Method for producing polyhydroxyalkanoate

Abstract

Provided is a method for producing a polyhydroxyalkanoate by microbial culture, which is capable of efficiently producing a polyhydroxyalkanoate despite using waste oil as a carbon source. The method for producing a polyhydroxyalkanoate comprises a step for producing a polyhydroxyalkanoate by culturing a polyhydroxyalkanoate-producing strain in a culture medium containing an oil or fat as a carbon source. The grease contains waste oil. And an anisidine value obtained by measuring the whole of the oil or fat is 180 or less. The anisidine value is more than 2. In addition, the amount of oil / fat polymer obtained by measuring the entire oil / fat is 25% or less.

Description

Technical Field

[0001] The present invention relates to a method for producing polyhydroxyalkanoate by culturing microorganisms. Background Art

[0002] With the world increasingly focused on sustainable development, as exemplified by the SDGs, and environmental issues such as the marine microplastics problem, efforts are underway to convert existing petroleum-derived, non-biodegradable plastics into biodegradable materials, primarily in industries such as packaging and food services, biomedicine, and agriculture. In recent years, examples of biodegradable materials that have become increasingly popular in industrial production include polylactic acid (PLA) and polyhydroxyalkanoates (PHA). PHA, among others, exhibits excellent biodegradability in a wide range of environments and is one of the few biodegradable materials that can even biodegrade in seawater. Consequently, expectations are growing that PHA will serve as a solution to the marine microplastics problem and other environmental issues.

[0003] PHA is a natural thermoplastic polyester produced and accumulated in the cells of many microbial species as an energy-storing substance. Generally, PHA is industrially produced by culturing PHA-accumulating microorganisms with nutrients such as carbon, nitrogen, and phosphorus sources. Carbon sources commonly used in PHA production include sugars such as glucose and fructose, vegetable oils such as palm oil and rapeseed oil, and free fatty acids and their salts.

[0004] However, these carbon sources are problematic due to their high carbon dioxide emissions during raw material production, placing a heavy environmental burden. Therefore, waste oils (also known as waste oil, waste cooking oil, waste edible oil, waste vegetable oil, used oil, etc.) have been proposed as carbon sources for PHA production.

[0005] Although there have been reports on the use of waste oil as a carbon source for the production of PHA (for example, see Patent Document 1), there has been no mention of the effect of waste oil on the productivity of PHA.

[0006] Prior art literature

[0007] Patent Literature

[0008] Patent Document 1: Japanese Patent Application Laid-Open No. 2004-254668 Summary of the Invention

[0009] Problems to be solved by the invention

[0010] As far as the inventors of the present application are aware, there has been no report on the quality of waste oil required for efficient cultivation and production of PHA.

[0011] As described in Comparative Example 1 below, the inventors of the present application obtained the following results: when a known PHA-producing strain was cultured using a certain waste oil as a carbon source, the PHA production amount decreased by approximately 40% compared to the culture using unused oil as a carbon source (Reference Examples 1 and 2).

[0012] This decrease in PHA productivity is undesirable, and it is desirable to achieve good PHA productivity while effectively utilizing waste oil as a carbon source.

[0013] In view of the above-mentioned situation, an object of the present invention is to provide a method for producing polyhydroxyalkanoate by microbial culture, which can efficiently produce polyhydroxyalkanoate while using waste oil as a carbon source.

[0014] Solutions to the Problem

[0015] The present inventors have discovered that when polyhydroxyalkanoate-producing microorganisms are cultured using fats and oils containing waste oil as a carbon source to produce polyhydroxyalkanoate, if the anisidine value obtained by measuring the entire fat and oil is 180 or less, a productivity similar to that obtained when only unused fat and oil is used can be achieved, thereby completing the present invention.

[0016] Specifically, the present invention relates to a method for producing polyhydroxyalkanoate, comprising: culturing a polyhydroxyalkanoate-producing strain in a culture medium containing oil as a carbon source to produce the polyhydroxyalkanoate;

[0017] The above grease includes waste oil,

[0018] The anisidine value measured for the entire fat and oil was 180 or less.

[0019] Effects of the Invention

[0020] According to the present invention, there can be provided a method for producing polyhydroxyalkanoate by microbial culture, which can efficiently produce polyhydroxyalkanoate while using waste oil as a carbon source.

