Method for assessing oil / fat for polyhydroxyalkanoic acid production

Abstract

This method for assessing whether an oil / fat can be used as a carbon source when culturing a polyhydroxyalkanoic acid–producing strain includes: a step for preparing a measured value for the oil / fat polymer content measured for the oil / fat; and a step for assessing that the oil / fat can be used as the carbon source when the measured value is equal to or less than an oil / fat polymer content threshold, and assessing that the oil / fat cannot be used as the carbon source when the measured value exceeds the oil / fat polymer content threshold.

Description

Method for determining fats and oils for producing polyhydroxyalkanoic acid

[0001] The present invention relates to a method for determining the usability of fats and oils in the cultivation of polyhydroxyalkanoic acid-producing microorganisms, and a method for producing polyhydroxyalkanoic acid using the determination method.

[0002] In response to growing global interest in sustainability, including the SDGs, and growing concern about environmental issues such as marine microplastics, efforts to convert existing petroleum-derived non-biodegradable plastics to biodegradable materials are being promoted, primarily in industries such as packaging, food service, biomedicine, and agriculture. Examples of biodegradable materials that have seen active industrial production in recent years include polylactic acid (PLA) and polyhydroxyalkanoic acid (hereinafter also referred to as PHA). Among these, PHA has excellent biodegradability in a wide range of environments and is one of the few biodegradable materials that can biodegrade in seawater, and as such, it is gaining increasing attention as a solution to the marine microplastics problem and other environmental issues.

[0003] PHA is a natural thermoplastic polyester that is produced and accumulated as an energy storage substance in the cells of many microbial species. Generally, PHA is industrially produced by culturing PHA-accumulating microorganisms while supplying them with nutrient sources such as carbon, nitrogen, and phosphorus. Carbon sources often used in PHA cultivation 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, the carbon sources mentioned above are problematic due to the large amount of carbon dioxide emitted during the raw material production process, which places a heavy burden on the environment. Therefore, in PHA production, the use of waste oil (also known as waste oil, waste cooking oil, waste vegetable oil, used oil, etc.) as a carbon source is considered promising.

[0005] There have been reports of culturing and producing PHA using waste oil as a carbon source (see, for example, Patent Document 1), but there has been no mention of the effect of waste oil on PHA productivity.

[0006] Japanese Patent Application Laid-Open No. 2004-254668

[0007] As shown in the experimental data presented below, when the present inventors cultured a known PHA-producing strain using a certain waste oil as a carbon source, the PHA productivity was sometimes reduced by about 20% compared to culture using unused oils and fats as the carbon source. On the other hand, even when cultured using waste oil, there were cases where PHA productivity comparable to that achieved when using unused oils and fats was achieved. From the above, it was found that when waste oil is used as a carbon source, PHA productivity varies greatly depending on the properties of the waste oil.

[0008] Needless to say, it is desirable to achieve good PHA productivity while effectively utilizing waste oil as a carbon source. However, there is no way to know in advance whether the oil to be used can achieve good PHA productivity before carrying out microbial culture. Due to the above circumstances, there are obstacles to using waste oil as a carbon source in a PHA production method aimed at high productivity.

[0009] In view of the above-mentioned current situation, the present invention aims to provide a method for determining, before microbial culture, whether a fat or oil to be evaluated is capable of achieving good productivity when used as a carbon source in the culture of a polyhydroxyalkanoic acid-producing microorganism. Another object of the present invention is to provide a method for producing polyhydroxyalkanoic acid by microbial culture in the presence of a fat or oil, in which it is determined in advance that the fat or oil is capable of achieving good productivity, and then microbial culture is carried out using the fat or oil.

[0010] The present inventors have found that the amount of oil polymers, which is one of the indicators for quantifying the degree of deterioration of oils and fats, correlates with PHA productivity in microbial culture. Based on this finding, they have found that whether or not good productivity can be achieved when using an oil or fat for culturing a PHA-producing strain can be predicted in advance based on the measured value of the amount of oil polymers of the oil or fat, and have arrived at the present invention.

