WO2020009232A1 - 脂肪酸含有油脂を分解する新規微生物 - Google Patents

脂肪酸含有油脂を分解する新規微生物 Download PDF

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Publication number
WO2020009232A1
WO2020009232A1 PCT/JP2019/026850 JP2019026850W WO2020009232A1 WO 2020009232 A1 WO2020009232 A1 WO 2020009232A1 JP 2019026850 W JP2019026850 W JP 2019026850W WO 2020009232 A1 WO2020009232 A1 WO 2020009232A1
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Prior art keywords
oil
fatty acids
oils
fats
burkholderia
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French (fr)
Japanese (ja)
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克敏 堀
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Nagoya University NUC
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Nagoya University NUC
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Priority to US17/258,132 priority Critical patent/US20210276904A1/en
Priority to JP2019557658A priority patent/JPWO2020009232A1/ja
Priority to EP19830561.7A priority patent/EP3819371A4/en
Publication of WO2020009232A1 publication Critical patent/WO2020009232A1/ja
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    • C—CHEMISTRY; METALLURGY
    • C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
    • C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
    • C02F3/00—Biological treatment of water, waste water, or sewage
    • C02F3/34—Biological treatment of water, waste water, or sewage characterised by the microorganisms used
    • C02F3/343—Biological treatment of water, waste water, or sewage characterised by the microorganisms used for digestion of grease, fat, oil
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor
    • C12N1/205—Bacterial isolates
    • C—CHEMISTRY; METALLURGY
    • C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
    • C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
    • C02F2103/00—Nature of the water, waste water, sewage or sludge to be treated
    • C02F2103/32—Nature of the water, waste water, sewage or sludge to be treated from the food or foodstuff industry, e.g. brewery waste waters
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales

Definitions

  • the present disclosure relates to a microorganism having an ability to decompose fats and / or fatty acids and use thereof. More specifically, the present invention relates to a novel microorganism that degrades trans fat-containing fats and oils.
  • Wastewater from food factories and oil and fat factories contains large amounts of oil.
  • This oil is used for various biological treatment functions, such as a decrease in treatment capacity by activated sludge, a failure in solid-liquid separation due to a decrease in sedimentation, membrane fouling in the membrane separation activated sludge method (MBR), and inhibition of methane fermentation in anaerobic digestion.
  • MLR membrane separation activated sludge method
  • oil is removed by, for example, a pressurized flotation device or the like as a preceding stage of biological treatment of oil-rich wastewater.
  • a grease strap for removing oil is installed.
  • Decomposition rate is a problem in oil removal by microorganisms, as described above. Especially, activity decrease due to low temperature in winter often makes it difficult to apply microorganisms. Particularly at low temperatures in winter, the rate of decomposition of fats and oils by microorganisms is extremely slow, and it is considered impossible to perform wastewater treatment and waste treatment by specific microorganisms.
  • microorganisms belonging to Burkholderia family
  • This microorganism can efficiently degrade fats and oils and / or fatty acids even at a low temperature of about 15 ° C.
  • the present disclosure also relates to applications of the microorganisms of the present disclosure, such as oil treatment.
  • the present disclosure provides a novel microorganism having an ability to decompose fats and oils and a method for decomposing fats and / or fatty acids using the microorganism.
  • the present disclosure provides the following.
  • Burkholderia bacteria having the ability to decompose oils and fats at 15 ° C.
  • the Burkholderia bacterium according to any one of items 1 to 4, wherein the ability to assimilate or degrade is maintained at 15 ° C.
  • Item 7 The fungus according to any one of Items 1 to 6, which is a bacterium belonging to the genus Burkholderia.
  • Item 8 The fungus according to any one of items 1 to 7, which is a microorganism belonging to Burkholderia arboris, Burkholderia ambifaria, or Burkholderia cepacia complex.
  • An oil disintegrant according to item 10 comprising an additional oil treating component.
  • a kit for decomposing oil comprising the bacterium according to any one of items 1 to 9 or the oleolytic agent according to item 10, and an additional oil treatment component.
  • An oil decomposition removal method comprising: causing the bacterium according to any one of items 1 to 9 or the oil decomposing agent according to item 10 or 11 to act on an object to be treated.
  • Item 14 Item 14. The method according to Item 13, wherein the object to be treated includes a trans fatty acid or a trans fatty acid-containing fat or oil.
  • the microorganism of the present disclosure and a composition containing the same are capable of decomposing trans fatty acids and fats and oils containing the same fatty acids. Can be used at lower temperatures than before, so it can be used in a wide range of applications such as purification of environmental pollution by oils and fats, waste treatment such as garbage disposal, composting treatment, wastewater treatment, and composting. It can be applied to various situations.
  • the present disclosure can also solve the problem of oils and fats that are difficult to decompose, that is, oil types.
  • the microorganism of the present disclosure and a composition containing the same are capable of decomposing trans-fatty acids generated in the hydrogenation step of fats and oils and fats and oils containing the same, and in particular, margarine, fat spread, shortening, etc., containing a large amount of such fats and oils containing fats and oils
  • the present disclosure provides an effect of achieving trans-fatty acid decomposition, which can be used at a practical level as a bacterium that becomes a leading microorganism in wastewater treatment and waste treatment.
  • the microorganism of the present disclosure and a composition containing the same may be capable of decomposing fats and oils and fatty acids at a low temperature.
  • FIG. 5 shows the activity of KH-1 to decompose elaidic acid-containing fats and oils (trielaidin). Culture was performed at 28 ° C. and 130 rpm for 5 days. It is the photograph which analyzed glyceride and fatty acid in the culture solution by TLC. The left column shows the results of TBS buffer, and the right column shows the results of KH-1. [Fig. 4] Fig. 4 shows the activity of decomposing elaidic acid-containing fats and oils (trielaidin) in the culture supernatant of KH-1. Incubation was performed at 28 ° C. and 130 rpm for 24 hours. It is a photograph which analyzed glyceride and fatty acid in a solution by TLC.
  • the left is the result of TBS buffer, and the right is the result of KH-1.
  • 4 shows a comparison of fat and oil resolution between KH-1 and BioRemove 3200 (BR3200) using actual wastewater.
  • the cells were cultured at 28 ° C., and samples were collected after 24 hours and 48 hours.
  • (B) shows the result of measuring the oil content equivalent to the normal hexane value using an oil content measurement kit.
  • FIG. 4 shows the degradation of canola oil by KH-1 (5 L volume fermenter, 250 rpm, 200 ml / min air reflux, pH 7.0) at 15 ° C. culture. It is the result of measuring the oil content equivalent to the normal hexane value using an oil content measurement reagent kit. From the left, the results at 0, 24, 48, and 72 hours after the start of culture are shown. The vertical axis shows the ratio with the standard deviation when the measured value at 0 hour is taken as 100%.
  • Fig. 4 shows the oil / fat degradability of KH-1 in 28 ° C culture. It is the result of measuring the oil content equivalent to the normal hexane value using an oil content measurement reagent kit. From the left, the results at 0, 24, and 48 hours after the start of culture are shown. The vertical axis shows the ratio with the standard deviation when the measured value at 0 hour is taken as 100%. 3 shows a comparison between KH-1 and a detergent.
  • FIG. 5 is a photograph in which a ventilation fan filter with oil stains is immersed in a KH-1 culture supernatant, a detergent for oil, and a general detergent and washed.
  • the left shows the state before performing the processing.
  • the middle row and the right row are KH-1 treatment (center row 30 minutes, right row 1 hour), middle row: detergent treatment for oil (center row 2 hours, right row 4 hours), and lower row: general detergent, respectively. Processing (center row 2 hours, right row 4 hours).
  • 3 shows a comparison of the palmiteridic and baccenolytic activities of KH-1 and BioRemove 3200 (BR3200) (Novozymes, Denmark).
  • FIG. 4 shows the degradation of canola oil by KH-1AL1 in 15 ° C. culture. The results at 0, 24, 48, 72 and 96 hours after the start of the culture are shown.
  • the left is a photograph in which residual oil in the culture solution was analyzed by TLC.
  • the upper right shows the result of quantifying the total fatty acids (total of the fatty acids in triglyceride and free fatty acids) by gas chromatography, and the vertical axis shows the total fatty acid concentration (error bars indicate standard deviation).
  • the lower right part shows the result of measuring the oil content equivalent to the normal hexane value using an oil content measurement kit, and the vertical axis shows the percentage of the residual oil content when the measured value at 0 hours is 100% (error bars are standard values). Deviation).
  • FIG. 4 shows the degradation of canola oil by KH-1AL2 in 15 ° C. culture. The results at 0, 24, 48, 72 and 96 hours after the start of the culture are shown. The left is a photograph in which residual oil in the culture solution was analyzed by TLC.