[0021] According to the present invention, waste oils unsuitable for efficient PHA production can be identified and eliminated before microbial culture. Furthermore, waste oils unsuitable for PHA production can be mixed with other oils to produce oils that are efficient for PHA production. Consequently, waste oils can be effectively used as a carbon source in PHA production cultures. DETAILED DESCRIPTION

[0022] Hereinafter, embodiments of the present invention will be described in detail.

[0023] One embodiment of the present invention relates to a method for producing polyhydroxyalkanoate, comprising the step of culturing a polyhydroxyalkanoate-producing strain in a culture medium containing oil or fat as a carbon source to produce the polyhydroxyalkanoate.

[0024] The PHA-producing strain of this embodiment is not particularly limited as long as it has the ability to produce polyhydroxyalkanoates. The strain may be a wild-type strain that already possesses a PHA synthase gene, a variant obtained by artificially mutating such a wild-type strain, or a strain into which an exogenous PHA synthase gene has been introduced using genetic engineering methods. The method for introducing the exogenous PHA synthase gene is not particularly limited, and the following methods may be selected: methods of directly inserting or replacing the gene on the host chromosome, methods of directly inserting or replacing the gene on a giant plasmid held by the host, or methods of configuring and introducing the gene on a vector such as a plasmid, phage, or phagemid. A combination of two or more of these methods may also be used. From the perspective of the stability of the introduced gene, methods of directly inserting or replacing the gene on the host chromosome or on a giant plasmid held by the host are preferred, and methods of directly inserting or replacing the gene on the host chromosome are more preferred.

[0025] Examples of the PHA-producing strain or host thereof according to the present embodiment include, for example, bacteria belonging to the genera Ralstonia, Cupriavidus, Wautersia, Burkholderia, Aeromonas, Escherichia, Alcaligenes, Pseudomonas, Bacillus, Azotobacter, Nocardia, Sphingomonas, and Comamonas, but are not limited thereto. From the viewpoint of safety and PHA productivity, bacteria belonging to the genera Ralstonia and Cupriavidus are more preferred, bacteria belonging to the genus Cupriavidus are even more preferred, and Cupriavidus necator is particularly preferred.

[0026] The PHA production strain of this embodiment is a strain with the ability to assimilate oils and fats. The strain can be a wild strain that originally has the ability to assimilate oils and fats, or a mutant strain obtained by artificially mutating such a wild strain, or a strain into which an enzyme group for assimilating oils and fats has been introduced using genetic engineering methods. The method for introducing an enzyme group for assimilating foreign oils and fats is not particularly limited, and the following methods can be selected: a method of directly inserting or replacing genes on the host's chromosome, a method of directly inserting or replacing genes on a giant plasmid held by the host, or a method of configuring and introducing genes on a vector such as a plasmid, phage, or phagemid, etc., or a combination of two or more of these methods can be used. Considering the stability of the introduced gene, a method of directly inserting or replacing genes on the host's chromosome or on a giant plasmid held by the host is preferred, and a method of directly inserting or replacing genes on the host's chromosome is more preferred.

[0027] (PHA synthase gene)

[0028] The PHA synthase gene possessed by the PHA-producing strain of this embodiment is not particularly limited and may be a PHA synthase gene native to the PHA-producing strain or a foreign PHA synthase gene. Specific examples of PHA synthase genes include PHA synthase genes derived from organisms similar to Ralstonia, Cupriavidus, Worsteria, Alcaligenes, Aeromonas, Pseudomonas, Nocardia, and Chromobacterium, as well as variants thereof. Such variants may include base sequences encoding PHA synthases in which one or more amino acid residues have been deleted, added, inserted, or substituted.

[0029] (PHA)

[0030] The type of PHA produced by the PHA-producing strain of the present embodiment is not particularly limited as long as it is a PHA that can be produced by the microorganism, but preferably is a homopolymer of one monomer selected from 3-hydroxyalkanoic acids having 4 to 16 carbon atoms, a copolymer of one monomer selected from 3-hydroxyalkanoic acids having 4 to 16 carbon atoms and another hydroxyalkanoic acid (for example, 2-hydroxyalkanoic acid, 4-hydroxyalkanoic acid, 5-hydroxyalkanoic acid, 6-hydroxyalkanoic acid, etc. having 4 to 16 carbon atoms), and a copolymer of two or more monomers selected from 3-hydroxyalkanoic acids having 4 to 16 carbon atoms. Examples include, but are not limited to, homopolymers of 3-hydroxybutyric acid (abbreviated as 3HB), copolymers of 3HB and 3-hydroxyvaleric acid (abbreviated as 3HV), copolymers of 3HB and 3-hydroxyhexanoic acid (abbreviated as 3HH), copolymers of 3HB and 4-hydroxybutyric acid (abbreviated as 4HB), and PHAs containing lactic acid (abbreviated as LA) as a component, such as copolymers of 3HB and LA, such as P(LA-co-3HB). Of these, P(3HB-co-3HH) is preferred from the perspective of its wide range of applications as a polymer. The type of PHA to be produced can be appropriately selected based on the intended purpose, including the type of PHA synthase gene possessed by the microorganism used or introduced separately, the type of metabolic genes involved in its synthesis, and culture conditions.