[0011] That is, the present invention relates to a method for determining whether an oil or fat can be used as a carbon source when culturing a polyhydroxyalkanoic acid-producing strain, the method comprising the steps of: preparing a measurement value of the amount of oil polymers measured for the oil or fat; and determining that the oil or fat can be used as the carbon source if the measurement value is equal to or less than a threshold amount of oil polymers, and determining that the oil or fat cannot be used as the carbon source if the measurement value exceeds the threshold amount of oil polymers. The present invention also relates to a method for producing polyhydroxyalkanoic acid by culturing a polyhydroxyalkanoic acid-producing strain, the method comprising the steps of: preparing a measurement value of the amount of oil polymers measured for an oil or fat (a); and adding the oil or fat (a) to a medium and culturing a polyhydroxyalkanoic acid-producing strain if the measurement value is equal to or less than the threshold amount of oil polymers.

[0012] According to the present invention, a method can be provided for determining, before microbial culture, whether a target oil or fat can achieve good productivity when used as a carbon source in the culture of a polyhydroxyalkanoic acid-producing microorganism. The present invention can also provide a production method for polyhydroxyalkanoic acid by microbial culture in the presence of an oil or fat, in which it is determined in advance that the oil or fat can achieve good productivity, and then microbial culture is carried out using the oil or fat. According to the present invention, oils or fats that are not suitable for efficient PHA production can be identified and eliminated in advance before microbial culture. Furthermore, oils or fats that are not suitable for PHA production (particularly waste oils) can be mixed with other oils or fats to prepare new oils or fats that can efficiently produce PHA. Therefore, waste oil can be effectively used as a carbon source in PHA-producing culture.

[0013]

[0014] The present invention relates to a method for determining whether or not a fat or oil can be used as a carbon source when culturing a polyhydroxyalkanoic acid-producing strain.

[0015] The present invention relates to a method for producing polyhydroxyalkanoic acid by culturing a polyhydroxyalkanoic acid-producing strain in the presence of a fat or oil.

[0014] The PHA-producing strain of this embodiment is not particularly limited as long as it is a microorganism capable of producing polyhydroxyalkanoic acid. The strain may be a wild-type strain inherently containing a PHA synthase gene, a mutant strain obtained by artificially mutating such a wild-type strain, or a strain into which an exogenous PHA synthase gene has been introduced by genetic engineering techniques. The method for introducing the exogenous PHA synthase gene is not particularly limited, and may include direct insertion or replacement of the gene onto the host chromosome, direct insertion or replacement of the gene onto a megaplasmid possessed by the host, or placement of the gene on a vector such as a plasmid, phage, or phagemid for introduction. Two or more of these methods may also be used in combination. Considering the stability of the introduced gene, direct insertion or replacement of the gene onto the host chromosome or onto a megaplasmid possessed by the host is preferred, and direct insertion or replacement of the gene onto the host chromosome is more preferred.

[0015] Preferred examples of the PHA-producing strain or its host according to this embodiment include, but are not limited to, bacteria belonging to the genera Ralstonia, Cupriavidus, Wautersia, Burkholderia, Aeromonas, Escherichia, Alcaligenes, Pseudomonas, Bacillus, Azotobacter, Nocardia, Sphingomonas, and Comamonas. From the viewpoints of safety and PHA productivity, bacteria belonging to the genus Ralstonia or Cupriavidus are more preferred, bacteria belonging to the genus Cupriavidus are even more preferred, and Cupriavidus necator is particularly preferred.

[0016] The PHA-producing strain according to this embodiment is a strain capable of assimilating fats and oils. The strain may be a wild-type strain inherently capable of assimilating fats and oils, a mutant strain obtained by artificially mutating such a wild-type strain, or a strain into which enzymes capable of assimilating fats and oils have been introduced by genetic engineering techniques. The method for introducing the exogenous enzymes capable of assimilating fats and oils is not particularly limited, and may include direct insertion or replacement of the gene onto the host chromosome, direct insertion or replacement of the gene onto a megaplasmid possessed by the host, or placement of the gene onto a vector such as a plasmid, phage, or phagemid for introduction. Two or more of these methods may also be used in combination. Considering the stability of the introduced gene, direct insertion or replacement of the gene onto the host chromosome or onto a megaplasmid possessed by the host is preferred, and direct insertion or replacement of the gene onto the host chromosome is more preferred.

[0017] (PHA synthase gene) The PHA synthase gene possessed by the PHA-producing strain according to this embodiment is not particularly limited, and may be a PHA synthase gene inherently possessed by the PHA-producing strain, or may be an exogenous PHA synthase gene. Specific examples of PHA synthase genes include PHA synthase genes derived from organisms similar to the genera Ralstonia, Capriavidus, Wautersia, Alcaligenes, Aeromonas, Pseudomonas, Norcadia, and Chromobacterium, as well as modified forms thereof. The modified forms may include a base sequence encoding a PHA synthase in which one or more amino acid residues have been deleted, added, inserted, or substituted.

