Novel microorganism having high ability to degrade oil at low temperature

Abstract

To provide a novel microorganism having an ester degrading ability.SOLUTION: 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.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present disclosure relates to microorganisms having ester (e.g., fats and oils) and / or fatty acid decomposition ability and uses thereof. More particularly, the present disclosure relates to Yarrowia yeast (e.g., Yarrowia lipolytica) that decomposes fats and oils that are difficult to decompose in the past, such as fats and oils containing trans fatty acids. The present disclosure also relates to Yarrowia yeast that improves the fat decomposition ability of Burkholderia bacteria. Furthermore, the present disclosure relates to combinations of microorganisms having fat decomposition ability and uses thereof. More particularly, the present disclosure relates to combinations of microorganisms having improved fat decomposition ability compared to at least one of the microorganisms in the combination alone. The present disclosure also relates to enhanced expression of lipase by combinations of microorganisms. More particularly, the present disclosure relates to combinations of Burkholderia bacteria and Yarrowia yeast. [Background technology]

[0002] Wastewater from food factories and oil and fat factories contains a large amount of oil. This oil causes various declines in biological treatment functions, such as a decrease in the treatment capacity of activated sludge, insufficient solid-liquid separation due to decreased sedimentation, membrane fouling in membrane separation activated sludge process (MBR), and inhibition of methane fermentation in anaerobic digestion. For this reason, oil is removed, for example, by a pressurized floatation separator as a pre-stage of biological treatment of wastewater containing a high oil content. In addition, kitchen wastewater from the food service industry also contains a lot of oil, so grease traps are installed to remove the oil. Both pressurized floatation separators and grease traps have problems such as being a source of odor and pests, the cost of collecting and transporting the separated oil and treating it as industrial waste, and the labor and cost of management and cleaning. As a means of solving such problems, oil decomposition technology using microorganisms has been considered, and several related microbial preparations are commercially available, but it is extremely difficult to reduce the oil concentration by microbial decomposition to the desired level within a settable retention time. For this reason, pressurized floatation separators and conventional grease traps are currently used in most cases.

[0003] In addition, in the fermentation treatment of food waste, if there is a large amount of oil, problems such as fermentation inhibition and high oil content in the wastewater from an elimination type treatment machine can occur. In addition, the oily sludge separated and collected by a pressurized flotation separation device or a grease trap becomes industrial waste, and its treatment is very costly. Therefore, the decomposition of these oils by microorganisms has been considered, but the reality is that there is a limit to the decomposition ability of microorganisms, just like the above-mentioned wastewater treatment.

[0004] As described above, the decomposition rate is an issue when using microorganisms to remove oil, but the decrease in activity due to low temperatures, especially in winter, often makes it difficult to apply microorganisms. In particular, at low temperatures in winter, the rate at which microorganisms decompose oils and fats is extremely slow, and it is thought that it is impossible to use certain microorganisms for wastewater treatment or waste treatment. [Prior art documents] [Non-patent literature]

[0005] [Non-Patent Document 1] Yoshikazu Kita et al., Hitachi Chemical Technical Report: No. 46: pp. 49-54 (2006) Summary of the Invention [Means for solving the problem]

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

[0007] Thus, the present disclosure provides: (Item A1) Yarrowia yeast has the ability to break down trans fatty acids. (Item A2) A Yarrowia yeast having the ability to assimilate trans fatty acids. (Item A3) A Yarrowia yeast having the ability to decompose oils and fats containing trans fatty acids. (Item A4) A Yarrowia yeast having the ability to assimilate oils and fats containing trans fatty acids. (Item A5) A Yarrowia yeast having the ability to degrade esters (e.g., fats and oils) and / or fatty acids at 15°C. (Item A6) A Yarrowia yeast having the ability to utilize esters (e.g., fats and oils) and / or fatty acids at 15°C. (Item A7) The Yarrowia yeast according to any one of the preceding items, wherein the ability to assimilate or decompose is maintained at 15°C. (Item A8) A Yarrowia yeast having the ability to decompose esters containing short- to medium-chain fatty acids (C2 to C12). (Item A9) A Yarrowia yeast that has the ability to decompose fats and oils containing short- to long-chain fatty acids (C2 or higher). (Item A10) A Yarrowia yeast having a higher decomposition activity for 4-nitrophenyl esters of short- to medium-chain fatty acids (C2 to C12) than for 4-nitrophenyl esters of long-chain fatty acids (C13 or longer), and having the ability to decompose triglycerides of long-chain fatty acids (C13 or longer). (Item A11) The Yarrowia yeast of any one of the preceding paragraphs, having two or more of the characteristics specified in paragraph A above. (Item A12) The yeast according to any one of the preceding items, which is Yarrowia lipolytica. (Item A13) 2. The Yarrowia yeast according to any one of the above items, which is the Yarrowia yeast KH-2 strain identified by accession number NITE BP-02732, or a derivative thereof, said derivative having the characteristics of the Yarrowia yeast according to any one or more of the above items A. (Item A14) An oil decomposition agent comprising the Yarrowia yeast according to any one of the preceding items. (Item A15) An oil decomposer according to any one of the preceding items, comprising a further oil treatment component. (Item A16) (a) To break down trans-fatty acids, (b) To break down fats and oils containing trans fatty acids. (c) for decomposing esters (e.g., fats and oils) and / or fatty acids at 15°C; (d) To decompose esters containing short- to medium-chain fatty acids (C2 to C12); and (e) To break down fats and oils containing short- and long-chain fatty acids (C2 or higher) A composition comprising the Yarrowia yeast described in any one of the above items or the oil decomposing agent described in any one of the above items for at least one selected from the group consisting of: (Item A17) A kit for decomposing esters (e.g., fats and oils), comprising the Yarrowia yeast described in any one of the above items or the oil decomposing agent described in any one of the above items, or the composition described in any one of the above items, and a further oil-treating component. (Item A18) A method for decomposing and removing esters (e.g., fats and oils), comprising applying the Yarrowia yeast described in any one of the above items, or the oil decomposing agent described in any one of the above items, or the composition described in any one of the above items to a subject to be treated. (Item A19) The method according to any one of the preceding items, wherein the target to be treated comprises a trans fatty acid or a trans fatty acid-containing oil or fat. (Item A20) (a) decomposing trans fatty acids; (b) decomposing trans fatty acid-containing oils and fats; (c) decomposing esters (e.g., fats and oils) and / or fatty acids at 15°C; (d) decomposing the short- to medium-chain fatty acid (C2 to C12)-containing ester; and (e) decomposing fats and oils containing short-chain to long-chain fatty acids (C2 or more); The method according to any one of the preceding items, comprising at least one step selected from the group consisting of: (Item B1) A composition for treating fats and oils comprising a Burkholderia bacterium in combination with a lipase-producing Yarrowia yeast and a lipase-producing Burkholderia bacterium. (Item B2) A composition for treating fats and oils comprising a Yarrowia yeast in combination with a lipase-producing Yarrowia yeast and a lipase-producing Burkholderia bacterium. (Item B3) A combination for treating fats and oils comprising a combination of a Burkholderia bacterium and a Yarrowia yeast, wherein both the Burkholderia bacterium and the Yarrowia yeast produce lipase. (Item B4) The composition or combination of any one of the preceding claims, wherein the Yarrowia yeast comprises Yarrowia lipolytica. (Item B5) The composition or combination described in any one of the above items, wherein the Burkholderia bacterium comprises a bacterium of the genus Burkholderia. (Item B6) The composition or combination of any one of the preceding items, wherein the Burkholderia bacterium comprises Burkholderia arboris, Burkholderia ambifaria, or Burkholderia cepacia complex. (Item B7) The composition or combination described in any one of the above items, wherein the combination of the Burkholderia bacterium and the Yarrowia yeast has a higher lipolytic ability than that calculated from the lipolytic ability of each of the bacteria cultured alone. (Item B8) The composition or combination according to any one of the preceding items, wherein the ratio of the cell number of the Burkholderia bacterium to the cell number of the Yarrowia yeast is 1:20 to 20:1. (Item B9) The composition or combination described in any one of the preceding items, wherein at least one of the Burkholderia bacterium and the Yarrowia yeast has the ability to decompose fatty acids at 15°C. (Item B10) The composition or combination according to any one of the preceding items, wherein the Burkholderia bacterium is Burkholderia arboris KH-1 strain (strain identified by accession number NITE BP-02731), Burkholderia ambifaria KH-1AL1 strain (strain identified by accession number NITE BP-02977), Burkholderia cepacia complex KH-1AL2 strain (strain identified by accession number NITE BP-02978) or Burkholderia cepacia complex KH-1AL3 strain (strain identified by accession number NITE BP-02979), or a derivative thereof. (Item B11) The composition or combination according to any one of the preceding items, wherein the Yarrowia yeast is Yarrowia lipolytica KH-2 strain (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. (Item B12) A composition or combination according to any one of the preceding items which is an oil decomposer. (Item B13) An oil decomposer according to any one of the preceding items, comprising a further oil treatment component. (Item B14) A method for decomposing and removing oil, comprising applying a composition or combination described in any one of the above items, or an oil decomposing agent described in any one of the above items to an object to be treated. (Item B15) A composition for improving lipase production in a lipase-producing Yarrowia yeast comprising a Burkholderia bacterium. (Item B16) A composition for improving lipase production in a lipase-producing Burkholderia bacterium, comprising a Yarrowia yeast. (Item B17) A composition for enhancing the ability of lipase-producing Yarrowia yeast to process fats and oils, comprising a Burkholderia bacterium. (Item B18) A composition for enhancing the ability of lipase-producing Burkholderia bacteria to process fats and oils, comprising a Yarrowia yeast. (Item B19) A method for improving lipase production by at least one of a Burkholderia bacterium and a Yarrowia yeast, comprising a step of mixing and culturing the Burkholderia bacterium and a Yarrowia yeast. (Item B20) Use of a Burkholderia bacterium in combination with a lipase-producing Yarrowia yeast and a lipase-producing Burkholderia bacterium for treating fats and oils. (Item B21) Use of a lipase-producing Yarrowia yeast in combination with a lipase-producing Burkholderia bacterium for treating fats and oils. (Item B22) 1. Use of a Burkholderia bacterium and a Yarrowia yeast in combination for treating fats and oils, wherein both the Burkholderia bacterium and the Yarrowia yeast produce lipase. (Item B23) The use according to any one of the preceding claims, wherein the Yarrowia yeast comprises Yarrowia lipolytica. (Item B24) The use according to any one of the preceding items, wherein the Burkholderia bacterium comprises a bacterium of the genus Burkholderia. (Item B25) The use according to any one of the preceding items, wherein the Burkholderia bacterium comprises Burkholderia arboris, Burkholderia ambifaria, or Burkholderia cepacia complex. (Item B26) The use according to any one of the preceding items, wherein the combination of the Burkholderia bacterium and the Yarrowia yeast has a higher lipolytic ability than that calculated from the lipolytic ability of each of the bacteria and the Yarrowia yeast cultures alone. (Item B27) The use according to any one of the preceding items, wherein the ratio of the number of cells of the Burkholderia bacterium to the number of cells of the Yarrowia yeast is 1:20 to 20:1. (Item B28) The use according to any one of the preceding items, wherein at least one of the Burkholderia bacterium and the Yarrowia yeast has the ability to decompose fatty acids at 15°C. (Item B29) The use according to any one of the preceding items, wherein the Burkholderia bacterium is Burkholderia genus bacteria KH-1 strain (strain specified by accession number NITE BP-02731), KH-1AL1 strain (strain specified by accession number NITE BP-02977), KH-1AL2 strain (strain specified by accession number NITE BP-02978) or KH-1AL3 strain (strain specified by accession number NITE BP-02979), or a derivative thereof. (Item B30) The use according to any one of the preceding items, wherein the Yarrowia yeast is Yarrowia lipolytica KH-2 strain (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. (Item B31) The use according to any one of the preceding items, in combination with a further oil treatment component. (Item B32) The use according to any one of the above items, comprising subjecting a subject to treatment to a combination of the Burkholderia bacterium and the Yarrowia yeast. (Item B33) Use of Burkholderia bacteria to improve lipase production in lipase-producing Yarrowia yeast. (Item B34) Use of Yarrowia yeast to improve lipase production in lipase-producing Burkholderia bacteria. (Item B35) Use of Burkholderia bacteria to enhance the ability of lipase-producing Yarrowia yeast to process fats and oils. (Item B36) Use of Yarrowia yeast to enhance the ability of lipase-producing Burkholderia bacteria to process fats and oils. (Item B37) Use of a Burkholderia bacterium and a Yarrowia yeast for improving lipase production in at least one of the Burkholderia bacterium and the Yarrowia yeast. (Item B38) A method for treating fats and oils with a combination of a lipase-producing Yarrowia yeast and a lipase-producing Burkholderia bacterium, comprising the step of contacting the Burkholderia bacterium with a subject. (Item B39) A method for treating fats and oils with a combination of lipase-producing Yarrowia yeast and lipase-producing Burkholderia bacteria, comprising the step of contacting the Yarrowia yeast with a locus. (Item B40) A method for treating fats and oils with a combination of lipase-producing Yarrowia yeast and lipase-producing Burkholderia bacteria, comprising the step of contacting a combination of Burkholderia bacteria and Yarrowia yeast with a subject. (Item B41) The method of any one of the preceding claims, wherein the Yarrowia yeast comprises Yarrowia lipolytica. (Item B42) The method according to any one of the preceding items, wherein the Burkholderia bacterium comprises a bacterium of the genus Burkholderia. (Item B43) The method of any one of the preceding claims, wherein the Burkholderia bacterium comprises Burkholderia arboris, Burkholderia ambifaria, or Burkholderia cepacia complex. (Item B44) The method according to any one of the preceding items, wherein the combination of the Burkholderia bacterium and the Yarrowia yeast has a higher lipolytic ability than that calculated from the lipolytic ability of each of the Burkholderia bacterium and the Yarrowia yeast cultured alone. (Item B45) The method according to any one of the preceding items, wherein the ratio of the number of cells of the Burkholderia bacterium to the number of cells of the Yarrowia yeast is 1:20 to 20:1. (Item B46) The method according to any one of the preceding 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 preceding items, wherein the Burkholderia bacterium is Burkholderia genus bacteria KH-1 strain (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 preceding items, wherein the Yarrowia yeast is Yarrowia lipolytica KH-2 strain (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. (Item B49) The method according to any one of the preceding claims, wherein an additional oil treatment component is used. (Item B50) The method according to any one of the preceding items, which is an oil decomposition and removal method. (Item B51) 1. A method for improving lipase production by a lipase-producing Yarrowia yeast, comprising the step of introducing a Burkholderia bacterium into a locus. (Item B52) 1. A method for improving lipase production by a lipase-producing Burkholderia bacterium, comprising the step of introducing a Yarrowia yeast into a locus. (Item B53) A method for enhancing the ability of a lipase-producing Yarrowia yeast to process fats and oils, comprising the step of introducing a Burkholderia bacterium into a subject. (Item B54) A method for enhancing the ability of lipase-producing Burkholderia bacteria to process fats and oils, comprising the step of introducing a Yarrowia yeast into a subject. (Item C1) Yarrowia yeast has the ability to break down trans fatty acids. (Item C2) A Yarrowia yeast having the ability to decompose oils and fats containing trans fatty acids. (Item C3) A Yarrowia yeast having the ability to decompose esters and / or fatty acids at 15°C. (Item C4) A Yarrowia yeast having the ability to decompose esters containing short- to medium-chain fatty acids. (Item C5) A Yarrowia yeast having the ability to decompose oils and fats containing short- to long-chain fatty acids. (Item C6) A Yarrowia yeast having a higher decomposition activity for 4-nitrophenyl esters of short- to medium-chain fatty acids (C2 to C12) than for 4-nitrophenyl esters of long-chain fatty acids (C13 or longer), and having the ability to decompose triglycerides of long-chain fatty acids (C13 or longer). (Item C7) A Yarrowia yeast having the ability to improve lipase production by lipase-producing Burkholderia bacteria. (Item C8) A Yarrowia yeast having the ability to impart to a Burkholderia bacterium a higher ability to decompose fats, oils or fatty acids than the ability of the Burkholderia bacterium to decompose fats, oils or fatty acids when cultured alone. (Item C9) The Yarrowia yeast according to any one of the preceding items, wherein the Burkholderia bacterium comprises a bacterium of the genus Burkholderia. (Item C10) The Yarrowia yeast of any one of the preceding paragraphs, wherein the Burkholderia bacterium comprises 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 to C6, and The characteristics of the Yarrowia yeast according to any one or more of items C7 to C10; The Yarrowia yeast having the structure (Item C12) The Yarrowia yeast according to any one of the preceding items, which is Yarrowia lipolytica. (Item C13) The Yarrowia yeast according to any one of the preceding items, which is Yarrowia lipolytica KH-2 strain (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, wherein the derivative has the characteristics of the Yarrowia yeast according to any one of the preceding items. (Item C14) An oil decomposition agent comprising the Yarrowia yeast according to any one of the preceding items. (Item C15) An oil decomposer according to any one of the preceding items, comprising a further oil treatment component. (Item C16) (a) To break down trans-fatty acids, (b) To break down fats and oils containing trans fatty acids. (c) decomposing esters and / or fatty acids at 15°C; (d) To decompose esters containing short- to medium-chain fatty acids (C2 to C12); and (e) To break down fats and oils containing short- and long-chain fatty acids (C2 or higher) A composition comprising the Yarrowia yeast or oil decomposing agent described in any one of the above items for at least one selected from the group consisting of: (Item C17) A kit for ester degradation comprising the Yarrowia yeast or oil decomposing agent described in any one of the preceding items, or the composition described in any one of the preceding items, and an additional oil treatment component. (Item C18) A method for decomposing and removing esters, comprising applying the Yarrowia yeast, oil decomposing agent, or composition described in any one of the above items to a subject to be treated. (Item C19) The method according to any one of the preceding items, wherein the target to be treated comprises a trans fatty acid or a trans fatty acid-containing oil or fat. (Item C20) (a) decomposing trans fatty acids; (b) decomposing trans fatty acid-containing oils and fats; (c) decomposing the esters and / or fatty acids at 15° C.; (d) decomposing the short- to medium-chain fatty acid (C2 to C12)-containing ester; and (e) decomposing fats and oils containing short-chain to long-chain fatty acids (C2 or more); The method according to any one of the preceding items, comprising at least one step selected from the group consisting of: (Item C21) A composition for treating oils or fatty acids comprising a Burkholderia bacterium in combination with a lipase-producing Yarrowia yeast and a lipase-producing Burkholderia bacterium. (Item C22) A composition for treating oils, fats or fatty acids comprising a Yarrowia yeast in combination with a lipase-producing Yarrowia yeast and a lipase-producing Burkholderia bacterium. (Item C23) A combination for treating oils or fatty acids comprising a combination of a Burkholderia bacterium and a Yarrowia yeast, wherein both the Burkholderia bacterium and the Yarrowia yeast produce lipase. (Item C24) The composition or combination of any one of the preceding claims, wherein the Yarrowia yeast comprises Yarrowia lipolytica. (Item C25) The composition or combination described in any one of the above items, wherein the Burkholderia bacterium comprises a bacterium of the genus Burkholderia. (Item C26) The composition or combination of any one of the preceding items, wherein the Burkholderia bacterium comprises Burkholderia arboris, Burkholderia ambifaria, or Burkholderia cepacia complex. (Item C27) The composition or combination described in any one of the above items, wherein the combination of the Burkholderia bacterium and the Yarrowia yeast has a higher oil or fatty acid decomposition ability than the oil or fatty acid decomposition ability calculated from the oil or fatty acid decomposition ability values ​​of each culture alone. (Item C28) The composition or combination according to any one of the preceding items, wherein the ratio of the cell number of the Burkholderia bacterium to the cell number of the Yarrowia yeast is 1:20 to 20:1. (Item C29) The composition or combination described in any one of the preceding 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 composition or combination according to any one of the preceding items, wherein the Burkholderia bacterium is Burkholderia genus bacteria KH-1 strain (strain specified by accession number NITE BP-02731), KH-1AL1 strain (strain specified by accession number NITE BP-02977), KH-1AL2 strain (strain specified by accession number NITE BP-02978) or KH-1AL3 strain (strain specified by accession number NITE BP-02979), or a derivative thereof. (Item C31) The composition or combination according to any one of the preceding items, wherein the Yarrowia yeast is Yarrowia lipolytica KH-2 strain (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. (Item C32) A composition or combination according to any one of the preceding items which is an oil decomposer. (Item C33) An oil decomposer according to any one of the preceding items, comprising a further oil treatment component. (Item C34) A method for decomposing and removing oil, comprising applying a composition or combination described in any one of the above items, or an oil decomposing agent to an object to be treated. (Item C35) A composition for improving lipase production in a lipase-producing Yarrowia yeast comprising a Burkholderia bacterium. (Item C36) A composition for improving lipase production in a lipase-producing Burkholderia bacterium, comprising a Yarrowia yeast. (Item C37) A composition for enhancing the ability of a lipase-producing Yarrowia yeast to process oils or fatty acids, comprising a Burkholderia bacterium. (Item C38) A composition for enhancing the ability of lipase-producing Burkholderia bacteria to process oils or fatty acids, comprising a Yarrowia yeast. (Item C39) A method for improving lipase production by at least one of a Burkholderia bacterium and a Yarrowia yeast, comprising a step of mixing and culturing the Burkholderia bacterium and a Yarrowia yeast.