[0031] PHA can be accumulated within the microorganisms by culturing PHA-producing strains. This culture is referred to as PHA production culture. Prior to PHA production culture, one or more pre-cultures may be performed for bacterial growth (also referred to as mother culture). In this embodiment, the process of culturing PHA-producing microorganisms can be carried out using conventional microbial culture methods in a culture medium containing a suitable carbon source. There are no particular limitations on the culture medium composition, culture scale, aeration and stirring conditions, culture temperature, and culture time for PHA production culture and pre-culture.

[0032] The method for adding a carbon source can be carried out according to known PHA production and culture techniques. Preferably, the carbon source is added at a point in time when the microbial proliferation leads to the depletion of nutrient sources such as phosphorus, nitrogen, or magnesium to increase the carbon source concentration. Generally speaking, under conditions where nutrient sources such as phosphorus, nitrogen, or magnesium are limited, microorganisms accumulate large amounts of PHA when the carbon source concentration is sufficiently high. The above-mentioned carbon source concentration is not particularly limited, and an appropriate concentration can be selected based on the microorganisms used, the culture medium, the carbon source, the aeration and stirring conditions, the culture temperature, etc. As a method for adding the carbon source to achieve the above-mentioned carbon source concentration, the carbon source can be added to the culture medium all at once, or it can be added to the culture medium continuously or intermittently, or a combination of the two can be used. More preferred is a method of continuous or intermittent addition.

[0033] (Carbon Source)

[0034] In this embodiment, in the PHA production culture, at least oil and fat is used as a carbon source, and the oil and fat includes at least waste oil.

[0035] Fats and oils refer to ester compounds of fatty acids and glycerol, and include at least one of triglycerides, diglycerides, and monoglycerides. Generally, the main component is triglycerides.

[0036] Waste oil refers to discarded fats and oils. Its name is not limited and includes oils called waste oil, waste cooking oil, waste edible oil, waste vegetable oil, used oil, etc. In addition, unused fats and oils refer to fats and oils that have not been used for a given purpose after production.

[0037] The source of waste oil is not particularly limited. For example, it can be oil used for cooking fried foods in ordinary households, restaurants, or food manufacturing companies, or oil that has been discarded due to being unsuitable for consumption due to exceeding its shelf life or expiration date. Alternatively, it can be oil discarded by companies such as oil manufacturing companies because it is judged to be unsuitable for its intended use. For example, when replacing the oil in an oil tank with oil A and then rinsing the oil tank or piping with oil B, it can also be used. Alternatively, it can be oil that has been discharged from one or more discharge sources and then collected by recycling personnel. Alternatively, it can be oil that has had moisture and impurities removed from the discharged or collected oil.

[0038] The original or unused types of oils and fats contained in the waste oil are not particularly limited. Examples include palm oil, palm kernel oil, or fractions thereof (e.g., separated low-melting-point fractions of soft palm oil, super soft palm oil, and soft palm kernel oil), corn oil, coconut oil, olive oil, soybean oil, rapeseed oil, jatropha oil, and their fractionated oils, or purified byproducts thereof. Furthermore, animal-derived oils and fats may be included. Furthermore, mixtures of the above-mentioned oils and fats may also be used.

[0039] The efficient production of PHA in this embodiment is related to the degree of deterioration of the waste oil. Generally speaking, when oils and fats are exposed to water, air, light containing ultraviolet rays, or heated to high temperatures, oxidation / hydrolysis will occur. This state is generally referred to as deterioration of oils and fats. For example, oils and fats react with oxygen to generate peroxides. In addition, peroxides can generate carbonyl compounds (aldehydes, ketones, etc.) when they decompose. The carbonyl compounds can be quantified using the methoxyaniline value and the carbonyl value as indicators. From the perspective of ease of measurement operation, it is preferred to quantify using the methoxyaniline value as an indicator. In addition, peroxides can polymerize to generate oil polymers. The oil polymers can be quantified using the amount of oil polymers as an indicator. It should be noted that there are various theories about the deterioration mechanism of oils and fats. As far as this embodiment is concerned, the deterioration mechanism of oils and fats is not limited.

