2AL1 strain, or KH-2AL3 strain, and the oil degradation ability was tested.

[0233] In an inorganic salt medium (the above composition, pH 7) containing 1% (v / v) of canola oil (Nisshin Canola Oil, Nisshin Oillio, Tokyo) at a final concentration, the KH-1 strain, KH-1AL1 strain, or KH-1AL3 strain alone, or a combination of these with the KH-2 strain, KH-2AL1 strain, or KH-2AL3 strain, was added so that the total cell concentration became 2×10 6 cells / mL, and the culture was carried out at 15°C. The culture supernatant obtained at the time of culturing for 48 hours was analyzed in the same manner as above using an oil content measurement reagent kit (Kyoritsu Chemical-Check Lab., Tokyo) (measurement reagent kit by the polynipum extract substance measurement method).

[0234] (Results) The results under each condition are shown in the following table.

Table 2

[0235] For any of the Burkholderia bacteria of the present disclosure, further improvement in oil degradation ability was observed when combined with the Yarrowia yeast of the present disclosure. Thus, the combination of the Burkholderia bacteria and Yarrowia yeast of the present disclosure achieves unexpectedly excellent oil degradation ability.

[0236] (Example 23: Decomposition of Trans-Fatty Acid-Containing Oil and Fat by Lipase) Examine the decomposition activities of trielaidin by the first and second lipases of the KH-1 strain in Example 19, and the decomposition activities of trielaidin by the first and second lipases of the KH-2 strain.

[0237] From the cultures of the KH-1 strain and / or the KH-2 strain, or the cultures of Escherichia coli strains transfected to express the lipase of interest, the first lipase of the KH-1 strain, the second lipase of the KH-1 strain, the first lipase of the KH-2 strain, and the second lipase of the KH-2 strain are purified by hydrophobic column chromatography or the like. These purified lipases are mixed with trielaidin to examine the decomposition of trielaidin.

[0238] It was shown that both the first and second lipases of the KH-1 strain and the first and second lipases of the KH-2 strain decompose trielaidin, which is a trans-form triglyceride. In the symbiotic system of the present disclosure, since the expression of the first and second lipases of the KH-1 strain and the first and second lipases of the KH-2 strain can be improved (Example 19), it is expected that various oils and fats including trans-fatty acid-containing oils and fats will be efficiently decomposed.

[0239] (Example 24: Confirmation Test) In addition to the above gas chromatography analysis, the confirmation of the decomposition of oils and fats may also be performed by oil analysis such as in Example 3, thin layer chromatography analysis, or the like.

[0240] Obtain the culture supernatant in the same manner as in Example 20, mix this with canola oil, and incubate. The culture supernatants of the symbiotic systems of the KH-1 strain and the KH-2 strain exhibit stronger lipase activity compared to the culture supernatant of the KH-1 strain alone.

[0241] In addition to the mixing ratios of Burkholderia bacteria and Yarrowia yeast tested in Example 17, for example, add them at a cell number ratio of 19:1, 1:19, etc. and culture at 15 °C or 28 °C. Similar to the above, oil decomposition superior to that of any single microorganism can be confirmed.

[0242] Growth curves of microorganisms of Burkholderia bacteria and Yarrowia yeast are created, and the amount of microorganisms obtained at a predetermined time, for example, 24 hours, is determined from the results. The lipolytic ability is measured by combining the amounts of each microorganism that can be prepared within the predetermined time. A combination that achieves a lipolytic ability exceeding the lipolytic ability calculated from the value of the lipolytic ability of each pure culture achieves an excellent lipolytic ability.

[0243] Burkholderia bacteria and Yarrowia yeast are co-grown by mixed culture. The mixed culture solution is sampled at regular intervals, and the cell concentration ratio of the microorganisms during mixed culture is determined by calculating the respective microorganism concentrations. Using the mixed culture solution obtained after culturing for a certain time, for example, 24 hours, the lipolytic ability at that mixing ratio is examined. It is confirmed that the lipolytic ability exceeds the lipolytic ability calculated from the value of the lipolytic ability of each pure culture.