[0008] It is contemplated that the present disclosure may provide one or more of the above features in combinations other than those specifically described. 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. Effect of the Invention

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

[0010] The present disclosure can also solve the problem of oils that are difficult to decompose, i.e., oil types. The microorganisms and compositions containing the microorganisms of the present disclosure can decompose trans fatty acids and oils and fats containing the trans fatty acids that are generated during the hydrogenation process into oil, and have the effect of being able to treat margarine, fat spreads, shortening, and other products that contain a large amount of such fatty acid-containing oils and fats that could not be treated with conventional microorganisms. In particular, the microorganisms and compositions containing the microorganisms of the present disclosure provide the effect of achieving trans fatty acid decomposition that can be used at a practical level as yeast (microorganisms) that are the main microorganisms in wastewater treatment and waste treatment. [Brief description of the drawings]

[0011] [Figure 1] These are photographs of the KH-2 strain after culturing for three days at 28°C on media supplemented with triolein (left), oleic acid (center), or canola oil (right). [Diagram 2] This is a photograph of the KH-2 strain after culturing it on a medium supplemented with elaidic acid at 15° C. for 14 days. [Figure 3A]Shown is the elaidic acid decomposition activity at 28°C by BioRemove3200 (BR3200) (Novozymes, Denmark) (left) or the KH-2 strain (right). (a) is a photograph showing fatty acids in the culture medium detected by thin-layer chromatography. (b) is the result of measuring the oil content equivalent to the normal hexane value using an oil content measurement reagent kit. The left shows the decomposition result by BR3200, and the right shows the decomposition result by the KH-2 strain. [Figure 3B] Shown is the elaidic acid decomposition activity at 15°C by BioRemove3200 (BR3200) (Novozymes, Denmark) (left) or the KH-2 strain (right). (a) is a photograph showing fatty acids in the culture medium detected by thin-layer chromatography. (b) is the result of measuring the oil content equivalent to the normal hexane value using an oil content measurement reagent kit. The left shows the decomposition result by BR3200, and the right shows the decomposition result by the KH-2 strain. [Figure 4] This shows the decomposition of trielaidin by the KH-2 strain. The photographs show the analysis of the residual oil content in the culture medium by thin layer chromatography. From the left, the photographs show the decomposition of trielaidin at 28°C and at 15°C. BS (inorganic salt medium) shows the control without the addition of microorganisms, and KH-2 shows the addition of the KH-2 strain. [Figure 5A] This shows the oil-decomposing ability of the KH-2 strain in actual wastewater. This is a photograph of the remaining oil content in the culture solution analyzed by thin-layer chromatography. The photograph on the left shows the results after 24 hours of culture, and the photograph on the right shows the results after 48 hours of culture. From the left, each column shows the results with no microorganism added (control), with BioRemove3200 (BR3200) added, and with KH-2 strain added. [Figure 5B] This shows the oil-decomposing ability of KH-2 strain in actual wastewater. The oil content equivalent to normal hexane value was measured using an oil measurement reagent kit. From the left, the results are shown for no microorganism added (control), with BioRemove3200 (BR3200) added, and with KH-2 strain added. The white bars show the results after 24 hours of incubation, and the black bars show the results after 48 hours of incubation. The vertical axis shows normal hexane value (mg / L). [Figure 6A]This shows the decomposition of canola oil by the KH-2 strain when cultured at 15°C (pH 7.0). At 0, 24, 48, and 72 hours after the start of culture, the oil content (residual oil content) equivalent to normal hexane was measured using an oil content measurement reagent kit, and the percentage calculated with the value measured at 0 hours taken as 100% (top), as well as the results of quantifying total fatty acids (the sum of fatty acids in triglycerides and free fatty acids) by gas chromatography (bottom). [Figure 6B] This shows the decomposition of canola oil by the KH-2 strain when cultured at 15°C (pH 7.0). This is a photograph of the remaining oil content in the culture medium analyzed by thin layer chromatography. Each column, from the left, shows the results at 0 hours, 24 hours, 48 ​​hours, and 72 hours after the start of culture. [Figure 7A] This shows the decomposition of canola oil by the KH-2 strain during cultivation at 28°C (pH 7.0). At 0, 12, 24, and 30 hours after the start of cultivation, the oil content equivalent to normal hexane (residual oil content) was measured using an oil content measurement reagent kit, and the percentage calculated with the value measured at 0 hours taken as 100% (top), as well as the results of quantifying total fatty acids (the sum of fatty acids in triglycerides and free fatty acids) by gas chromatography (bottom). [Figure 7B] This shows the decomposition of canola oil by the KH-2 strain when cultured at 28°C (pH 7.0). This is a photograph of the analysis of the remaining oil content in the culture medium by thin layer chromatography. Each column, from the left, shows the results at 0 hours, 12 hours, 24 hours, and 30 hours after the start of culture. [Figure 8A] The figure shows the elaidic acid decomposition activity at 28°C by the germ-free group, KH-2 strain, KH-2AL1 strain, and KH-2AL3 strain. (a) is a photograph showing fatty acids in the culture medium detected by thin-layer chromatography. (b) shows the results of measuring the oil content equivalent to the normal hexane value using an oil content measurement reagent kit. From the left, the decomposition results are those by the germ-free group, KH-2 strain, KH-2AL1 strain, and KH-2AL3 strain. [Figure 8B]The figure shows the elaidic acid decomposition activity at 15°C by the germ-free group, KH-2 strain, KH-2AL1 strain, and KH-2AL3 strain. (a) is a photograph showing fatty acids in the culture medium detected by thin-layer chromatography. (b) shows the results of measuring the oil content equivalent to normal hexane using an oil content measurement reagent kit. From the left, the decomposition results are those by the germ-free group, KH-2 strain, KH-2AL1 strain, and KH-2AL3 strain. [Figure 9] The figure shows the trielaidin decomposition activity at 28°C by the germ-free area, KH-2 strain, KH-2AL1 strain, and KH-2AL3 strain. The oil content equivalent to normal hexane was measured using an oil content measurement reagent kit. From the left, the decomposition results are for the germ-free area, KH-2 strain, KH-2AL1 strain, and KH-2AL3 strain. [Figure 10A] This shows the decomposition of canola oil by the KH-2AL1 strain during cultivation at 15°C (pH 7.0). At 0, 24, 48, and 72 hours after the start of cultivation, the oil content equivalent to normal hexane (residual oil content) was measured using an oil content measurement reagent kit, and the percentage calculated with the value measured at 0 hours taken as 100% (top), as well as the results of quantifying total fatty acids (the sum of fatty acids in triglycerides and free fatty acids) by gas chromatography (bottom). [Figure 10B] This shows the decomposition of canola oil by the KH-2AL1 strain when cultured at 15°C (pH 7.0). This is a photograph of the analysis of the remaining oil content in the culture medium by thin layer chromatography. Each column, from the left, shows the results at 0 hours, 24 hours, 48 ​​hours, and 72 hours after the start of culture. [Figure 11A] This shows the decomposition of canola oil by the KH-2AL3 strain when cultured at 15°C (pH 7.0). At 0, 24, 48, and 72 hours after the start of culture, the oil content (residual oil) equivalent to normal hexane was measured using an oil content measurement reagent kit, and the percentage calculated with the value measured at 0 hours taken as 100% (top), as well as the results of quantifying total fatty acids (the sum of fatty acids in triglycerides and free fatty acids) by gas chromatography (bottom). [Figure 11B]This shows the decomposition of canola oil by the KH-2AL3 strain when cultured at 15°C (pH 7.0). This is a photograph of the analysis of the remaining oil content in the culture medium by thin layer chromatography. Each column, from the left, shows the results at 0 hours, 24 hours, 48 ​​hours, and 72 hours after the start of culture. [Figure 12] This shows the ability of the KH-2 strain to assimilate trielaidin or elaidic acid at 15°C. The photograph on the left shows the results of cultivation in a medium containing elaidic acid, and the photograph on the right shows the results of cultivation in a medium containing trielaidin. The results are shown for the conditions with the addition of the KH-2 strain (left) and without the addition of microorganisms (control, right). [Figure 13] This shows a comparison of the cleaning power of KH-2 strain and detergent. The photographs show an oil-stained ventilation fan filter that was soaked in culture supernatant of KH-2 strain, detergent for oil, and general detergent for cleaning. The left photo shows the filter before treatment. The right column shows the filter treated with KH-2 strain culture supernatant (top), the filter treated with detergent for oil (middle), and the filter treated with general detergent (bottom). [Figure 14] Comparison of palmitellidic acid and vaccenic acid degradation activities of KH-2 strain and BioRemove3200 (BR3200) (Novozymes, Denmark) is shown. After culturing at 15°C for 48 or 72 hours, the fatty acids in the culture medium were analyzed by thin-layer chromatography. The panels, from the left, are palmitellidic acid culture for 48 hours, vaccenic acid culture for 48 hours, palmitellidic acid culture for 72 hours, and vaccenic acid culture for 72 hours. In each panel, the right is a negative control (BS), the center is BR3200, and the left is KH-2 strain. [Figure 15] Comparison of palmitelaidic acid and vaccenic acid decomposition activity of KH-2 strain and BioRemove3200 (BR3200) (Novozymes, Denmark) is shown. After 24 hours of cultivation at 28°C, the fatty acids in the culture medium were analyzed by thin layer chromatography. The left panel shows palmitelaidic acid culture, and the right panel shows vaccenic acid culture. In each panel, the right is a negative control (BS), the center is BR3200, and the left is KH-2 strain. [Figure 16]The decomposition of canola oil at various mixing ratios of KH-1 and KH-2 strains at 15°C is shown. The reduction in total fatty acids (total fatty acids in triglycerides and free fatty acids) (amount of decomposition) at 48 hours after the start of culture at each ratio of KH-1 strain:KH-2 strain = 10:0, 9:1, 5:5, 1:9, and 0:10 at a total cell concentration of 5 x 105 cells / mL was quantified by gas chromatography, and the results are compared with the lipolytic ability calculated from the lipolytic ability of each monoculture. The vertical axis shows the relative lipolytic ability at each mixing ratio compared to the lipolytic ability (100%) calculated from the lipolytic ability of each monoculture, and the horizontal axis shows the KH-2 strain content (based on cell number). [Figure 17] Decomposition of canola oil at various mixing ratios of KH-1 and KH-2 strains at 28°C. The results of gas chromatography quantification of the reduction (decomposition amount) of total fatty acids (total fatty acids in triglycerides and free fatty acids) 18 hours after the start of culture at each ratio (cell number basis) of KH-1 strain:KH-2 strain = 10:0, 9:1, 5:5, 1:9, and 0:10 at a total cell concentration of 5 x 105 cells / mL are shown. The vertical axis shows the decomposition amount of total fatty acids, and the horizontal axis shows the KH-1 strain:KH-2 strain mixing ratio (cell number basis). The straight dashed line shows the fat decomposition ability calculated from the fat decomposition ability value of each monoculture. When the results were calculated in comparison with the fat decomposition ability calculated from the fat decomposition ability value of each monoculture as in Figure 16, the same tendency as in Figure 16 was shown. [Figure 18] The figure shows the decomposition of oleic acid at various mixing ratios of KH-1 and KH-2 strains at 28°C. The figure shows the results of quantifying the reduction (decomposition) of total fatty acids 18 hours after the start of culture at ratios (cell number basis) of KH-1 strain:KH-2 strain = 10:0, 9:1, 5:5, 1:9, and 0:10 at a total cell concentration of 5 x 105 cells / mL, using gas chromatography. The vertical axis shows the total fatty acid decomposition amount, and the horizontal axis shows the KH-1 strain:KH-2 strain mixing ratio (cell number basis). The straight dashed line shows the fatty acid decomposition ability calculated from the fatty acid decomposition ability value of each monoculture. [Figure 19]The expression level of each microbial gene in the symbiotic system of KH-1 strain + KH-2 strain is shown. The left panel shows the results of KH-1 strain cultured for 71 hours, and the vertical axis shows the relative expression level of the gene in the symbiotic system culture of KH-1 strain + KH-2 strain when the RNA expression level of each gene in the case of KH-1 strain monoculture is set to 1. The first lipase of KH-1 strain is represented by the nucleotide sequence of SEQ ID NO: 1 and the amino acid sequence of SEQ ID NO: 2. The second lipase of KH-1 strain is represented by the nucleotide sequence of SEQ ID NO: 3 and the amino acid sequence of SEQ ID NO: 4. The right panel shows the results of KH-2 strain cultured for 48 hours, and the vertical axis shows the relative expression level of the gene in the symbiotic system culture of KH-1 strain + KH-2 strain when the RNA expression level of each gene in the case of KH-2 strain monoculture is set to 1. The first lipase of KH-2 strain is represented by the nucleotide sequence of SEQ ID NO: 5 and the amino acid sequence of SEQ ID NO: 6. The second lipase of the KH-2 strain is shown by the nucleotide sequence of SEQ ID NO:7 and the amino acid sequence of SEQ ID NO:8. [Figure 20] The decomposition activity of model substrates (4-nitrophenyl butyrate, 4-nitrophenyl palmitate) by culture supernatants of KH-1 strain alone or a symbiotic system of KH-1 strain + KH-2 strain is shown. The left panel shows the activity evaluated with 4-nitrophenyl butyrate, and the right panel shows the activity evaluated with 4-nitrophenyl palmitate. In each panel, the left panel shows the results after 48 hours of culture, and the right panel shows the results after 71 hours of culture. The vertical axis shows the relative value of the absorbance of the symbiotic system culture of KH-1 strain + KH-2 strain, assuming the absorbance at 410 nm for the KH-1 strain culture alone to be 1. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0012] The present disclosure will be described below while showing the best mode. Throughout this specification, the expression of the singular form should be understood to include the concept of the plural form, unless otherwise specified. Therefore, the singular article (for example, in the case of English, "a", "an", "the", etc.) should be understood to include the concept of the plural form, unless otherwise specified. In addition, it should be understood that the terms used in this specification are used in the sense commonly used in the field, unless otherwise specified. Therefore, unless otherwise defined, all technical terms and scientific and technical terms used in this specification have the same meaning as commonly understood by those skilled in the art to which this disclosure belongs. In case of conflict, the present specification (including definitions) will take precedence.

[0013] The definitions of terms particularly used in this specification and / or basic technical content are appropriately explained below. Note that in this specification, the term "strain" may be omitted for strain names such as KH-1 strain, but those skilled in the art will appropriately understand that the term "strain" is used depending on the context.

[0014] (definition, etc.) As used herein, "esterase" refers to a hydrolase that decomposes an ester into an acid and an alcohol through a chemical reaction with water. Typically, as used herein, esterase refers to a hydrolase that decomposes a fatty acid ester into a fatty acid and an alcohol through a chemical reaction with water.

[0015] As used herein, the term "lipase" refers to a type of esterase, an enzyme that reversibly catalyzes the hydrolysis of neutral fats (glycerol esters) into fatty acids and glycerol. For example, lipase includes triglycerol lipase, which is classified into EC3.1.1.3 by enzyme number (EC number).

[0016] As used herein, the term "symbiotic system" refers to a combination of multiple types of microorganisms present in the same environment or system. In addition, "symbiotic" refers to a combination of multiple types of microorganisms present in the same environment or system. For example, the term "symbiotic system" is used to mean any of a combination of multiple types of microorganisms present in a mixture in a composition, a combination of multiple types of microorganisms present on a carrier in a composition, and a combination of multiple types of microorganisms formed as a result of each microbial species being separately introduced into an environment. In one embodiment, each microbial species in the symbiotic system may be in a state in which they can contact each other (including fusion and encapsulation). In one embodiment, the symbiotic system may be in a state in which another microbial species in the symbiotic system can utilize the environment created by the action (e.g., release, decomposition) of at least one microbial species in the symbiotic system, in which case the microbial species may or may not be in contact with each other (e.g., each microorganism is placed upstream and downstream in the system). Examples of environments created by the action of at least one microbial species in a symbiotic system include an environment in which a certain component is reduced, an environment in which a certain component (a component produced by decomposition by a certain microbial species, a secondary component produced by the action (e.g., reaction) of this component, etc.) is increased, and an environment in which a certain factor (such as pH) is changed. In particular, a typical example of a symbiotic system is when a product produced by the decomposition of a chemical substance by one or more microorganisms constituting the symbiotic system becomes a growth substrate such as a carbon source for other microorganisms. In addition, in a mixed system of microorganisms, microorganisms that have the ability to decompose and assimilate the same growth substrate generally compete with each other for the substrate, so it is considered that a symbiotic system is difficult to establish. In this specification, "capable of symbiosis" refers to the ability of a combination of multiple types of microorganisms to form a symbiotic system.

[0017] In this specification, the term "lipolytic ability calculated from the value of the lipolytic ability of each monoculture" refers to a value calculated based on the value of the lipolytic ability of each microbial strain constituting the combination of microorganisms (e.g., Burkholderia bacteria and Yarrowia yeast) alone, taking into account the contribution of each mixture. A representative example is a value of lipolytic ability calculated from the sum of values ​​obtained by multiplying the values ​​of the lipolytic ability obtained based on the values ​​obtained from the results of culturing each strain under the same conditions (medium composition, time, temperature, etc.) by a simple ratio, but is not limited thereto. In this case, it can be expressed as "the sum of the lipolytic ability calculated from the value of the lipolytic ability of each monoculture". In addition, a value considering the contribution based on the initial ratio at the time of mixing can be used. In this case, it can be expressed as "the lipolytic ability calculated from the initial value of the lipolytic ability of each monoculture". For example, when the total cell concentration of one type of Burkholderia bacteria and one type of Yarrowia yeast is 1×10 5 The "lipolytic activity calculated from the lipolytic activity of each monoculture" for the combination of cells / mL is [(1×10 5 (1 x 10 cells / mL) x (mixture ratio of this Burkholderia bacterium) + (1 x 10 Yarrowia yeast) 5 The Yarrowia yeast can be calculated by the following formula: (oil-decomposing ability when 1000 cells / mL are cultured alone) × (mixing ratio of this Yarrowia yeast). The cell concentration during monoculture, which is the basis of the calculation, is not particularly limited and can be appropriately selected by a person skilled in the art, and can be calculated by multiplying by an appropriate coefficient (which may be 1). As another example, the calculation may be performed from the value of the oil-decomposing ability based on the amount of microorganisms initially input (the growth rate of the microorganisms may be taken into consideration, if necessary). The "fatty acid decomposing ability calculated from the value of the fatty acid decomposing ability of each monoculture" and the "sum of the fatty acid decomposing ability calculated from the value of the fatty acid decomposing ability of each monoculture" are also understood in the same context.

[0018] In this specification, "oils and fats" refers to oily substances, and includes ester group-containing compounds formed by dehydration condensation of a hydroxyl group-containing compound and a fatty acid. A typical example of the hydroxyl group-containing compound is glycerin, but other examples include polyglycerin. In this specification, an ester group-containing compound formed by dehydration condensation of glycerin and a fatty acid is called a "glyceride" in the same sense as that usually used in this technical field. When this hydroxyl group-containing compound has multiple hydroxyl groups, if at least one of the hydroxyl groups is dehydration condensed with a fatty acid to form an ester, it corresponds to the ester group-containing compound in this specification. In this specification, the oils and fats may include cis fatty acid-containing oils and fats, trans fatty acid-containing oils and fats, or both.

[0019] Grease traps and pressurized flotation separators, which are treatment equipment for removing oil and fat by solid-liquid separation, are sources of odors and pests, and have many problems such as the labor and cost required for maintenance such as collection, transportation, and cleaning of the separated oil, and the cost of the coagulant required for this. The combination of microorganisms disclosed herein and the composition or combination that provides the combination can be used to eliminate oil in grease traps and industrial wastewater treatment equipment.

[0020] The present disclosure provides a microbial preparation for grease traps and industrial wastewater. In particular, when applied to industrial wastewater, it can reduce the operating rate of pressurized flotation separation equipment or even replace it. Kitchen wastewater from the food service industry not only contains high concentrations of fats and oils, usually 1 g / L or more, and sometimes 10 g / L or more, but the residence time of wastewater in most grease traps is extremely short, about 10 minutes, but the microorganisms disclosed in the present disclosure can also be used in such environments.

[0021] Grease and oil are also found in food waste, livestock waste, sludge from wastewater treatment plants, etc. Microorganisms are often used to treat such solid waste, but if the waste contains a large amount of oil, the treatment becomes difficult or the oil remains. The combination of microorganisms disclosed herein and the composition or combination that provides the combination can also be applied to the decomposition treatment of oil in such waste.

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

[0023] In this specification, a "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 linear, but may be branched or may 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 sense commonly used in this technical field, and refers to an unsaturated fatty acid having a trans double bond.

[0024] In this specification, "trans fatty acid-containing fats and oils" refers to compounds formed by dehydration condensation of trans fatty acids and compounds containing hydroxyl groups. Trans fatty acids include elaidic acid, vaccenic acid, etc., but there is no particular restriction on the type of trans fatty acid when referred to in this specification. There is no particular restriction on the ratio of trans fatty acids present in trans fatty acid-containing fats and oils. The "trans type" and "cis type" of double bonds are used in the sense commonly used in this technical field, and refer to the following structure in which four substituents (R1, R2, R3 and R4) are bonded to the two carbon atoms forming the double bond. [ka] In the above formula, when R1 and R2 or R3 and R4 are groups other than hydrogen and the remaining two substituents are hydrogen atoms, it is called cis type, and when R1 and R4 or R3 and R2 are groups other than hydrogen and the remaining two substituents are hydrogen atoms, it is called trans type. Trans fatty acids exist in small amounts in nature as conjugated linoleic acid and vaccenic acid, and are relatively abundant in the fat of ruminants, for example. Trans fatty acids can be produced during the hydrogenation process to produce saturated fatty acids from unsaturated fatty acids, and during the refining of vegetable oils that contain a lot of unsaturated fatty acids. Therefore, margarine, fat spreads, shortening, etc. may contain relatively large amounts of trans fatty acids.

[0025] In this specification, "short-medium chain fatty acid-containing ester" refers to an ester of a fatty acid containing one or more 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 sense usually used in this technical field, and respectively mean fatty acids having 2 to 6 carbon atoms, 7 to 12 carbon atoms and 13 or more carbon atoms. 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), palmitelidic 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), and the like.

[0026] In this specification, the "normal hexane value" refers to the amount of non-volatile substances extracted by normal hexane, and is an index showing the amount of oil (oils and fats, their hydrolyzates, etc.) in water. The normal hexane value can be determined, for example, according to JIS K 0102. It can also be determined using a simplified measurement reagent kit for measuring polynipam extract substances.