[0040] In this embodiment, an index that quantifies the degree of oil degradation is used as a criterion to identify oils and fats that can efficiently produce PHA. The anisidine value is used as such an index. In addition to the anisidine value, the amount of oil polymers can also be used.

[0041] The present inventors have discovered that the anisidine value, an indicator for quantifying the degree of deterioration of fats and oils, correlates with PHA productivity during microbial culture. The lower the anisidine value of a fat or oil, the higher the PHA productivity when using that fat or oil. In particular, they have discovered that good PHA productivity can be achieved when the anisidine value is 180 or less.

[0042] The amount of oil and fat polymers also correlates with PHA productivity. The lower the amount of oil and fat polymers, the higher the PHA productivity when using that oil. In particular, it was discovered that good PHA productivity can be achieved when the amount of oil and fat polymers is 25% or less.

[0043] Therefore, in addition to the anisidine value, the amount of polymer in an oil can also be used to predict in advance the PHA productivity that the oil can achieve.

[0044] Meanwhile, other known indicators for quantifying the degree of oil and fat degradation include peroxide value, color, and acid value. However, no correlation has been found between these indicators and PHA productivity. Therefore, it is clear that peroxide value, color, and acid value cannot be used to identify oils and fats capable of efficient PHA production.

[0045] The fats and oils used in this embodiment have an aniline value of 180 or less, as measured for the entire fat and oil, including the waste oil. This allows efficient PHA production despite the waste oil being used as a carbon source in microbial culture. From the perspective of further improving PHA productivity, the aniline value is preferably 120 or less, more preferably 100 or less, even more preferably 80 or less, even more preferably 70 or less, and particularly preferably 60 or less.

[0046] A lower methoxyaniline value indicates less deterioration of the oil. However, some available waste oils have little deterioration and exhibit methoxyaniline values comparable to unused oils. Therefore, the lower limit of the methoxyaniline value is not particularly limited and may be 1 or greater, or 2 or greater. Alternatively, it may be 5 or greater, 10 or greater, or 20 or greater.

[0047] The method for measuring the anisidine value in this embodiment is carried out in accordance with the Standard Oil and Fats Analysis Test Method 2.5.3-2013 established by the Japan Oil Chemists' Society.

[0048] In addition, regarding the fats and oils used in this embodiment, in addition to the anisidine value, the fat polymer content measured for the entire fat and oil including the waste oil is preferably 25% or less from the viewpoint of PHA productivity. The fat polymer content is more preferably 10% or less, further preferably 9% or less.

[0049] It is further preferably 8% or less, particularly preferably 7% or less, and most preferably 6% or less.

[0050] The lower limit of the amount of the oil and fat polymer is not particularly limited, and may be 0.05% or more, or 0.1% or more, or 0.3% or more, 0.5% or more, or 0.8% or more.

[0051] The method for measuring the amount of oil and fat polymers in this embodiment is carried out in accordance with Provisional Method 16 of the Standard Oil and Fat Analysis Test Method 2.5.7-2013.

[0052] In this embodiment, the oil and fat used as the carbon source for PHA production culture may be composed solely of waste oil. In this case, waste oil having an anisidine value of 180 or less is used as the waste oil.

[0053] Furthermore, the oil used as a carbon source for PHA production culture may include, in addition to waste oil, unused oil that has not deteriorated. In this case, the anisidine value of the oil and fat combined, including the waste oil and unused oil, can be 180 or less, as described above. Such a mixed oil and fat can be easily prepared by appropriately adjusting the anisidine value of the unused oil and fat and the mixing ratio of the waste oil and unused oil. Therefore, when used in combination with unused oil, the anisidine value of the waste oil is not particularly limited and may exceed 180.

[0054] In this case, waste oil that is unsuitable for the production of PHA when used alone can be mixed with other oils and fats to become an oil and fat suitable for the production of PHA.

[0055] From the perspective of reducing environmental burden, the proportion of waste oil contained in the entire fat and oil used as a carbon source is preferably 10% by weight or more, more preferably 50% by weight or more, and even more preferably 80% by weight or more. It may also be 90% by weight or more.