[0244] (Example 25: Acquisition of additional alternative strains of Burkholderia bacteria and Yarrowia yeast useful in symbiotic systems) Alternative strains of microorganisms that can be used in the combinations of microorganisms of the present disclosure can be obtained as follows. PBS is added to various separation sources such as a microorganism preservation library, soil, river water, lake water, and activated sludge to prepare a dilution series, which is spread on an inorganic salt agar medium using canola oil as the sole carbon source. Colonies are obtained by statically culturing the inoculated agar medium at 15 °C or 28 °C. Among them, those that form a clear zone around the colonies are picked up and inoculated into an inorganic salt medium supplemented with canola oil together with the KH-1 strain, KH-1AL1 strain, KH-1AL3 strain, KH-2 strain, KH-2AL1 strain, or KH-2AL3 strain, and cultured at 15 °C or 28 °C. After culturing, microorganisms having excellent lipolytic ability are obtained by analyzing the degree of decomposition of the oil and fat in the culture supernatant by thin layer chromatography, as compared with the KH-1 strain, KH-1AL1 strain, KH-1AL3 strain, KH-2 strain, KH-2AL1 strain, or KH-2AL3 strain alone.

[0245] (Example 26: Use in Equipment) Bullkhoderia bacteria and Yarowia yeast are cultured in the same manner as in the above examples to obtain a stock culture solution. This is diluted to obtain a microbial preparation. This is refrigerated and stored in the microbial storage tank of the automatic amplification input device to serve as an inoculum. This inoculum is automatically inoculated in appropriate amounts into the inorganic salt medium in the culture amplification tank of the same device, and cultured until the number of microorganisms reaches the same cell concentration as that of the microbial preparation. By appropriately introducing this into the drainage volume of the oil decomposition treatment tank, the microbial concentration of the decomposing bacteria in the oil-treated water is adjusted to the target concentration, and the drainage from a food factory that discharges wastewater containing a large amount of oil and fat is decomposed and treated. As a result, a significant reduction in the normal hexane value is observed compared to the control example where no microorganisms are introduced.

[0246] (Example 27: Additional Application Examples) An appropriate amount of Bullkhoderia bacteria and Yarowia yeast are inoculated into a disposable food waste processor and treated at an appropriate temperature. The normal hexane value in the drainage from the food waste processor is measured. A significant reduction in the value is observed compared to the control example where no microorganisms are introduced.

[0247] The oily sludge recovered by floatation separation is put into a culture tank, and an appropriate amount of inorganic salt medium is added. An appropriate amount of Bullkhoderia bacteria and Yarowia yeast are inoculated herein, and incubated at an appropriate temperature while stirring and aerating. Then, the amount of decomposition and reduction of the oily sludge is examined by measuring the amount of oil based on the normal hexane value of the treated liquid, or by measuring the weight of the residue mainly composed of the oil remaining after evaporating the water. As a result, significant decomposition of the oily sludge is observed compared to the control example where no microorganisms are introduced.

[0248] Carriers such as charcoal, various plastics, and ceramic pieces are put into a grease trap, and an appropriate amount of Bullkhoderia bacteria and Yarowia yeast are automatically introduced every day after the operation of the cafeteria ends. Over time, water is sampled immediately before the start of the operation, and the normal hexane value is analyzed. In addition to a significant decrease in the normal hexane value being observed compared to the control example where no microorganisms are introduced, effects such as a reduction in the adhesion and floating of oil are also observed in the appearance of the grease trap itself.

[0249] (Note) As described above, the present disclosure has been illustrated using preferred embodiments of the present disclosure. However, it is understood that the scope of the present invention should be construed only by the claims. It is understood that patents, patent applications, and other documents cited herein should be incorporated by reference herein as if the contents thereof were specifically set forth herein.

[0250] This application claims the benefit of priority to Japanese Patent Application No. 2019-163252, filed with the Japan Patent Office on September 6, 2019, and Japanese Patent Application No. 2020-001744, filed with the Japan Patent Office on January 8, 2020, the entire contents of which are incorporated herein by reference.