[0027] As used herein, "assimilation" refers to using something as a nutrient source, and the substance that is the subject of assimilation (for example, fats and oils) is broken down and, as a result, disappears or is reduced.

[0028] As used herein, the term "decomposition", when used in reference to esters (e.g., fats and oils) and / or fatty acids, refers to the breaking down of the target esters (e.g., fats and oils) and / or fatty acids into smaller molecules, for example, into glycerol and (free) fatty acids. Decomposition also refers to the conversion of fatty acids into fatty acids with fewer carbon atoms, or even into carbon dioxide and water.

[0029] In this specification, "ability to decompose esters" or "esterase activity" refers to the activity of hydrolyzing esters into alcohols and acids (e.g., free fatty acids). For example, "ability to decompose esters" or "esterase activity" may be measured by contacting a microorganism, a combination of microorganisms, or a culture supernatant thereof with an ester of 4-nitrophenol and a fatty acid and measuring the amount of 4-nitrophenol generated by the hydrolysis reaction, or may be measured in the same manner as the measurement of the ability to decompose fats and oils described in this specification. It is also understood that "ability to decompose esters containing short- to medium-chain fatty acids" and "ability to decompose fats and oils containing short- to long-chain fatty acids", which are more specific abilities of "ability to decompose esters" or "esterase activity", can also be measured in the same manner.

[0030] In this specification, the term "having lipase activity" (of a microorganism, etc.) refers to the activity of hydrolyzing fats and oils produced by 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 or not a microorganism has lipase activity can be confirmed by the reduction of fats and oils (animal and vegetable oils such as canola oil, triolein, etc.) contained in a medium to which the microorganism has been added.

[0031] In this specification, the expression "producing lipase" by a microorganism refers to the microorganism producing lipase extracellularly or intracellularly. In particular, when lipase is produced extracellularly, it is said to secrete lipase or secrete and produce lipase. In this case, however, the secreted lipase may leave the bacterial cell and be released 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 decomposition ability is demonstrated in any of the following tests, and decomposition does not necessarily have to be observed in all of the tests. A test to confirm whether or not the organism has the ability to assimilate fats and oils at a specified temperature (e.g., 15°C, 28°C). A test to confirm whether a clear zone is observed around the colony formed on an agar medium containing oil at a specified temperature (e.g., 15°C, 28°C). A test in which fats and oils are provided as a carbon source, the bacteria are cultivated at a specified temperature (e.g., 15°C, 28°C), and the reduction in normal hexane levels in the culture supernatant is measured. A test in which fats and oils are given as a carbon source, the bacteria are cultivated at a specified temperature (e.g., 15°C, 28°C), and the change over time in the amount 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, the bacteria has the ability to decompose fats and oils. Alternatively, if the amount of free fatty acids increases, the bacteria has the ability to decompose fats and oils. A test in which fats and oils are given as a carbon source, the bacteria are cultivated at a specified temperature (e.g., 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, and high performance liquid chromatography. Once the concentration of free fatty acids increases, it can be said that the bacteria have the ability to decompose them. A test in which sample water containing oil and fat as the main organic matter (e.g., 70% or more by weight of the total organic matter) is prepared and the biochemical oxygen demand (BOD) is measured. The proteins in the culture supernatant or cultured cells are analyzed by electrophoresis or mass spectrometry to confirm the presence of lipase. -Confirm the expression of microbial lipase genes by quantifying or detecting their mRNA. The "lipase activity" of a microorganism, a combination of microorganisms, or their culture supernatant is analyzed in accordance with the method for measuring "esterase activity" described above. More detailed methods for each measurement are provided herein, but a person skilled in the art can also perform these measurements using any other equipment and conditions.

[0032] In this specification, the term "ability to decompose esters at 15°C" refers to the ability of a microorganism or the like to hydrolyze esters into alcohols and acids at low temperatures. The ability to decompose fats and oils at 15°C may be measured by contacting a microorganism (including a derivative of the KH-2 strain) of the present disclosure with an ester of 4-nitrophenol and an acid (e.g., free fatty acid) at 15°C and measuring the amount of 4-nitrophenol produced by the hydrolysis reaction, or may be measured in the same manner as in the measurement of the ability to decompose fats and oils at 15°C described in this specification.

[0033] In this specification, "the ability to decompose fats and oils at 15°C" refers to the ability of a microorganism or the like 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 sufficient if the decomposition ability is demonstrated in any of the following tests, and it is not necessary that decomposition is observed in all of the tests. A test to check whether the substance has the ability to assimilate fats and oils at 15℃. A test to confirm whether a clear zone is observed around the colony formed on an agar medium containing oil at 15℃. A test in which fats and oils are provided as a carbon source, the bacteria are cultivated at 15°C, and the reduction in normal hexane levels in the culture supernatant is measured. A test in which fats and oils are given as a carbon source, the bacteria are cultured at 15°C, and the changes over time in the amount of fats and oils and free fatty acids in the culture supernatant are measured by thin-layer chromatography. If the amount of fats and oils decreases over time, the bacteria has the ability to decompose fats and oils. Alternatively, if the amount of free fatty acids increases, the bacteria has the ability to decompose fats and oils. A test in which fats and oils are given as a carbon source and cultured at 15°C, and the concentration of free fatty acids in the culture supernatant is measured using instrumental analysis such as gas chromatography, gas chromatography mass spectrometry, and high performance liquid chromatography. Once the concentration of free fatty acids increases, it can be said that the bacteria has the ability to break down fatty acids. A test in which sample water containing oil and fat as the main organic matter (e.g., 70% or more by weight of the total organic matter) is prepared and the biochemical oxygen demand (BOD) is measured. More detailed methods for each measurement are provided herein, but a person skilled in the art can also perform these measurements using any other equipment and conditions.

[0034] In this specification, "ability to assimilate trans fatty acid-containing oils" refers to the activity of assimilating trans fatty acid-containing oils. In this specification, "assimilating trans fatty acid-containing oils" is used in the sense commonly used in this technical field, and means that a microorganism takes in trans fatty acid-containing oils as a nutrient source such as a carbon source. When "assimilating", it includes not only hydrolysis to glycerol and free fatty acids, but also conversion to some other substances. The ability to assimilate trans fatty acid-containing oils can be measured and identified by the following tests. It is sufficient that assimilation ability is demonstrated in any of the following tests, and assimilation does not necessarily have to be confirmed in all tests. A test to confirm whether the trans fatty acid-containing oil can grow in a medium containing the oil as the only carbon source. A test to confirm whether or not colonies are formed in a medium containing trans fatty acid-containing oils as the only carbon source. A test to measure the decrease in n-hexane levels in the culture supernatant accompanying proliferation. A test in which the total fatty acids in the culture supernatant (the sum of fatty acids in fats and oils and free fatty acids) are converted to methyl esters as the bacteria grow, and the total amount is measured by gas chromatography. A test to measure the amount of fats and free fatty acids in the culture supernatant during proliferation using thin layer chromatography. More detailed methods for each measurement are provided herein, but a person skilled in the art can also perform these measurements using any other equipment and conditions.

[0035] In this specification, the term "ability to decompose trans-fatty acid-containing fats and oils" refers to the activity of hydrolyzing trans-fatty acid-containing fats and oils into glycerol and free fatty acids. The ability to decompose trans-fatty acid-containing fats and oils can be measured and identified by the following tests. It is sufficient that the decomposition ability is demonstrated in any of the following tests, and it is not necessary that decomposition is observed in all of the tests. A test to confirm the ability to assimilate oils containing trans fatty acids. A test to measure the amount of fats and free fatty acids in the culture supernatant using thin layer chromatography. A test in which oils containing trans fatty acids are fed as a carbon source, and the reduction in normal hexane levels in the culture supernatant is measured. A test in which the free fatty acids contained in the culture supernatant and the fatty acids in the oils and fats are converted to methyl esters, and their concentrations are measured by gas chromatography. A test to measure the concentration of free fatty acids in the culture supernatant using gas chromatography-mass spectrometry. A test to measure the concentration of free fatty acids in the culture supernatant using high performance liquid chromatography. A test to confirm whether a clear zone is observed around the colonies formed on an agar medium containing oils containing trans fatty acids. A test to prepare sample water containing trans fatty acid-containing oils as the main organic matter (e.g., 70% or more by weight of the total organic matter) and measure the biochemical oxygen demand (BOD). More detailed methods for each measurement are provided herein, but a person skilled in the art can also perform these measurements using any other equipment and conditions.

[0036] In this specification, "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 if the ability to assimilate is demonstrated by any of the following tests, and assimilation does not necessarily have to be confirmed by all of the tests. A test to determine whether the animal can grow in a medium containing trans fatty acids as the only carbon source. A test to determine whether or not colonies form on a medium containing trans fatty acids as the only carbon source. A test in which the bacteria are cultured using trans fatty acids as a carbon source and the decrease in normal hexane levels in the culture supernatant is measured as the bacteria grow. A test to measure the concentration of trans fatty acids in the culture supernatant during proliferation using gas chromatography. A test to measure the concentration of trans fatty acids in the culture supernatant during growth using gas chromatography-mass spectrometry. A test to measure the concentration of trans fatty acids in the culture supernatant during proliferation using high performance liquid chromatography. A test to measure the amount of trans fatty acids in the culture supernatant during growth using thin layer chromatography. More detailed methods for each measurement are provided herein, but a person skilled in the art can also perform these measurements using any other equipment and conditions.

[0037] In this specification, "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 if the decomposition ability is demonstrated by any of the following tests, and degradation does not necessarily have to be observed by all of the tests. A test to determine whether or not there is the ability to assimilate trans fatty acids. A test to measure the amount of trans fatty acids in the culture supernatant using thin layer chromatography. A test in which the bacteria are cultured using trans fatty acids as a carbon source and the reduction in normal hexane levels in the supernatant is measured. A test to measure the concentration of trans fatty acids in the culture supernatant using gas chromatography. A test to measure the concentration of trans fatty acids in the culture supernatant using gas chromatography-mass spectrometry. A test to measure the concentration of trans fatty acids in the culture supernatant using high performance liquid chromatography. A test to confirm whether a clear zone can be observed around the colonies formed on an agar medium containing trans fatty acids. A test to prepare sample water containing trans fatty acids as the main organic matter (e.g., 70% or more by weight of the total organic matter) and measure the biochemical oxygen demand (BOD). More detailed methods for each measurement are provided herein, but a person skilled in the art can also perform these measurements using any other equipment and conditions.

[0038] In this specification, "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 sense commonly used in this technical field, and means that a microorganism takes in fats and oils or their decomposition products as a nutrient source such as a carbon source. "Assimilation" includes not only hydrolysis into glycerol and free fatty acids, but also conversion into some other substances. The ability to assimilate fats and oils can be measured and identified by the following tests. It is sufficient if assimilation ability is demonstrated in any of the following tests, and assimilation does not necessarily have to be confirmed in all tests. A test to confirm whether the animal can grow in a medium containing fats and oils as the only carbon source. A test to confirm whether colonies are formed on a medium containing fats and oils as the only carbon source. A test to measure the decrease in n-hexane levels in the culture supernatant accompanying proliferation. A test in which the total fatty acids in the culture supernatant (the sum of fatty acids in fats and oils and free fatty acids) are converted to methyl esters as the bacteria grow, and the total amount is measured by gas chromatography. A test to measure the amount of fats and free fatty acids in the culture supernatant during proliferation using thin layer chromatography. More detailed methods for each measurement are provided herein, but a person skilled in the art can also perform these measurements using any other equipment and conditions.

[0039] In this specification, the term "ability to decompose fats and oils" refers to the activity of hydrolyzing fats and oils into glycerol and free fatty acids. The ability to decompose fats and oils can be measured and identified by the following tests. It is sufficient that the decomposition ability is demonstrated in any of the following tests, and it is not necessary that decomposition is observed in all of the tests. A test to check whether there is the ability to assimilate fats and oils. A test to measure the amount of fats and free fatty acids in the culture supernatant using thin layer chromatography. A test in which oils and fats are used as a carbon source to culture the bacteria, and the reduction in normal hexane levels in the culture supernatant is measured. A test in which the free fatty acids contained in the culture supernatant and the fatty acids in the oils and fats are converted to methyl esters, and their concentrations are measured by gas chromatography. A test to measure the concentration of free fatty acids in the culture supernatant using gas chromatography-mass spectrometry. A test to measure the concentration of free fatty acids in the culture supernatant using high performance liquid chromatography. A test to confirm whether a clear zone can be observed around the colonies formed on an agar medium containing oil or fat. A test in which sample water containing fatty acid-containing fats and oils as the main organic matter (e.g., 70% or more by weight of the total organic matter) is prepared and the biochemical oxygen demand (BOD) is measured. More detailed methods for each measurement are provided herein, but a person skilled in the art can also perform these measurements using any other equipment and conditions.

[0040] In this specification, the term "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 if the ability to assimilate is demonstrated by any of the following tests, and assimilation does not necessarily have to be confirmed by all of the tests. A test to determine whether the animal can grow on a medium containing fatty acids as the only carbon source. A test to determine whether colonies form on a medium containing fatty acids as the only carbon source. A test in which fatty acids are provided as a carbon source and cultivation is carried out, and the decrease in normal hexane levels in the culture supernatant is measured as the bacteria grow. A test to measure the concentration of fatty acids in the culture supernatant during proliferation using gas chromatography. A test to measure the concentration of fatty acids in the culture supernatant during proliferation using gas chromatography-mass spectrometry. A test to measure the concentration of fatty acids in the culture supernatant during proliferation using high performance liquid chromatography. A test to measure the amount of fatty acids in the culture supernatant during proliferation using thin layer chromatography. More detailed methods for each measurement are provided herein, but a person skilled in the art can also perform these measurements using any other equipment and conditions.

[0041] In this specification, the term "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 demonstrated in any one of the following tests, and it is not necessary that decomposition is observed in all of the tests. A test to check whether there is the ability to assimilate fatty acids. A test to measure the amount of fatty acids in the culture supernatant using thin layer chromatography. A test in which fatty acids are fed as a carbon source, cultivation is carried out, and the reduction in normal hexane levels in the supernatant is measured. A test to measure the concentration of fatty acids in the culture supernatant using gas chromatography. A test to measure the concentration of fatty acids in the culture supernatant using gas chromatography-mass spectrometry. A test to measure the concentration of fatty acids in the culture supernatant using high performance liquid chromatography. A test to confirm whether a clear zone can be observed around the colonies formed on an agar medium containing fatty acids. A test in which sample water containing fatty acids as the main organic matter (e.g., 70% or more by weight of the total organic matter) is prepared and the biochemical oxygen demand (BOD) is measured. More detailed methods for each measurement are provided herein, but a person skilled in the art can also perform these measurements using any other equipment and conditions.

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

[0043] In the present specification, the term "oil decomposer" refers to a preparation that contains at least one of the microorganisms of the present disclosure or the combination of microorganisms of the present disclosure as an active ingredient and is capable of decomposing oils and / or fatty acids alone or in the combination of microorganisms of the present disclosure. A preparation that is capable of decomposing oils and / or fatty acids in the combination of microorganisms of the present disclosure is a composition that contains only one of the microorganisms of the combination of microorganisms of the present disclosure (e.g., either Burkholderia bacteria or Yarrowia yeast), and even if the composition does not exhibit the desired oil and / or fatty acid decomposition ability by itself, when it is used to form the combination of microorganisms of the present disclosure, this composition can be an oil decomposer if it exhibits the desired oil and / or fatty acid decomposition ability. In the present disclosure, the oil decomposer may be used in combination with an oil treatment component. In this case, the timing of the combined use of the oil decomposer and the oil treatment component may be simultaneous or one of them may be used first. Furthermore, the oil decomposing agent may further contain components (e.g., carbon sources, nitrogen sources) that enhance the activity of the microbial strain or esterase (e.g., lipase) derived from the microbial strain used, surfactants, drying protection agents, components for maintaining the microorganisms for a long period of time, preservatives, excipients, strengthening agents, antioxidants, etc.

[0044] As used herein, a "derived strain," "analogous strain," or "mutant strain" preferably includes, but is not limited to, a gene that includes a region that is substantially homologous to the DNA of the microorganism of interest (e.g., 16S rDNA or 26S rDNA), and in various embodiments, such a strain has a full genome sequence that is at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or 99% identical when compared to the sequence of the full genome of the original strain by alignment using computer homology programs known in the art. This refers to a microorganism that has been modified by gene mutation, substitution, deletion, and / or addition, and whose derived strain still exhibits the biological function of the original microorganism, although not necessarily to the same degree. In one embodiment, such a derived strain, analogous strain, or mutant strain is provided in the present disclosure as a microorganism that has the same or higher biological function (e.g., oil decomposition ability, ability to enhance lipase production or oil decomposition ability of another microorganism, etc.) as the original microorganism. For example, genetic mutations can be introduced using any known mutagen, UV, plasma, etc. In one embodiment, the "derived strain", "similar strain" or "mutant strain" is a strain of the same genus and / or species as the original strain. For example, the biological function of such microorganisms can be examined by suitable and available in vitro assays described herein or known in the art. As used herein, the "similarity" of a gene or base sequence refers to the degree of similarity of two or more gene sequences to each other, and refers to a high degree of similarity of other sequences with identity. "Similarity" is a numerical value that takes into account similar bases in addition to identity, where similar bases refer to cases where a portion of the mixed 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) are identical.

[0045] As used herein, "protein," "polypeptide," "oligopeptide," and "peptide" are used interchangeably herein to refer to a polymer of amino acids of any length. The polymer may be linear, branched, or cyclic. The amino acids may be natural, non-natural, or modified. The term may also include those assembled into a complex of multiple polypeptide chains. The term also includes naturally or artificially modified amino acid polymers. Such modifications include, for example, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification (e.g., conjugation with a labeling moiety). The definition also includes, for example, polypeptides containing one or more analogs of amino acids (e.g., including non-natural amino acids), peptide-like compounds (e.g., peptoids), and other modifications known in the art. As used herein, "amino acid" is a general term for an organic compound having an amino group and a carboxyl group. When a protein or enzyme according to an embodiment of the present disclosure includes a "specific amino acid sequence," any amino acid in the amino acid sequence may be chemically modified. Any of the amino acids in the amino acid sequence may form a salt or a solvate. Any of the amino acids in the amino acid sequence may be L-type or D-type. In such cases, the protein according to the embodiment of the present disclosure may contain the above-mentioned "specific amino acid sequence". Examples of chemical modifications that amino acids contained in proteins undergo in vivo include N-terminal modifications (e.g., acetylation, myristoylation, etc.), C-terminal modifications (e.g., amidation, glycosylphosphatidylinositol addition, etc.), and side chain modifications (e.g., phosphorylation, sugar chain addition, etc.). The amino acids may be natural or unnatural as long as they satisfy the purpose of the present disclosure.

[0046] As used herein, "polynucleotide", "oligonucleotide" and "nucleic acid" are used interchangeably to refer to a polymer of nucleotides of any length. This term also includes "oligonucleotide derivative" or "polynucleotide derivative". "Base sequence" or "nucleic acid sequence" refers to the order of consecutive nucleic acid bases in a "polynucleotide", "oligonucleotide" or "nucleic acid". "Oligonucleotide derivative" or "polynucleotide derivative" refers to an oligonucleotide or polynucleotide that contains a derivative of nucleotides or has a bond between nucleotides that is different from the usual one, and are used interchangeably. Specific examples of such oligonucleotides include 2'-O-methyl-ribonucleotides, oligonucleotide derivatives in which the phosphodiester bond in an oligonucleotide has been converted to a phosphorothioate bond, oligonucleotide derivatives in which the phosphodiester bond in an oligonucleotide has been converted to an N3'-P5' phosphoramidate bond, oligonucleotide derivatives in which the ribose and phosphodiester bond in an oligonucleotide have been converted to a peptide nucleic acid bond, oligonucleotide derivatives in which the uracil in an oligonucleotide has been replaced with C-5 propynyl uracil, oligonucleotide derivatives in which the uracil in an oligonucleotide has been replaced with C-5 thiazole uracil, oligonucleotide derivatives in which the cytosine in an oligonucleotide has been replaced with C-5 propynyl cytosine, oligonucleotide derivatives in which the cytosine in an oligonucleotide has been replaced with phenoxazine-modified cytosine, oligonucleotide derivatives in which the ribose in DNA has been replaced with 2'-O-propyl ribose, and oligonucleotide derivatives in which the ribose in an oligonucleotide has been replaced with 2'-methoxyethoxy ribose. Unless otherwise indicated, a particular base sequence is also intended to encompass conservatively modified variants thereof (e.g., degenerate codon substitutions) and complementary sequences, as well as the sequence explicitly indicated.Specifically, degenerate codon substitutions can be achieved by creating sequences in which the third position of one or more selected (or all) codons is substituted with mixed-base and / or deoxyinosine residues (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, the term "gene" refers to a factor that determines a genetic trait, and may refer to a "polynucleotide," an "oligonucleotide," and a "nucleic acid."

[0048] As used herein, the term "homology" of a gene refers to the degree of identity between two or more gene sequences, and generally, "homology" refers to a high degree of identity or similarity. Thus, the higher the homology between two genes, the higher the identity or similarity between their sequences. Whether two genes have homology can be determined by direct comparison of sequences, or, in the case of nucleic acids, by hybridization methods under stringent conditions. When two gene sequences are directly compared, the genes have homology when the DNA sequences between the gene sequences are typically at least 50% identical, preferably at least 70% identical, and more preferably at least 80%, 90%, 95%, 96%, 97%, 98% or 99% identical.

[0049] Amino acids may be referred to herein by either their commonly known three-letter symbols or the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Nucleotides may also be referred to by their commonly accepted one-letter codes. In this specification, the comparison of similarity, identity, and homology of amino acid and base sequences is calculated using the sequence analysis tool BLAST with default parameters. Identity searches can be performed, for example, using NCBI's BLAST2.7.1 (published 2017.10.19). The value of "identity" in this specification usually refers to the value when aligned under default conditions using the above BLAST. However, if a higher value is obtained by changing the parameters, the highest value is taken as the identity value. If identity is evaluated in multiple regions, the highest value among them is taken as the identity value. "Similarity" is a value that takes into account similar amino acids in addition to identity.