[0018] (PHA) The type of PHA produced by the PHA-producing strain according to this embodiment is not particularly limited as long as it is a PHA that can be produced by a microorganism, but preferred are homopolymers of one monomer selected from 3-hydroxyalkanoic acids having 4 to 16 carbon atoms, copolymers of one monomer selected from 3-hydroxyalkanoic acids having 4 to 16 carbon atoms and other hydroxyalkanoic acids (e.g., 2-hydroxyalkanoic acids, 4-hydroxyalkanoic acids, 5-hydroxyalkanoic acids, 6-hydroxyalkanoic acids, etc. having 4 to 16 carbon atoms), and copolymers of two or more monomers selected from 3-hydroxyalkanoic acids having 4 to 16 carbon atoms. Examples of PHAs include, but are not limited to, P(3HB), which is a homopolymer of 3-hydroxybutyric acid (abbreviation: 3HB), P(3HB-co-3HV), a copolymer of 3HB and 3-hydroxyvaleric acid (abbreviation: 3HV), P(3HB-co-3HH), a copolymer of 3HB and 3-hydroxyhexanoic acid (abbreviation: 3HH), P(3HB-co-4HB), a copolymer of 3HB and 4-hydroxybutyric acid (abbreviation: 4HB), and PHAs containing lactic acid (abbreviation: LA) as a constituent, such as P(LA-co-3HB), a copolymer of 3HB and LA. Among these, P(3HB-co-3HH) is preferred from the viewpoint of its wide range of applications as a polymer. The type of PHA produced can be appropriately selected depending on the purpose, the type of PHA synthase gene possessed by the microorganism used or introduced separately, the type of metabolic system gene involved in its synthesis, the culture conditions, and the like.

[0019] By culturing a PHA-producing strain, PHA can be accumulated in the cells. This culture is called PHA-producing culture. Prior to PHA-producing culture, pre-culture (also called seed culture) for cell growth may be performed one or more times. In this embodiment, the step of culturing a PHA-producing microorganism can be performed according to a conventional microbial culture method, and the culture may be performed in a medium containing an appropriate carbon source. There are no particular limitations on the medium composition, culture scale, aeration and agitation conditions, culture temperature, culture time, etc. for the PHA-producing culture and pre-culture.

[0020] The carbon source can be added according to known PHA production culture techniques. It is preferable to add the carbon source so that the carbon source concentration increases when nutrient sources such as phosphorus, nitrogen, or magnesium are depleted as the microorganisms grow. Generally, when the carbon source concentration is sufficiently high under conditions where nutrient sources such as phosphorus, nitrogen, or magnesium are limited, microorganisms actively accumulate PHA. The carbon source concentration is not particularly limited, and an appropriate concentration can be selected depending on the microorganism, medium, carbon source, aeration and agitation conditions, culture temperature, etc. used. The carbon source may be added to the medium all at once, continuously, or intermittently, or a combination of both, to achieve the carbon source concentration. A continuous or intermittent addition method is more preferred.

[0021] (Oils and fats) In this embodiment, at least oils and fats are used as a carbon source in the PHA production culture. The oils and fats may include either unused oils and fats, waste oils, or both. However, this embodiment allows the PHA productivity of the oils and fats to be predicted in advance before carrying out microbial culture, and waste oils may have lower PHA productivity than unused oils and fats, so it is preferable that the oils and fats used in this embodiment include at least waste oil.

[0022] The term "oils and fats" refers to ester compounds of fatty acids and glycerin, and includes at least one of triacylglycerol, diacylglycerol, and monoacylglycerol. Usually, the main component is triacylglycerol.

[0023] Waste oil refers to discarded oils and fats, but the term is not limited to this and includes what is called waste oil, waste cooking oil, waste vegetable oil, used oil, etc. Furthermore, unused oils and fats refer to oils and fats that have not yet been used for a specified purpose after production.

