Method for producing polyhydroxyalkanoate

Abstract

To provide a method for producing polyhydroxyalkanoate through microbial culture, allowing efficient production of polyhydroxyalkanoate even when waste oil is used as a carbon source.SOLUTION: A method for producing polyhydroxyalkanoate comprises the step of culturing a polyhydroxyalkanoate-producing strain in a medium containing oil or fat as a carbon source to produce polyhydroxyalkanoate. The oil or fat comprises waste oil. The amount of oil or fat polymers measured for the oil or fat as a whole is 10% or less. The amount of oil or fat polymers may be 0.1% or more.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to a method for producing polyhydroxyalkanoic acid by microbial culture. [Background technology]

[0002] In response to growing global interest in sustainability, including the SDGs, and growing concern about environmental issues such as marine microplastics, efforts are underway to convert existing petroleum-derived non-biodegradable plastics to biodegradable materials, primarily in industries such as packaging, food service, biomedicine, and agriculture. Biodegradable materials that have seen active industrial production in recent years include polylactic acid (PLA) and polyhydroxyalkanoates (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. Therefore, PHA is expected to be 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 providing them with nutrients such as carbon, nitrogen, and phosphorus. Carbon sources often 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, 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, the use of waste oil (also known as waste oil, waste cooking oil, waste vegetable oil, used oil, etc.) as a carbon source for PHA production 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). However, there has been no mention of the effect of waste oil on PHA productivity. [Prior art documents] [Patent documents]

[0006] [Patent Document 1] Japanese Patent Application Laid-Open No. 2004-254668 Summary of the Invention [Problem to be solved by the invention]

[0007] To the best of the inventors' knowledge, there have been no reports to date on the quality of waste oil required for efficient cultivation and production of PHA.

[0008] As shown in Comparative Examples 1 and 2 below, the present inventors cultured a known PHA-producing strain using a certain waste oil as a carbon source, and found that PHA productivity was reduced by approximately 20% compared to cultures using unused oils and fats as a carbon source (Reference Examples 1 and 2). Such a decrease in PHA productivity is undesirable, and it is desirable to achieve good PHA productivity while effectively utilizing waste oil as a carbon source.

[0009] In view of the above-described current situation, the present invention aims to provide a method for producing polyhydroxyalkanoic acid by microbial culture, which is capable of efficiently producing polyhydroxyalkanoic acid even when waste oil is used as a carbon source. [Means for solving the problem]

[0010] The present inventors have discovered that when polyhydroxyalkanoic acid-producing microorganisms are cultured to produce polyhydroxyalkanoic acid, productivity close to that achieved when only virgin oils and fats are used can be achieved if the amount of oil and fat polymer measured for the entire oil and fat is 10% or less when oils and fats containing waste oil are used as a carbon source, and have arrived at the present invention.

[0011] That is, the present invention provides a method for producing polyhydroxyalkanoic acid, which comprises a step of culturing a polyhydroxyalkanoic acid-producing strain in a medium containing fats and oils as a carbon source to produce polyhydroxyalkanoic acid, The oils and fats include waste oil, The present invention relates to a method for producing a polyhydroxyalkanoic acid, in which the amount of oil polymers measured for the entire oil is 10% or less. [Effects of the Invention]

[0012] According to the present invention, there can be provided a method for producing polyhydroxyalkanoic acid by microbial culture, which is capable of efficiently producing polyhydroxyalkanoic acid even when waste oil is used as a carbon source. 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 and fats to produce oils and fats suitable for efficient PHA production. Therefore, waste oils can be effectively used as a carbon source in PHA production culture. DETAILED DESCRIPTION OF THE INVENTION

[0013] Hereinafter, embodiments of the present invention will be described in detail. One embodiment of the present invention relates to a method for producing polyhydroxyalkanoic acid, which comprises a step of culturing a polyhydroxyalkanoic acid-producing strain in a medium containing oil or fat as a carbon source to produce polyhydroxyalkanoic acid.

[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 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.

[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 of 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 that assimilate fats and oils have been introduced by genetic engineering techniques. The method for introducing the exogenous enzymes that assimilate 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 of 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 versions thereof. Examples of the modified versions include nucleotide sequences encoding PHA synthases 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 of 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), a homopolymer of 3-hydroxybutyric acid (abbreviated as 3HB), P(3HB-co-3HV), a copolymer of 3HB and 3-hydroxyvaleric acid (abbreviated as 3HV), P(3HB-co-3HH), a copolymer of 3HB and 3-hydroxyhexanoic acid (abbreviated as 3HH), P(3HB-co-4HB), a copolymer of 3HB and 4-hydroxybutyric acid (abbreviated as 4HB), and PHAs containing lactic acid (abbreviated as 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 genes involved in its synthesis, the culture conditions, etc.