[0050] In one embodiment of the present disclosure, the numerical value of identity, etc., "70% or more" 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 of the starting numerical values. The above-mentioned "identity" is calculated by calculating the ratio of the number of homologous amino acids or bases in two or more amino acid or base sequences according to a known method as described above. Specifically, before calculating the ratio, the amino acid or base sequences of the amino acid or base sequence group to be compared are aligned, and if necessary, gaps are introduced into a part of the amino acid or base sequence to maximize the ratio of identical amino acids or bases. Methods for alignment, methods for calculating the ratio, methods for comparison, and computer programs related thereto are conventionally well known in the art (for example, BLAST, etc., as described above). In this specification, "identity" and "similarity" can be expressed as values ​​measured by NCBI's BLAST, unless otherwise specified. When comparing amino acid or base sequences with BLAST, the algorithm Blastp can be used with default settings. Measurement results are quantified as Positives or Identities. In this case, when "similarity" is used instead of "identity", the value also takes into consideration those that fall under the definition of "similar" "amino acids" or "bases" described in this specification. In this specification, polynucleotides, oligonucleotides, nucleic acids, peptides, or proteins that have homology, identity, and / or similarity between their sequences may be referred to as "variants" of each other.

[0051] As used herein, "polynucleotides that hybridize under stringent conditions" refer to well-known conditions commonly used in the art. Such polynucleotides can be obtained by colony hybridization, plaque hybridization, Southern blot hybridization, or the like, using a polynucleotide selected from the polynucleotides disclosed herein as a probe. Specifically, the polynucleotide refers to a polynucleotide that can be identified by hybridizing a filter on which DNA derived from a colony or plaque is immobilized in the presence of 0.7 to 1.0 M NaCl at 65°C, and then washing the filter at 65°C using a 0.1 to 2-fold concentration of SSC (saline-sodium citrate) solution (the composition of a 1-fold concentration SSC solution is 150 mM sodium chloride and 15 mM sodium citrate). For example, the following conditions can be adopted as "stringent conditions." (1) using low ionic strength and high temperature for washing (e.g., 0.015 M sodium chloride / 0.0015 M sodium citrate / 0.1% sodium dodecyl sulfate at 50°C), (2) using a denaturing agent such as formamide during hybridization (e.g., 50% (v / v) formamide with 0.1% bovine serum albumin / 0.1% Ficoll / 0.1% polyvinylpyrrolidone / 50 mM sodium phosphate buffer pH 6.5, with 750 mM sodium chloride, 75 mM sodium citrate at 42°C), or (3) incubating overnight at 37°C in a solution containing 20% ​​formamide, 5x SSC, 50 mM sodium phosphate (pH 7.6), 5x Denhardt's solution, 10% dextran sulfate, and 20 mg / ml denatured sheared salmon sperm DNA, followed by washing the filters with 1x SSC at approximately 37-50°C. The formamide concentration may be 50% or more, and the washing time may be 5, 15, 30, 60, or 120 minutes or more.Factors that affect the stringency of the hybridization reaction include temperature, salt concentration, and more. For details, see Ausubel et al., Current Protocols in Molecular Biology, Wiley Interscience Publishers, (1995). Examples of "highly stringent conditions" are 0.0015M sodium chloride, 0.0015M sodium citrate, 65-68°C, or 0.015M sodium chloride, 0.0015M sodium citrate, and 50% formamide, 42°C. Hybridization can be performed according to the method described in experimental books 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 that contain only A or T sequences. Moderately stringent conditions can be readily determined by one of skill in the art based on, for example, the length of the DNA, and are set forth in Sambrook et al., Molecular Cloning: A Laboratory Manual, No. 3, Vol. 1, pp. 7.42-7.45 Cold Spring Harbor Laboratory Press, 2001, and include the use of, for nitrocellulose filters, a pre-wash solution of 5xSSC, 0.5% SDS, 1.0 mM EDTA (pH 8.0), hybridization conditions of about 50% formamide, 2xSSC-6xSSC (or other similar hybridization solutions, such as Stark's solution in about 50% formamide at about 42°C) at about 40-50°C, and wash conditions of 0.5xSSC, 0.1% SDS at about 60°C.Thus, a polypeptide as used in this disclosure also includes a polypeptide encoded by a nucleic acid molecule that hybridizes under high or moderate stringency conditions to a nucleic acid molecule encoding a polypeptide specifically described in this disclosure.

[0052] As used herein, the term "biological function" refers to a specific function that a microorganism may have, including, but not limited to, the decomposition of esters (e.g., fats and oils) (e.g., decomposition at low temperatures), lipase production, oil decomposition, the ability to enhance lipase production in other microorganisms, the ability to enhance the oil decomposition activity of other microorganisms, and the like. As used herein, a biological function may be exerted by a corresponding "biological activity." As used herein, "biological activity" refers to an activity that a microorganism may have in a certain environment, and includes the activity of exerting various functions (e.g., oil decomposition activity at 15°C). Such biological activity can be measured by techniques well known in the art. Thus, "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 also include, for example, a measure of the amount of upstream or downstream proteins or other similar functions of the microorganisms of the present disclosure after some stimulus or event.

[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 a volume of the sample. However, the amount also contemplates a relative amount compared to the amount of another analyte. For example, the amount of an analyte in a sample may be an amount that is greater than a control level or normal level of the analyte that is normally present in the sample.

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

[0055] As used herein, the term "kit" refers to a unit in which the parts to be provided (e.g., a composition containing the microorganism of the present disclosure, additional components, buffer solution, instructions, etc.) are provided, usually in two or more compartments. This kit form is preferred when the purpose is to provide a composition that should not be provided in a mixed state for stability or the like, but is preferably mixed and used immediately before use. Such a kit is preferably provided with instructions or instructions that describe how to use or process the parts to be provided (e.g., a composition containing the microorganism, additional components, etc.). When the term "kit" is used herein, the kit usually includes instructions that describe how to use the microorganism or composition of the present disclosure.

[0056] In the present specification, the term "instructions" refers to instructions written to explain to a user how to use the microorganism or combination of microorganisms disclosed herein. The instructions contain a statement instructing how to use the microorganism or combination of microorganisms disclosed herein. If necessary, the instructions are prepared in accordance with a format specified by the supervisory agency of the country in which the method disclosed herein is carried out (e.g., the Ministry of Health, Labor and Welfare or the Ministry of Agriculture, Forestry and Fisheries in Japan, the Food and Drug Administration (FDA), the Department of Agriculture (USDA), etc. in the United States), and it is clearly stated that the instructions have been approved by the supervisory agency. The instructions may be provided in paper form, but are not limited thereto, and may also be provided in the form of, for example, electronic media (e.g., a homepage provided on the Internet, e-mail).

[0057] (Preferred embodiment) The preferred embodiments of the present disclosure are described below. The embodiments provided below are provided for a better understanding of the present disclosure, and it is understood that the scope of the present disclosure should not be limited to the following description. Therefore, it is clear that a person skilled in the art can make appropriate modifications within the scope of the present disclosure in light of the description 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 a microorganism having a newly discovered ability to degrade oils and / or fatty acids. In particular, the microorganism of the present disclosure has lipase activity for triglycerides containing long-chain fatty acids (fatty acids containing 13 or more carbon atoms, for example 14 to 22 carbon atoms), has higher hydrolysis activity for 4-nitrophenyl esters of short- to medium-chain fatty acids (fatty acids containing 2 to 12 carbon atoms) than for 4-nitrophenyl esters of long-chain fatty acids, has the ability to degrade oils and fats containing short- to long-chain fatty acids (fatty acids containing 2 or more carbon atoms), has the ability to degrade esters containing short- to medium-chain fatty acids (fatty acids containing 2 to 12 carbon atoms), has the ability to assimilate esters (e.g., oils and fats) and / or fatty acids at 15° C., and has the ability to degrade esters ( For example, the microorganism has one or more characteristics of having an ability to decompose esters (e.g., fats and oils) and / or fatty acids, having an ability to utilize trans-fatty acid-containing fats and oils, having an ability to decompose trans-fatty acid-containing fats and oils, having an ability to utilize trans-fatty acids (e.g., elaidic acid, palmitelaidic acid, and / or vaccenic acid), and / or having an ability to decompose trans-fatty acids (e.g., elaidic acid, palmitelaidic acid, and / or vaccenic acid), having an ability to improve lipase production of Burkholderia bacteria, and having an ability to improve fatty acid and / or fat and oil decomposition ability of Burkholderia bacteria. In one aspect, the present disclosure provides a new microorganism with ester (e.g., fat and oil) decomposition ability. The microorganism of the present disclosure may have a wide range of hydrolysis activity against fats and oils of short-chain to long-chain fatty acids. In one aspect, the present disclosure provides a new microorganism with an ability to improve lipase production and / or oil (including fatty acids and fats and oils) decomposition 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 a cell wall. 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 present inventor identified a new microbial strain discovered by examining the trans-fatty acid decomposition / assimilation ability and low-temperature ester (e.g., fats and oils) decomposition ability as Yarrowia lipolytica, and deposited it at the National Institute of Technology and Evaluation Patent Microorganisms Deposit Center, which was received on June 4, 2018, and a deposit certificate was issued on June 12, 2018. The deposit number is NITE BP-02732. In addition, further strains of Yarrowia lipolytica (KH-2AL1 strain and KH-2AL3 strain) were further identified and deposited at the National Institute of Technology and Evaluation, Patent Microorganisms Deposit Center, where they were received on December 23, 2019, and a certificate of deposit was issued on January 16, 2020. The accession numbers are NITE BP-03091 and NITE BP-03092, respectively. In one embodiment, the microorganism of the present disclosure is Yarrowia yeast KH-2 strain (strain identified by accession number NITE BP-02732), KH-2AL1 strain (strain identified by accession number NITE BP-03091), or KH-2AL3 strain (strain identified by accession number NITE BP-03092), or a derivative thereof. In further aspects of the disclosure, the Yarrowia yeast of the disclosure can be a yeast of the genus Candida, Zygoascus, Ogataea, Pichia, or Aciculoconidium.Such yeasts of the genus Candida include deformans, oslonensis, galli, phangngensis, hollandica, alimentaria, hispaniensis, and incommunis, those of the genus Zygoascus include steatolyticus var. steatolyticus, and those of the genus Aciculoconidium include aculeatum.

[0060] In one embodiment, the microorganism of the present disclosure is a derivative of the Yarrowia yeast KH-2 strain (a strain identified by accession number NITE BP-02732), KH-2AL1 strain (a strain identified by accession number NITE BP-03091), or KH-2AL3 strain (a strain identified by accession number NITE BP-03092). Here, the derivative does not necessarily have to be a strain derived from the Yarrowia yeast KH-2 strain, KH-2AL1 strain, or KH-2AL3 strain, and refers to a microorganism that exhibits the biological functions of the Yarrowia yeast KH-2 strain, KH-2AL1 strain, or KH-2AL3 strain, although not necessarily to the same degree. In one embodiment, a microorganism that is a derivative strain of the present disclosure, like the Yarrowia yeast KH-2, KH-2AL1, or KH-2AL3 strain, exhibits a biological function selected from the group consisting of having lipase activity, an ability to utilize (decompose) esters (e.g., trans fatty acid-containing fats and oils) 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.), an ability to utilize (decompose) trans fatty acids (e.g., elaidic acid, palmitelaidic acid, and / or vaccenic acid), an ability to improve lipase production in Burkholderia bacteria, and an ability to improve fatty acid and / or fat and oil decomposition ability in Burkholderia bacteria; however, the degree of the biological function may be different from that of the KH-2, KH-2AL1, or KH-2AL3 strain. In one embodiment, the microorganism that is the derivative of the present disclosure may be a yeast of the genus Yarrowia, and more specifically, Yarrowia lipolytica.

[0061] The microorganisms of the present disclosure (including derivatives of the KH-2, KH-2AL1, or KH-2AL3 strains) may be isolated on an inorganic salt agar medium containing an ester (e.g., fat or oil) as the only carbon source and adjusted to a pH of 6 to 8. In one embodiment, the microorganisms of the present disclosure (including derivatives of the KH-2, KH-2AL1, or KH-2AL3 strains) may be distinguished by confirming the formation of a clear zone (halo) around the colonies formed on an agar medium containing dispersed esters (e.g., fat or oil). In one embodiment, the microorganisms of the present disclosure (including derivatives of the KH-2, KH-2AL1, or KH-2AL3 strains) can form colonies and grow on an agar medium containing 10 g / L of canola oil as a carbon source at 15° C. or 28° C., preferably at 15° C. Without intending to be limiting, it may be useful for the microorganisms of the present disclosure to have minimal ester (e.g., fat) assimilation (decomposition) ability or ester (e.g., fat) tolerance.

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

[0063] In one embodiment, the microorganisms of the present disclosure (including derivatives of strain KH-2, strain KH-2AL1, or strain KH-2AL3) are grown in mineral salts medium supplemented with 1% (v / v) canola oil at an initial cell optical density (OD 660=0.05, and then cultured at 28°C for 24 hours, 48 ​​hours at 28°C, or 48 hours at 15°C. After centrifugation to remove the cells, 1 ml of the supernatant was mixed with 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) aqueous Triton (registered trademark) X-100 solution and 1 ml of a 150 mM GTA buffer (pH 7.0). When the absorbance at 410 nm was monitored for 1 minute with stirring, the hydrolysis activity of the strain against 4-nitrophenyl esters of short- to medium-chain fatty acids (fatty acids containing 2 to 12 carbon atoms) was 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 4-nitrophenyl esters of long-chain fatty acids (fatty acids containing 13 or more carbon atoms).

[0064] In one embodiment, the microorganisms of the present disclosure (including derivatives of the KH-2, KH-2AL1, or KH-2AL3 strains) have the ability to utilize trans fatty acids (e.g., elaidic acid, palmitic acid, and / or vaccenic acid) or oils and fats containing trans fatty acids. In one embodiment, the microorganisms of the present disclosure (including derivatives of the KH-2, KH-2AL1, or KH-2AL3 strains) have the ability to degrade trans fatty acids (e.g., elaidic acid, palmitic acid, and / or vaccenic acid). In one embodiment, the microorganisms of the present disclosure (including derivatives of the KH-2, KH-2AL1, or KH-2AL3 strains) have the ability to degrade oils and fats containing trans fatty acids.

[0065] In one embodiment, the microorganisms of the present disclosure (including derivatives of the KH-2, KH-2AL1, or KH-2AL3 strains) have lipase activity that degrades triolein. In one embodiment, the microorganisms of the present disclosure (including derivatives of the KH-2, KH-2AL1, or KH-2AL3 strains) have lipase activity that degrades trielaidin.

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

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

[0068] In one embodiment, the microorganisms of the present disclosure, including derivatives of strain KH-2, KH-2AL1, or KH-2AL3, grow at an OD of 0.25% by weight in a mineral salts medium containing Triton® X-100 and elaidic acid at concentrations of 0.25% by weight and 0.2% by weight, respectively. 660 The microorganism was inoculated at a cell optical density of 0.5 or 0.8, and cultured at pH 7.0 and 28° C. The elaidic acid concentration in the supernatant after 24 or 46 hours was measured (for example, the OD 660 The microorganisms were inoculated at a cell optical density of 0.5 and cultured at pH 7.0 and 28°C. The elaidic acid concentration in the supernatant after 24 hours was measured, or the OD was measured in an inorganic salt medium containing Triton® X-100 and elaidic acid at concentrations of 0.25% by weight and 0.2% by weight, respectively. 660When the microorganism is inoculated at a bacterial cell optical density such that the bacterial cell optical density is 0.8 and 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, the trans fatty acid decomposition ability is such that It is preferable that the microorganisms of the present disclosure (including derivatives of the KH-2 strain, KH-2AL1 strain, or KH-2AL3 strain) have 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, and such microorganisms having the ability to decompose trans fatty acids can be beneficially used in various applications of the present disclosure.

[0069] In one embodiment, the microorganisms of the present disclosure, including derivatives of strain KH-2, KH-2AL1, or KH-2AL3, grow at an OD of 0.25% by weight in a mineral salts medium containing Triton® X-100 and elaidic acid at concentrations of 0.25% by weight and 0.2% by weight, respectively. 660When the microorganism is inoculated at a bacterial cell optical density such that the bacterial cell optical density is 0.8 and 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, the trans fatty acid decomposition ability is such that It is preferable that the microorganisms of the present disclosure (including derivatives of the KH-2 strain, KH-2AL1 strain, or KH-2AL3 strain) have 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 determined under these conditions, and such microorganisms having the ability to decompose trans fatty acids can be beneficially used in various applications of the present disclosure.

[0070] In one embodiment, the microorganisms of the present disclosure, including derivatives of strain KH-2, KH-2AL1, or KH-2AL3, grow at an OD of 0.25% by weight and 0.1% by weight of Triton® X-100 and trielaidin, respectively. 660When the microorganism is inoculated at a bacterial cell optical density such that the bacterial cell optical density is 0.8 and 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 has an ability to decompose trans fatty acids-containing oils and fats. It is preferable that the microorganisms of the present disclosure (including derivatives of the KH-2 strain, KH-2AL1 strain, or KH-2AL3 strain) have 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 determined under these conditions, and such microorganisms 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 microorganisms of the present disclosure (including derivatives of strain KH-2, strain KH-2AL1, or strain KH-2AL3) are grown in mineral salts medium containing 1% (v / v) canola oil to a final concentration of OD 660 =0.05 and 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. The microorganisms of the present disclosure (including derivatives of the KH-2, KH-2AL1, or KH-2AL3 strains) preferably have an oil-decomposing ability such that the residual oil content when cultured at 15°C 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, and such microorganisms having low-temperature oil-decomposing ability can be beneficially used in various applications of the present disclosure.

[0072] In one embodiment, the microorganisms of the present disclosure (including derivatives of strain KH-2, strain KH-2AL1, or strain KH-2AL3) are grown in mineral salts medium containing 1% (v / v) canola oil to a final concentration of OD 660 When the microorganism is inoculated at a bacterial cell optical density of 0.05 and 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% at the start of culture. The microorganisms of the present disclosure (including derivatives of KH-2, KH-2AL1, or KH-2AL3 strains) preferably have an oil-decomposing ability of less than 20%, less than 17%, less than 15%, less than 12%, or less than 10%, particularly less than 10%, when cultured at 28° C. under these conditions, and such microorganisms having high-speed oil-decomposing ability can be beneficially used in various applications of the present disclosure.

[0073] In one embodiment, the microorganisms of the present disclosure (including derivatives of strain KH-2, strain KH-2AL1, or strain KH-2AL3) are grown in mineral salts medium containing 1% (v / v) canola oil to a final concentration of OD 660=0.05, and cultured at pH 7.0, 15°C and 28°C, respectively. The oil content corresponding to the normal hexane value in the supernatant after 24 hours of culture 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 of the oil content corresponding to the normal hexane value in the supernatant after 24 hours of culture at 28°C. When judged under these conditions, the microorganisms of the present disclosure (including derivatives of the KH-2, KH-2AL1, or KH-2AL3 strains) preferably have an oil-decomposing ability such that the oil residual rate when cultured at 15°C is 800% or less, 700% or less, 600% or less, 500% or less, 400% or less, and particularly 500% or less, compared to when cultured at 28°C. Such microorganisms having low-temperature oil-decomposing ability can be beneficially used in various applications of the present disclosure.

[0074] In one embodiment, the microorganisms of the present disclosure (including derivatives of strain KH-2, strain KH-2AL1, or strain KH-2AL3) are grown in mineral salts medium containing 1% (v / v) canola oil to a final concentration of OD 660 =0.05, and culturing is initiated at pH 7.0, 15°C and 28°C, respectively. The total fatty acid decomposition rate in culture 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 culture at 28°C. When judged under these conditions, the microorganisms of the present disclosure (including derivatives of the KH-2, KH-2AL1, or KH-2AL3 strains) preferably have an oil-decomposing ability such that the total fatty acid decomposition rate when cultured 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 when cultured at 28°C. Such microorganisms having 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 lipase production in a Burkholderia bacterium that produces lipase. In one embodiment, the Yarrowia yeast of the present disclosure has the ability to impart a higher lipolytic ability to the Burkholderia bacterium than the lipolytic ability of the Burkholderia bacterium when cultured alone. In one embodiment, the Yarrowia yeast of the present disclosure has the ability to impart a higher fatty acid decomposition ability to the Burkholderia bacterium than the fatty acid decomposition ability of the Burkholderia bacterium when cultured alone. In one embodiment, the Burkholderia bacterium is any of the Burkholderia bacteria in the microbial combinations of the present disclosure. Any feature of the microbial combinations of the present disclosure described herein below is also intended to be a description of the ability of the Yarrowia yeast of the present disclosure to impart to the Burkholderia bacterium of the present disclosure. For example, when the Burkholderia bacterium and the Yarrowia yeast are mixed in a 1:1 ratio (cell number basis) in an inorganic salt medium supplemented with 1% (v / v) canola oil, the total number of cells is 5×10 5 Disclosure of a combination of microbial strains of Burkholderia bacteria and Yarrowia yeast that, when mixed and inoculated at a cell concentration of 100% or more, is greater than the lipolytic ability calculated from the lipolytic ability of each of the individual monocultures based on gas chromatography analysis when cultured at pH 7.0 and 28°C for 18 hours, is also disclosure of the ability of the Yarrowia yeast to confer the same lipolytic ability to the Burkholderia bacteria under the same culture conditions and in the same mixture ratio. Similarly, disclosure of the characteristics of the combination of microorganisms of the present disclosure is equivalent to describing the ability that the Yarrowia yeast of the present disclosure confer to the Burkholderia bacteria of the present disclosure. In one embodiment, the Yarrowia yeast of the present disclosure has both one or more abilities based on the Yarrowia yeast alone described herein (described above) and the ability that the combination of microorganisms of the present disclosure confer to the Burkholderia bacteria (described below). A Yarrowia yeast that combines these different aspects of abilities is particularly unexpected. Examples of Yarrowia yeast strains having such different capabilities include, but are not limited to, the KH-2 strain, the KH-2AL1 strain, and the KH-2AL3 strain.