[0024] The origin of the waste oil is not particularly limited, but it may be, for example, oils and fats that have been used in ordinary households, restaurants, or food manufacturing companies for cooking fried foods or that have passed their expiration date or best-before date and are no longer suitable for consumption and are discarded. It may also be oils and fats that have been determined to be unsuitable for their intended use and discarded by companies such as oil and fat manufacturing companies. For example, this includes co-washing oils generated when switching from oil and fat A to another type of oil and fat B in a tank and co-washing the tank or piping with oil and fat B. It may also be oils and fats that have been discharged from one or more sources and collected by a collection company. Alternatively, it may be oils and fats from which moisture and impurities have been removed from discharged or collected oil and fat.

[0025] The type of original fat or oil contained in the waste oil or the type of unused fat or oil are not particularly limited, and may be, for example, palm oil, palm kernel oil, or fractionated oils thereof (e.g., palm olein, palm double olein, palm kernel olein, etc., which are fractionated low-melting point fractions), corn oil, coconut oil, olive oil, soybean oil, rapeseed oil, jatropha oil, or other fats or oils or fractionated oils thereof, or refinery by-products thereof. Furthermore, fats or oils derived from animal oils may also be included. Furthermore, the waste oil may be a mixture of the fats and oils exemplified above.

[0026] The degree of deterioration of waste oil is related to the efficient production of PHA. Generally, fats and oils are oxidized and hydrolyzed when exposed to water, air, light including ultraviolet light, or when heated to high temperatures. This state is generally referred to as fat and oil deterioration. For example, peroxides can be produced when fats and oils react with oxygen. Furthermore, peroxides can polymerize to produce fat and oil polymers. The fat and oil polymers can be quantified using the amount of fat and oil polymers as an indicator. There are various theories about the mechanism of fat and oil deterioration, and the mechanism of fat and oil deterioration in this embodiment is not limited.

[0027] In the first aspect, first, a measurement value of the amount of fat polymer measured for the fat to be evaluated is prepared, and then, based on the magnitude of the measurement value, it is predicted in advance whether or not the fat can achieve good PHA productivity when used as a carbon source for culturing a PHA-producing strain. In this application, preparing a measurement value includes both performing the measurement yourself to obtain the measurement value and obtaining a measurement value obtained by a third party.

[0028] The present inventors have found that there is a correlation between the amount of oil polymer, which is an index for quantifying the degree of deterioration of oils and fats, and PHA productivity in microbial culture, and that the smaller the value of the amount of oil polymer exhibited by an oil and fat, the more likely it is that PHA productivity will improve when that oil and fat is used.

[0029] Therefore, a threshold value for the amount of oil and fat polymer is set based on the desired PHA productivity, and if the measured value of the amount of oil and fat polymer of the oil and fat to be evaluated is below the threshold value, the oil and fat is determined to be usable as a carbon source, whereas if the measured value exceeds the threshold value, the oil and fat is determined to be unusable as the carbon source.

[0030] On the other hand, other indicators for quantifying the degree of deterioration of fats and oils are also known, such as peroxide value, color, and acid value. However, no correlation was found between these indicators and PHA productivity. Therefore, it was found that peroxide value, color, and acid value cannot be used to predict PHA productivity in advance.

[0031] The specific value of the threshold amount of the oil / fat polymer described above is not particularly limited and may be appropriately set based on the desired PHA productivity. However, from the viewpoint of increasing PHA productivity while using waste oil as a carbon source in microbial culture, the threshold amount of the oil / fat polymer is preferably 10%. Furthermore, 8% is more preferable, 4% is even more preferable, and 2% is particularly preferable.

[0032] Since the smaller the amount of fat polymer, the more PHA productivity tends to improve, there is no need to set a threshold value for the lower limit of the amount of fat polymer. However, the lower limit of the amount of fat polymer in the fat may be, for example, 0.05% or more, 0.1% or more, or 0.3% or more.

[0033] It should be noted that a smaller value for the amount of oil and fat polymer means less deterioration of the oil and fat, but there is also available waste oil that has hardly deteriorated and has an amount of oil and fat polymer that is about the same as that of unused oil and fat.

[0034] The amount of fat polymer refers to the proportion (percentage) of polymers contained in fats and oils, and can be quantified by gel permeation chromatography. Here, fat polymer refers to a substance that elutes before triacylglycerol. Specifically, Provisional Method 16 of the Standard Methods for the Analysis of Fats, Oils, and Related Materials 2.5.7-2013 can be followed. However, this method is not limited thereto, and other test methods may also be used.