[0019] PHA can be accumulated in the cells by culturing a PHA-producing strain. 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, microorganisms actively accumulate PHA when the carbon source concentration is sufficiently high under conditions where nutrient sources such as phosphorus, nitrogen, or magnesium are limited. 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. Continuous or intermittent addition is more preferred.

[0021] (carbon source) In this embodiment, at least fats and oils are used as a carbon source in the PHA production culture, and the fats and oils include at least waste oil.

[0022] Fats and oils refer to ester compounds of fatty acids and glycerin, and contain 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 their production.

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

[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 (for example, fractionated low-melting-point fractions such as palm olein, palm double olein, and palm kernel olein), corn oil, coconut oil, olive oil, soybean oil, rapeseed oil, or jatropha oil, 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 efficient production of PHA in this embodiment is related to the degree of deterioration of waste oil. 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 deterioration of fats and oils. 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 this embodiment, fats and oils that can be used to efficiently produce PHA through culture are specified using an index that quantifies the degree of deterioration of the fat and oil, and the amount of fat and oil polymer is used as such an index.

[0028] The present inventors have found that there is a correlation between the amount of fat polymer, which is an index for quantifying the degree of fat deterioration, and PHA productivity in microbial culture, and that the smaller the value of the amount of fat polymer exhibited by a fat, the more likely it is that PHA productivity will be improved when that fat is used. In particular, they have found that good PHA productivity can be achieved when the amount of fat polymer is 10% or less.

[0029] On the other hand, other indicators known to quantify the degree of deterioration of fats and oils include 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 define fats and oils that can efficiently produce PHA through cultivation.

[0030] The fats and oils used in this embodiment are those in which the amount of fat and oil polymers measured for the entire fat and oil, including waste oil, is 10% or less. This allows efficient production of PHA while using waste oil as a carbon source in microbial culture. From the viewpoint of further increasing PHA productivity, the amount of fat and oil polymers is preferably 8% or less, more preferably 4% or less, and particularly preferably 2% or less.

[0031] The smaller the amount of oil and fat polymer, the less the deterioration of the oil and fat. However, there is also available waste oil that has hardly deteriorated and has an amount of oil and fat polymer equivalent to that of unused oil and fat. Therefore, the lower limit of the amount of oil and fat polymer is not particularly limited, but may be 0.05% or more, 0.1% or more, or 0.3% or more.

[0032] The method for measuring the amount of fat and oil polymers in this embodiment complies with Provisional Method 16 of the Standard Methods for the Analysis of Fats, Oils and Related Materials 2.5.7-2013.

[0033] In this embodiment, the fat or oil used as a carbon source in the PHA-producing culture may consist solely of waste oil, and in this case, the waste oil used should contain 10% or less of fat or oil polymers.

[0034] Furthermore, the oils and fats used as a carbon source in the PHA-producing culture may contain not only waste oil but also unused, undegraded oil. In this case, the amount of oil and fat polymers in the entire oil and fat mixture, including the waste oil and unused oil and fat, is required to be 10% or less, as described above. Such mixed oils and fats can be easily prepared by appropriately adjusting the amount of oil and fat polymers in the unused oil and fat or the mixing ratio of the waste oil and unused oil and fat. Therefore, the amount of oil and fat polymers in the waste oil used in combination with unused oil and fat is not particularly limited and may exceed 10%. In this case, waste oil that is not suitable for PHA production when used alone can be made suitable for PHA production by mixing it with other oils and fats.

[0035] From the viewpoint of reducing the environmental load, the proportion of waste oil contained in the total fats and oils used as the carbon source is preferably 10% by weight or more, more preferably 50% by weight or more, even more preferably 80% by weight or more, and may be 90% by weight or more.

[0036] From the viewpoint of the culture production yield, the moisture content of the oils and fats including the waste oil 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 in this embodiment is measured in accordance with Standard Fats and Oils Analysis Test Method 2.1.3.2-2013. Furthermore, the oils and fats may be subjected to a treatment to reduce the moisture content before use in the PHA production culture.

[0037] Furthermore, the carbon source used in the PHA production culture may contain, in addition to fats and oils including waste oil, 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.

[0038] 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, and their fractionated oils (e.g., fractionated low-melting-point fractions such as palm olein, palm double olein, and palm kernel 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 myristic acid, as well as their derivatives; and glycerol. Mixtures of the above-listed oils and fats may also be used.