[0056] From the perspective of culture production yield, the moisture content of the oils and fats including the waste oils is preferably 10% or less, more preferably 5% or less, more preferably 2% or less, more preferably 1% or less, more preferably 0.5% or less, and even more preferably 0.2% or less. The moisture content measurement method of this embodiment is performed in accordance with Standard Oil and Fats Analysis Test Method 2.1.3.2-2013. Furthermore, prior to using the oils and fats for PHA production culture, a treatment to reduce moisture content may be performed.

[0057] In addition to fats and oils including waste oils, the carbon source used in PHA production culture may further include carbon sources other than fats and oils (e.g., sugars, fatty acids, glycerol, etc.). However, from the perspective of reducing environmental burden, the proportion of fats and oils in the carbon source is preferably 10% by weight or more, more preferably 50% by weight or more, and even more preferably 80% by weight or more. It may also be 90% by weight or more.

[0058] It should be noted that the carbon source for the pre-culture is not particularly limited, and waste oil may or may not be used. Examples of usable carbon sources include sugars such as glucose, fructose, and sucrose; palm oil, palm kernel oil, or their fractions (e.g., soft palm oil, super soft palm oil, and soft palm kernel oil as separated low-melting-point fractions); oils and fats such as corn oil, coconut oil, olive oil, soybean oil, rapeseed oil, and jatropha oil, and their fractions, or their purified byproducts; fatty acids such as lauric acid, oleic acid, stearic acid, palmitic acid, and myristic acid, and their derivatives; and glycerol. Furthermore, mixtures of the oils and fats listed above may also be used.

[0059] In the production of PHA according to this embodiment, the microorganisms described above are preferably cultured using a culture medium containing the aforementioned carbon source, a nitrogen source as a nutrient source other than the carbon source, inorganic salts, and other organic nutrient sources. Examples of nitrogen sources, although not limited to the following, include ammonia; ammonium salts such as ammonium chloride, ammonium sulfate, and ammonium phosphate; peptone, meat extract, and yeast extract. Examples of inorganic salts include potassium dihydrogen phosphate, disodium hydrogen phosphate, magnesium phosphate, magnesium sulfate, and sodium chloride. Examples of other organic nutrient sources include amino acids such as glycine, alanine, serine, threonine, and proline; and vitamins such as vitamin B1, vitamin B12, and vitamin C.

[0060] After culturing for an appropriate period of time to allow PHA to accumulate within the cells, PHA can be recovered from the cells using known methods. The recovery method is not particularly limited, but for example, the following method can be used: after the culture is completed, the cells are separated from the culture medium using a centrifuge, a separation membrane, or the like, and then dried. PHA is then extracted from the dried cells using an organic solvent such as chloroform. Cellular components are removed from the organic solvent solution containing PHA by filtration, a poor solvent such as methanol or hexane is added to the filtrate to precipitate PHA, and the supernatant is removed by filtration or centrifugation and dried to recover PHA. Alternatively, cellular components other than PHA can be dissolved in water using a surfactant, an alkali, an enzyme, or the like, followed by filtration or centrifugation to separate PHA particles from the aqueous phase and drying for recovery.

[0061] According to this embodiment, PHA can be efficiently produced in PHA production cultures using waste oil as a carbon source. Furthermore, the carbon source used in PHA production cultures can be converted from unused oils and fats, which have a high environmental burden, to waste oil, thereby reducing the environmental burden of PHA production cultures.

[0062] The following items list preferred embodiments of the present disclosure, but the present invention is not limited to the following items. [Item 1] 1. A method for producing a polyhydroxyalkanoate, the method comprising:

[0063] A step of producing polyhydroxyalkanoate by culturing a polyhydroxyalkanoate-producing strain in a medium containing oil as a carbon source,

[0064] The grease comprises waste oil,

[0065] The anisidine value measured for the entire fat and oil was 180 or less.

[0066] [Project 2]

[0067] The manufacturing method according to item 1, wherein:

[0068] The anisidine value is 2 or more.

[0069] [Item 3]

[0070] The manufacturing method according to item 1 or 2, wherein

[0071] The amount of oil and fat polymers measured in the entire oil and fat is 25% or less.

[0072] [Item 4]

[0073] The manufacturing method according to item 3, wherein:

[0074] The amount of the oil and fat polymer is 0.1% or more.