  • the upper right shows the result of quantifying the total fatty acids (total of the fatty acids in triglyceride and free fatty acids) by gas chromatography, and the vertical axis shows the total fatty acid concentration (error bars indicate standard deviation).
  • the lower right part shows the result of measuring the oil content equivalent to the normal hexane value using an oil content measurement kit, and the vertical axis shows the percentage of the residual oil content when the measured value at 0 hours is 100% (error bars are standard values). Deviation).
  • FIG. 4 shows the degradation of canola oil by KH-1AL3 in 15 ° C. culture. The results at 0, 24, 48, 72 and 96 hours after the start of the culture are shown. The left is a photograph in which residual oil in the culture solution was analyzed by TLC.
  • FIG. 2 is a photograph of TEL analysis of the degradation of trielaidin by the culture supernatant of KH-1, KH-1AL1, KH-1AL2 and KH-1AL3 in 28 ° C. culture.
  • FIG. 2 shows the assimilation and degradation of elaidic acid by KH-1, KH-1AL1, KH-1AL2 and KH-1AL3 in 28 ° C. culture.
  • the left is a photograph of residual elaidic acid in the culture solution analyzed by TLC.
  • the right side shows the result of measuring the oil content corresponding to the normal hexane value using an oil content measurement kit, and the vertical axis shows the residual oil content concentration (mg / L).
  • FIG. 2 shows the assimilation and degradation of elaidic acid by KH-1, KH-1AL1, KH-1AL2 and KH-1AL3 in 15 ° C. culture.
  • the left is a photograph of residual elaidic acid in the culture solution analyzed by TLC.
  • the right side shows the result of measuring the oil content corresponding to the normal hexane value using an oil content measurement kit, and the vertical axis shows the residual oil content concentration (mg / L).
  • the results for the sterile group (control), KH-1, KH-1AL1, KH-1AL2 and KH-1AL3 are shown, respectively (error bars indicate standard deviation).
  • lipase is a type of esterase, and refers to an enzyme that reversibly catalyzes the reaction of hydrolyzing neutral fats (glycerol esters) to decompose them into fatty acids and glycerol.
  • lipase includes triglycerol lipase classified into EC 3.1.1.3 by enzyme number (EC number).
  • oil and fat refers to an oily substance, and an oil and fat includes an ester group-containing compound formed by dehydration condensation of a hydroxyl group-containing compound and a fatty acid.
  • the compound containing the hydroxyl group is glycerin, but other examples include polyglycerin.
  • an ester group-containing compound formed by dehydration-condensation of glycerin and a fatty acid is referred to as “glyceride”, similarly to the meaning commonly used in this technical field.
  • the compound containing a hydroxyl group has a plurality of hydroxyl groups, if at least one of the hydroxyl groups forms an ester by dehydration condensation with a fatty acid, the compound falls under the category of the ester group-containing compound in the present specification. .
  • Oils and fats are contained in, for example, kitchen wastewater of the food service industry, wastewater of food factories, and the like, and grease traps and pressure flotation devices, which are processing equipment that removes them by solid-liquid separation, are sources of odors and pests.
  • grease traps and pressure flotation devices which are processing equipment that removes them by solid-liquid separation, are sources of odors and pests.
  • the labor and costs involved in the maintenance such as collection and transportation of separated oil, cleaning, etc., the cost of coagulants, etc.
  • the microorganisms of the present disclosure or the present disclosure in order to extinguish the oil in the grease trap or in the factory wastewater treatment equipment.
  • the composition is used.
  • This disclosure provides microbial formulations for grease traps and industrial effluents that include the lipolytic bacteria of the present disclosure.
  • Food factory effluents contain trans fatty acids, which are often not ultimately removed and can be problematic as soil residues, but the microorganisms of the present disclosure can also solve them.
  • kitchen wastewater in the food service industry usually contains not less than 1 g / L, and if it is high, not less than 10 g / L of high-concentration fats and oils.
  • the retention time of wastewater in many grease traps is as extremely as about 10 minutes.
  • Fats and oils are also abundant in garbage, livestock waste, and sludge from wastewater treatment plants. Many of them contain trans fatty acids. Microorganisms are often used for the treatment of such solid waste, but if the oil content is large, the treatment becomes difficult or the oil remains. The microorganisms or compositions of the present disclosure are also applicable to the treatment of decomposing oil in such waste.
  • fatty acid is a compound having 2 to 100 carbon atoms and having at least one carboxyl group.
  • the carbon chain in the fatty acid is straight, but it may be branched or contain a ring.
  • a fatty acid contains one carboxyl group, but may contain more than one carboxyl group.
  • trans fatty acid-containing fat or oil refers to a compound formed by the dehydration condensation of a trans fatty acid and a compound containing a hydroxyl group.
  • the trans fatty acid includes elaidic acid, vaccenic acid and the like, but when referred to in the present specification, there is no particular limitation on the type of trans fatty acid.
  • the ratio of trans fatty acid present in the trans fatty acid-containing fat or oil is not particularly limited.
  • trans form and “cis form” of a double bond are used in a meaning commonly used in the art, and four substituents (R 1 , R 2 , R 3 and R 4 ) are bonded to the following structure
  • R 1 , R 2 , R 3 and R 4 substituents
  • R 1 and R 2 or R 3 and R 4 are groups other than hydrogen and the remaining two substituents are hydrogen atoms
  • trans type the case where 2 is a group other than hydrogen and the remaining two substituents are hydrogen atoms.
  • Trans fatty acids are naturally present in trace amounts as conjugated linoleic acid or vaccenic acid, and are relatively high in, for example, ruminant fat.
  • Trans fatty acids can occur during the hydrogenation step to produce saturated fatty acids from unsaturated fatty acids and during the refining of vegetable oils rich in unsaturated fatty acids. Therefore, margarine, fat spreads, shortenings, etc. may contain relatively high amounts of trans fatty acids.
  • normal hexane (n-Hex) value refers to the amount of a non-volatile substance extracted by normal hexane, and is an index indicating the amount of oil (such as oils and fats and hydrolysis products thereof) in water. Point.
  • the n-Hex value can be determined according to, for example, JIS ⁇ K ⁇ 0102. In addition, it can also be determined using a simple measurement reagent kit for measuring a polynipam extract substance.
  • assimilation refers to utilization as a nutrient source, and a substance (eg, fat or oil) subject to assimilation is lost as a result.
  • composition when used for fats and oils and / or fatty acids, means that the subject fats and oils and / or fatty acids become smaller molecules, for example, divided into glycerol and (free) fatty acids. That is, the conversion of fatty acids into fatty acids having fewer carbon atoms and the conversion to carbon dioxide and water are also called decomposition.
  • the term “ability to assimilate trans-fatty acid-containing fats and oils” refers to the activity of assimilating trans-fatty acid-containing fats and oils.
  • "utilizing trans fatty acid-containing fats and oils” is used in a meaning commonly used in the art, and means that microorganisms take in trans fat-containing fats and oils as a nutrient source such as a carbon source.
  • "Utilization” includes hydrolysis to glycerol and free fatty acids, as well as conversion to part of other substances. The ability to assimilate trans-fatty acid-containing fats and oils can be measured and identified by the following tests.
  • -A test to confirm whether it is possible to grow on a medium containing trans fatty acid-containing fats and oils as the sole carbon source.
  • -A test to confirm whether colonies are formed in a medium containing trans fat containing fats and oils as a sole carbon source.
  • ⁇ Test to measure decrease in normal hexane value in culture supernatant with growth.
  • -A test in which all fatty acids (sum of fatty acids and free fatty acids in fats and oils) in the culture supernatant are converted to methyl esters as the cells grow, and the total amount is measured by gas chromatography.
  • a test in which the amount of fats and oils and free fatty acids in the culture supernatant is measured by thin-layer chromatography as the cells grow. Individual, more detailed measurement methods are provided herein, and one of skill in the art can perform these measurements using any other equipment and conditions.
  • the ability to decompose trans-fatty acid-containing fats and oils refers to the activity of hydrolyzing trans-fatty acid-containing fats and oils to glycerol and free fatty acids.
  • the ability to degrade trans fatty acids-containing fats and oils can be measured and identified in the following tests. -A test to confirm the ability to assimilate trans-fatty acid-containing fats and oils.
  • a test in which trans fatty acid-containing fats and oils are fed as a carbon source and cultured, and the amount of decrease in normal hexane value in the culture supernatant is measured.
  • -A test for measuring the concentration of free fatty acids in the culture supernatant by gas chromatography.
  • -A test for measuring the concentration of free fatty acids in the culture supernatant by GC-MS.
  • ⁇ Test for measuring the concentration of free fatty acids in the culture supernatant by HPLC.
  • -A test to confirm whether a clear zone is observed around a colony formed on an agar medium containing a trans fatty acid-containing fat or oil.