[0076] (Combination of oil-degrading microorganisms) In one aspect, the present disclosure provides a combination of Burkholderia bacteria and Yarrowia yeast that degrades fats and / or oils (also referred to herein as the "microbial combination of the present disclosure"). As used herein, "Burkholderia bacteria" refers to bacteria including microorganisms of the order Burkholderiales, and details will be described later in this specification. As used herein, "Yarrowia yeast" refers to yeast including microorganisms of the genus Yarrowia, and details will be 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 decomposition ability. As shown in the examples, an improvement in oil decomposition ability was observed by the combination of Burkholderia bacteria and Yarrowia yeast. Without wishing to be bound by theory, it is expected that a substance produced as a result of the Yarrowia yeast decomposing oil acts as an inducer to improve the oil decomposition ability of the Burkholderia bacteria, and therefore a microorganism having a similar oil decomposition ability can be suitably used as the Yarrowia yeast, and a strain having a similar sensitivity can be suitably used as the Burkholderia bacteria. Similarly, it is expected that a substance produced as a result of the Burkholderia bacteria decomposing oil acts as an inducer to improve the oil decomposition ability of the Yarrowia yeast, and therefore a microorganism having a similar oil decomposition ability can be suitably used as the Burkholderia bacteria, and a strain having a similar sensitivity can be suitably used as the Yarrowia yeast.

[0077] In one embodiment, at least one of the Burkholderia bacteria and the Yarrowia yeast in the microbial combination of the present disclosure has the ability to produce lipase. In one embodiment, both the Burkholderia bacteria and the Yarrowia yeast in the microbial combination of the present disclosure have the ability to produce lipase. The inventors have found that a combination of Burkholderia bacteria and Yarrowia yeast, each of which produces lipase alone, unexpectedly exhibits high oil decomposition ability, even though it was expected that the Burkholderia bacteria and the Yarrowia yeast, each of which produces lipase alone, would compete with each other and suppress each other's abilities (reducing the overall oil decomposition ability) due to their similar properties. In one embodiment, the Burkholderia bacteria and the Yarrowia yeast in the microbial combination of the present disclosure can live symbiotically. In one embodiment, the Yarrowia yeast in the microbial combination of the present disclosure can improve 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 lipase expression and / or production of the Yarrowia yeast. The microorganisms in the microbial combination of the present disclosure can be produced by culturing them using any suitable method.

[0078] In one aspect, the combination of microorganisms disclosed herein provides an oil decomposition ability that exceeds the oil decomposition ability of each microorganism used in the combination (e.g., ability to decompose oils and / or fatty acids at 28°C, ability to decompose oils and / or fatty acids at 15°C, etc.). In one aspect, the combination of microorganisms disclosed herein provides an oil decomposition ability that exceeds the sum of the oil decomposition abilities of each microorganism used in the combination (e.g., ability to decompose oil at 28°C, ability to decompose oil at 15°C, etc.). In particular, if any of the oil decomposition abilities exceeds the oil decomposition ability calculated from the oil decomposition ability value of each monoculture, the combination of microorganisms can be usefully used even if another ability is reduced. In one embodiment, the combination of microorganisms disclosed herein 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 the Burkholderia bacteria and / or Yarrowia yeasts used in the combination.

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

[0081] The cell number ratio or cell concentration can be determined and / or adjusted using any suitable method, one example being the ratio of the cell number concentration of the microorganism to the optical density (OD 660 By preparing a calibration curve in advance for the relationship between the OD and the cell number concentration, 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, 660 =0.01 KH-1 strain is approximately 1 × 10 6 cells / mL, OD 660 =0.01 KH-2 strain is approximately 1 × 10 5 Can be converted to 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 ~1:100, approx. 50:1~1:50, approx. 50:1~1:20, approx. 50:1~1:10, approx. 50:1~1:5, approx. 50:1~1:2, approx. 50:1~1:1, approx. 50:1~2:1, approx. 50:1~5:1, approx. 20:1~1:10, approx. 20:1~1:50, approx. 20:1~1:20, approx. 20:1~1:10, approx. 20:1~1:5, approx. 20:1~1:2, approx. 20:1~1:1, Approx. 20:1-2:1, Approx. 20:1-5:1, Approx. 10:1-1:100, Approx. 10:1-1:50, Approx. 10:1-1:20, Approx. 10:1-1:10, Approx. 10:1-1:5, Approx. 10:1-1:2, Approx. 10:1-1:1, Approx. 10:1-2:1, Approx. 10:1-5:1, Approx. 5:1-1:100, Approx. 5:1-1:50, Approx. 5:1-1:20, Approx. 5:1-1:10, Approx. 5:1-1:5 , 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, or about 1:100.

[0083] In one embodiment, the combination of microorganisms disclosed herein has a higher oleolytic ability than that calculated from the oleolytic ability value of each monoculture when cultured in mineral salts medium supplemented with 1% (v / v) canola oil at a predetermined temperature (e.g., 15°C or 28°C). In one embodiment, the combination of microorganisms disclosed herein has a higher oleolytic ability than either the Burkholderia bacteria alone or the Yarrowia yeast alone in the combination at the same cell number as the total cell number of the combination when cultured in mineral salts medium supplemented with 1% (v / v) canola oil at a predetermined temperature (e.g., 28°C). In one embodiment, the combination of microorganisms disclosed herein has a higher fatty acid decomposition ability than that calculated from the fatty acid decomposition value of each monoculture when cultured in mineral salts medium supplemented with 1% (v / v) oleic acid at a predetermined temperature (e.g., 28°C). In one embodiment, the combination of microorganisms disclosed herein has a higher fatty acid decomposition ability than either the Burkholderia bacteria alone or the Yarrowia yeast alone in the combination at the same cell number as the total cell number of the combination when cultured at a given temperature in a mineral salt medium supplemented with 1% (v / v) oleic acid. The ability to decompose fats and / or fatty acids can also be tested by thin layer chromatography as described herein, and can be determined, for example, by collecting culture supernatant over time and comparing the time it takes for spots corresponding to fats and / or fatty acids to disappear.

[0084] In one embodiment, a total of 5×10 Burkholderia bacteria and Yarrowia yeast were grown in mineral salts medium supplemented with 1% (v / v) canola oil. 5The combination and mixing ratio of microbial strains of Burkholderia bacteria and Yarrowia yeast are such that, when inoculated at a cell concentration of 100000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000

[0085] In one embodiment, Burkholderia bacteria and Yarrowia yeast were cultured in mineral salts medium supplemented with 1% (v / v) canola oil at a total cell count of 5×10 5 The combination and mixing ratio of microbial strains of Burkholderia bacteria and Yarrowia yeast are used such that, when the strains are inoculated together at a cell concentration of 100 cells / mL and cultured at pH 7.0 and 15°C for 48 hours, the lipolysis ability 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 the lipolysis ability values ​​of the respective monocultures based on gas chromatography analysis.

[0086] In one embodiment, a total of 5×10 Burkholderia bacteria and Yarrowia yeast were grown in mineral salts medium supplemented with 1% (v / v) canola oil. 5 The combination and mixing ratio of microbial strains of Burkholderia bacteria and Yarrowia yeast are such that, when inoculated at a cell concentration of 100000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000

[0087] In one embodiment, Burkholderia bacteria and Yarrowia yeast were cultured in mineral salts medium supplemented with 1% (v / v) canola oil at a total cell count of 5×10 5 The combination and mixing ratio of microbial strains of Burkholderia bacteria and Yarrowia yeast are used such that, when the strains are inoculated together at a cell concentration of 100 cells / mL and cultured at pH 7.0 and 28°C for 18 hours, the lipolysis ability 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 the lipolysis ability values ​​of the respective monocultures based on gas chromatography analysis.

[0088] In one embodiment, a total of 5×10 Burkholderia bacteria and Yarrowia yeast were grown in mineral salts medium supplemented with 1% (v / v) oleic acid. 5 The combination and mixing ratio of microbial strains of Burkholderia bacteria and Yarrowia yeast are such that, when inoculated at a cell concentration of 100000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000

[0089] In one embodiment, Burkholderia bacteria and Yarrowia yeast were cultured in mineral salts medium supplemented with 1% (v / v) oleic acid at a total cell count of 5×10 5The combination and mixing ratio of microbial strains of Burkholderia bacteria and Yarrowia yeast are used such that, when the strains are inoculated together at a cell concentration of 100 cells / mL and cultured at pH 7.0 and 15°C for 48 hours, the fatty acid degradation ability is 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 compared to the fatty acid degradation ability values ​​of each culture alone based on gas chromatography analysis.

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

[0091] In one embodiment, the microbial combination of the present disclosure is grown at an OD of 1% (v / v) in mineral salts medium supplemented with 1% (v / v) canola oil. 660 When each microorganism was inoculated at a cell body optical density such that the measured value of OD was 0.018:0.02 for Burkholderia bacteria:Yarrowia yeast, and cultured at pH 7.0 and 15°C, total RNA was extracted from the cells 71 hours after the start of culture and quantitative PCR analysis was performed for a specific Burkholderia lipase. The expression level was OD 660 The combination is such that the bacterial cell optical density is 6-fold or more, 7-fold or more, or 8-fold or more, compared to the case where only the Burkholderia bacteria is inoculated, so that the measured value of the bacterial cell optical density is 0.02.

[0092] In one embodiment, the combination of microorganisms disclosed herein is a combination that improves lipase expression and / or production in Yarrowia yeast in the combination of microorganisms disclosed herein. In one embodiment, the lipase in Yarrowia yeast includes a first lipase (e.g., nucleotide sequence: SEQ ID NO:5, amino acid sequence: SEQ ID NO:6, or a variant thereof) and / or a second lipase (e.g., nucleotide sequence: SEQ ID NO:7, amino acid sequence: SEQ ID NO:8, or a variant thereof). In one embodiment, the improvement in lipase expression and / or production can be determined by comparing the results of lipase gene expression (e.g., RNA amount, produced protein amount, secreted protein amount) when using the combination of microorganisms disclosed herein compared to a Yarrowia yeast culture alone.

[0093] In one embodiment, the microbial combination of the present disclosure is cultured at 2.5×10 5 cells / mL of Burkholderia bacteria and 2.5 x 10 5 When Yarrowia yeast was inoculated at 1000 cells / mL and cultured at pH 7.0 and 15°C, total RNA was extracted from the cells 48 hours after the start of culture and quantitative PCR analysis was performed on a specific Yarrowia yeast lipase. The expression level was 5×10 5 The combination is such that the number of cells / mL is 1.1 or more, 1.2 or more, 1.5 or more, 1.7 or more, 2 or more, 5 or more, 10 or more, or 20 or more times higher than when Yarrowia yeast is inoculated alone.

[0094] In one aspect, the present disclosure provides a composition for improving lipase production in a lipase-producing Burkholderia bacterium, comprising a Yarrowia yeast. Alternatively, the present disclosure provides a Yarrowia yeast for improving lipase production in a lipase-producing Burkholderia bacterium. In another aspect, the present disclosure provides a composition for improving lipase production in a lipase-producing Yarrowia yeast, comprising a Burkholderia bacterium. Alternatively, the present disclosure provides a Burkholderia bacterium for improving lipase production in a lipase-producing Yarrowia yeast. As described herein, the combined effect of two microorganisms is observed in the present disclosure, but from another perspective, this can be said to be an invention based on the discovery that a new function, ability, or use of the Yarrowia yeast can improve lipase production in a lipase-producing Burkholderia bacterium, or an invention based on the discovery that a new function, ability, or use of the Burkholderia bacterium can improve lipase production in a lipase-producing Yarrowia yeast. Therefore, in these aspects, it is understood that the matters relating to the combination of microorganisms of the present disclosure described herein are equally applicable to these novel uses.

[0095] In another aspect, the present disclosure provides a composition for enhancing the ability of a lipase-producing Burkholderia bacterium to process fats and / or oils and / or fatty acids, comprising a Yarrowia yeast. Alternatively, the present disclosure provides a Yarrowia yeast for enhancing the ability of a lipase-producing Burkholderia bacterium to process fats and / or oils and / or fatty acids. In yet another aspect, the present disclosure provides a composition for enhancing the ability of a lipase-producing Yarrowia yeast to process fats and / or oils and / or fatty acids, comprising a Burkholderia bacterium. Alternatively, the present disclosure provides a Burkholderia bacterium for enhancing the ability of a lipase-producing Yarrowia yeast to process fats and / or oils and / or fatty acids. In the present disclosure, the combined effect of two types of microorganisms is observed, but from another perspective, this can be said to be an invention based on the discovery that a new function, ability, or use of Yarrowia yeast is to enhance the ability of a lipase-producing Burkholderia bacterium to process fats and / or oils, or an invention based on the discovery that a new function, ability, or use of Burkholderia bacteria is to enhance the ability of a lipase-producing Yarrowia yeast to process fats and / or oils, or oils and / or fatty acids. Therefore, in these aspects, it is understood that the matters related to the combination of microorganisms of the present disclosure described herein can be similarly applied to these new uses.

[0096] Without wishing to be bound by theory, in one preferred embodiment, it is preferable to provide a composition for improving lipase production in a lipase-producing Burkholderia bacterium comprising Yarrowia yeast, and / or a composition for enhancing the ability of a lipase-producing Burkholderia bacterium comprising Yarrowia yeast to process oils and / or fatty acids, because the ability of Burkholderia bacteria, which are relatively easy to prepare, can be utilized to achieve an equivalent or greater oil and / or fatty acid decomposition effect by utilizing a small amount of Yarrowia yeast. Without 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, while the doubling time of Yarrowia yeast is about 1.5 hours, so that Burkholderia bacteria grow faster. The inventors have discovered that the combination of microorganisms disclosed herein can have an oil and / or fatty acid decomposition ability that exceeds the oil and / or fatty acid decomposition ability calculated from the oil and / or fatty acid decomposition values ​​of each culture alone. Based on this finding, by using the combination of microorganisms disclosed herein, the oil and / or fatty acid decomposition ability of fast-growing Burkholderia bacteria is improved, accelerating the overall oil and / or fatty acid decomposition, thereby enabling the efficient and stable use of microorganisms.

[0097] In one embodiment, the Burkholderia bacteria in the microbial combination of the present disclosure are from the order Burkholderiales. In one embodiment, the Burkholderia bacteria in the microbial combination of the present disclosure are from the family Burkholderiaceae. In one embodiment, the Burkholderia bacteria in the microbial combination of the present disclosure are from the genus Burkholderia. The genus Burkholderia is a gram-negative, non-spore-forming, aerobic, polar-flagellated bacillus and is the type genus of the family Burkholderiaceae. In one embodiment, the Burkholderia bacteria in the microbial combination 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 microbial combination of the present disclosure is a microorganism belonging to the Burkholderia cepacia complex, which is a classification of microorganisms in the genetically closely related genus Burkholderia, including amphifaria, 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 microbial combinations of the present disclosure may be metallica, seminalis, anthina, amphifaria, diffusa, ubonensis, multivorans, latens, cenocepacia, vietnamiensis, pyrrocinia, stabilis, glumae, gladioli, plantarii, oklahomensis, thailandensis, mallei, pseudomallei, or phytofirmans. In a further aspect of the present disclosure, the Burkholderia bacteria in the microbial combinations of the present disclosure may be a bacterium of the genus Ralstonia or Pseudomonas. The present inventors identified a new microbial strain (KH-1 strain) as Burkholderia arboris by determining the base sequence of 16S ribosomal DNA and phylogenetic analysis, and deposited it at the National Institute of Technology and Evaluation Patent Microorganism Deposit Center, 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 bacteria of the genus Burkholderia (KH-1AL1 strain, KH-1AL2 strain, and KH-1AL3 strain) were further identified, and deposited them at the National Institute of Technology and Evaluation Patent Microorganism Deposit Center, 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 bacterium in the microbial combination of the present disclosure is Burkholderia strain KH-1 (strain identified by accession number NITE BP-02731), KH-1AL1 (strain identified by accession number NITE BP-02977), KH-1AL2 (strain identified by accession number NITE BP-02978) or KH-1AL3 (strain identified by accession number NITE BP-02979), or a derivative thereof.

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

[0099] The Burkholderia bacteria in the combination of microorganisms of the present disclosure may be isolable on an inorganic salt agar medium containing oil or fat as the only carbon source and adjusted to a pH of 6 to 8. In one embodiment, the Burkholderia bacteria in the combination of microorganisms of the present disclosure produce lipase. In one embodiment, the Burkholderia bacteria in the combination of microorganisms of the present disclosure may be distinguishable by confirming the formation of a clear zone (halo) around the colony formed on the agar medium.

[0100] In one embodiment, the Burkholderia bacteria in the combination of microorganisms of the present disclosure have the ability to degrade oils and / or fatty acids at 15° C. In one embodiment, the Burkholderia bacteria in the combination of microorganisms of the present disclosure retain the ability to produce lipase and / or the ability to assimilate or degrade fatty acids or oils and oils at 15° C.

[0101] In one embodiment, the Burkholderia bacteria in the microbial combination of the present disclosure may secrete a biosurfactant when cultured in a medium containing an oil or fatty acid.

[0102] In one embodiment, the Burkholderia bacteria in the microbial combination of the present disclosure are cultured at 3×10 6 cells / mL or 2 x 10 6 When the microorganism is inoculated at a cell concentration of 2×10 6 cells / mL and cultured under air reflux of 200 ml / min at pH 7.0 and 15° C., the oil content equivalent to the normal hexane value in the supernatant after 24 or 48 hours is 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. In particular, the Burkholderia bacteria in the combination of microorganisms disclosed herein have an oil-decomposing ability of 2×10 6 cells / mL when determined under these conditions. 6It is preferable that the bacteria have an ability to decompose fats and oils, which reduces the oil content equivalent to normal hexane to less than about 6 g / L when inoculated at a cell concentration of 3×10 cells / mL and cultured for 48 hours, and 6 It is particularly preferable that the microorganism has an oil-decomposing ability that reduces the oil content equivalent to a normal hexane value to less than 6 g / L when inoculated at a cell concentration of 1000 cells / mL and cultured for 24 hours, and such a microorganism having low-temperature oil-decomposing ability can be advantageously used in various applications of the present disclosure.

[0103] In one embodiment, the Burkholderia bacteria in the microbial combination of the present disclosure are cultured in mineral salts medium containing 10 g / L canola oil at a final concentration of 3×10 6 When the microorganism is inoculated at a cell concentration of 1000 cells / mL and cultured under air reflux of 200 ml / min at pH 7.0, at 15°C and at 28°C, the oil content equivalent to the normal hexane value in the supernatant after 24 hours of culture 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 of the oil content equivalent to the normal hexane value in the supernatant after 24 hours of culture at 28°C. When judged under these conditions, the Burkholderia bacteria in the combination of microorganisms disclosed herein preferably have an oil-decomposing ability such that the oil residual rate when cultured at 15°C is 800% or less, 700% or less, 600% or less, 500% or less, 400% or less, and particularly 700% or less compared to when cultured at 28°C. Such microorganisms having low-temperature oil-decomposing ability can be beneficially used in various applications of the present disclosure.

[0104] In one embodiment, the Burkholderia bacteria in the microbial combination of the present disclosure are cultured in mineral salts medium containing 10 g / L canola oil at a final concentration of 3×10 6When the microorganism is inoculated at a cell concentration of 1000 cells / mL and culture is started under air reflux of 200 ml / min at pH 7.0, 15 ° C. and 28 ° C., the total fatty acid decomposition rate in 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 than that in 28 ° C. culture. The Burkholderia bacterium in the combination of microorganisms disclosed herein preferably has an oil-decomposing ability of 50% or more, 40% or more, 30% or more, 20% or more or 10% or more, particularly 30% or more than that in 15 ° C. culture when judged under these conditions, compared to 28 ° C. culture, and such microorganisms having low-temperature oil-decomposing ability can be beneficially used in various applications of the present disclosure.

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

[0106] At least one microorganism in the combination of microorganisms of the present disclosure may be isolable on an inorganic salt agar medium containing fats and oils as the only carbon source and adjusted to a pH of 6 to 8. In one embodiment, at least one microorganism in the combination of microorganisms of the present disclosure may be distinguishable by confirming the formation of a clear zone (halo) around a colony formed on an agar medium. In one embodiment, one microorganism in the combination of microorganisms of the present disclosure is preferably capable of forming and growing colonies on an agar medium to which 10 g / L of canola oil is added as a carbon source at 15° C. or 28° C., preferably at 15° C., and more preferably, both the Burkholderia bacteria and the Yarrowia yeast in the combination of microorganisms of the present disclosure are capable of forming and growing colonies under the same conditions. In the combination of microorganisms of the present disclosure, it may be useful that both the Burkholderia bacteria and the Yarrowia yeast have a minimum level of fat and oil assimilation (decomposition) ability.

[0107] In one embodiment, the ability of a microorganism to decompose and assimilate oils and fatty acids can be evaluated by quantifying the fatty acids contained in the oils and fatty acids remaining in the medium and the free fatty acids generated by decomposition by gas chromatography. The specific quantitative procedure is as follows: first, 3 mL of culture supernatant is acidified with hydrochloric acid, and an equal amount of ethyl acetate is added. After stirring for 5 minutes, the mixture is centrifuged, and 1 mL of the ethyl acetate layer is transferred to a separate container and the solvent is evaporated to concentrate. The mixture is dissolved in 1 mL of chloroform, and 4 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 fatty acids. After that, a solution of chloroform:pure water = 1:1 is added and stirred, and the chloroform layer is mixed with 0.5% methyl octanoate in a ratio of 1:1, and analyzed by gas chromatography to quantify the methyl esters of all fatty acids. The amount of reagents and the type of extraction solvent can be appropriately changed, and for example, the following alternative procedure is possible. 1 mL of culture supernatant is acidified with hydrochloric acid, and 2 mL of chloroform is added. After stirring for 2 minutes, centrifuge, and transfer 1 mL of the chloroform layer to a separate container and evaporate the solvent to concentrate. Add 2 mL of methanolysis solution (methanol:sulfuric acid = 17:3) and heat at 100°C for 2 hours to methylate the fatty acids and free fatty acids in the oil. Then, add 2 mL of chloroform and 1 mL of pure water and stir. After that, analyze the chloroform layer by gas chromatography to quantify the methyl esters of all fatty acids.