[0035] When the measured amount of oil / fat polymers for the target oil / fat (a) is equal to or less than the threshold amount of oil / fat polymers, it is predicted that PHA productivity will be good when the oil / fat (a) is used, and therefore the oil / fat (a) is added to a medium to culture a PHA-producing strain to produce PHA. Such a PHA production method constitutes a second aspect of the present invention. According to this production method, it is possible to identify oils and fats of various origins that are expected to have good PHA productivity, and then perform PHA production culture.

[0036] Furthermore, when the measured amount of oil polymers for the target oil (a) exceeds the threshold amount of oil polymers, it is desirable to avoid performing PHA production culture using only the oil (a). However, the oil (a) can be mixed with another oil (b) to prepare a mixed oil (c) in which the amount of oil polymers is equal to or less than the threshold amount of oil polymers, and such a mixed oil (c) can be added to a medium to culture a PHA-producing strain to produce PHA.

[0037] As the other fat (b), a fat having a lower amount of fat polymer than the fat (a) may be appropriately selected. This fat (b) may be either waste oil or unused fat, but unused fat is preferred. The mixed fat (c) can be easily prepared by appropriately adjusting the amount of fat polymer in the other fat (b) or the mixing ratio of the fat (a) and the other fat (b).

[0038] According to the above manufacturing method, fats and oils that cannot be expected to produce good PHA productivity when used alone can be converted into fats and oils that can achieve good PHA productivity by mixing them with other fats and oils, making it possible to effectively utilize fats and oils that cannot be expected to produce good PHA productivity when used alone in PHA production culture.

[0039] The proportion of waste oil contained in the entire fat and oil to be evaluated is not particularly limited and may be 0% by weight, but from the viewpoint of reducing the environmental load, it 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. The fat and oil to be evaluated may consist only of waste oil.

[0040] From the viewpoint of the culture production yield, the water content of the oil or fat 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 water content measurement method in this embodiment follows Standard Methods for Analysis of Fats, Oils, and Related Materials 2.1.3.2-2013. Furthermore, the oil or fat may be subjected to a treatment to reduce the water content before use in the PHA production culture.

[0041] Furthermore, the carbon source used in the PHA production culture may further contain, in addition to fats and oils, a carbon source other than fats and oils (for example, sugars, fatty acids, glycerol, etc.). However, from the viewpoint of reducing the environmental load, the proportion of fats and oils contained 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.

[0042] The carbon source used in the pre-culture is not particularly limited, and waste oil may or may not be used. Usable carbon sources include, for example, sugars such as glucose, fructose, and sucrose; oils and fats such as palm oil, palm kernel oil, or their fractionated oils (e.g., fractionated low-melting-point fractions such as palm olein, palm double olein, and palm kernel oil olein), corn oil, coconut oil, olive oil, soybean oil, rapeseed oil, and jatropha oil, as well as their fractionated oils and their refined by-products; fatty acids such as lauric acid, oleic acid, stearic acid, palmitic acid, and myrinsic acid, as well as their derivatives, and glycerol. Mixtures of the above-listed oils and fats may also be used.

[0043] In the production of PHA in the second embodiment, it is preferable to culture the microorganism using a medium containing the 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 include, but are not limited to, 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.

[0044] After culturing for an appropriate time to accumulate PHA in the cells, PHA can be recovered from the cells using a known method. The recovery method is not particularly limited, but for example, after the culture is completed, the cells are separated from the culture solution using a centrifuge or a separation membrane, etc., and dried, and then PHA is extracted from the dried cells using an organic solvent such as chloroform, and cellular components are removed from the organic solvent solution containing PHA by filtration or the like, and 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 the PHA can be recovered by drying. Alternatively, cellular components other than PHA can be dissolved in water using a surfactant, alkali, enzyme, etc., and then PHA particles can be separated from the aqueous phase by filtration or centrifugation, dried, and recovered.

[0045] According to this embodiment, in PHA production culture using oils and fats as a carbon source, the effect of the degree of oil and fat degradation on PHA productivity can be predicted in advance. Therefore, it is possible to identify oils and fats that are expected to provide good PHA productivity in advance and use them in PHA production culture. Furthermore, oils and fats that are not expected to provide good PHA productivity can be converted into oils and fats that can achieve good PHA productivity and effectively utilized. As a result, it is possible to efficiently use waste oil, which has a low environmental impact, as a carbon source in PHA production culture, thereby reducing the environmental impact of PHA production culture.