  • -A test in which a test water containing trans fatty acid-containing fats and oils as a main organic substance (for example, 70% by weight or more of all organic substances) is prepared, and a biochemical oxygen demand (BOD) is measured.
  • BOD biochemical oxygen demand
  • the ability to assimilate trans fatty acids refers to the ability to assimilate trans fatty acids.
  • the ability to assimilate trans fatty acids can be measured and identified in the following tests. -A test to determine if growth is possible on media containing trans fatty acids as the sole carbon source. -A test to confirm whether colonies are formed in a medium containing trans fatty acids as the sole carbon source. ⁇ Test to measure decrease in normal hexane value in culture supernatant with growth. -A test in which the concentration of trans fatty acid in the culture supernatant is measured by gas chromatography with the growth. -A test for measuring the concentration of trans fatty acid in the culture supernatant by HPLC with proliferation. -A test in which the amount of trans fatty acid in the culture supernatant is measured by thin-layer chromatography during growth. Individual, more detailed measurement methods are provided herein, and one of skill in the art can perform these measurements using any other equipment and conditions.
  • the ability to degrade trans fatty acids refers to the ability to degrade trans fatty acids.
  • the ability to degrade trans fatty acids can be measured and identified in the following tests. -A test to determine if there is an ability to assimilate trans fatty acids. -A test for measuring the amount of trans fatty acid in the culture supernatant by thin-layer chromatography. A test in which trans fatty acids are fed as a carbon source and cultured, and a decrease in the normal hexane value in the supernatant is measured. -A test for measuring the concentration of trans fatty acid in the culture supernatant by gas chromatography. -Test for measuring the concentration of trans fatty acid in the culture supernatant by GC-MS.
  • test to measure the concentration of trans fatty acid in the culture supernatant by HPLC.
  • a test to confirm whether a clear zone is observed around a colony formed on an agar medium containing trans fatty acids.
  • BOD biochemical oxygen demand
  • the ability to decompose fats and oils at 15 ° C.” refers to the activity of hydrolyzing fats and oils to 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 test. Degradation ability may be shown in any of the following tests, and not necessarily in all tests. A test to confirm whether or not there is an ability to use fats and oils at 15 ° C. -A test to confirm whether a clear zone is observed around a colony formed on an agar medium containing oil at 15 ° C.
  • a test in which fats and oils are given as a carbon source and cultured at 15 ° C., and a decrease in normal hexane value in the culture supernatant is measured.
  • a test in which fats and oils are given as a carbon source and cultured at 15 ° C., and changes in the amounts of fats and fats and free fatty acids in the culture supernatant with time are measured by thin-layer chromatography. If the amount of fats and oils decreases over time, there is a decomposition ability. Alternatively, it can be said that once the amount of free fatty acid increases, it has the ability to degrade.
  • oil treatment component means a component that assists in assimilation and decomposition of fats and oils and / or fatty acids. Specifically, components such as biosurfactants that promote the dispersing of fats and oils and / or fatty acids, components that break down fats and oils into fatty acids and glycerol, those that break down fatty acids, those that break down glycerol, That are adsorbed and removed from the object to be treated.
  • the oil processing component may include a biosurfactant produced by the microorganism of the present disclosure.
  • oil disintegrant refers to a preparation capable of decomposing fats and oils and / or fatty acids, using the microorganism of the present disclosure as an active ingredient.
  • an 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 used simultaneously or one of them may be used first.
  • the lipolytic agent includes a component to enhance the activity of the bacterial strain or a lipase derived from the bacterial strain to be used (for example, a carbon source or a nitrogen source), a surfactant, a desiccant, a component for maintaining bacteria for a long time, Preservatives, excipients, fortifiers, antioxidants and the like may be further included.
  • a component to enhance the activity of the bacterial strain or a lipase derived from the bacterial strain to be used for example, a carbon source or a nitrogen source
  • a surfactant for example, a carbon source or a nitrogen source
  • a desiccant for maintaining bacteria for a long time
  • Preservatives, excipients, fortifiers, antioxidants and the like may be further included.
  • an "derived strain”, “similar strain” or “mutant” preferably, but not limited to, comprises a region substantially homologous to the DNA of the microorganism of interest.
  • a gene eg, 16S ⁇ rDNA
  • such strains may, in various embodiments, be aligned by a computer homology program known in the art and compared to the entire genome sequence of the original strain, It has a whole genome sequence that is at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or 99% identical.
  • gene mutations can be introduced using any known mutagen, UV, plasma, and the like.
  • an "derived strain,” “similar strain,” or “mutant” is a strain that is of the same genus and / or species as the original strain.
  • the biological function of such microorganisms can be determined by suitable and available in vitro assays described herein or known in the art.
  • homology of a gene refers to the degree of identity between two or more gene sequences, and generally having “homology” means that the degree of identity or similarity is high. Say. Thus, the higher the homology of a given two genes, the higher the identity or similarity of their sequences. Whether two genes have homology can be determined by direct sequence comparison or, in the case of nucleic acids, by hybridization under stringent conditions. When two gene sequences are directly compared, the DNA sequences between the gene sequences are typically at least 50% identical, preferably at least 70% identical, more preferably at least 80%, 90% , 95%, 96%, 97%, 98% or 99% identical, the genes are homologous.
  • Amino acids may be referred to herein by either their commonly known three-letter symbols or by the one-letter symbols recommended by IUPAC-IUB Biochemical Nomenclature Commission. Nucleotides may also be referred to by the generally recognized one-letter code.
  • BLAST a sequence analysis tool
  • the search for identity can be performed, for example, using NCBI's BLAST 2.7.1 (issued 2017.10.19).
  • the value of “identity” usually refers to a value when the above-mentioned BLAST is used and aligned under default conditions.
  • Similarity is a numerical value calculated for similar amino acids in addition to identity.
  • “70% or more” which is a numerical value of identity or the like is, 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, and may be in the range of any two of the starting values.
  • the “identity” is calculated by calculating the ratio of the number of homologous amino acids or bases in two or more amino acids or base sequences according to the known method as described above.
  • the amino acids or base sequences of the amino acid or base sequence group to be compared are aligned, and if necessary to maximize the ratio of the same amino acid or base, the amino acid or base is used. Introduce gaps in part of the array. Alignment methods, percentage calculation methods, comparison methods, and their associated computer programs are well known in the art (eg, BLAST, etc., described above).
  • “identity” and “similarity” can be represented by values measured by BLAST of NCBI unless otherwise specified. Blastp can be used with default settings for the algorithm when comparing amino acid or base sequences in BLAST. The measurement result is quantified as Positives or Identities.
  • the term “similarity” instead of “identity” is a numerical value that also takes into account the definition of “similar” “amine acid” or “base” described in the present specification.
  • the decomposition of trans-fatty acid-containing fats and oils there can be mentioned, but not limited to, the decomposition of cis fatty acids-containing fats and oils, the decomposition of cis fatty acids, the decomposition of saturated fatty acids-containing fats and oils, and the decomposition of saturated fatty acids. .
  • a biological function may be performed by a corresponding “biological activity”.
  • biological activity refers to an activity that a certain microorganism may have in a certain environment, and includes an activity that exerts various functions (for example, an activity of decomposing trans-fatty acid-containing fats and oils). You. Such a biological activity can be measured by techniques well known in the art.
  • “activity” refers to various measurable indicators that affect the response (ie, have a measurable effect in response to some exposure or stimulus), for example, some stimuli of the microorganisms of the present disclosure. Measures of the amount of protein upstream or downstream after or after an event or other similar function may also be included.
  • the “amount” of an analyte in a sample generally refers to an absolute value that reflects the mass of the analyte that can be detected in the volume of the sample. However, amounts also contemplate relative amounts as compared to another analyte amount. For example, the amount of the analyte in the sample may be greater than the control or normal levels of the analyte normally present in the sample.
  • kit refers to a kit which is usually divided into two or more compartments and provided with a portion to be provided (eg, a composition containing the microorganism of the present disclosure, additional components, buffers, instructions, and the like). Refers to the unit provided. This kit form is preferred when it is intended to provide a composition that should not be provided as a mixture for stability and the like, but is preferably used by mixing immediately before use. Such kits preferably include instructions or instructions describing how to use or provide the provided portions (eg, compositions containing microorganisms, additional components) and the like. It is advantageous to have instructions. When a kit is used in the present specification, the kit usually contains instructions and the like describing how to use the microorganism, the composition and the like of the present disclosure.
  • the “instruction” describes a method for using the present disclosure to a user.
  • the instruction includes a statement instructing how to use the present disclosure.
  • This directive may be provided, if necessary, to the competent authority of the country in which this disclosure is implemented (eg, the Ministry of Health, Labor and Welfare or the Ministry of Agriculture, Forestry and Fisheries in Japan, the Food and Drug Administration (FDA) in the United States, the Ministry of Agriculture (USDA) ), Etc.), and specify that they have been approved by the competent authority.