[0108] In one embodiment, the ability of a microorganism to decompose and assimilate fats and oils and fatty acids can be evaluated by analyzing fats and oils remaining in the medium and fatty acids that are decomposition products by thin layer chromatography. Specifically, fats and oils are extracted by adding an equal amount 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 in a volume ratio of 96:4:1, respectively. The ratio of the developing solvent can be appropriately changed, and good results can be obtained even if, for example, chloroform, acetone, and methanol are used in a volume ratio of 96:4:2, respectively. The plate is treated with molybdophosphate n-hydrate to color the fats and oils and / or fatty acids.

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

[0110] In one embodiment, a microorganism in the combination of microorganisms of the present disclosure is capable of producing an esterase (e.g., a lipase).

[0111] In one embodiment, lipase activity can be determined by performing an enzyme reaction using 4-nitrophenyl palmitate (4-NPP), an ester of palmitic acid and 4-nitrophenol, as a substrate, and measuring the absorbance at 410 nm of the amount of p-nitrophenol produced by hydrolysis of the ester. 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 (150 mM 3,3-dimethylglutaric acid, 150 mM Tris, and 150 mM 2-amino-2-methyl-1,3-propanediol, adjusted to pH 7.0 by adding NaOH or HCl) are placed in a cell and incubated at 28° C. for 5 minutes. 0.1 mL of culture supernatant is added to the cell, and the value at 410 nm is measured while stirring. Lipase activity is measured by defining the amount of enzyme required to produce 1 μmole of 4-nitrophenol as 1 unit (U), and units per 1 mL of culture supernatant are calculated.

[0112] In one embodiment, the microorganisms in the combination of microorganisms disclosed herein may be capable of growing and decomposing oil under weakly acidic conditions (eg, pH of about 5.5 to 6.0).

[0113] In one embodiment, the proliferation ability of a microorganism can be examined by a method of measuring the absorbance (turbidity) at 660 nm as the optical density of the bacterial body, a method of measuring colony forming units (CFU), etc. In the latter method, a certain amount of the original culture solution and a diluted 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-mentioned measurement methods to test derivatives of the KH-1, KH-1AL1, KH-1AL2, and KH-1AL3 strains or the KH-2, KH-2AL1, and KH-2AL3 strains to obtain derivatives having the above-mentioned biological functions (and their degrees).

[0115] In one embodiment, the Burkholderia bacteria and / or Yarrowia yeast in the combination of microorganisms disclosed herein can be selected based on the criteria that the results of the KH-1 strain plus the Yarrowia yeast or the Burkholderia bacteria plus the KH-2 strain are equivalent to or better than the KH-1 strain alone or the KH-2 strain alone.

[0116] In one embodiment, for example, a total of 5×10 KH-1 strain + Yarrowia yeast or Burkholderia bacteria + KH-2 strain was cultured in mineral salts medium supplemented with 1% (v / v) canola oil. 5 The Burkholderia bacterium and / or Yarrowia yeast can be suitably used when the total fatty acid value determined by gas chromatography analysis of the culture supernatant after inoculation at a cell concentration of 100000000 cells / mL and culturing at pH 7.0 and 15°C for 48 hours or at pH 7.0 and 28°C for 18 hours 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% of the total fatty acid value of the KH-1 strain + KH-2 strain under the same conditions.

[0117] In one embodiment, OD was measured in mineral salts medium supplemented with 1% (v / v) canola oil. 660 Each microorganism is inoculated at a bacterial cell optical density such that the measured value of KH-1 strain:Yarrowia yeast or Burkholderia bacteria:KH-2 strain = 0.018:0.02, and the cells are cultured at pH 7.0 and 15°C for 71 hours. Total RNA is extracted from the cells and quantitative PCR analysis is performed for a specific Burkholderia bacterium lipase. If 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, or 200% or more compared to the expression level of the Burkholderia bacterium lipase in a mixed culture of the KH-1 strain and the KH-2 strain under the same conditions, then the Burkholderia bacterium and / or Yarrowia yeast can be suitably used.

[0118] In one embodiment, the KH-1 strain: Yarrowia yeast or Burkholderia bacteria: KH-2 strain = 2.5 x 10 5Cells / mL:2.5×10 5 Each microorganism was inoculated at a concentration of 1000 cells / mL and cultured at pH 7.0 and 15°C for 48 hours. Total RNA was extracted from the cells and quantitative PCR analysis was performed on a specific Yarrowia yeast lipase. If 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, or 200% or more compared to the expression level of the Yarrowia yeast lipase in a mixed culture of the KH-1 strain and the KH-2 strain under the same conditions, then the Burkholderia bacterium and / or Yarrowia yeast can be suitably used.

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

[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 embodiment, the combination of microorganisms of the present disclosure is provided in a composition (optionally in combination with a microorganism that is 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 decomposition agent. In one embodiment, the composition of the present disclosure is a composition for improving the expression and / or production of lipase of the other of the Burkholderia bacterium and the Yarrowia yeast, which comprises one of the Burkholderia bacterium and the Yarrowia yeast in the combination of microorganisms of the present disclosure, and in one embodiment, the lipase comprises a first lipase of the Burkholderia bacterium (base sequence: SEQ ID NO: 1, amino acid sequence: SEQ ID NO: 2, or a variant thereof, etc.), a second lipase (base sequence: SEQ ID NO: 3, amino acid sequence: SEQ ID NO: 4, or a variant thereof, etc.), a first lipase of the Yarrowia yeast (base sequence: SEQ ID NO: 5, amino acid sequence: SEQ ID NO: 6, or a variant thereof, etc.) and / or a second lipase (base sequence: SEQ ID NO: 7, amino acid sequence: SEQ ID NO: 8, or a variant thereof, etc.). In one embodiment, the composition is an oil decomposing agent for decomposing oils and / or fatty acids at a predetermined temperature (e.g., 15°C, 28°C), for decomposing oils and / or fats containing short-chain to medium-chain fatty acids (C2 to C12), and / or for decomposing oils and fats containing short-chain to long-chain fatty acids (C2 or more). Even if the oil decomposing agent contains only one type of microorganism (e.g., either Burkholderia bacteria or Yarrowia yeast) of the combination of microorganisms disclosed herein and does not exhibit the desired oil and / or fatty acid decomposition ability by itself, it is sufficient that the oil and / or fatty acid decomposition ability is exhibited when used to form the combination of microorganisms disclosed herein. In one embodiment, the composition is a fatty acid decomposing agent. By processing with the fatty acid decomposing agent disclosed herein, a compound containing fewer carbons than the number of carbons contained in the fatty acid can be generated.

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

[0123] In a specific embodiment, the fats and oils to which the oil-splitting agent of the present disclosure is applied include fats and oils containing trans fatty acids (e.g., elaidic acid, palmitellidic acid, and / or vaccenic acid), and examples of such fats and oils include, but are not limited to, processed products such as fats and oils produced by hydrogenation (margarine, shortening, butter, etc.). The addition of hydrogen reduces the number of double bonds in unsaturated fatty acids and increases the proportion of saturated fatty acids, which may result in the production of trans fatty acids. Trans fatty acids are said to be contained in margarine, fat spreads, and shortening produced by hydrogenation, as well as in Western confectioneries such as bread, cakes, and donuts, and fried foods that use these products as raw materials. In the process of refining oils extracted from plants and fish, high temperature treatment is performed to remove undesirable odors. At this time, trans fatty acids are produced from cis-type unsaturated fatty acids contained in the oil, so refined vegetable oils such as salad oils are also said to contain trace amounts of trans fatty acids.

[0124] The oil decomposer or fatty acid decomposer of the present disclosure may be applied to any of a variety of objects, including, but not limited to, industrial wastewater, household wastewater, industrial waste, household waste (such as food waste), livestock waste, aquaculture farms (and their wastewater), livestock barns (and their wastewater), slaughterhouses (and their wastewater), soil contaminated with oil, water contaminated with oil (such as the sea, ponds, rivers, and drinking water for animals), animal body surfaces, aquariums (for aquaculture, ornamental use, etc.), any oil-contaminated products (such as tableware, machine parts, etc.), grease traps installed in kitchens, drainage pipes, fatbergs, insulating oil leaked from transformers, and deteriorated insulating oil. A "grease trap" is a device for separating and collecting oil in wastewater, and is typically composed of three tanks. The first tank is equipped with a basket and captures food particles and leftover food. In the second tank, oil and water are separated. The wastewater separated from the oil is sent to the third tank, and sedimentary garbage and the like are removed. Grease traps are mandatory for commercial kitchens such as restaurants, hospitals, and hotels. When applying this method to a grease trap, a separate decomposition treatment tank may be provided, but the oil decomposition agent and microorganisms may be directly added to the grease trap to perform decomposition treatment within the grease trap.

[0125] The combination of microorganisms disclosed in the present disclosure is also highly efficient at low temperature, so low temperature treatment can be a desirable embodiment.For example, industrial wastewater, household wastewater, industrial waste, household waste (such as food waste), oil-contaminated soil, oil-contaminated water (sea, pond, river, animal drinking water, etc.), etc., which are assumed to be treated at temperatures below 20°C (for example, 15°C), are also preferred examples of the target to be treated by the combination of microorganisms disclosed in the present disclosure.

[0126] Specifically, a preparation or the like is introduced or added, or a carrier on which one or more of the combinations of microorganisms disclosed herein are immobilized is placed in a drainage path, a drainage storage tank, a grease trap, etc. A dedicated decomposition treatment tank may be provided separately outside the grease trap.

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

[0128] (Usage form) The form of the microbial combination, combination, or composition of the present disclosure may be, for example, in a liquid state or a solid state. Examples of the microbial combination, combination, or composition in a liquid state include a microbial culture solution, and a microorganism collected from the culture solution by centrifugation or the like and then dispersed again in water, a buffer solution, or a culture solution. Examples of the microorganism or composition in a solid state include a dehydrated one by centrifugation or press compression, a paste state or mayonnaise state that is intermediate between a solid and a liquid, and a dried body that has been dried (for example, dried under reduced pressure or freeze-dried). Examples of the solid form include a powder, granule, tablet, and the like. In addition, the microbial combination of the present disclosure may be provided in a state in which the microorganism or the culture supernatant is fixed to a carrier.

[0129] In one embodiment, the microbial combinations disclosed herein and compositions or combinations providing the same have a total of about 1×10 8 cells / mL, approximately 1×10 7 cells / mL, approximately 1×10 6 cells / mL, approximately 1×10 5 cells / mL, approximately 1×10 4 cells / mL, approximately 1×10 3 cells / mL, approximately 1×10 2 The liquid may be added to a cell concentration of about 10 cells / mL, about 10 cells / mL, or about 1 cell / mL.

[0130] (Applicable environment) The microbial combination of the present disclosure and the composition or combination that provides the same can be used in any suitable environment. In one embodiment, the microbial combination of the present disclosure and the composition or combination that provides 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 compositions or combinations providing the same may be used in an environment of pH 4-12, pH 5-11, pH 6-10, pH 7-9, pH 5.5-8.5, 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 disclosed herein and compositions or combinations providing the same may be used in environments with dissolved oxygen concentrations (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 combination of microorganisms disclosed herein and the composition or combination providing the same can be used in an environment (e.g., in wastewater) with a normal hexane value of 100 to 40,000 mg / L, 200 to 30,000 mg / L, or 300 to 30,000 mg / L. In solid waste (which may contain water) such as sludge slurry or food waste treatment, oils and fats may be present at higher concentrations, and in one embodiment, the combination of microorganisms disclosed herein and the composition or combination providing the same can be usefully applied to such solid waste.

[0134] In one embodiment, the microorganisms, compositions or combinations of the present disclosure may be added to a locus that contains 50% or more, 20% or more, 10% or more, 7% or more, 5% or more, 2% or more, 1% or more, 0.7% or more, 0.5% or more, 0.2% or more, 0.1% or more, 0.07% or more, 0.05% or more, 0.02% or more, 0.01% or more, 0.007% or more, 0.005% or more, 0.002% or more, or 0.001% or more by weight of trans fatty acids.

[0135] In one embodiment, the microorganism, composition or combination of the present disclosure may be added to a target having an ester (e.g., fat or oil) containing trans fatty acids of 50% by weight or more, 20% by weight or more, 10% by weight or more, 7% by weight or more, 5% by weight or more, 2% by weight or more, 1% by weight or more, 0.7% by weight or more, 0.5% by weight or more, 0.2% by weight or more, 0.1% by weight or more, 0.07% by weight or more, 0.05% by weight or more, 0.02% by weight or more, 0.01% by weight or more, 0.007% by weight or more, 0.005% by weight or more, 0.002% by weight or more, or 0.001% by weight or more.

[0136] In one embodiment, the subject to which the microorganism, combination of microorganisms, and composition or combination providing the same of the present disclosure are added may have nitrogen present in a form available to the microorganism, preferably in the form of ammonium salt, nitrate, sulfate, or organic nitrogen compound, more preferably in the form of ammonium sulfate, urea, amino acid, or peptide such as peptone, tryptone, or casamino acid. The amount of nitrogen present may be in the range of C / N=2 to 50, preferably C / N=2 to 30, more preferably C / N=2 to 20. Here, C / N is the weight ratio of carbon atoms derived from normal hexane and 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, the microorganism, combination of microorganisms, and composition or combination providing the same of the present disclosure may be added to a target in which phosphorus (P) is 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 an amount relative to nitrogen such that N / P=1 to 20, where N / P is the weight ratio of nitrogen atoms to phosphorus atoms contained in wastewater. In one embodiment, phosphorus may be further added so as to fall within these ranges.

[0138] In one embodiment, the microorganisms, combinations of microorganisms and compositions or combinations providing the same of the present disclosure may be used in the presence of salt, surfactants, light, electric current, agitation, aeration, or any combination thereof.

[0139] In one embodiment, the microorganisms, combinations of microorganisms and compositions or combinations providing the same of the present disclosure may be applied after removal of substances that kill or inhibit the growth of the microorganisms of the present disclosure (e.g., chlorine, antibiotics, etc.).

[0140] In one embodiment, the microorganisms, combinations of microorganisms, and compositions or combinations providing the same of the present disclosure may be used together with a carrier capable of immobilizing the microorganisms. Washout can be effectively avoided by using such a carrier. The material of the carrier is not particularly limited as long as it can immobilize the microorganisms, and examples thereof include carbon fiber (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 efficiency of the action of the microorganisms, it is preferable to use a porous or fibrous carrier. The microorganisms may also be included in a gel-like carrier. The shape of the carrier may be, for example, a cube, a rectangular parallelepiped, a cylinder, a sphere, a disk, a sheet, a membrane, etc. For information on microbial immobilization technology, see, for example, "Wastewater Treatment by Microbial Immobilization Method" (edited by Ryuichi Sudo, Industrial Water Research Association) or "Water Treatment by Microbial Immobilization Method - Carrier Immobilization Method, Entrapment Immobilization Method, Biological Activated Carbon Method (New Water Treatment Series (1))" (written by Kazuhiro Mochizuki, Katsutoshi Hori, and Hideki Tachimoto, NTS Co., Ltd.).

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

[0142] In one embodiment, the additional components include, but are not limited to, components that enhance the activity of the microorganism used (e.g., carbon source, nitrogen source), surfactants, drying protection agents, components for maintaining the microorganism for a long period of time, preservatives, excipients, strengthening agents, antioxidants, and other microorganisms, and any suitable components can be used.

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

[0144] Examples of microorganisms that decompose (assimilate) glycerol include eubacteria, yeasts, and filamentous fungi. Candida yeasts are preferably used. A specific example of Candida yeast is Candida cylindracea SL1B2 (deposited at the Patent Microorganisms Depositary Center of the National Institute of Technology and Evaluation under the accession number NITE P-714). This strain has excellent glycerol assimilation ability. By using a microorganism that decomposes (assimilates) glycerol in combination, it is possible to prevent a decrease in the rate of fat and oil decomposition caused by the accumulation of glycerol, and to achieve more efficient fat and oil decomposition.

[0145] (Method 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, comprising acting on a subject to be treated with a microorganism, composition, or combination of the present disclosure. The subject 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 subject to be treated may be any subject to be treated described herein to which the microorganism, composition, or combination 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 microorganism, composition, or combination 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 may include a step of decomposing trans fatty acids (e.g., elaidic acid, palmitelaidic acid, and / or vaccenic acid), a step of decomposing fats and oils containing trans fatty acids, a step of decomposing esters (e.g., fats and oils) and / or fatty acids at 15° C., a step of decomposing esters containing short- to medium-chain fatty acids (C2 to C12), and / or a step of decomposing fats and oils containing short- to long-chain fatty acids (C2 or more). The method for decomposing and removing esters (e.g., fats and oils) and / or fatty acids of the present disclosure may use any of the additional components described herein that may be used in combination with the microorganisms, compositions, or combinations of the present disclosure.

[0146] In one aspect, the present disclosure provides a method for decomposing and removing fats and / or fatty acids, comprising applying the combination of microorganisms of the present disclosure and a composition or combination that provides the combination to a subject to be treated. In one aspect, the present disclosure provides a method for improving lipase production of at least one of the Burkholderia bacteria and the Yarrowia yeast, comprising a step of mixing (e.g., symbiotically culturing) 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 application of each microorganism (or a composition containing the same) results in the combination of microorganisms of the present disclosure, and in this specification, any embodiment is described as applying, applying, or introducing the combination of microorganisms of the present disclosure.

[0147] In one embodiment, the method for decomposing and removing esters (e.g., fats and oils) and / or fatty acids of the present disclosure includes a step of feeding the microorganisms, combinations of microorganisms, and compositions or combinations providing the same of the present disclosure into a fat and oil decomposition tank, and the feeding may be continuous or sequential. The HRT (hydraulic retention time) of the fat and oil decomposition tank is usually 12 hours or more, preferably 18 hours or more, more preferably 20 hours or more, and even more preferably 24 hours or more. For wastewater with a normal hexane value of more than 10,000 mg / L, when a reduction in the normal hexane value of 80% or more is expected, the HRT may be usually 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 a reduction in the normal hexane value of 80% or more is expected, the HRT may be usually 8 hours or more, preferably 12 hours or more, and more preferably 18 hours or more.

[0148] The microbial concentration in the fat / oil decomposition tank may depend on the concentration of esters (e.g. fats and oils) and / or fatty acids in the wastewater, and the higher the fat / oil and / or fatty acid concentration, the higher the bacterial concentration can be maintained. If foaming occurs in the fat / oil decomposition tank, measures can be taken to reduce the HRT, perform showering, add a defoaming agent, or take other measures. However, since defoaming agents can inhibit the growth of microorganisms, it is desirable to set the amount of defoaming agent to be added taking this into consideration.

[0149] The normal hexane value of the effluent from the oil and fat decomposition tank is preferably 60 mg / L or less, more preferably 30 mg / L or less, in the case of low-concentration wastewater with a normal hexane value of about 300 mg / L or less. In the case of medium-concentration wastewater with a normal hexane value of about 3000 mg / L, it is preferably 600 mg / L or less, more preferably 300 mg / L or less, even 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 about 10000 mg / L, it is preferably 1000 mg / L or less, more preferably 500 mg / L or less, even 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 about 30000 mg / L or more, it is preferably 3000 mg / L or less, more preferably 1000 mg / L or less, and even more preferably 300 mg / L or less.

[0150] In one embodiment, the method of the present disclosure can reduce the normal hexane value of wastewater containing fats and oils and / or fatty acids by preferably 80% or more, more preferably 90% or more, even more preferably 95% or more, and most preferably 99% or more. As a result, in many wastewaters, it is also possible to reduce the normal hexane value of the effluent from the fat and oil decomposition tank to less than 30 mg / L, which is the standard value for discharge into sewerage in many municipalities. If this standard value is achieved, focusing only on the normal hexane value, even the main treatment such as the subsequent activated sludge treatment may not be necessary.

[0151] The amount of the microorganisms added in the outflow water from the oil / fat decomposition tank does not need to be increased. The amount of the microorganisms added in the outflow water is preferably 0.01 times or more, more preferably 0.1 times or more, even more preferably 0.5 times or more, and most preferably 1 times or more, relative to the amount added.

[0152] The method for decomposing and removing fats and / or fatty acids disclosed herein may include additional steps in addition to the steps described above. Such steps include, for example, returning all or a part of the effluent water from the fat / oil decomposition tank back to the fat / oil decomposition tank. However, in the method disclosed herein, a sufficient fat / oil and / or fatty acid decomposition effect can be obtained without such return treatment, so it is not essential to return all or a part of the effluent water from the fat / oil decomposition tank back to the fat / oil decomposition tank.

[0153] (General technology) The molecular biological techniques, biochemical techniques, and microbiological techniques used herein are well known and commonly used in the art, and are described in, 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, Ishii Seiji, Okumura Hiroshi, Matsubara Chiyo, Ninomiya Fumi, Yoshioka Hiroshi, 2004, "Simple method for measuring oil content in water using thermosensitive polymers," Vol. 46, No. 12, "Water and Wastewater," and the relevant parts (which may be in their entirety) are incorporated by reference in this specification.

[0154] (Note) In this specification, "or" is used when "at least one or more" of the items listed in the sentence can be employed. The same applies to "alternative." In this specification, when "within the range of" two values ​​is specified, the range includes the two values ​​themselves.

[0155] All references cited herein, including scientific literature, patents, patent applications, and the like, are hereby incorporated by reference in their entirety to the same extent as if each was specifically set forth.