[0075] [Item 5]

[0076] The production method according to any one of items 1 to 4, wherein

[0077] The polyhydroxyalkanoate-producing strain is a microorganism belonging to the genus Cupriavidus. [Item 6] The production method according to any one of Items 1 to 5, wherein

[0078] The polyhydroxyalkanoate is a copolymer of two or more hydroxyalkanoic acids.

[0079] [Item 7] The manufacturing method according to Item 6, wherein

[0080] The polyhydroxyalkanoate is a copolymer containing 3-hydroxyhexanoic acid as a monomer unit.

[0081] [Item 8] The manufacturing method according to Item 7, wherein

[0082] The polyhydroxyalkanoate is a copolymer of 3-hydroxybutyric acid and 3-hydroxyhexanoic acid.

[0083] [Item 9] The production method according to any one of Items 1 to 8, wherein

[0084] The anisidine value is 120 or less.

[0085] [Item 10]

[0086] The production method according to any one of items 1 to 8, wherein

[0087] The anisidine value is 70 or less.

[0088] [Item 11]

[0089] A fat used as a carbon source in the cultivation of a polyhydroxyalkanoate-producing strain, the fat comprising waste oil, wherein:

[0090] The anisidine value measured for the entire fat and oil was 180 or less.

[0091] [Item 12]

[0092] The oil and fat according to item 11, wherein

[0093] The anisidine value is 2 or more.

[0094] [Item 13]

[0095] The fat or oil according to item 11 or 12, wherein

[0096] The amount of oil and fat polymers measured in the entire oil and fat is 25% or less.

[0097] [Item 14]

[0098] The manufacturing method according to item 13, wherein:

[0099] The amount of the oil and fat polymer is 0.1% or more.

[0100] [Item 15]

[0101] A method for adjusting waste oil, comprising:

[0102] Mix the waste oil with other oils and fats so that the anisidine value of the mixed oils and fats is below 180.

[0103] When the mixed oil and fat is used as a carbon source in the cultivation of a polyhydroxyalkanoate-producing strain, the productivity of polyhydroxyalkanoate is improved compared to the waste oil.

[0104] Example

[0105] Hereinafter, the present invention will be described in more detail based on examples, but the present invention is not limited to these examples.

[0106] In the following Reference Examples, Comparative Examples, and Examples, the KNK-005 strain was used as a PHA-producing microorganism. KNK-005 is a well-known PHA-producing microorganism, prepared according to the method described in U.S. Patent No. 7,384,766. It is a transformant of the Cupriavidus necator H16 strain into which a PHA synthase gene derived from Aeromonas cavie has been introduced into the chromosome. When using oils and fats as raw materials, this transformant can produce P(3HB-co-3HH), a type of PHA.

[0107] (Reference Example 1) PHA production using soft palm oil as a carbon source

[0108] Cultivation studies using the KNK-005 strain were conducted under the following conditions. Commercially available soft palm oil (unused oil) was used as a carbon source in the PHA production culture. The anisidine value, oil polymer content, peroxide value, and color of the soft palm oil used are shown in Table 1. The anisidine value was determined in accordance with the Standard Oil and Fats Analysis Test Method 2.5.3-2013 established by the Japan Oil Chemists' Association. The oil polymer content was determined in accordance with Provisional Method 16 of the Standard Oil and Fats Analysis Test Method 2.5.7-2013. The peroxide value was determined in accordance with the Standard Oil and Fats Analysis Test Method 2.5.2.2-2013. The color was determined in accordance with the Standard Oil and Fats Analysis Test Method 2.2.1.3-2013.

[0109] (Culture medium)

[0110] The composition of the mother culture medium is 1w / v% Meat-extract, 1w / v% Bacto-Tryptone, 0.2w / v% Yeast-extract, 0.9w / v% Na2HPO4·12H2O, 0.15w / v% KH2PO4, (pH 6.8).

[0111] The preculture medium consisted of 1.1 w / v% Na₂HPO₄·12H₂O, 0.19 w / v% KH₂PO₄, 1.29 w / v% (NH₄)₂SO₄, 0.1 w / v% MgSO₄·7H₂O, 2.5 w / v% soft palm oil, and 0.5 v / v% trace metal salt solution (1.6 w / v% FeCl₃·6H₂O, 1 w / v% CaCl₂·2H₂O, 0.02 w / v% CoCl₂·6H₂O, 0.016 w / v% CuSO₄·5H₂O, and 0.012 w / v% NiCl₂·6H₂O dissolved in 0.1N hydrochloric acid). Soft palm oil was added as a carbon source at a concentration of 10 g / L.