  • the instruction sheet may be provided in a paper medium, but is not limited thereto.
  • the instruction sheet may be provided in a form such as an electronic medium (for example, a homepage provided on the Internet, an e-mail).
  • the present disclosure provides a new microorganism capable of degrading fat.
  • the microorganisms of the present disclosure have the ability to assimilate trans-fatty acid-containing fats, assimilate trans-fatty acids, degrade trans-fatty acids, degrade trans-fatty acid-containing fats and / or oils and fats at 15 ° C. And / or has one or more characteristics of its ability to assimilate and / or degrade fatty acids.
  • the microorganism of the disclosure is a bacterium of the family Burkholderiaceae. In one embodiment, the microorganism of the present disclosure is a bacterium of the genus Burkholderia. Burkholderia is a gram-negative, non-spore-forming aerobic polar flagellar bacillus and is the reference genus of the Burkholderia family. In one embodiment, the microorganism of the present disclosure is Burkholderia arboris, Burkholderia ambifaria or Burkholderia cepacia, preferably Burkholderia arboris or Burgholderia ⁇ Ambifaria.
  • the microorganism of the present disclosure is a microorganism belonging to Burkholderia cepacia complex.
  • Burkholderia cepacia complex is a classification of microorganisms of the genus Burkholderia that are genetically very close, ambifaria, anthina, arboris, cenocepacia, cepacia, contaminans, diffusa, dolosa, lata, latens, metallica, multivorans, pseudomultivorans, puraquae, pyrrocinia.
  • a Burkholderia bacterium of the present disclosure comprises a metallica, seminalis, anthina, ambifaria, diffusa, ubonensis, multivorans, latens, cenocepacia, vietnamiensis, pyrrocinia, stabilis, glumee, gladioli, plantarii, oklahomensis, thailandensis, mallei.
  • the present inventor identified a new microorganism (KH-1) as Burkholderia arboris by determining the base sequence of 16S ribosomal DNA and analyzing the phylogenetic sequence, and submitted it to the Patent Organism Depositary of the National Institute of Technology and Evaluation, the Patent Microorganisms Depositary. Deposited, received on June 4, 2018, and issued a deposit certificate on June 12, 2018. The receipt number is NITE @ ABP-02731. Further, bacteria belonging to the genus Burkholderia (KH-1AL1, KH-1AL2 and KH-1AL3) were further identified and deposited with the National Institute of Technology and Evaluation, Patent Microorganisms Depositary, and incorporated on June 28, 2019.
  • the microorganism of the present disclosure is a Burkholderia strain KH-1 (accession number: NITE @ BP-02731 / a strain identified by accession number NITE @ ABP-027331), a KH-1AL1 strain (accession number NITE).
  • the microorganism of the present disclosure is a Burkholderia strain KH-1 (strain identified by accession number NITE @ BP-02731 / accession number NITE @ ABP-027331), strain KH-1AL1 (accession number NITE @ ABP). Strain derived from KH-1AL2 (strain specified by accession number NITE @ ABP-02978) or KH-1AL3 strain (strain specified by accession number NITE @ ABP-02979).
  • the derivative strain does not need to be a strain obtained based on the strains of Burkholderia KH-1, KH-1AL1, KH-1AL2 or KH-1AL3.
  • the microorganism that is the derivative strain of the present disclosure is a low-temperature (eg, 25 ° C. or lower), similar to the Burkholderia KH-1, KH-1AL1, KH-1AL2, or KH-1AL3 strain. , 20 ° C. or less, 15 ° C. or less, 10 ° C. or less, 5 ° C. or less, etc .; Shows a biological function selected from the group, but the degree of the biological function may be different from the KH-1, Strain KH-1AL1, Strain KH-1AL2 or Strain KH-1AL3.
  • the microorganism that is the derivative strain of the present disclosure is a bacterium belonging to the family Burkholderia (Burkholderiaceae), more specifically, a bacterium belonging to the genus Burkholderia, and more specifically, Is a microorganism belonging to Burkholderia arboris, Burkholderia ambifaria, or Burkholderia cepacia complex.
  • Burkholderia Burkholderiaceae
  • Is a microorganism belonging to Burkholderia arboris, Burkholderia ambifaria, or Burkholderia cepacia complex.
  • the microorganism of the present disclosure contains an oil or fat as a sole carbon source and has an inorganic pH adjusted to 6 to 8. It may be isolable on a salt agar medium.
  • the microorganism of the present disclosure (including a derivative of KH-1, KH-1AL1, KH-1AL2 or KH-1AL3) is cleared around colonies formed on an agar medium. It may be possible to determine by confirming that a zone (halo) is formed.
  • the microorganism of the present disclosure (including a derivative of KH-1, KH-1AL1, KH-1AL2 or KH-1AL3) has the ability to assimilate trans-fatty acids or trans-fatty acid-containing fats and oils. Having.
  • a microorganism of the present disclosure (including a derivative of KH-1, KH-1AL1, KH-1AL2 or KH-1AL3) has the ability to degrade trans fatty acids.
  • the microorganism of the present disclosure is capable of degrading trans fatty acid-containing fats and oils.
  • the microorganism of the present disclosure including a derivative of KH-1, KH-1AL1, KH-1AL2 or KH-1AL3, has the ability to degrade fats and / or fatty acids at 15 ° C. Having.
  • the microorganism of the present disclosure (including the KH-1, Strain KH-1AL1, Strain KH-1AL2 or derived strain of KH-1AL3) comprises trans fatty acids or fats and oils containing trans fatty acids. The ability to crack or decompose is maintained at 15 ° C.
  • the microorganism of the present disclosure (including a derivative of KH-1, KH-1AL1, KH-1AL2 or KH-1AL3) is cultured in a medium containing fats and oils, Surfactants can be secreted.
  • the oil content equivalent to the normal hexane value in the subsequent supernatant 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, and less than 2 g / L.
  • It is preferable to have an oil / fat degrading ability to reduce the oil content corresponding to the normal hexane value, and to reduce the oil content corresponding to the normal hexane value to less than 6 g / L when inoculated at a density of OD 660 0.03 and cultured for 24 hours. It is particularly preferred to have a resolution, and microorganisms having such a low-temperature fat / oil resolution can be beneficially used in various applications of the present disclosure.
  • a microorganism of the present disclosure (including a derivative of KH-1, KH-1AL1, KH-1AL2 or KH-1AL3) is a trans fatty acid (eg, elaidic acid, palmiteridic acid) , (Vaccenic acid) -containing fats and oils capable of assimilating and / or decomposing the trans-fatty acid-containing fats and oils in a medium at pH 7.0 and 15 ° C. or 28 ° C.
  • a trans fatty acid eg, elaidic acid, palmiteridic acid
  • (Vaccenic acid) -containing fats and oils capable of assimilating and / or decomposing the trans-fatty acid-containing fats and oils in a medium at pH 7.0 and 15 ° C. or 28 ° C.
  • the microorganism of the present disclosure (including a derivative of KH-1, KH-1AL1, KH-1AL2, or KH-1AL3) comprises Triton X-100 and elaidic acid at 0.25 each.
  • the elaidic acid concentration in the supernatant in% by weight is less than 0.15%, less than 0.12%, less than 0.1%, less than 0.09%, less than 0.08%, less than 0.07%, 0.
  • microorganisms of the present disclosure include KH-1, KH-1AL1, KH-1AL2 or KH-1AL3 derived strains) have less than 0.15% and less than 0.12% as determined under these conditions.
  • it has the ability to reduce elaidic acid concentration in the supernatant to less than 0.1%, or less than 0.05%, especially less than 0.12%, and microorganisms having such trans fatty acid degradability are disclosed herein. Can be beneficially used in various applications.
  • the elaidic acid concentration in the supernatant in% by weight is less than 0.15%, less than 0.12%, less than 0.1%, less than 0.09%, less than 0.08%, less than 0.07%, 0.
  • microorganisms of the present disclosure include KH-1, KH-1AL1, KH-1AL2 or KH-1AL3 derived strains
  • the microorganisms of the present disclosure have less than 0.15% and less than 0.12% as determined under these conditions. , Preferably less than 0.1%, less than 0.05%, less than 0.02% or less than 0.01%, in particular less than 0.1%.
  • Microorganisms having such trans fatty acid degradability can be beneficially used in various applications of the present disclosure.
  • the microorganism of the present disclosure (including a derivative of KH-1, KH-1AL1, KH-1AL2 or KH-1AL3) is placed in an inorganic salt medium containing 10 g / L of canola oil.
  • an inorganic salt medium containing 10 g / L of canola oil.