[0046] The following items are preferred embodiments of the present disclosure, but the present invention is not limited to them. [Item 1] A method for determining whether an oil or fat can be used as a carbon source when culturing a polyhydroxyalkanoic acid-producing bacterial strain, comprising the steps of: preparing a measurement value of the amount of oil polymer measured for the oil or fat; and determining that the oil or fat can be used as the carbon source if the measurement value is equal to or less than a threshold amount of oil polymer, and determining that the oil or fat cannot be used as the carbon source if the measurement value exceeds the threshold amount of oil polymer. [Item 2] The method according to Item 1, wherein the threshold amount of oil polymer is 10%. [Item 3] The method according to Item 1 or 2, wherein the oil or fat includes waste oil. [Item 4] The method according to any one of Items 1 to 3, wherein the polyhydroxyalkanoic acid-producing bacterial strain is a microorganism belonging to the genus Capriavidus. [Item 5] The method according to any one of Items 1 to 4, wherein the polyhydroxyalkanoic acid is a copolymer of two or more types of hydroxyalkanoic acids. [Item 6] The method for determining the amount of polyhydroxyalkanoic acid according to Item 5, wherein the polyhydroxyalkanoic acid is a copolymer containing 3-hydroxyhexanoic acid as a monomer unit. [Item 7] The method for determining the amount of polyhydroxyalkanoic acid according to Item 6, wherein the polyhydroxyalkanoic acid is a copolymer of 3-hydroxybutyric acid and 3-hydroxyhexanoic acid. [Item 8] A method for producing polyhydroxyalkanoic acid by culturing a polyhydroxyalkanoic acid-producing strain, comprising the steps of: preparing a measurement value of an amount of oil / fat polymer measured for an oil / fat (a); and, if the measurement value is equal to or less than a threshold amount of oil / fat polymer, adding the oil / fat (a) to a medium and culturing the polyhydroxyalkanoic acid-producing strain. [Item 9] The method for producing polyhydroxyalkanoic acid according to Item 8, further comprising, if the measurement value exceeds the threshold amount of oil / fat polymer, mixing the oil / fat (a) with another oil / fat (b) to prepare a mixed oil / fat (c) in which the amount of oil / fat polymer is equal to or less than the threshold amount, and adding the mixed oil / fat (c) to a medium and culturing the polyhydroxyalkanoic acid-producing strain. [Item 10] The production method according to Item 8 or 9, wherein the threshold value of the amount of fat or oil polymer is 10%.

[0047] The present invention will be described in more detail below with reference to examples, although the present invention is not limited to these examples.

[0048] In the following examples, the KNK-005 strain was used as the PHA-producing microorganism. The KNK-005 strain is a known PHA-producing microorganism, and is a transformant of the Capriavidus necator H16 strain, prepared according to the method described in U.S. Pat. No. 7,384,766, in which a PHA synthase gene derived from Aeromonas caviae has been introduced onto the chromosome. The KNK-005 strain is a transformed microorganism capable of producing P(3HB-co-3HH), a type of PHA, when fats and oils are used as raw materials.

[0049] (Example 1) PHA production using palm olein oil as a carbon source A culture study was conducted using the KNK-005 strain under the following conditions. Commercially available palm olein oil (virgin oil) was used as the carbon source in the PHA production culture. The amount of fat polymer, iodine value, acid value, peroxide value, and color of the palm olein oil used are shown in Table 1. The amount of fat polymer was measured in accordance with Provisional Method 16 of the Standard Analysis of Fats, Oils, and Related Materials 2.5.7-2013. The iodine value was measured in accordance with the Standard Analysis of Fats, Oils, and Related Materials 2.3.4.1-2013. The acid value was measured in accordance with the Standard Analysis of Fats, Oils, and Related Materials 2.3.1-2013. The peroxide value was measured in accordance with the Standard Analysis of Fats, Oils, and Related Materials 2.5.2.2-2013. The color was measured in accordance with the Standard Analysis of Fats, Oils, and Related Materials 2.2.1.3-2013.

[0050] (Culture medium) The composition of the seed culture medium is 1 w / v% Meat extract, 1 w / v% Bacto-Tryptone, 0.2 w / v% Yeast extract, 0.9 w / v% Na ２ HPO ４ ・12H ２ O, 0.15w / v% KH ２ P.O. ４ , (pH 6.8).