[0156] The present disclosure has been described above by showing preferred embodiments for ease of understanding. The present disclosure will be described below based on examples, but the above description and the following examples are provided for illustrative purposes only and are not provided 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 in this specification, but is limited only by the scope of the claims. EXAMPLES

[0157] Examples are described below. When necessary, the handling of the organisms used in the following examples complied with the standards stipulated by Nagoya University, the regulatory authorities, and the Cartagena Protocol. The reagents used were specifically the products described in the examples, but equivalent products from other manufacturers (Sigma-Aldrich, Fujifilm, Wako Pure Chemical Industries, Nakarai, R&D Systems, USCN Life Science INC, Kanto Chemical, Funakoshi, Tokyo Kasei, Merck, etc.) can also be used. In addition, unless otherwise stated, the concentration of various oils and fatty acids added to the medium refers to the final concentration of the substance in the medium, and the percentage notation indicates volume / volume (v / v%) for oils and fatty acids in liquid form, and weight / volume (w / v%) for solid form.

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

[0159] We also examined the ability of each microorganism to degrade fats and oils at low temperatures (15°C). A colony of each microorganism was inoculated with a toothpick into 20 mL of mineral salts 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 Na2MoO4 0.25 mg / L) containing 10 g / L canola oil as the sole carbon source, and cultured in a 100 mL Erlenmeyer flask. As a result, microorganisms were discovered that can break down, assimilate, and grow on fats and oils at low temperatures.

[0160] Next, the ability of each microorganism to degrade and assimilate trans fatty acids was examined. The final cell optical density OD 660 Each microorganism was inoculated so that the β-amino acid ratio was 0.04. The microorganisms were cultured in 15 mL Harmony centrifuge tubes (LM S, Tokyo) at 28°C and 130 rpm for 24 hours. As a result, microorganisms capable of decomposing, assimilating, and growing the trans-fatty acid elaidic acid were found.

[0161] Through these tests, it was found that one of the isolated microbial strains could be cultivated in a low-temperature environment using fats and oils as a nutrient source, was able to assimilate (break down) fats and oils at low temperatures, and was able to break down and assimilate trans fatty acid-containing fats and oils as well as trans fatty acids; this new strain was named KH-2.

[0162] Subsequent analysis revealed that the KH-2 strain was Yarrowia lipolytica. Here, the analysis was focused on the KH-2 strain, but the lipid and / or fatty acid decomposition ability of other strains can also be identified by examining the lipid and / or fatty acid decomposition ability through similar tests.

[0163] (Example 2: Ability of KH-2 strain to assimilate and decompose fats and oils and fatty acids) For the KH-2 strain, the bacteria were streaked onto an agar medium containing triolein, oleic acid, or canola oil (above) as the sole carbon source (final concentration of fat or oil: 1%, final concentration of Triton (registered trademark) X-100: 0.25%, final concentration of polyvinyl alcohol: 0.5%, inorganic salts agar medium (inorganic salts medium containing 1.5% agar) (pH 7.0)), and cultured at 28°C for 3 days. The composition of the inorganic salt medium was 3.5g Na2HPO4 / L for KH-2 strain, 2.0g PO4 / L, 4.0g (NH4)2SO4 / L, 0.34g MgCl2·6H2O / L, 2.8mg FeSO4·7H2O / L, 2.4mg MnSO4·5H2O / L, 2.4mg CoCl2·6H2O / L, 1.7mg CaCl2·2H2O / L, 0.2mg CuCl2·2H2O / L, 0.3mg ZnSO4·7H2O / L, and 0.25mg Na2MoO4 / L. The results are shown in Figure 1. Colonies were formed on all agar media, demonstrating that the KH-2 strain is capable of decomposing, assimilating, and growing on canola oil, a typical oil-containing vegetable oil, triolein, a triglyceride oil, and oleic acid, a typical example of a free fatty acid that is the decomposition product of these oils.

[0164] Furthermore, for the KH-2 strain, the bacterium was streaked onto an agar medium containing elaidic acid as the sole carbon source (elaidic acid final concentration 0.1%, Triton (registered trademark) X-100 final concentration 0.25%, inorganic salt agar medium (pH 7.0)) 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 strain was capable of decomposing and assimilating elaidic acid, a typical trans-fatty acid, and growing.

[0165] (Example 3A: Trans fatty acid decomposition ability of KH-2 strain at 28°C) The trans-fatty acid decomposition abilities of the KH-2 strain and BioRemove3200 (BR3200) (Novozymes, Denmark) at 28°C were compared. The cells were cultured in 20 mL of inorganic salt medium (composition as above, pH 7.0) containing 0.2% elaidic acid and 0.25% Triton (registered trademark) X-100 until the final cell optical density OD 660= 0.5 (HITACHI U-2810 spectrophotometer (Hitachi, Tokyo)) of KH-2 strain or 5 times the manufacturer's recommended concentration, 5 × 10 6 The bacteria were inoculated to a CFU / ml concentration of BR3200 and cultured at 28° C. with shaking at 130 rpm for 24 hours.

[0166] The lipids in the supernatant after the culture were analyzed by thin layer chromatography. Specifically, an equal amount of chloroform was added to the culture supernatant, and after stirring, 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, the fatty acids were visualized by spraying molybdophosphate n-hydrate solution (2.4 g / 60 ml ethanol) and heating at 110 °C for 12 minutes, and the amount of fatty acids remaining in the medium was compared (Figure 3A (a)).

[0167] In addition, 0.5 mL of the 24-h culture supernatant was analyzed using an oil content measurement reagent kit (Kyoritsu Chemical Research Institute, Tokyo) (a measurement reagent kit using the Polynipum extract substance measurement method) according to the manufacturer's recommended protocol (Figure 3A(b)).

[0168] The KH-2 strain was found to have a strong ability to degrade trans fatty acids.

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

[0170] The fats and oils in the supernatant after the culture were analyzed by thin-layer chromatography. Specifically, 1 / 2 the volume of chloroform was added to the culture supernatant, and after stirring, 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, the fatty acids were visualized by spraying molybdophosphate n-hydrate solution (2.4 g / 60 ml ethanol) and heating at 110 °C for 12 minutes, and the amount of fatty acids remaining in the medium was compared (Figure 3B (a)).

[0171] In addition, after 48 hours of culture, 0.5 mL of the supernatant was analyzed using an oil content measurement reagent kit (Kyoritsu Chemical Research Institute, Tokyo) (a measurement reagent kit using the Polynipum extract substance measurement method) according to the manufacturer's recommended protocol (Figure 3B(b)).

[0172] The KH-2 strain was found to have strong trans-fatty acid decomposition ability even at low temperatures (15°C).

[0173] (Example 4A: Trans fatty acid-containing fat decomposition ability of KH-2 strain) The ability of the KH-2 strain to decompose oils containing trans fatty acids at 15°C and 28°C was tested. 660 The KH-2 strain was inoculated at 0.8 (HITACHI U-2810 spectrophotometer (Hitachi, Tokyo)) and cultured at 15°C or 28°C for 6 days with shaking at 130 rpm. Comparison was made with a control sample to which no microorganism was added.

[0174] The fats and oils in the supernatant after the culture were analyzed by thin layer chromatography. Specifically, an equal amount of chloroform was added to the culture supernatant and stirred, after which 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, the fats and oils and free fatty acids were visualized by spraying molybdophosphate n-hydrate solution (2.4 g / 60 ml ethanol) and heating at 110°C for 12 minutes, and the amount of fats and oils remaining in the medium and fatty acids, which are their decomposition products, were compared (Figure 4). It was found that the KH-2 strain has the ability to decompose oils and fats containing trans fatty acids at both room temperature (28°C) and low temperature (15°C).

[0175] (Example 5: Oil-decomposition ability of KH-2 strain in actual wastewater) The oil-degrading ability of KH-2 strain was compared with that of BioRemove3200 (BR3200) (Novozymes, Denmark) using actual wastewater (Figure 5). A wastewater sample containing a large amount of trans-fatty acid-containing oils from a food factory using hydrogenated oils was cultured by adding nitrogen (ammonium sulfate) and phosphorus equivalent to the inorganic salt medium (above). The KH-2 strain was cultured in LB medium, washed twice with inorganic salt medium, and then the bacterial optical density OD 660 = 0.1 (HITACHI U-2810 spectrophotometer (Hitachi, Tokyo)), and BR3200 was inoculated at 1 × 10 7 The bacteria were inoculated at CFU / ml. Comparison was also made with a control sample to which no microorganisms were added. The samples were incubated at 28°C, and samples were taken after 24 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 molybdophosphate n-hydrate) (Figure 5A) and an oil measurement reagent kit (normal hexane value measurement, as above) (Figure 5B). While BR3200 showed slow decomposition even in excess, KH-2 strain showed excellent decomposition ability.

[0176] (Example 6: Lipid decomposition ability of KH-2 strain at 15°C) The cells of the KH-2 strain were soaked in an inorganic salt medium (pH 7) containing 1% canola oil (Nissin Canola Oil, Nisshin Oillio) until the final cell optical density (OD 660= 0.05 (HITACHI U-2810 spectrophotometer (Hitachi, Tokyo)), and cultured in a fermenter at 15 ° C. Samples were taken at 0, 24, 48 and 72 hours of culture, and the cells were removed from the sampled culture solution by centrifugation. The oil content equivalent to the normal hexane value in the supernatant was measured using an oil content measurement reagent kit (normal hexane extraction, as described above) (Figure 6A, upper part), and total fatty acids (total amount of fatty acids in triglycerides 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 molybdophosphate 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 amount of ethyl acetate was added. After stirring for 5 minutes, the mixture was centrifuged, and 1 ml of the ethyl acetate layer was transferred to an organic solvent-resistant tube and completely evaporated. The mixture was dissolved in 1 ml of chloroform, and 4 ml of a methanolysis solution made of a mixture of methanol and sulfuric acid at a ratio of 17:3 was added, and the mixture was heated at 100°C for 2 hours to methylate all fatty acids. After adding a solution made of a mixture of chloroform and pure water at a ratio of 1:1 and stirring thoroughly, 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. These results confirmed that the KH-2 strain has high lipid decomposition and utilization ability even at low temperatures.

[0177] (Example 7: Lipid decomposition ability of KH-2 strain at 28°C) The cells of the KH-2 strain were soaked in an inorganic salt medium (pH 7) containing 1% canola oil (Nissin Canola Oil, Nisshin Oillio) until the final cell optical density (OD 660= 0.05 (HITACHI U-2810 spectrophotometer (Hitachi, Tokyo)), and cultured in a fermenter at 28 ° C. Samples were taken at 0, 12, 24, and 30 hours into the culture, and the cells were removed from the sampled culture solution by centrifugation. The oil content equivalent to the normal hexane value was measured for the supernatant in the same manner as in Example 6 (Figure 7A, upper panel), total fatty acids were quantified by gas chromatography (Figure 7A, lower panel), and analyzed by thin layer chromatography (Figure 7B). The KH-2 strain decomposed fats and oils extremely rapidly at 28°C; within 12 hours, more than 70% of the 1% fats and oils initially contained in the medium was decomposed, and within 24 hours, both the fats and oils and fatty acids had almost completely disappeared.

[0178] Example 8: Fatty Acid and Oil Degradation by Additional 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 examined to see if they could produce lipase in a low-temperature environment (15°C) and decompose oils and fats. As a result, microorganisms capable of decomposing oils and fats at low temperatures were found. These microbial strains were named KH-2AL1 and KH-2AL3, respectively.

[0179] To further characterize strains KH-2AL1 and KH-2AL3, 26S rDNA gene sequence analysis was performed. The KH-2AL1 strain was identified as Yarrowia lipolytica because the partial base sequence of the 26S rDNA was 100% identical to that of Yarrowia lipolytica. The KH-2AL3 strain was identified as Yarrowia lipolytica because the partial base sequence of the 26S rDNA was 100% identical to that of Yarrowia lipolytica.

[0180] These strains were also examined for their ability to decompose trans fatty acids, trans fatty acid-containing oils, and oils.

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

[0182] The KH-2AL1 and KH-2AL3 strains were found to have trans fatty acid decomposition ability similar to that of the KH-2 strain.

[0183] (Decomposition of fats and oils containing trans fatty acids) Similarly to Example 4, the KH-2AL1 and KH-2AL3 strains were also tested for their ability to decompose trans-fatty acid-containing oils and fats. 660 The KH-2, KH-2AL1 or KH-2AL3 strains were inoculated at 0.8 (HITACHI U-2810 spectrophotometer (Hitachi, Tokyo)) and cultured for 5 days at 28°C with shaking at 130 rpm. Comparison was made with a control sample to which no microorganisms were added. As in Example 3, fat and oil decomposition in the culture supernatant was investigated. The analysis results using the oil content measurement reagent kit are shown in Figure 9.

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

[0185] (fat decomposition) The cells of the KH-2AL1 or KH-2AL3 strain were soaked in a mineral salt medium (pH 7) containing 1% canola oil (Nissin Canola Oil, Nisshin Oillio) until the final cell optical density OD 660 = 0.05 (HITACHI U-2810 spectrophotometer (Hitachi, Tokyo)) and cultured in a fermenter at 15 ° C. for 72 hours. The culture supernatant was used for analysis with an oil content measurement reagent kit (Figures 10A and 11A, upper panels), total fatty acid analysis by gas chromatography (Figures 10A and 11A, lower panels), and thin layer chromatography analysis (Figures 10B and 11B).

[0186] These results confirmed that the Yarrowia yeast of the present disclosure has high oil-decomposing and fat-assimilating ability even at low temperatures.

[0187] (Example 9: Ability of KH-2 strain to assimilate trans fatty acids and fats and oils containing trans fatty acids at 15°C) The ability of the KH-2 strain to assimilate trans fatty acids and oils containing trans fatty acids at 15°C was tested. The final cell optical density OD 660 The KH-2 strain was inoculated with 0.08 μg / ml (HITACHI U-2810 spectrophotometer (Hitachi, Tokyo)) and cultured at 15°C for 5 days with shaking at 130 rpm. The results were compared with a control sample to which no microorganisms were added. The results are shown in Figure 12. In the sample inoculated with the KH-2 strain, the medium was observed to be turbid, which indicated that the KH-2 strain can grow even at 15°C in an environment with elaidic acid or trielaidin as the sole carbon source and has the ability to assimilate these compounds.

[0188] (Example 10: Comparison of cleaning power between KH-2 strain and detergent) The oil-stained ventilation fan filter was then immersed in a culture supernatant of the KH-2 strain (culture conditions: 1% canola oil-added inorganic salt medium (composition as above), initial cell optical density OD 660= 0.01, and cultured at 28°C for 70 hours. After culture, the supernatant was centrifuged to remove the bacterial cells, and the pH was adjusted to 8.0 using sodium hydroxide solution), oil detergent (natural enzyme detergent for kitchen use Niko Eco (Niko Eco, Nagano), diluted 143 times with water according to the instructions), and general detergent (Family (registered trademark) (Kao, Tokyo), diluted 666 times according to the instructions) at room temperature (25°C) for washing (Figure 13). The washing times were 30 minutes, 2 hours, and 4 hours for the KH-2 strain culture supernatant, oil detergent, and general detergent, respectively. Although it was not possible to completely remove oil stains using general detergent and oil detergent, even after soaking for 2 and 4 hours, respectively, the filter became as white as new after 30 minutes of soaking with KH-2 strain.

[0189] (Example 11: Decomposition of various esters) The substrate specificity of the esters decomposed in the culture supernatant of the KH-2 strain was investigated. Inorganic salt medium (composition as above) supplemented with 1% canola oil, initial cell optical density OD 660 The KH-2 strain was inoculated so that the β-glucose concentration became 0.05, and the mixture was cultured at 28°C for 24 hours, 28°C for 48 hours, or 15°C for 48 hours. The mixture was then centrifuged to remove the bacterial cells, and the supernatant was used.

[0190] Substrate solutions were prepared by mixing 0.05 mol of any of the 4-nitrophenyl esters (substrates) of five types of fatty acids (acetic acid (C2), butyric acid (C4), octanoic acid (C8), lauric acid (C12), and palmitic acid (C16)) with 12 ml of a 3% (v / v) aqueous Triton (registered trademark) X-100 solution and melting at 70°C. The substrate solution, 150 mM GTA buffer (pH 7.0), and each supernatant were mixed in an amount of 1 ml, and the absorbance at 410 nm (corresponding to 4-nitrophenol generated by hydrolysis) was monitored for 1 minute while stirring.

[0191] The results are shown in the table below. The amount of 4-nitrophenol liberated by each substrate is shown as a percentage, with the substrate showing the maximum decomposition (4-nitrophenol liberation) among the five substrates taken as 100%. [Table 1]

[0192] In general, the lipase activity of microorganisms is measured by the hydrolysis activity of 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, is yellow, it can be easily quantitatively evaluated by a colorimetric method. In this case, in order to correlate with the decomposition activity of animal and vegetable oils and fats (triglycerides), an ester of 4-nitrophenol and a long-chain fatty acid (e.g., C16 or more) constituting animal and vegetable oils and fats is usually used. However, it was found that the KH-2 strain has low activity on an ester substrate of a long-chain fatty acid and 4-nitrophenol, and high activity on an ester substrate of a short-chain fatty acid (C6 or less) or a medium-chain fatty acid (C7 to 12). In other words, based on an experiment usually performed by a person skilled in the art (an experiment to examine the decomposition ability of an ester of a long-chain fatty acid and 4-nitrophenol), the 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 inventors have found that the KH-2 strain has high activity in decomposing triglycerides of long-chain fatty acids, which is a completely unexpected finding. Furthermore, from this result, it is predicted that the microorganism of the present disclosure may have a wide range of lipase activity against oils and fats containing short- to long-chain fatty acids.

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

[0194] The KH-2 strain and BR3200 strain were each added to 5 mL of inorganic salt medium (composition as above, pH 7) prepared so that the final concentration of palmitelaidic acid or vaccenic acid was 0.2% and the final concentration of Triton (registered trademark) X-100 was 0.25%, and the final concentration was measured by measuring the OD using a HITACHI U-2810 spectrophotometer (Hitachi, Tokyo). 660 = 0.5, and the KH-2 strain was inoculated at 5 × 10 6 BR3200 was inoculated to a concentration of CFU / ml. This was cultured at 15°C for 48 or 72 hours, or at 28°C for 24 hours with shaking at 130 rpm, and the culture supernatant of each strain was obtained. A control sample in which no microorganism was used was also prepared.

[0195] The remaining oil content in the culture medium was then analyzed by thin layer chromatography. Specifically, fatty acids were extracted with chloroform in an amount half that of the sample, and 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, fatty acids were visualized by a color reaction with molybdophosphate n-hydrate 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 degrade palmitelaidic 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 degrade these fatty acids. Thus, the microorganisms disclosed herein may be able to assimilate and degrade various trans fatty acids and trans fatty acid-containing oils and fats.

[0197] (Example 13: Obtaining related strains) Related strains can be obtained as follows. PBS is added to various isolation sources such as yeast storage libraries, soil, rivers, lakes, and activated sludge to prepare a dilution series, which is then spread on an inorganic salt agar medium containing trielaidin or elaidic acid as the only carbon source. Alternatively, 1 to 10% by weight of the above isolation source is added to an inorganic salt medium containing 0.3 to 1% by weight of shortening, and cultured 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 liquid 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, colonies that have formed a clear zone around them are picked up and inoculated into about 2 mL of inorganic salt agar medium (containing the above-mentioned concentrations of trielaidin or trielaidic acid), and cultured at 15°C or 28°C. After the cultivation, the degree of decomposition of fats and oils in the culture supernatant is analyzed by thin layer chromatography to obtain microorganisms capable of decomposing fats and oils at low temperatures or capable of decomposing fats and oils containing trans fatty acids.

[0198] Example 14: Use in a device The KH-2 strain was grown at 2 × 10 9 This is then cultured to 2 × 10 cells / mL to prepare the stock culture solution. 8 The amount of the seed bacteria is set at 1 / 100 of the amount of the cells per mL. This is stored in a refrigerated tank in the automatic amplification and injection device to be used as the seed bacteria. This seed bacteria is automatically inoculated into the inorganic salt medium in the culture amplification tank of the same device at 1 / 100th the amount every day, and is cultivated until the number of microorganisms increases 100-fold, i.e., until the cell concentration is the same as that of the microbial preparation. By injecting this into the oil decomposition treatment tank at 1 / 1000 of the amount of wastewater, the microbial concentration of decomposing bacteria in the oil treatment water is increased to 2 x 10 5 The experiment was carried out with 1000 cells / mL for 24 hours to decompose wastewater from a food factory that discharges wastewater containing a large amount of trans fatty acid-containing oils. The experiment took place in winter, and the water temperature during the treatment fluctuated between 12 and 17°C. As a result, a significant reduction in normal hexane values ​​was observed compared to a control case in which no microorganisms were added.

[0199] Example 15: Other embodiments 1×10 of KH-2 strain was added to the food waste disposal machine.6 The bacteria are inoculated at 37.5 cells / mL and treated at 25-35°C for 12-24 hours. The n-hexane value in the wastewater from the food waste processor is measured. A significant reduction in the value is observed compared to the control case where the KH-2 strain is not added.

[0200] The oily sludge collected by flotation separation is put into a culture tank, and then 10 to 1000% of the weight of the put-in sludge is added to the inorganic salt medium. 5 The bacteria are inoculated at 37.5 cells / mL and incubated at 20-35°C for 12-240 hours with stirring and aeration. The amount of oily sludge decomposed and reduced is then examined by measuring the amount of oil in the treated liquid using the normal hexane value, or by measuring the weight of the residue, mainly oil, remaining after the water has been evaporated. As a result, significant oily sludge decomposition is observed compared to a control case in which the KH-2 strain was not added.