  • the oil equivalent to the normal hexane value in the supernatant after 24 hours is 1000% or less, 800% or less, 600% or less, and 400% compared to the oil equivalent to the normal hexane value in the supernatant after 24 hours in 28 ° C. culture.
  • the microorganisms of the present disclosure (including the derivative strain of the KH-1 strain), when determined under these conditions, have a fat / oil residual ratio of 800% or less, 700% or less, and 600% or less at 15 ° C culture compared to 28 ° C culture.
  • a fat and oil decomposability of 500% or less, 400% or less, and particularly 700% or less, and a microorganism having such a low temperature fat and oil decomposability can be advantageously used in various applications of the present disclosure.
  • the microorganism of the present disclosure (including a derivative of KH-1, KH-1AL1, KH-1AL2 or KH-1AL3) is placed in an inorganic salt medium containing 10 g / L of canola oil.
  • culture compared to 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, It has a fat and oil resolution of 50% or more, 40% or more, 30% or more, 20% or more, 10% or more, or 5% or more.
  • the microorganism of the present disclosure (including the derivative strain of the KH-1 strain) has a total fatty acid degradation rate of 50% or more, 40% or more, and 30% or more at 15 ° C. culture as compared with 28 ° C. culture, as determined under these conditions. % Or more, preferably 20% or more, or 10% or more, particularly 30% or more. Microorganisms having such a low-temperature fat / oil resolution can be advantageously used in various applications of the present disclosure. .
  • the ability of microorganisms to decompose and assimilate fats and oils and fatty acids can be evaluated by analyzing the fats and oils remaining in the culture medium and fatty acids, which are decomposition products, by thin-layer chromatography (TLC). Specifically, first, an oil or fat is extracted by adding an equal amount of chloroform to a culture supernatant. 5 ⁇ l of the extract is developed on a silica gel-coated plate using a developing solvent containing chloroform, acetone and methanol at a volume ratio of 96: 4: 1, respectively. The plate is treated with molybdate n-hydrate to develop oils and / or fatty acids.
  • TLC thin-layer chromatography
  • the ability of microorganisms to decompose and assimilate fats and oils and fatty acids can be evaluated by examining the ability to grow on a medium using each as a sole carbon source.
  • a microorganism of the present disclosure may be capable of secreting lipase, It may not have the ability to secrete lipase.
  • the microorganism of the present disclosure (including a derivative of KH-1, KH-1AL1, KH-1AL2, or KH-1AL3) is under mildly acidic conditions (eg, a pH of about 5.5 to about 5.5). 6.0) may be capable of growing and decomposing fats and oils.
  • the growth ability of the microorganism can be examined by a method of measuring the absorbance (turbidity) (OD 660 ) at 660 nm as an optical density of the cells, a method of measuring a colony forming unit (CFU), or the like. it can. In the latter, a fixed amount of a stock solution and a diluent of a culture solution are spread on an agar medium, and colonies formed by stationary culture are counted.
  • a method of measuring the absorbance (turbidity) (OD 660 ) at 660 nm as an optical density of the cells a method of measuring a colony forming unit (CFU), or the like.
  • the microorganism of the present disclosure may secrete lipase.
  • the ability of a microorganism to secrete lipase can be evaluated by measuring the lipase activity of a culture supernatant obtained by centrifuging a microorganism culture solution.
  • the lipase activity is determined by measuring the amount of 4-nitrophenol produced by performing an enzymatic reaction using 4-nitrophenyl palmitate (4-NPP), which is an ester of palmitic acid and 4-nitrophenol, as a substrate and hydrolyzing the ester.
  • 4-NPP 4-nitrophenyl palmitate
  • composition containing microorganisms provides a composition comprising a microorganism of the present disclosure.
  • the present disclosure provides a composition comprising a culture supernatant of the microorganism of the present disclosure.
  • the microorganism of the present disclosure can be produced by culturing by any appropriate method.
  • the composition is an oil disintegrant.
  • the composition is a fatty acid decomposer. By treating with the fatty acid decomposer of the present disclosure, a compound containing fewer carbon atoms than the number of carbon atoms contained in the fatty acid can be produced.
  • the composition is a trans fatty acid decomposer.
  • the fats and oils to which the oil disintegrant of the present disclosure is applied include, for example, vegetable fats (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 (lard, beef tallow, milk fat, etc.), fish oil, processed products of these fats (margarine, shortening, butter, etc.), insulating oils, lubricating oils, etc., but are not limited thereto. Not done.
  • the fat or oil may be present in the form of an emulsion or in a free state.
  • fats and oils to which the oil decomposer of the present disclosure is applied include fats and oils containing trans fatty acids, and such fats and oils include processed products such as fats and oils produced by hydrogenation (margarine). , Shortening, butter, etc.), but are not limited thereto.
  • hydrogenation (margarine). , Shortening, butter, etc.), but are not limited thereto.
  • the addition of hydrogen reduces the number of double bonds in unsaturated fatty acids and increases the proportion of saturated fatty acids, which may produce trans fatty acids. It is said that trans-fatty acids are contained in margarine, fat spread, shortening produced by hydrogenation, Western confectionery such as bread, cake and donut using such raw materials, and fried foods.
  • treatment is carried out at high temperatures to remove unwanted odors.
  • trans-fatty acids are formed from cis-type unsaturated fatty acids contained in the oil, refined vegetable oils such as salad oil also contain a small amount of trans-fatty acids.
  • the target to which the oil decomposer or fatty acid decomposer of the present disclosure is applied is not particularly limited.
  • industrial wastewater domestic wastewater, industrial waste, household waste (such as garbage), livestock waste, aquaculture (and Wastewater), barn (and its wastewater), and slaughterhouses (and its wastewater), soil contaminated with grease, water contaminated with grease (sea, ponds, rivers, potable water for animals, etc.), animal body Insulation oil or deterioration leaked from tables, aquariums (for aquaculture, appreciation, etc.), any oil-and-fat contaminated products (tableware, machine parts, etc.), grease traps, fatbergs, drain pipes, transformers installed in kitchens, etc. But not limited thereto.
  • any of these subjects may include trans-fatty acid-containing fats and / or trans-fatty acids, and the oil disintegrant or fatty acid decomposer of the present disclosure may be suitably applied.
  • "Grease trap” is a device for separating and collecting oil in drainage, and is typically composed of three tanks. The first tank has a basket and captures food pieces, leftovers, and the like. Oil and water are separated in the second tank. The wastewater separated from the oil is sent to the third tank to remove sedimentary debris and the like. Grease straps are required to be installed in commercial kitchens such as restaurants, hospitals and hotels. When applied to the grease trap, a separate decomposition treatment tank may be provided, but the fat and oil decomposing agent and microorganisms may be directly charged into the grease trap to perform the decomposition treatment in the grease trap.
  • the microorganisms of the present disclosure have high efficiency of low-temperature treatment, there may be embodiments in which low-temperature treatment is desirable.
  • low-temperature treatment For example, industrial wastewater, domestic wastewater, industrial waste, domestic waste (such as garbage), soil contaminated with fats and oils, water contaminated with fats (sea, ponds, rivers, drinking water for animals, etc.)
  • An example in which a process at a temperature lower than 20 ° C. (for example, 15 ° C.) is assumed is a preferable example as a target to be processed according to the present disclosure.
  • a preparation or the like is charged or added, or a carrier or the like on which the microorganism of the present disclosure is immobilized is placed in a drainage channel, a drainage storage tank, a grease trap, or the like.
  • a dedicated decomposition treatment tank may be separately provided outside the grease strap.
  • the wastewater includes, but is not limited to, wastewater from restaurants, hospitals, hotels, etc., domestic wastewater, industrial wastewater discharged from food processing factories, oil and fat processing plants, and the like.
  • Examples of the form of the microorganism or the composition of the present disclosure include a liquid state and a solid state.
  • the liquid-state microorganism or composition include a culture solution of the microorganism, a microorganism collected from the culture solution by centrifugation or the like, and then dispersed again in water, a buffer solution, a culture solution, or the like.
  • Microorganisms or compositions in the solid state include those dehydrated by centrifugation or press compression, those in the paste or mayonnaise state between the solid and liquid, and those dried (eg, dried under reduced pressure and freeze-dried). And the like.
  • the solid form include powder, granules, tablets and the like. Further, the composition may be provided in a state where the microorganism or the culture supernatant is fixed on a carrier.
  • the microorganism or composition of the present disclosure comprises about 1 ⁇ 10 8 cells / mL, about 1 ⁇ 10 7 cells / mL, about 1 ⁇ 10 6 cells / mL, about 1 ⁇ 10 5 cells / mL. , About 1 ⁇ 10 4 cells / mL, about 1 ⁇ 10 3 cells / mL, about 1 ⁇ 10 2 cells / mL, or about 10 cells / mL.