[0112] The composition of the PHA production medium is 0.385w / v% Na2HPO4·12H2O, 0.067w / v% KH2PO4, 0.291w / v% (NH4)2SO4, 0.1w / v% MgSO4·7H2O, and 0.5v / v% trace metal salt solution (a solution of 1.6w / v% FeCl3·6H2O, 1w / v% CaCl2·2H2O, 0.02w / v% CoCl2·6H2O, 0.016w / v% CuSO4·5H2O, and 0.012w / v% NiCl2·6H2O dissolved in 0.1N hydrochloric acid).

[0113] (PHA production and cultivation)

[0114] PHA production culture was performed as follows: First, glycerol stock (20 μl) of the KNK-005 strain was inoculated into a seed culture medium (20 ml) and cultured for 24 hours to perform seed culture.

[0115] Next, the mother culture was inoculated at 1.0 v / v% into a 3L fermenter (MDL-300, manufactured by Marubi Bioengineering) containing 1.8L of pre-culture medium. Pre-culture was performed under operating conditions: a culture temperature of 33°C, a stirring rate of 500 rpm, an aeration rate of 1.8 L / min, and a 24-hour incubation period while controlling the pH to no less than 6.4. A 14% aqueous ammonium hydroxide solution was used for pH control.

[0116] Next, the pre-culture solution was inoculated into a 5L fermenter (MDS-U50, manufactured by Marubi Bioengineering) containing 2.5L of PHA production medium at 5.0v / v%. The operating conditions were set to a culture temperature of 34±1°C, a stirring speed of 420rpm, an aeration volume of 2.1L / min, and a pH of no less than 6.4. A 25% aqueous ammonium hydroxide solution was used to control the pH. A carbon source was added intermittently, and the rate of addition of the carbon source was controlled so that the carbon source concentration in the culture solution was within the range (0.2-5%) for efficient PHA production.

[0117] (purification)

[0118] After the culture was completed, the culture medium was weighed in a centrifuge tube and the weight of the culture medium was measured. The bacteria were recovered by centrifugation and suspended in a 3.3 wt / v% sodium dodecyl sulfate (SDS) aqueous solution. The cellular components of the bacteria were broken using an ultrasonic disruptor to extract PHA. PHA was recovered by centrifugation, washed with water and ethanol in sequence, and then vacuum-dried at 60°C for 3 hours to obtain dry PHA. The weight of the obtained dry PHA was measured and divided by the weight of the culture medium initially measured to calculate the average PHA weight per 1 g of culture medium obtained 72 hours after the start of culture.

[0119] (Calculation method of PHA productivity)

[0120] The PHA productivity (%) was calculated as the ratio of the average PHA weight (g) per 1 g of the culture solution obtained 72 hours after the start of culture in each example to the average PHA weight (g) per 1 g of the culture solution obtained 72 hours after the start of culture in Reference Example 1 using the following formula.

[0121] PHA productivity (%) = [Average PHA weight per 1g of culture solution obtained in each example (g)] / [Average PHA weight per 1g of culture solution obtained in Reference Example 1 (g)] × 100

[0122] Table 1 shows the measurement results of the PHA productivity (%) in Reference Example 1.

[0123] (Reference Example 2) PHA production using rapeseed oil as a carbon source

[0124] Cultivation studies were conducted under the same conditions as in Reference Example 1, except that the carbon source used in PHA production culture was changed from soft palm oil to commercially available rapeseed oil (unused oil). The anisidine value, oil polymer content, peroxide value, and color of the rapeseed oil used are shown in Table 1. Furthermore, the results of measurement of PHA productivity (%) are shown in Table 1.

[0125] (Comparative Example 1 and Examples 1-10) PHA Production Using Waste Oil as Carbon Source

[0126] Cultivation studies were conducted under the same conditions as in Reference Example 1, except that the carbon source used in PHA production culture was changed from soft palm oil to waste oils A to K. Furthermore, waste oils A to I were obtained by collecting oils discharged from multiple discharge sources and treating them to remove a certain amount of water and impurities from the collected oils.

[0127] In addition, waste oils J and K are rinse oils that are determined to be waste by oil and fat manufacturing personnel.

[0128] The anisidine value, oil and fat polymer content, peroxide value, and color of waste oils A to K are shown in Table 1. In addition, the measurement results of PHA productivity (%) are shown in Table 1.