[0201] Add an appropriate amount of carrier (e.g., 1×10) such as charcoal, various plastics, or ceramic pieces to the grease trap. 5 KH-2 strain (1000 cells / mL) is automatically added every day after the cafeteria operation is finished. Water is sampled every day just before operation starts, and the normal hexane value is analyzed. After one week, a significant decrease in normal hexane value is observed compared to the control case where KH-2 strain is not added, and the appearance of the glue trap itself shows effects such as reduced adhesion and floating of oil.

[0202] (Example 16: Combination of microorganisms with useful oil-degrading properties) There are strains of Burkholderia bacteria and Yarrowia yeast that have the ability to decompose fats and oils. By combining these strains with another microbial strain that has the ability to decompose fats and oils, a combination of microorganisms with excellent fat-and-oil decomposition ability can be obtained.

[0203] In the examples, the ability to degrade fats and oils by co-culture was tested using representative combinations of Burkholderia bacteria and several Yarrowia yeasts. Excellent ability to degrade fats and oils was observed in the combination of Burkholderia bacteria and Yarrowia yeasts. This will be described in detail below.

[0204] A combination of Burkholderia bacteria and Yarrowia yeast, both of which have lipase secretion ability and fat / oil decomposition ability, is co-cultured to test their fat / oil decomposition ability, and a combination with excellent fat / oil decomposition ability is obtained when co-cultured. The symbiotic system of Burkholderia bacteria and Yarrowia yeast was cultured in a mineral 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 1.7 mg / L, ZnSO4·7H2O 1.7 mg / L, and ZnSO4·7H2O 1.7 mg / L) containing canola oil (Nissin Canola Oil, Nisshin Oillio, Tokyo, Japan) at a final concentration of 1% (v / v). The bacteria were co-cultured in a 0.3 mg / L NaMoO3 solution and 0.25 mg / L NaMoO4 solution, and the culture supernatant was obtained and the degradation of fats and oils was tested by thin-layer chromatography, gas chromatography, and other methods. As a result, a symbiotic system of Burkholderia bacteria and Yarrowia yeast with excellent fat-degrading ability was found. Microbial strains that can be usefully used in this symbiotic system, for example, strain KH-1 (Burkholderia arboris) and strain KH-2 (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 investigating the fat-decomposing ability through similar tests.

[0206] Example 17: Further analysis of the lipid decomposition ability of the symbiotic system The lipid-decomposing ability of the KH-1 and KH-2 strains in various mixing ratios at various temperatures was tested.

[0207] (Analysis at 15°C) The KH-1 and KH-2 strains were cultured in an inorganic salt medium (composition as above, pH 7) containing 1% (v / v) canola oil (Nissin Canola Oil, Nisshin Oillio, Tokyo) at a total cell concentration of 5 × 10 5The KH-1 strain:KH-2 strain were added at ratios of 10:0, 9:1, 5:5, 1:9, and 0:10 (based on cell number) to obtain cells / mL, and cultured at 15° C. The culture supernatant obtained after 48 hours of culture was subjected to total fatty acid analysis by gas chromatography according to the following procedure.

[0208] Total fatty acid analysis by gas chromatography 3 ml of culture supernatant was acidified with hydrochloric acid, and an equal volume of ethyl acetate was added. After stirring for 5 minutes and centrifuging, 1.5 ml of the ethyl acetate layer was transferred to an organic solvent-resistant tube and completely evaporated. The mixture was dissolved in 1 ml of chloroform, and 4 ml of a methanolysis solution of methanol:sulfuric acid = 17:3 was added, and the mixture was heated at 100 °C for 2 hours to methylate all fatty acids. After adding a solution of chloroform:pure water = 1:1 and stirring thoroughly, the chloroform layer was mixed with 0.5% methyl octanoate (internal standard) in a 1:1 ratio, and analyzed by gas chromatography (GC-17A (Shimadzu Corporation, Kyoto)) equipped with an FID detector.

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

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

[0211] The KH-1 and KH-2 strains were cultured in an inorganic salt medium (composition as above, pH 7) containing 1% (v / v) canola oil (Nissin Canola Oil, Nisshin Oillio, Tokyo) at a total cell concentration of 5 × 10 5 The KH-1 strain and KH-2 strain were added at ratios of 10:0, 9:1, 5:5, 1:9, and 0:10 (based on cell number) to obtain cells / mL, and cultured at 28° C. The culture supernatant obtained after 18 hours of culture was subjected to total fatty acid analysis by gas chromatography in the same manner as above.

[0212] (result) The results of the gas chromatography analysis are shown in Figure 17. The symbiotic system of KH-1 and KH-2 strains showed a higher ability to decompose fats and oils than that calculated from the values ​​of the fat decomposition abilities of each of the monocultures at various mixing ratios. Thus, the symbiotic system of KH-1 and KH-2 strains has a high ability to decompose fats and oils at various temperature ranges.

[0213] (Example 18: Analysis of fatty acid decomposition ability of symbiotic system) The fatty acid decomposition ability of the KH-1 and KH-2 strains in combination at various mixing ratios was tested. (Analysis at 28°C) The lipid-decomposing ability of the combination of strains KH-1 and KH-2 was tested at 28°C.

[0214] The KH-1 and KH-2 strains were cultured in a mineral salt medium (composition as above, pH 7) containing oleic acid at a final concentration of 1% (v / v) at a total cell concentration of 5 × 10 5 The KH-1 strain and KH-2 strain were added at ratios of 10:0, 9:1, 5:5, 1:9, and 0:10 (based on cell number) to obtain cells / mL, and cultured at 28° C. The culture supernatant obtained after 18 hours of culture was subjected to total fatty acid analysis by gas chromatography in the same manner as above.

[0215] (result) The results of the gas chromatography analysis are shown in Figure 18. The symbiotic system of the KH-1 and KH-2 strains showed a fatty acid decomposition ability that exceeded that of each strain in monoculture at various mixing ratios. Thus, the symbiotic system of the KH-1 and KH-2 strains was found to be a very favorable combination with high fatty acid decomposition ability.

[0216] Example 19: Gene expression in symbiotic systems Changes in gene expression in the symbiotic system of KH-1 and KH-2 strains were examined.

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

[0218] The procedure was as follows. The KH-1 and KH-2 strains were cultured overnight in LB medium, and then washed twice with PBS buffer to remove medium components. The OD 660 The KH-1 strain alone, which had an OD of 0.02, or 660 Each microorganism was inoculated at a bacterial cell optical density such that the measured value of KH-1 strain:KH-2 strain = 0.018:0.02, and then cultured in a fermenter at 15°C. After 71 hours of culture, total RNA was extracted using the Cica geneus RNA Prep Kit (For Tissue) (Kanto Chemical).

[0219] Total RNA was used as a template and PrimeScript TM Genomic DNA was removed and cDNA was synthesized using the RT reagent Kit with gDNA Eraser Perfect Real Time (Takara Bio). The original cDNA solution was then diluted 3-fold using the dilution solution provided with the kit. Synthetic primers specific to the genes encoding the first and second lipases of the KH-1 strain were used to synthesize cDNA using Applied Biosystems (registered trademark) StepOnePlus TMQuantitative real-time RT-PCR was performed using a PCR kit (Applied Biosystems). Here, the first and second lipases of the KH-1 strain refer to those whose representative amino acid sequences and nucleotide sequences are shown in SEQ ID NOs: 1 to 4. PCR reactions were performed using a PowerUp TM PCR was carried out in a 20 μl solution containing SYBR® Green Master Mix (Thermo Fisher Scientific) (10 μl), each primer (final concentration 0.5 μM), and cDNA (1 μl). PCR was carried out in fast cycling mode with one cycle of denaturation at 95°C for 2 minutes, followed by 40 cycles of 95°C for 3 seconds and 60°C for 30 seconds.

[0220] The expression levels were normalized to the expression levels of RNA polymerase, Sigma 70 (rpoD). After confirming that the melting curves were single-peaked, the data were analyzed by the comparative Ct method (ΔΔCt method).

[0221] (result) The results are shown in the left panel of FIG. 19. In the symbiotic system of the KH-1 and KH-2 strains, the expression of the first and second lipases 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 enhanced lipase expression in the KH-1 strain. Without wishing to be bound by theory, it is expected that the fat and oil decomposition products produced 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 fat and oil decomposition abilities can be suitably used as the Yarrowia yeast, and strains having similar sensitivity can be suitably used as the Burkholderia bacteria.

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

[0223] As described above, the KH-1 and KH-2 strains were cultured overnight in LB medium, and then washed twice with PBS buffer to remove medium components. 5 × 10 5 KH-2 strain alone or 2.5 × 10 cells / mL 5 KH-1 strain + 2.5 x 10 cells / mL 5 The KH-2 strain was inoculated to give a concentration of 100 cells / mL, and then cultured in a fermenter at 15°C for 48 hours.

[0224] As in the case of the 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 and second lipases of the KH-2 strain. Here, the first and second lipases of the KH-2 strain refer to those whose representative amino acid sequences and base sequences are shown in SEQ ID NOs: 5 to 8. PCR reaction was performed in the same manner as in the case of the KH-1 strain.

[0225] The expression levels were normalized to the expression levels of alpha-1,2-mannosyltransferase (alg9). Data were analyzed by the comparative Ct method (ΔΔCt method) after confirming that the melting curves were single peaked.

[0226] (result) The results are shown in the right panel of FIG. 19. In the symbiotic system of the KH-1 and KH-2 strains, the expression of the first and second lipases 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 the enhancement of lipase expression in the KH-2 strain, and it is expected that the fat and oil decomposition products produced by the KH-1 strain function as inducers to activate the expression of lipase in the KH-2 strain. Therefore, it is expected that microorganisms having similar fat and oil decomposition abilities can be preferably used as the Burkholderia bacteria, and strains having similar sensitivity can be preferably used as the Yarrowia yeast.

[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, and 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 a bacterial cell optical density such that the measured value of KH-1 strain:KH-2 strain=0.018:0.02, and then cultured in a fermenter at 15°C. The supernatant was obtained 48 hours and 71 hours after the start of culture. 4-Nitrophenyl palmitate or 4-nitrophenyl butyrate was added to a 3% (v / v) Triton (registered trademark) X-100 aqueous 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 at room temperature for 1 minute.

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

[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 examined to see if they could produce lipase in a low-temperature environment (15°C) and decompose oils and fats. 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 strains KH-1AL1, KH-1AL2, and KH-1AL3, 16S rDNA gene sequence analysis was performed. The KH-1AL1 strain was identified as Burkholderia ambifaria because the partial base sequence of the 16S rDNA was 100% identical to that of Burkholderia ambifaria. The partial base sequence of the 16S rDNA of the KH-1AL2 strain was 99.9% identical to that of Burkholderia contaminans, and the strain was classified in the same group as B. seminalis, B. territorii, and B. cepacia (the partial base sequence of the 16S rDNA was 99.7%, 99.7%, and 99.8%, respectively) in the molecular phylogenetic tree. As a result, the KH-1AL2 strain was identified as a bacterium of the Burkholderia cepacia complex. The partial base sequence of the 16S rDNA of the KH-1AL3 strain was 99.9% identical to that of Burkholderia contaminans, and the strain was classified in the same group as B. seminalis, B. territorii, and B. cepacia (the partial base sequence of the 16S rDNA was 99.7%, 99.7%, and 99.8%, respectively) in 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 closely related organisms in the genus Burkholderia, which includes amphifaria, 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, various bacteria belonging to the Burkholderia cepacia complex showed high lipid and / or fatty acid decomposition ability, so bacteria belonging to the Burkholderia cepacia complex are expected to be particularly useful.

[0232] (Example 22: Symbiotic systems using other combinations) As in Example 17, the alternative strains identified in Examples 8 and 21 were added, and the lipid decomposition abilities were tested by comparing KH-1, KH-1AL1, and KH-1AL3 strains alone or in mixed cultures in which one-tenth of the cell number of each strain was replaced with KH-2, KH-2AL1, or KH-2AL3 strain.

[0233] The KH-1, KH-1AL1 or KH-1AL3 strains alone or in combination with the KH-2, KH-2AL1 or KH-2AL3 strains were cultured in an inorganic salt medium (composition as above, pH 7) containing 1% (v / v) canola oil (Nissin Canola Oil, Nisshin Oillio, Tokyo) at a total cell concentration of 2 × 10 6 The cells were added at a concentration of 1000 cells / mL and cultured at 15° C. The culture supernatant obtained after 48 hours of culture was analyzed in the same manner as above using an oil content measurement reagent kit (Kyoritsu Chemical Research Institute, Tokyo) (a measurement reagent kit using the polynipam extract substance measurement method).

[0234] (result) The results for each condition are shown in the table below. [Table 2]

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

[0236] (Example 23: Decomposition of fats and oils containing trans fatty acids by lipase) The decomposition activity of trielaidin by the first and second lipases of the KH-1 strain in Example 19, and the decomposition activity of trielaidin by the first and second lipases of the KH-2 strain are examined.

[0237] From a culture of the KH-1 strain and / or the KH-2 strain, or a culture of an E. coli strain genetically introduced to express a target lipase, 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, etc. These purified lipases are mixed with trielaidin to examine the degradation 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, a trans triglyceride. In the symbiotic system of the present disclosure, 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), so it is expected that various oils and fats, including oils and fats containing trans fatty acids, will be efficiently decomposed.

[0239] Example 24: Confirmatory Test The decomposition of fats and oils may be confirmed by the oil analysis as in Example 3, thin layer chromatography analysis, or the like, other than the above gas chromatography analysis.

[0240] The culture supernatant is obtained in the same manner as in Example 20, and is mixed with canola oil and incubated. The culture supernatant of the symbiotic system of the KH-1 and KH-2 strains shows stronger lipase activity than the culture supernatant of the KH-1 strain alone.

[0241] Other than the mixture ratio of Burkholderia bacteria and Yarrowia yeast tested in Example 17, other cell number ratios such as 19:1, 1:19, etc. can also be added and cultured at 15° C. or 28° C. As above, superior oil and fat decomposition can be confirmed compared to either microorganism alone.

[0242] A growth curve is prepared for each of the Burkholderia bacteria and the Yarrowia yeast, and the amount of microorganisms obtained in a given time, for example, 24 hours, is determined from the results. The amount of each microorganism that can be prepared within a given time is combined to measure the oil-decomposing ability. A combination that achieves an oil-decomposing ability that exceeds the oil-decomposing ability calculated from the oil-decomposing ability of each culture alone achieves excellent oil-decomposing ability.

[0243] Burkholderia bacteria and Yarrowia yeast are grown simultaneously in a mixed culture. The mixed culture solution is sampled at regular intervals, and the concentration of each microorganism is calculated to determine the cell concentration ratio of the microorganisms during the mixed culture. The mixed culture solution obtained after a certain period of culture, for example 24 hours, is used to examine the lipid decomposition ability at that mixture ratio. It is confirmed that the lipid decomposition ability exceeds that calculated from the lipid decomposition ability of each culture alone.

[0244] (Example 25: Obtaining additional alternative strains of Burkholderia bacteria and Yarrowia yeast useful in symbiotic systems) Alternative strains of microorganisms that can be used in the microbial combinations of the present disclosure can be obtained as follows. PBS is added to various isolation sources such as microbial storage libraries, soil, rivers, lakes, and activated sludge to prepare a dilution series, which are then spread onto an inorganic salt agar medium with canola oil as the only carbon source. The inoculated agar medium is then subjected to static culture at 15°C or 28°C to obtain colonies. Among these, colonies that have formed clear zones around them are picked up and inoculated into an inorganic salt medium supplemented with canola oil together with KH-1, KH-1AL1, KH-1AL3, KH-2, KH-2AL1, or KH-2AL3 strains, and cultured at 15°C or 28°C. After the culture, the degree of fat and oil decomposition in the culture supernatant is analyzed by thin-layer chromatography to obtain microorganisms with superior fat and oil decomposition ability compared to KH-1, KH-1AL1, KH-1AL3, KH-2, KH-2AL1, or KH-2AL3 strains alone.

[0245] (Example 26: Use in device) As in the above examples, Burkholderia bacteria and Yarrowia yeast are cultured to obtain a stock culture solution. This is diluted to obtain a microbial preparation. This is stored in a refrigerated microbial storage tank of an automatic amplification feeding device to obtain a seed culture. An appropriate amount of this seed culture is automatically inoculated into an inorganic salt medium in a culture amplification tank of the same device, and cultured until the number of microorganisms reaches the same cell concentration as the microbial preparation. By feeding an appropriate amount of this to the amount of wastewater in the oil decomposition treatment tank, the microbial concentration of the decomposition bacteria in the oil treatment water is adjusted to the target concentration, and the wastewater from a food factory that discharges wastewater containing a large amount of oil and fat is decomposed. As a result, a significant reduction in normal hexane value is observed compared to a control example in which no microorganisms are fed.

[0246] (Example 27: Additional Application Examples) An appropriate amount of Burkholderia bacteria and Yarrowia yeast are inoculated into an elimination type food waste processor and processed at an appropriate temperature. The normal hexane value in the wastewater discharged from the food waste processor is measured. A significant reduction in the value is observed compared to a control case in which no microorganisms are added.

[0247] The oily sludge recovered by flotation separation is placed in a culture tank and an appropriate amount of inorganic salt medium is added. An appropriate amount of Burkholderia bacteria and Yarrowia yeast are inoculated into this and incubated at an appropriate temperature with stirring and aeration. The amount of oily sludge decomposed and reduced is then examined by measuring the amount of oil in the treated liquid based on its normal hexane value, or by measuring the weight of the residue, mainly composed of oil, remaining after the water has been evaporated. As a result, significant oily sludge decomposition is observed compared to a control case in which no microorganisms were added.

[0248] Carriers such as charcoal, various plastics, and ceramic pieces are added to the grease trap, and appropriate amounts of Burkholderia bacteria and Yarrowia yeast are automatically added each day after operations in the cafeteria are completed. Water samples are taken over time just before operations begin, and the normal hexane levels are analyzed. A significant drop in normal hexane levels is observed compared to a control case in which no microorganisms are added, and the appearance of the glue trap itself also shows effects such as reduced adhesion and floating of oil.

[0249] (Note) Although the present disclosure has been illustrated using the preferred embodiments thereof, it is understood that the present invention should be interpreted in scope only by the claims. It is understood that the patents, patent applications, and other documents cited in this specification are incorporated by reference into this specification in the same manner as if the contents themselves 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 20, 2020, the contents of which are incorporated herein by reference in their entireties. [Industrial Applicability]

[0251] The present disclosure provides microorganisms having the ability to decompose esters (e.g., fats and oils) and / or fatty acids and compositions containing the same, and by using such microorganisms or compositions, it is possible to reduce the environmental burden caused by food factory wastewater containing a large amount of esters (e.g., fats and oils) and / or fatty acids. The present disclosure also provides a combination of microorganisms having the ability to decompose fats and / or fatty acids and by using such a combination of microorganisms, it is possible to reduce the environmental burden caused by food factory wastewater containing a large amount of fats and oils and / or fatty acids. [Accession 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 List Free Text]

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

Claims

1. A composition for treating oils or fatty acids comprising a Burkholderia bacterium in combination with a lipase-producing Yarrowia yeast and a lipase-producing Burkholderia bacterium.

2. A composition for treating oils, fats or fatty acids comprising a Yarrowia yeast in combination with a lipase-producing Yarrowia yeast and a lipase-producing Burkholderia bacterium.

3. A combination for treating oils, fats or fatty acids comprising a combination of a Burkholderia bacterium and a Yarrowia yeast, wherein both the Burkholderia bacterium and the Yarrowia yeast produce lipase.

4. The composition or combination of any one of claims 1 to 3, wherein the Yarrowia yeast comprises Yarrowia lipolytica.

5. The composition or combination according to any one of claims 1 to 4, wherein the Burkholderia bacterium comprises a bacterium of the genus Burkholderia.

6. 6. The composition or combination of any one of claims 1 to 5, wherein the Burkholderia bacterium comprises Burkholderia arboris, Burkholderia ambifaria, or Burkholderia cepacia complex.

7. The composition or combination according to any one of claims 1 to 6, wherein the combination of the Burkholderia bacterium and the Yarrowia yeast has a higher ability to decompose fats or fatty acids than that calculated from the fat or fatty acid decomposition values ​​of each of the bacteria and the yeast cultures alone.

8. The composition or combination according to any one of claims 1 to 7, wherein the cell number of the Burkholderia bacterium:the cell number of the Yarrowia yeast is 1:20 to 20:

1.

9. The composition or combination according to any one of claims 1 to 8, wherein at least one of the Burkholderia bacterium and the Yarrowia yeast has the ability to decompose fatty acids at 15°C.

10. The composition or combination according to any one of claims 1 to 9, wherein the Burkholderia bacterium is Burkholderia genus bacteria KH-1 strain (strain specified by accession number NITE BP-02731), KH-1AL1 strain (strain specified by accession number NITE BP-02977), KH-1AL2 strain (strain specified by accession number NITE BP-02978) or KH-1AL3 strain (strain specified by accession number NITE BP-02979), or a derivative thereof.

11. The composition or combination according to any one of claims 1 to 10, wherein the Yarrowia yeast is Yarrowia lipolytica KH-2 strain (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.

12. The composition or combination according to any one of claims 1 to 11, which is an oil decomposer.

13. 13. The oil disintegrator of claim 12, further comprising an oil treatment component.

14. A method for decomposing and removing oil, comprising applying the composition or combination according to any one of claims 1 to 11, or the oil decomposing agent according to claim 12 or 13 to an object to be treated.

15. A composition for improving lipase production in a lipase-producing Yarrowia yeast comprising a Burkholderia bacterium.

16. A composition for improving lipase production in a lipase-producing Burkholderia bacterium, comprising a Yarrowia yeast.

17. A composition for enhancing the ability of a lipase-producing Yarrowia yeast to process oils or fatty acids, comprising a Burkholderia bacterium.

18. A composition for enhancing the ability of lipase-producing Burkholderia bacteria to process oils or fatty acids, comprising a Yarrowia yeast.

19. A method for improving lipase production by at least one of a Burkholderia bacterium and a Yarrowia yeast, comprising a step of mixing and culturing the Burkholderia bacterium and a Yarrowia yeast.

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