[0051] The composition of the pre-culture medium was 1.1 w / v% Na ２ HPO ４ ・12H ２ O, 0.19w / v%KH ２ P.O. ４ , 1.29 w / v% (NH４ ) ２ SO ４ , 0.1w / v% MgSO ４ ・7H ２ O, 2.5 w / v% palm olein oil, 0.5 v / v% trace metal salt solution (1.6 w / v% FeCl in 0.1 N hydrochloric acid ３ ・6H ２ O, 1w / v% CaCl ２ ・2H ２ O, 0.02w / v% CoCl ２ ・6H ２ O, 0.016w / v% CuSO ４ ・5H ２ O, 0.012w / v% NiCl ２ ・6H ２ Palm olein oil was added as a carbon source at a concentration of 10 g / L all at once.

[0052] The composition of the PHA production medium was 0.385 w / v% Na ２ HPO ４ ・12H ２ O, 0.067w / v% KH ２ P.O. ４ , 0.291w / v% (NH ４ ) ２ SO ４ , 0.1w / v% MgSO ４ ・7H ２ 0, 0.5 v / v% trace metal salt solution (1.6 w / v% FeCl in 0.1 N hydrochloric acid ３ ・6H ２ O, 1w / v% CaCl ２ ・2H ２ O, 0.02w / v% CoCl ２ ・6H ２ O, 0.016w / v% CuSO ４ ・5H ２ O, 0.012w / v% NiCl ２ ・6H ２ O was dissolved).

[0053] (PHA production culture) PHA production culture was carried out as follows. First, a glycerol stock (20 μl) of the KNK-005 strain was inoculated into a seed medium (20 ml) and cultured for 24 hours to carry out seed culture. Next, the seed culture solution was inoculated at 1.0 v / v% into a 3 L jar fermenter (MDL-300 model, manufactured by Marubishi Bioengine) containing 1.8 L of preculture medium. The operating conditions were a culture temperature of 33°C, a stirring speed of 500 rpm, and an aeration rate of 1.8 L / min, and the culture was continued for 24 hours while controlling the pH so that it did not fall below 6.4 to carry out preculture. A 14% aqueous ammonium hydroxide solution was used to control the pH.

[0054] Next, the preculture solution was inoculated at 5.0 v / v% into a 5 L jar fermenter (Marubishi Bioengine MDS-U50 model) containing 2.5 L of PHA production medium. The operating conditions were a culture temperature of 34±1°C, an agitation speed of 420 rpm, and an aeration rate of 2.1 L / min, and the pH was controlled so as not to fall below 6.4. A 25% aqueous ammonium hydroxide solution was used for pH control. The 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%) in which PHA could be efficiently produced.

[0055] (Purification) After the culture was completed, the culture solution was weighed into a centrifuge tube and the weight of the culture solution was measured. The bacterial cells were collected by centrifugation and suspended in a 3.3 wt / v% aqueous solution of sodium lauryl sulfate (SDS), and the cellular components of the bacterial cells were disrupted using an ultrasonic disrupter to extract PHA. The PHA was collected by centrifugation, washed with water and ethanol in turn, and then vacuum dried at 60 ° C for 3 hours to obtain dried PHA. The weight of the obtained dried PHA was measured and divided by the weight of the culture solution measured initially to calculate the weight of PHA per 1 g of culture solution obtained 72 hours after the start of culture.

[0056] (Method of calculating PHA productivity) PHA productivity (%) was calculated using the following formula as the ratio of the PHA weight (g) per gram of culture solution obtained in each example 72 hours after the start of culture to the PHA weight (g) per gram of culture solution obtained 72 hours after the start of culture in Example 1: PHA productivity (%) = [PHA weight (g) per gram of culture solution obtained in each example] / [PHA weight (g) per gram of culture solution obtained in Example 1] × 100 The measurement results of PHA productivity (%) in Example 1 are shown in Table 1.

[0057] (Example 2) PHA production using rapeseed oil as a carbon source Cultivation was carried out under the same conditions as in Example 1, except that the carbon source used in the PHA production culture was changed from palm olein oil to commercially available rapeseed oil (virgin oil). The amount of oil and fat polymer, iodine value, acid value, peroxide value, and color of the rapeseed oil used are shown in Table 1. The measurement results of PHA productivity (%) are also shown in Table 1.