Industrial Applicability

[0251] The present disclosure provides microorganisms having ester (e.g., fats and oils) and / or fatty acid-degrading ability and compositions containing the same. By using such microorganisms or compositions, it is possible to reduce the environmental load caused by food factory wastewater containing a large amount of ester (e.g., fats and oils) and / or fatty acids. Further, the present disclosure provides combinations of microorganisms having fats and oils and / or fatty acid-degrading ability, and by using such combinations of microorganisms, it is possible to reduce the environmental load caused by food factory wastewater containing a large amount of fats and oils and / or fatty acids.

Deposit Number

[0252] KH-1 (NITE BP-02731) KH-1AL1 (NITE BP-02977) KH-1AL2 (NITE BP-02978) KH-1AL3 (NITE BP-02979) KH-2 (NITE BP-02732) KH-2AL1 (NITE BP-03091) KH-2AL3 (NITE BP-03092)

Sequence Listing Free-Text

[0253] Mature nucleotide sequence of the first lipase of strain KH-1, SEQ ID NO: 1 Mature amino acid sequence of the first lipase of strain KH-1, SEQ ID NO: 2 Mature nucleotide sequence of the second lipase of strain KH-1, SEQ ID NO: 3 Mature amino acid sequence of the second lipase of strain KH-1, SEQ ID NO: 4 Mature sequence of the representative nucleotide sequence of the first lipase of strain KH-2, SEQ ID NO: 5 Mature sequence of the representative amino acid sequence of the first lipase of strain KH-2, SEQ ID NO: 6 Mature sequence of the representative nucleotide sequence of the second lipase of strain KH-2, SEQ ID NO: 7 Mature sequence of the representative amino acid sequence of the second lipase of strain KH-2, SEQ ID NO: 8

Claims

1. An oil decomposer for decomposing trans fatty acids, comprising Yarrowia yeast having the ability to decompose trans fatty acids or a culture thereof, which is Yarrowia lipolytica.

2. An oil decomposer for decomposing oils and fats containing trans fatty acids, comprising Yarrowia yeast having the ability to decompose oils and fats containing trans fatty acids or a culture thereof, which is Yarrowia lipolytica.

3. Yarrowia yeast or a culture thereof, which is Yarrowia lipolytica strain KH-2 (a microbial strain identified by accession number NITE BP-02732), Yarrowia lipolytica strain KH-2AL1 (a microbial strain identified by accession number NITE BP-03091), or Yarrowia lipolytica strain KH-2AL3 (a microbial strain identified by accession number NITE BP-03092).

4. An oil decomposer comprising the Yarrowia yeast or a culture thereof according to Claim 3.

5. The oil decomposer according to Claim 1, 2 or 4, further comprising an additional oil treatment component.

6. (a) for decomposing esters and / or fatty acids at 15°C, (b) for decomposing esters containing short-chain to medium-chain fatty acids (C2-C12), and (c) for decomposing oils and fats containing short-chain to long-chain fatty acids (C2 or more) The oil decomposer according to Claim 1, 2, 4 or 5, for at least one further selected from the group consisting of.

7. A kit for decomposing trans fatty acids and / or oils and fats containing trans fatty acids, comprising the Yarrowia yeast or a culture thereof according to Claim 3, or the oil decomposer according to Claim 1, 2, 4 or 5, and an additional oil treatment component.

8. A method for decomposing and removing trans fatty acids and / or oils and fats containing trans fatty acids, comprising allowing the Yarrowia yeast or a culture thereof according to Claim 3, or the oil decomposer according to Claim 1, 2, 4 or 5 to act on a treatment target.

9. (a) a step of decomposing esters and / or fatty acids at 15°C, (b) a step of decomposing esters containing short-chain to medium-chain fatty acids (C2-C12), and (c) a step of decomposing oils and fats containing short-chain to long-chain fatty acids (C2 or more) The method according to Claim 8, further comprising at least one step selected from the group consisting of.

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