[0129] (Example 11) Production of PHA using a mixture of waste oil and unused oil as a carbon source

[0130] Cultivation studies were conducted under the same conditions as in Reference Example 1, except that the carbon source used in PHA production culture was changed from soft palm oil to mixed oil A. Mixed oil A was prepared by mixing waste oil A used in Comparative Example 1 and soft palm oil used in Reference Example 1 at a volume ratio of 1:3. The anisidine value, oil polymer content, peroxide value, and color of mixed oil A are shown in Table 1. Furthermore, the results of PHA productivity (%) are also shown in Table 1.

[0131] The obtained PHA was reacted in a mixture of methanol and sulfuric acid under high temperature and high pressure and then subjected to HPLC to confirm that the PHA obtained in all reference examples, comparative examples, and examples was P(3HB-co-3HH).

[0132] [Table 1]

[0133] As shown in Table 1, the PHA productivity of Comparative Example 1, which used waste oil with an anisidine value exceeding 180 as a carbon source, was reduced by about 40% compared to Reference Examples 1 and 2, which used unused oil as a carbon source.

[0134] In contrast, Examples 1 to 10, which used waste oils with an methoxyaniline value of 180 or less as a carbon source, showed improved PHA productivity compared to Comparative Example 1, resulting in relatively good results. Specifically, Examples 3 to 10, which used waste oils with an methoxyaniline value of 120 or less as a carbon source, showed only a 10% decrease in PHA productivity compared to Reference Examples 1 and 2. However, Examples 4 to 10, which used waste oils with an methoxyaniline value of 70 or less as a carbon source, achieved PHA productivity comparable to that of Reference Examples 1 and 2, demonstrating particularly good PHA productivity.

[0135] In Example 11, waste oil A used in Comparative Example 1 was mixed with unused oil used in Reference Example 1 to prepare a mixed oil having an anisidine value of 70 or less, and this mixed oil was used as a carbon source for culture. Despite using waste oil A from Comparative Example 1, this example achieved the same PHA productivity as Reference Example 1. This demonstrates that even waste oils with significantly low PHA productivity when used alone, as shown in Comparative Example 1, can improve PHA productivity by adjusting the anisidine value by mixing with other oils and fats.

[0136] The above situation applies not only to the anisidine value, but also to the amount of oil polymers.

[0137] On the other hand, regarding the peroxide value and color, it was found that there was no clear correlation between these values and the PHA productivity.

Claims

1. A method for producing a polyhydroxyalkanoate, the method comprising: A step of producing polyhydroxyalkanoate by culturing a polyhydroxyalkanoate-producing strain in a medium containing oil as a carbon source, The grease comprises waste oil, The anisidine value measured for the entire fat and oil was 180 or less.

2. The manufacturing method according to claim 1, wherein The anisidine value is 2 or more.

3. The manufacturing method according to claim 1 or 2, wherein: The amount of oil and fat polymers measured in the entire oil and fat is 25% or less.

4. The manufacturing method according to claim 3, wherein: The amount of the oil and fat polymer is 0.1% or more.

5. The manufacturing method according to claim 1 or 2, wherein: The polyhydroxyalkanoate-producing strain is a microorganism belonging to the genus Cupriavidus.

6. The manufacturing method according to claim 1 or 2, wherein: The polyhydroxyalkanoate is a copolymer of two or more hydroxyalkanoic acids.

7. The manufacturing method according to claim 6, wherein: The polyhydroxyalkanoate is a copolymer containing 3-hydroxyhexanoic acid as a monomer unit.

8. The manufacturing method according to claim 7, wherein: The polyhydroxyalkanoate is a copolymer of 3-hydroxybutyric acid and 3-hydroxyhexanoic acid.

9. The manufacturing method according to claim 1, wherein: The anisidine value is 120 or less.

10. The manufacturing method according to claim 1, wherein: The anisidine value is 70 or less.

11. A fat used as a carbon source in the cultivation of a polyhydroxyalkanoate-producing strain, the fat comprising waste oil, wherein: The anisidine value measured for the entire fat and oil was 180 or less.

12. The grease according to claim 11, wherein The anisidine value is 2 or more.

13. A method for conditioning waste oil, the method comprising: Mix the waste oil with other oils and fats so that the anisidine value of the mixed oils and fats is below 180. When the mixed oil and fat is used as a carbon source in the cultivation of a polyhydroxyalkanoate-producing strain, the productivity of polyhydroxyalkanoate is improved compared to the waste oil.

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