  • the microorganisms or compositions of the present disclosure can be used in any suitable environment.
  • the microorganism or composition of the present disclosure is 0-100 ° C., 5-70 ° C., less than 10-50 ° C., 15-40 ° C., 20-35 ° C., less than 70 ° C., less than 60 ° C., 50 Less than 40C, less than 40C, less than 30C, less than 25C, less than 20C, less than 15C, less than 10C, less than 5C, less than 0C, about 70C, about 60C, about 50C, about 40C. , About 30 ° C, about 25 ° C, about 15 ° C, about 10 ° C, about 5 ° C or about 0 ° C.
  • the microorganism or composition of the present disclosure has a pH of 3-13, pH 4-12, pH 5-11, pH 6-10, pH 7-9, pH 5.5-8.5, about pH 3, about pH 4, about 4 It can be used in an environment of pH 5, about pH 6, about pH 7, about pH 8, about pH 9, about pH 10, about pH 11, about pH 12, or about pH 13.
  • the microorganism or composition of the present disclosure is in an environment with a dissolved oxygen concentration (DO) of 0.05 mg / L or more, 0.1 mg / L or more, 0.5 mg / L or more, or 1 mg / L or more.
  • DO dissolved oxygen concentration
  • the microorganisms or compositions of the present disclosure may be used in wastewater with normal hexane values of 100 to 40000 mg / L, 200 to 30000 mg / L, 300 to 30000 mg / L.
  • solid wastes which may include water
  • sludge slurries and garbage disposal higher concentrations of oils and fats may be present, but in one embodiment, the microorganisms or compositions of the present disclosure may comprise It can also be applied to such solid wastes.
  • the microorganism or composition of the present disclosure comprises 50% or more, 20% or more, 10% or more, 7% or more, 5% or more, 2% or more, 1% or more, 0.7 wt% or more, 0.5 wt% or more, 0.2 wt% or more, 0.1 wt% or more, 0.07 wt% or more, 0.05 wt% or more, 0.02 wt% or more, 0 It may be added to a subject containing at least 0.01% by weight, at least 0.007% by weight, at least 0.005% by weight, at least 0.002% by weight, or at least 0.001% by weight of trans fatty acids.
  • the ratio of trans fatty acids (total of free fatty acids and fatty acids in the ester group-containing compound) in the fats and oils contained is 50% by weight or more, and 20% by weight. % 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% or more 0.1 wt% or more, 0.07 wt% or more, 0.05 wt% or more, 0.02 wt% or more, 0.01 wt% or more, 0.007 wt% or more, 0.005 wt% or more, 0.1 wt% or more. It may be added to a subject that is at least 002 wt% or at least 0.001 wt%.
  • the microorganism or composition of the present disclosure is added to a form in which nitrogen is available to the microorganism, preferably an ammonium salt, nitrate, sulfate, or organic nitrogen compound, more preferably ammonium sulfate, urea.
  • nitrogen is available to the microorganism, preferably an ammonium salt, nitrate, sulfate, or organic nitrogen compound, more preferably ammonium sulfate, urea.
  • C / N is the weight ratio of carbon atoms derived from n-Hex and nitrogen atoms contained in the wastewater.
  • nitrogen may be further added to be in these ranges.
  • the phosphorus (P) is present in a form available to the microorganism, preferably in the form of phosphate or nucleic acid, more preferably in the form of phosphate. It may be.
  • N / P is the weight ratio of nitrogen atoms to phosphorus atoms contained in the wastewater.
  • phosphorus may be further added to be in these ranges.
  • the microorganisms or compositions of the present disclosure may be used in the presence of salts, surfactants, light, current, agitation, aeration, or any combination thereof.
  • the microorganisms or compositions of the present disclosure may be applied after killing the microorganisms of the present disclosure and removing substances that inhibit growth (chlorine, antibiotics, etc.).
  • the microorganism or composition of the present disclosure may be used with a carrier capable of immobilizing the microorganism. Washout can be effectively avoided by using such a carrier.
  • the material of the carrier is not particularly limited as long as it can fix microorganisms.
  • carbon fiber PAN-based, pitch-based, phenolic-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 (zeolites, diatomaceous earth, etc.), and composites of these Is mentioned.
  • a porous or fibrous carrier in order to increase the immobilization rate of microorganisms and the efficiency of action of microorganisms.
  • microorganisms may be included in the gel carrier.
  • the shape of the carrier include a cubic shape, a rectangular parallelepiped shape, a columnar shape, a spherical shape, a disk shape, a sheet shape, and a film shape.
  • the technology for immobilizing microorganisms include, for example, “Wastewater treatment by microorganism immobilization method (edited by Ryuichi Sudo, Industrial Water Research Committee)” and “Water treatment by microorganism immobilization method-carrier immobilization method comprehensive immobilization method bioactive carbon method (New water treatment series (1)) (Kazuhiro Mochizuki, Katsutoshi Horizaki, Hideki Tatemoto (author), NTT Co., Ltd.).
  • the microorganisms or compositions of the present disclosure may be used in combination with additional components.
  • the additional components may be added to the composition, or used separately from the microorganism or composition, and if used separately, provided as a kit. Is also good.
  • additional components include components that enhance the activity of the microorganisms used (eg, carbon sources, nitrogen sources), surfactants, desiccants, components for maintaining the microorganisms for long periods of time, preservatives, emulsifiers.
  • components that enhance the activity of the microorganisms used eg, carbon sources, nitrogen sources
  • surfactants e.g., surfactants, desiccants
  • components for maintaining the microorganisms for long periods of time e.g., ocogen, ocants, emulsifiers.
  • the other microorganisms include microorganisms having an ability to decompose fats and oils, microorganisms that produce lipase, microorganisms that degrade (utilize) fatty acids and / or glycerol (which is a degradation product of fats and oils by lipase), proteins, Examples include microorganisms that degrade (utilize) amino acids, nucleic acids, or polysaccharides (eg, cellulose). Other microorganisms are preferably compatible with the microorganisms of the present disclosure.
  • Perilipase-producing microorganisms include eubacteria, yeasts, filamentous fungi and the like, preferably eubacteria and yeasts, more preferably Gram-positive bacteria and proteobacteria.
  • the true bacteria include, for example, Bacillus bacteria, Corynebacterium bacteria, Rhodococcus bacteria, Burkholderia bacteria, Acinetobacter bacteria, and Pseudomonas pseudomonas.
  • proteobacteria for example, alpha bacteria, beta bacteria, and gamma bacteria can be used.
  • Candida yeast is used.
  • a specific example of the yeast of the genus Candida is Candida albicans strain SL1B2 (deposited with the National Institute of Technology and Evaluation, Patent Microorganism Depositary under the accession number NITE @ P-714).
  • This strain has not only excellent glycerol assimilation ability, but also a property that it can be symbiotic with Burkholderia arboris. Therefore, the combined use of Candida albicans SL1B2 strain is particularly preferable in an embodiment using Burgholderia alboris.
  • Microorganisms that degrade (assimilate) fatty acids include eubacteria, yeasts, filamentous fungi, and the like, preferably eubacteria and yeasts, and more preferably yeast.
  • yeast include yeast of the genus Yarrowia, yeast of the genus Cryptococcus, yeast of the genus Trichosporon, and yeast of the genus Hansenula.
  • the present disclosure provides a method for removing fats and oils and / or fatty acids, which comprises applying the microorganism or composition of the present disclosure to a treatment target.
  • This treatment target may include trans fatty acids or trans fatty acids containing fats and oils.
  • the subject to be treated can be any of the subjects described herein to which the microorganisms or compositions of the present disclosure can be applied.
  • the fat and / or fatty acid degradation removal methods of the present disclosure can be performed in any of the environments described herein to which the microorganisms or compositions of the present disclosure can be applied.
  • the fat and / or fatty acid degradation removal method of the present disclosure may employ any of the additional components described herein that may be used in combination with the microorganisms or compositions of the present disclosure.
  • the method for removing fats and oils and / or fatty acids according to the present disclosure includes a step of introducing the microorganisms or the composition of the present disclosure into a fat-decomposing tank, and the input may be continuous or sequential. There may be.
  • the HRT (hydraulic residence 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 further preferably 24 hours or more.
  • the HRT can be usually 18 hours or more, preferably 20 hours or more, more preferably 24 hours or more.
  • the HRT is usually at least 8 hours, preferably at least 12 hours, more preferably at least 18 hours. obtain.
  • the concentration of microorganisms in the fat and oil decomposition tank may depend on the concentration of fats and oils and / or fatty acids in the wastewater, and the higher the concentration of fats and oils and / or fatty acids, the higher the cell concentration can be maintained.
  • a defoaming operation such as shortening the HRT, showering, and adding an antifoaming agent can be performed.