[0039] In the production of PHA in this embodiment, it is preferable to culture the microorganisms 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.

[0040] After culturing for an appropriate period of time to allow PHA to accumulate in the cells, PHA can be recovered from the cells using well-known methods. The recovery method is not particularly limited. For example, after culturing, the cells can be separated from the culture medium using a centrifuge or a separation membrane, dried, and then PHA can be extracted from the dried cells using an organic solvent such as chloroform. Cell components can be removed from the organic solvent solution containing PHA by filtration or other methods. A poor solvent such as methanol or hexane can be added to the filtrate to precipitate PHA, and the supernatant can be removed by filtration or centrifugation. The PHA can also be recovered by drying. Alternatively, cell components other than PHA can be dissolved in water using surfactants, alkalis, enzymes, etc., and then PHA particles can be separated from the aqueous phase by filtration or centrifugation, dried, and then recovered.

[0041] According to this embodiment, PHA can be efficiently produced in a PHA production culture using waste oil as a carbon source. Furthermore, the carbon source used in the PHA production culture can be changed from unused oils and fats, which have a high environmental impact, to waste oil, thereby reducing the environmental impact of the PHA production culture.

[0042] The following items list preferred aspects of the present disclosure, but the present invention is not limited to the following items. [Item 1] A method for producing polyhydroxyalkanoic acid, comprising a step of culturing a polyhydroxyalkanoic acid-producing strain in a medium containing fat or oil as a carbon source to produce polyhydroxyalkanoic acid, The oils and fats include waste oil, A method for producing a polyhydroxyalkanoic acid, wherein the amount of oil and fat polymer measured for the entire oil and fat is 10% or less. [Item 2] Item 2. The method according to item 1, wherein the amount of the oil / fat polymer is 0.1% or more. [Item 3] 3. The method according to item 1 or 2, wherein the polyhydroxyalkanoic acid-producing strain is a microorganism belonging to the genus Capriavidus. [Item 4] 4. The method according to any one of items 1 to 3, wherein the polyhydroxyalkanoic acid is a copolymer of two or more kinds of hydroxyalkanoic acids. [Item 5] 5. The method according to item 4, wherein the polyhydroxyalkanoic acid is a copolymer containing 3-hydroxyhexanoic acid as a monomer unit. [Item 6] Item 6. The method according to item 5, wherein the polyhydroxyalkanoic acid is a copolymer of 3-hydroxybutyric acid and 3-hydroxyhexanoic acid. [Example]

[0043] The present invention will be described in more detail below with reference to examples, although the present invention is not limited to these examples.

[0044] In the following Reference Examples, Comparative Examples, and Examples, the KNK-005 strain was used as the PHA-producing microorganism. The KNK-005 strain is a known PHA-producing microorganism, a transformant of the Capriavidus necator H16 strain, prepared according to the method described in U.S. Patent No. 7,384,766, in which a PHA synthase gene derived from Aeromonas caviae has been introduced into the chromosome. This transformed microorganism is capable of producing P(3HB-co-3HH), a type of PHA, when fats and oils are used as raw materials.

[0045] (Reference Example 1) PHA production using palm olein oil as a carbon source A culture study using the KNK-005 strain was conducted under the following conditions. Commercially available palm olein oil (virgin oil) was used as the carbon source for PHA production culture. The amount of oil polymer, iodine value, acid value, peroxide value, and color of the palm olein oil used are shown in Table 1. The amount of oil polymer was measured according to Provisional Method 16 of the Standard Analysis of Fats, Oils, and Related Materials 2.5.7-2013. The iodine value was measured according to the Standard Analysis of Fats, Oils, and Related Materials 2.3.4.1-2013. The acid value was measured according to the Standard Analysis of Fats, Oils, and Related Materials 2.3.1-2013. The peroxide value was measured according to the Standard Analysis of Fats, Oils, and Related Materials 2.5.2.2-2013. The color was measured according to the Standard Analysis of Fats, Oils, and Related Materials 2.2.1.3-2013.

[0046] (Culture medium) The composition of the seed culture medium was 1 w / v% meat extract, 1 w / v% bacto-tryptone, 0.2 w / v% yeast extract, 0.9 w / v% Na2HPO4·12H2O, and 0.15 w / v% KH2PO4 (pH 6.8).

[0047] The pre-culture 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% palm olein 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 0.1 N hydrochloric acid). Palm olein oil was added as a carbon source at a concentration of 10 g / L.

[0048] The PHA production medium consisted of 0.385 w / v% Na2HPO4·12H2O, 0.067 w / v% KH2PO4, 0·291 w / v% (NH4)2SO4, 0.1 w / v% MgSO4·7H2O, 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 0.1 N hydrochloric acid).