[0058] (Examples 3 to 12) PHA production using waste oil as a carbon source Cultivation studies were conducted under the same conditions as in Example 1, except that the carbon source used in the PHA production culture was changed from palm olein oil to waste oils A to J. Note that waste oils A to I are oils and fats collected by a collection company from oils and fats discharged from multiple sources and processed to remove moisture and impurities to some extent. Furthermore, waste oil J is co-wash oil that an oil and fat manufacturer has determined to be discarded. The amount of oil and fat polymers, iodine value, acid value, peroxide value, and color of waste oils A to J are shown in Table 1. Furthermore, the measurement results of PHA productivity (%) are also shown in Table 1.

[0059] In addition, the obtained PHA was reacted in a mixed solution of methanol and sulfuric acid under high temperature and pressure, and then subjected to HPLC, and it was confirmed that the obtained PHA was P(3HB-co-3HH) in all cases.

[0060]

[0061] Table 1 shows that there is a correlation between the amount of fat and oil polymer and PHA productivity, and that the smaller the value of the amount of fat and oil polymer, the more improved the PHA productivity when using the fat and oil. In particular, in Examples 3 and 4, in which waste oil containing more than 10% fat and oil polymer was used as a carbon source, PHA productivity was significantly reduced. Based on this result, 10% can be adopted as the threshold value for the amount of fat and oil polymer. In this case, fats and oils containing 10% or less fat and oil polymer (other than Examples 3 and 4) are predicted to have relatively good PHA productivity, and are therefore judged to be usable as a carbon source when culturing a polyhydroxyalkanoic acid-producing strain. On the other hand, fats and oils containing more than 10% fat and oil polymer (Examples 3 and 4) are predicted to have significantly lower PHA productivity, and are therefore judged to be unusable as the carbon source.

[0062] However, the threshold value of the amount of fat and oil polymer is not limited to 10%. Since a difference in PHA productivity is also observed between Example 8 and Example 9, the threshold value of the amount of fat and oil polymer may be set to, for example, 4% between the amounts of fat and oil polymer in Example 8 and Example 9. Similarly, the threshold value of the amount of fat and oil polymer may be set to, for example, 2% between the amounts of fat and oil polymer in Example 10 and Example 11.

[0063] On the other hand, it is clear that there is no clear correlation between the iodine value, acid value, peroxide value, and color and the PHA productivity. This means that these indices cannot be used to predict the PHA productivity that can be achieved by fats and oils.

Claims

1. A method for determining whether or not an oil or fat can be used as a carbon source when culturing a polyhydroxyalkanoic acid-producing bacterial strain, the method comprising the steps of: preparing a measurement value of the amount of oil or fat polymer measured for the oil or fat; and determining that the oil or fat can be used as the carbon source if the measurement value is equal to or less than a threshold amount of oil or fat polymer, and determining that the oil or fat cannot be used as the carbon source if the measurement value exceeds the threshold amount of oil or fat polymer.

2. The method of claim 1, wherein the threshold value of the amount of oil and fat polymer is 10%.

3. The method of claim 1 or 2, wherein the oils and fats include waste oil.

4. The method of claim 1 or 2, wherein the polyhydroxyalkanoic acid-producing strain is a microorganism belonging to the genus Capriavidus.

5. The method of claim 1 or 2, wherein the polyhydroxyalkanoic acid is a copolymer of two or more types of hydroxyalkanoic acid.

6. The method according to claim 5, wherein the polyhydroxyalkanoic acid is a copolymer containing 3-hydroxyhexanoic acid as a monomer unit.

7. The method according to claim 6, wherein the polyhydroxyalkanoic acid is a copolymer of 3-hydroxybutyric acid and 3-hydroxyhexanoic acid.

8. A method for producing polyhydroxyalkanoic acid by culturing a polyhydroxyalkanoic acid-producing strain, comprising: a step of preparing a measurement value of the amount of oil polymer measured for an oil (a); and a step of adding the oil (a) to a medium and culturing the polyhydroxyalkanoic acid-producing strain when the measurement value is equal to or less than a threshold value for the amount of oil polymer.

9. The production method described in claim 8, further comprising the steps of: if the measured value exceeds a threshold for the amount of oil-and-fat polymer, mixing the oil-and-fat (a) with another oil-and-fat (b) to prepare a mixed oil-and-fat (c) in which the amount of oil-and-fat polymer is equal to or less than the threshold; and adding the mixed oil-and-fat (c) to a culture medium and culturing a polyhydroxyalkanoic acid-producing strain.

10. The manufacturing method according to claim 8 or 9, wherein the threshold value of the amount of oil and fat polymer is 10%.

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