  • an antifoaming agent can inhibit the growth of microorganisms, it is desirable to set the amount to be added in consideration of such matters.
  • the n-Hex value of the effluent from the oil / fat decomposition tank is preferably 60 mg / L or less, more preferably 30 mg / L or less in the case of low-concentration wastewater having an inflow water n-Hex value of about 300 mg / L or less.
  • the n-Hex value of the influent water is medium concentration wastewater of about 3000 mg / L, it is preferably 600 mg / L or less, more preferably 300 mg / L or less, further preferably 150 mg / L or less, and most preferably 30 mg / L or less. is there.
  • high-concentration wastewater having an n-Hex value of influent water of about 10000 mg / L preferably 1000 mg / L or less, more preferably 500 mg / L or less, still more preferably 100 mg / L or less, and most preferably 30 mg / L or less. is there.
  • high-concentration wastewater having an n-Hex value of inflow water of about 30,000 mg / L or more it is preferably 3,000 mg / L or less, more preferably 1,000 mg / L or less, and still more preferably 300 mg / L or less.
  • the method of the present disclosure reduces the n-Hex value of oil and / or fatty acid containing wastewater to preferably 80% or more, more preferably 90% or more, even more preferably 95% or more, and most preferably 99% or more. % Or more.
  • the n-Hex value of the effluent from the fat and oil decomposition tank is less than 30 mg / L, which is the standard value for discharge to sewerage in many municipalities.
  • this reference value is achieved, even if this treatment such as the activated sludge treatment at the subsequent stage is unnecessary, focusing only on the n-Hex value, it may be unnecessary.
  • the amount of microorganisms introduced in the effluent is preferably 0.01 times or more, more preferably 0.1 times or more, still more preferably 0.5 times or more, and most preferably 1 time or more, based on the amount introduced. is there.
  • the method for removing fats and oils and / or fatty acids by decomposition according to the present disclosure may include additional steps in addition to the above steps.
  • Such a step includes, for example, a step of returning all or a part of the effluent from the fat / oil decomposition tank to the fat / oil decomposition tank again.
  • all or a part of the effluent from the fat and oil decomposition tank is again transferred to the fat and oil decomposition tank. It is not mandatory to return.
  • Example 1 Identification of microorganisms capable of assimilating and decomposing trans-fatty acid-containing fats and oils
  • a sample was taken from a river near a food factory from which oil-and-fat effluent flows out, and microorganisms were separated therefrom.
  • microorganisms that can be cultured at low temperatures using oils and fats as a nutrient source were found.
  • the cells were cultured in a 15 mL harmony centrifuge tube (LMS, Tokyo) at 28 ° C. and 130 rpm for 24 hours. As a result, a microorganism capable of decomposing and assimilating trans-fatty acid elaidic acid and growing was found.
  • one isolated microorganism strain can be cultured in a low-temperature environment using fats and oils as a nutrient source, can assimilate (decompose) fats and oils at low temperatures, and decompose and assimilate trans fatty acids.
  • this strain was named KH-1.
  • KH-1 was identified as Burkholderia arboris.
  • RAST Rapid Annotation using Subsystem Technology, http://rast.nmpdr.org
  • SNPs / INDELs analysis using CLC ⁇ Genomics ⁇ server ⁇ 9.0 detected variants at several hundred locations, indicating that KH-1 is a new strain of Burkholderia ⁇ arboris.
  • Burkholderia arboris strain KH-1 was streaked on an agar medium containing canola oil (Nisshin Canola Oil, Nisshin Oillio, Tokyo) as the sole carbon source and cultured at 15 ° C. for 5 days. The result is shown in FIG. Since the KH-1 strain grew well, it is considered that the KH-1 strain is suitable for survival in an environment containing oils and fats.
  • canola oil Nisshin Canola Oil, Nisshin Oillio, Tokyo
  • Example 2 Comparison of resolution of fats and oils containing trans fatty acids with other microorganisms
  • Trans Fatty Acid Degrading Activity The activity of KH-1 for degrading the trans fatty acid elaidic acid (trans form of oleic acid) was compared with that of BioRemove 3200 (BR3200) (Novozymes, Denmark) (FIG. 2).
  • a stock solution was prepared by dissolving 2% elaidic acid in a 2.5% Triton X-100 (Sigma-Aldrich) solution. Since elaidic acid is solid at room temperature, then heated at 65 ° C.
  • the stock solution of 4 ml, mineral salts medium of 40ml (Na 2 HPO 4 3.5g / L, KH 2 PO 4 2.0g / L, (NH 4) 2 SO 4 4.0g / L , MgCl 2 ⁇ 6H 2 O 0.34g / L, FeSO 4 ⁇ 7H 2 O 2.8mg / L, MnSO 4 ⁇ 5H 2 O 2.4mg / L, CoCl 2 ⁇ 6H 2 O 2.4mg / L, CaCl 2 ⁇ 2H 2 O 1.7mg / L, CuCl 2 ⁇ 2H 2 O 0.2mg / L, ZnSO 4 ⁇ 7H 2 O 0.3mg / L, and NaMoO 4 0.
  • Example 3 Comparison of resolution of trans fatty acid-containing fats and oils
  • a HITACHI U-2810 spectrophotometer (Hitachi, Tokyo, Japan) was added to a TBS buffer (20 mM Tris, 150 mM NaCl, pH 7.0) to which trielaidin was added to a final concentration of 0.1%.
  • TBS buffer (20 mM Tris, 150 mM NaCl, pH 7.0)
  • trielaidin was added to a final concentration of 0.1%.
  • OD 660 0.04. This was cultured in a Falcon tube (130 rpm, 28 ° C.), and a sample was collected 5 days later.
  • the sample was extracted with an equal volume of chloroform, and 5 ⁇ l was subjected to TLC.
  • the sample was applied to a TLC (silica gel plate, developed with a chloroform: acetone: methanol (96: 4: 2) solution, and then visualized with molybphosphoric acid n-hydrate (2.4 g in 60 ml ethanol) (FIG. 3B). From these results, it was confirmed that trans-fatty acid-containing fats and oils (trielaidin) could be decomposed into trans-fatty acids (elaidic acid) even when the supernatant of KH-1 was used.
  • KH-1 and BR3200 were compared (FIG. 4).
  • Nitrogen (ammonium sulfate) and phosphorus equivalent to an inorganic salt medium were added to a wastewater sample containing a large amount of trans-fatty acid-containing fats and oils from a food factory using hydrogenated fats and oils, and cultured.
  • KH-1 was cultured in LB medium, washed twice with PBS medium, and inoculated at 1 ⁇ 10 6 cells / ml
  • BR3200 was inoculated at 1 ⁇ 10 8 cells / ml, which is 10 times the recommended concentration of the manufacturer. Inoculated.
  • the cells were cultured at 28 ° C., and samples were collected after 24 hours and 48 hours.
  • the sample was applied to a TLC (silica gel plate, developed with a chloroform: acetone: methanol (96: 4: 1) solution, and then visualized with molybphosphoric acid n-hydrate) (FIG. 4A) and an oil measurement reagent kit (equivalent to normal hexane extraction) , Supra) (FIG. 4B).
  • BR3200 showed a slow progression of decomposition even in excess, whereas KH-1 showed excellent decomposition ability.
  • Example 4 Decomposition ability of KH-1 strain at 15 ° C.
  • the oil content corresponding to the normal hexane value in this supernatant was measured using an oil content measurement kit (normal hexane extraction, described above) (FIG. 5A).
  • Total fatty acids (total amount of fatty acids and free fatty acids in triglycerides) were quantified by gas chromatography (FIG. 5B). Specifically, 3 ml of the culture supernatant was acidified with hydrochloric acid, and twice the 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.
  • Example 4A assimilation ability of trans fatty acid of KH-1 strain and oil and fat containing the same at 15 ° C.
  • the assimilation ability of the KH-1 strain was evaluated by culturing the KH-1 strain in a medium containing elaidic acid or trielaidin as the sole carbon source.
  • An inorganic salt medium (the composition described above, prepared by adding a stock solution of elaidic acid or trielaidin (2.5% aqueous solution of Triton X-100) to adjust the final concentration of elaidic acid or trielaidin to 0.1%.
  • Example 5 Ability of KH-1 strain to decompose fats and oils at 28 ° C.
  • FIG. 6A The residual oil in the culture was analyzed by thin layer chromatography (TLC) (FIG. 6A). Specifically, an equal amount of ethyl acetate was added to 3 ml of the sample supernatant, and the mixture was stirred for 5 minutes. After the ethyl acetate layer was separated and the solvent was completely evaporated, it was dissolved in 300 ⁇ L of chloroform.