[0049] (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 (Marubishi Bioengine MDL-300 model) containing 1.8 L of pre-culture medium. The operating conditions were a culture temperature of 33°C, an agitation speed of 500 rpm, and an aeration rate of 1.8 L / min. Pre-culture was carried out for 24 hours while controlling the pH so that it did not fall below 6.4. A 14% aqueous ammonium hydroxide solution was used to control the pH.

[0050] 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 that it did not fall below 6.4. A 25% aqueous solution of ammonium hydroxide was used to control the pH. The carbon source was added intermittently, and the rate of addition was controlled so that the carbon source concentration in the culture solution remained within the range (0.2-5%) that allowed for efficient PHA production.

[0051] (purification) After the cultivation was completed, the culture medium was weighed into a centrifuge tube and the weight of the culture medium was measured. The bacterial cells were collected by centrifugation and suspended in a 3.3 wt / v% aqueous solution of sodium lauryl sulfate (SDS). The cellular components of the bacterial cells were disrupted using an ultrasonic disrupter, and PHA was extracted. 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 medium measured initially to calculate the weight of PHA per 1 g of culture medium obtained 72 hours after the start of cultivation.

[0052] (Calculation method for PHA productivity) PHA productivity (%) was calculated using the following formula as the ratio of the PHA weight (g) per gram of culture medium obtained in each example 72 hours after the start of culture to the PHA weight (g) per gram of culture medium obtained in Reference Example 1 72 hours after the start of culture. PHA productivity (%)=[PHA weight (g) per 1 g of culture medium obtained in each example] / [PHA weight (g) per 1 g of culture medium obtained in Reference Example 1]×100 The measurement results of PHA productivity (%) in Reference Example 1 are shown in Table 1.

[0053] (Reference Example 2) PHA production using rapeseed oil as a carbon source Cultivation was carried out under the same conditions as in Reference 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. Table 1 also shows the measurement results of PHA productivity (%).

[0054] (Comparative Examples 1-2 and Examples 1-8) PHA production using waste oil as a carbon source Cultivation was examined under the same conditions as in Reference 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. Each of the waste oils A to I is oil that has been collected by a collection company from oils and fats discharged from multiple sources and processed to remove moisture and impurities to a certain extent. In addition, waste oil J is co-washing oil that has been determined to be discarded by the oil and fat manufacturer. The amount of oil and fat polymer, iodine value, acid value, peroxide value, and color of waste oils A to J are shown in Table 1. Table 1 also shows the measurement results of PHA productivity (%).

[0055] In addition, the obtained PHA was reacted in a mixture 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 reference examples, comparative examples, and examples.

[0056] [Table 1]

[0057] Table 1 shows that in Comparative Examples 1 and 2, in which waste oil containing more than 10% oil polymer was used as the carbon source, PHA productivity decreased by approximately 20% compared to Reference Examples 1 and 2, in which unused oil was used as the carbon source.

[0058] In contrast, in Examples 1 to 8, in which waste oil containing 10% or less of fat and oil polymers was used as the carbon source, the PHA productivity was only about 10% lower than in Reference Examples 1 and 2, and was improved and relatively good compared to Comparative Examples 1 and 2. Among these, in Examples 5 to 8, in which waste oil containing 4% or less of fat and oil polymers was used as the carbon source, PHA productivity almost equivalent to that of Reference Examples 1 and 2 was achieved, and it was found that the PHA productivity was particularly good.

[0059] 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.

Claims

1. A method for producing polyhydroxyalkanoic acid, comprising a step of culturing a polyhydroxyalkanoic acid-producing strain in a medium containing fat or oil as a carbon source to produce polyhydroxyalkanoic acid, The oils and fats include waste oil, The method for producing polyhydroxyalkanoic acid, wherein the amount of oil and fat polymer measured for the entire oil and fat is 10% or less.

2. The method according to claim 1, wherein the amount of the oil / fat polymer is 0.1% or more.

3. 3. The method according to claim 1, wherein the polyhydroxyalkanoic acid-producing strain is a microorganism belonging to the genus Capriavidus.

4. The method according to claim 1 or 2, wherein the polyhydroxyalkanoic acid is a copolymer of two or more kinds of hydroxyalkanoic acids.

5. The method according to claim 4, wherein the polyhydroxyalkanoic acid is a copolymer containing 3-hydroxyhexanoic acid as a monomer unit.

6. The method according to claim 5, wherein the polyhydroxyalkanoic acid is a copolymer of 3-hydroxybutyric acid and 3-hydroxyhexanoic acid.

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