  • Example 4 the total fatty acids (total amount of fatty acids and free fatty acids in triglycerides) were quantified by gas chromatography even at 28 ° C. (FIG. 6B). Similarly to Example 4, residual oils and fats were measured at 28 ° C. using the oil content measurement kit (FIG. 6C). KH-1 retained a good fat and oil decomposability even at 28 ° C.
  • fatty acids were extracted with a half amount of chloroform of the sample, 6 ⁇ l of the extract was applied to a silica gel plate, and developed with a chloroform: acetone: methanol (96: 4: 2) solution. After the development, the fatty acids were visualized with a 12-molybdo (IV) phosphoric acid ethanol solution in the same manner as in Example 5, and the amount of the fatty acids remaining in the medium was compared (FIGS. 8 and 9). KH-1 was able to completely decompose palmiteridic acid and vaccenic acid within 24 hours at 28 ° C and within 72 hours at 15 ° C. On the other hand, BR3200 had no ability to decompose these fatty acids. Thus, the microorganisms of the present disclosure may be able to degrade various trans fatty acids.
  • Example 7 Comparison between KH-1 strain and detergent
  • time culture After culturing, the supernatant was used after centrifugation to remove bacterial cells), detergent for oil (natural enzyme detergent for Nico Eco kitchen (Nico Eco, Nagano) diluted 143 times with water according to the instructions), general detergent (Family (registered trademark) (Kao, Tokyo), diluted 666 times according to the instructions) at room temperature (25 ° C.) and washed (FIG. 7).
  • detergent for oil natural enzyme detergent for Nico Eco kitchen (Nico Eco, Nagano) diluted 143 times with water according to the instructions
  • general detergent Fluor (registered trademark) (Kao, Tokyo), diluted 666 times according to the instructions) at room temperature
  • Example 8 Acquisition of another strain
  • a sample was taken from a river near a food factory from which oil-and-fat effluent flows out, and microorganisms were separated therefrom.
  • the isolated microorganisms were examined for their ability to degrade trans-fatty acids or trans-fatty acid-containing fats and oils in a low-temperature environment (15 ° C.).
  • microorganisms capable of decomposing trans fatty acids and trans fatty acids containing fats and oils at low temperatures were found.
  • These microbial strains were named KH-1AL1, KH-1AL2, and KH-1AL3, respectively.
  • KH-1AL1 was identified as Burkholderia ambifaria because the partial nucleotide sequence of 16rDNA matched Burkholderia ambifaria with a homology of 100%.
  • KH-1AL2 has a partial nucleotide sequence of 16 rDNA that is identical to Burkholderia contaminans with a homology of 99.9%, and that on the molecular phylogenetic tree, B. seminalis, B. territorii, and B. cepacia (each has a homology in the partial nucleotide sequence of 16 rDNA).
  • KH-1AL2 was identified as a bacterium of Burkholderia cepacia complex.
  • KH-1AL3 has a partial nucleotide sequence of 16 rDNA that is identical to Burkholderia contaminans with a homology of 99.9%.
  • B. seminalis, B. territorii, and B. cepacia were identified as a bacterium of Burkholderia cepacia complex.
  • Burkholderia cepacia complex is a classification of microorganisms of the genus Burkholderia that are genetically very close, ambifaria, anthina, arboris, cenocepacia, cepacia, contaminans, diffusa, dolosa, lata, latens, metallica, multivorans, pseudomultivorans, puraquae, pyrrocinia. , Seminalis, stabilis, stagnalis, territorii, ubonensis, and vietnamiensis (Martina P et al., Int J Syst Evol Microbiol. 2018 Jan; 68 (1): 14-20.). As a result of the analysis, various bacteria belonging to Burkholderia cepacia complex showed high ability to degrade fats and / or fatty acids, so that bacteria belonging to Burkholderia cepacia complex are expected to be particularly useful.
  • Example 10A Comparison between strains of trans-fatty acid-containing fats and oils at 28 ° C resolution by culture supernatant
  • KH-1, KH-1AL1, KH-1AL2, and KH-1AL3 were each added to an inorganic salt medium (the above composition, pH 7) in which the final concentration of trielaidin was adjusted to 0.2%.
  • the final concentrations of trielaidin and Triton X100 are adjusted to 0.4% and 0.5%, respectively. And incubated at 37 ° C. with shaking at 130 rpm for 24 hours. Then, the residual oil in the culture solution was analyzed by thin layer chromatography (TLC). Specifically, fats and oils and free fatty acids were extracted with a half amount of chloroform as a sample, 5 ⁇ l of the extract was applied to a silica gel plate, and developed with a chloroform: acetone: methanol (96: 4: 2) solution. .
  • TLC thin layer chromatography
  • Example 10B Comparison between strains of assimilation and resolution of trans fatty acid at 28 ° C.
  • a control sample in which no microorganism was used was also prepared. Then, the residual oil in the culture solution was analyzed by thin layer chromatography (TLC). Specifically, fatty acids were extracted with a half amount of chloroform of the sample, 5 ⁇ l of the extract was applied to a silica gel plate, and developed with a chloroform: acetone: methanol (96: 4: 2) solution. After the development, free fatty acids were visualized with a 12-molybdo (IV) phosphoric acid ethanol solution as in Example 5, and the amount of fatty acids remaining in the medium was compared (left in FIG. 14).
  • TLC thin layer chromatography
  • Example 10A the residual oil equivalent to the normal hexane value was measured by an oil content measuring reagent kit in the same procedure as in Example 4 (right in FIG. 14).
  • KH-1, KH-1AL1, KH-1AL2, and KH-1AL3 can efficiently degrade trans fatty acids, and that these strains are all useful for removing oil containing trans fatty acids.
  • trans-fatty acid-containing fats and oils eg, trielaidin
  • Example 10C Comparison between strains of assimilation and resolution of trans fatty acid at 15 ° C.
  • HITACHI U-2810 spectrophotometer Hitachi, Tokyo
  • a control sample in which no microorganism was used was also prepared.
  • the residual oil in the culture solution was analyzed by thin layer chromatography (TLC). Specifically, free fatty acids were extracted with a half amount of chloroform of the sample, 5 ⁇ l of the extract was applied to a silica gel plate, and developed with a chloroform: acetone: methanol (96: 4: 2) solution. After the development, free fatty acids were visualized with a 12-molybdo (IV) phosphoric acid-ethanol solution in the same manner as in Example 5, and the amount of fatty acids remaining in the medium was compared (FIG. 15, left). Further, the residual oil equivalent to the normal hexane value was measured by the oil content measuring reagent kit in the same procedure as in Example 4 (right in FIG. 15).
  • TLC thin layer chromatography
  • KH-1 was inoculated into a disappearing garbage disposer at a concentration of 1.2 ⁇ 10 7 cells / mL and treated at 30 ° C. for 24 hours.
  • the normal hexane value in the wastewater discharged from the garbage disposer was measured.
  • the garbage in the disappearing garbage processing machine was obtained by removing bones and shells from garbage discharged from a restaurant kitchen.
  • the garbage disposal capacity of the extinguishable garbage disposal machine was 20 kg / day, and the amount of water injected / discharged was 38.4 L / day.
  • KH-1 is cultured to 2 ⁇ 10 10 cells / mL in an inorganic salt medium containing 10 mL / L of canola oil to obtain a stock culture solution. This is diluted 10-fold to obtain a microbial preparation (2 ⁇ 10 9 cells / mL). This is refrigerated and stored in a microorganism storage tank of the automatic amplification and injection device to be used as a seed. This inoculum is automatically inoculated in an inorganic salt medium in a culture amplification tank of the same apparatus in an amount of 1/100 every day, and cultured until the number of microorganisms becomes 100 times, that is, the cell concentration becomes the same as that of the microorganism preparation.
  • the microorganism concentration of the decomposing bacteria in the oil treatment water is set to 2 ⁇ 10 6 cells / mL, and the wastewater containing a large amount of trans fatty acid-containing fats and oils is discharged for 24 hours.
  • Example 13 Another embodiment
  • a carrier such as charcoal, various plastics, and ceramic pieces is put into the grease trap, and an appropriate amount (for example, 1 ⁇ 10 6 cells / mL) of KH-1 is automatically put in every day after the operation of the canteen. Every day, water is collected just before the start of operation, and the normal hexane value is analyzed. One week later, in addition to the remarkable decrease in the normal hexane value as compared with the control example in which KH-1 was not added, the effect of the grease trap itself was also observed, such as reduced oil adhesion and floating.
  • the present disclosure provides a microorganism having an ability to degrade fats and oils and a composition containing the same, and by using such microorganisms or the composition, it is possible to reduce the environmental load caused by food factory wastewater containing a large amount of fats and oils.
  • KH-1 strain (NITE BP-02731) KH-1AL1 strain (NITE ABP-02977) KH-1AL2 strain (NITE ABP-02978) KH-1AL3 strain (NITE ABP-02979)

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