Method for producing poly(3-hydroxyalkanoate)

Abstract

In the culturing poly(3-hydroxyalkanoate)-producing microorganisms, fats A and fats B are used as carbon sources, and the amount the fats B used relative to the total amount of the fats A and the fats B used in the entire culture is greater than or equal to 10 wt%. Fats A: refers to all fats in which the amount of poly(3-hydroxyalkanoate) accumulated in the poly(3-hydroxyalkanoate)-producing microorganisms reaches up to 16 wt%, and in all of the fats A, the average content ratio of unsaturated fatty acids, which are constituent fatty acids, is at least 25 wt% and less than 75 wt%. Fats B: the content ratio of unsaturated fatty acids, which are constituent fatty acids of fats B, is higher than the average content ratio of the unsaturated fatty acids in all of the fats A.

Description

[Technical Field]

[0001] The present invention relates to a method for producing poly(3-hydroxyalkanoate) by culturing a poly(3-hydroxyalkanoate)-producing microorganism. [Background technology]

[0002] Poly(3-hydroxyalkanoates) (hereinafter sometimes referred to as PHA) are biopolyesters stored within the cells of microorganisms. They are used as plastic materials and have attracted attention in recent years as environmentally friendly materials due to their biodegradability after use.

[0003] PHA is produced by culturing microorganisms capable of producing PHA and allowing the microorganisms to accumulate PHA. During the cultivation, it is necessary to provide a carbon source that can be suitably utilized by the microorganisms. Typical carbon sources include carbohydrates, oils and fats, and free fatty acids.

[0004] For example, Patent Document 1 describes culturing microorganisms capable of producing PHA using palm oil as a carbon source. [Prior art documents] [Patent documents]

[0005] [Patent Document 1] Special Publication No. 2013-510572 Summary of the Invention [Problem to be solved by the invention]

[0006] It is known that PHA can be efficiently produced by culturing PHA-producing microorganisms using palm oil as a carbon source. However, because palm oil is a relatively expensive oil, there is a need to produce PHA using other readily available oils as a carbon source.

[0007] Therefore, when attempts were made to cultivate PHA-producing microorganisms using various oils and fats as carbon sources, it was found that some oils could achieve a PHA production rate similar to that of palm oil, while others had a PHA production rate that was clearly slower than that of palm oil.Furthermore, it was found that the oils that produced a slower PHA production rate generally had a relatively high content of unsaturated fatty acids among their constituent fatty acids.

[0008] Such oils and fats having a relatively high content of unsaturated fatty acids, such as rapeseed oil, are widely used in food applications, and a large amount of used edible oil is discarded. From the viewpoint of effectively utilizing such edible oils, it is desirable to use oils and fats having a relatively high content of unsaturated fatty acids as a carbon source in PHA production.

[0009] In view of the above, an object of the present invention is to achieve a high PHA production rate while using fats and oils having a relatively high content of unsaturated fatty acids as a carbon source when culturing a PHA-producing microorganism to produce PHA. [Means for solving the problem]

[0010] The present inventors have discovered that by using both fat A, which has a relatively low content of unsaturated fatty acids such as palm oil, and fat B, which has a relatively high content of unsaturated fatty acids such as rapeseed oil, as carbon sources for PHA-producing microorganisms, and by using fat A in the early stages of cultivation, it is possible to achieve a PHA production rate as high as that achieved when fat A is used alone, despite the use of fat B, and have arrived at the present invention.

[0011] Specifically, the present invention relates to a method for producing poly(3-hydroxyalkanoate) by culturing a poly(3-hydroxyalkanoate)-producing microorganism in the presence of a carbon source, wherein fats and oils A and B are used as carbon sources in the culturing, and the amount of fats and oils B used is 10% by weight or more relative to the total amount of fats and oils A and B used throughout the culturing. Oil and fat A: refers to the total of oils and fats used until the amount of poly(3-hydroxyalkanoate) accumulated in the poly(3-hydroxyalkanoate)-producing microorganism reaches 16% by weight, and the average content of unsaturated fatty acids, which are the constituent fatty acids, in the entire oil and fat A is 25% by weight or more but less than 75% by weight. Oil / fat B: The content of unsaturated fatty acids, which are constituent fatty acids in oil / fat B, is higher than the average content of unsaturated fatty acids in oil / fat A as a whole. Preferably, the carbon source used after a certain point in time when the amount of poly(3-hydroxyalkanoate) accumulated in the poly(3-hydroxyalkanoate)-producing microorganism becomes more than 16% by weight and less than 85% by weight is oil or fat B. Preferably, the fat / oil B is a fat / oil having an unsaturated fatty acid content of 60% by weight or more and 98% by weight or less. Preferably, the amount of fat B used relative to the total amount of fat A and fat B used throughout the culture is 40% by weight or more. Preferably, the poly(3-hydroxyalkanoate)-producing microorganism is cultured until the amount of poly(3-hydroxyalkanoate) accumulated in the poly(3-hydroxyalkanoate)-producing microorganism reaches 80% by weight or more. Preferably, the culture is carried out while continuously adding fat / oil A and / or fat / oil B to a medium containing the poly(3-hydroxyalkanoate)-producing microorganism. Preferably, the culture is carried out while continuously adding fat / oil A to a medium containing the poly(3-hydroxyalkanoate)-producing microorganism, and then the culture is continued while continuously adding fat / oil B. Preferably, the poly(3-hydroxyalkanoate) comprises at least 3-hydroxybutyrate units. Preferably, the poly(3-hydroxyalkanoate) comprises a homopolymer of 3-hydroxybutyrate units or a copolymer of 3-hydroxybutyrate units with other hydroxyalkanoate units, more preferably 3-hydroxyhexanoate units. Preferably, the poly(3-hydroxyalkanoate)-producing microorganism is a bacterium, and more preferably, the poly(3-hydroxyalkanoate)-producing microorganism is a bacterium belonging to the genus Capriavidus. [Effects of the Invention]

[0012] According to the present invention, when PHA-producing microorganisms are cultured to produce PHA, a high PHA production rate can be achieved while using fats and oils having a relatively high content of unsaturated fatty acids as a carbon source. DETAILED DESCRIPTION OF THE INVENTION

[0013] Specific embodiments of the present invention will be described in detail below, but the present invention is not limited to these embodiments.

[0014] The present embodiment relates to a method for producing PHA by culturing a PHA-producing microorganism in the presence of a carbon source.

[0015] The PHA in the present disclosure is not particularly limited as long as it is a poly(3-hydroxyalkanoate) that can be produced by a microorganism, but is preferably a homopolymer of one monomer selected from 3-hydroxyalkanoic acids having 4 to 16 carbon atoms, a copolymer of at least one monomer selected from 3-hydroxyalkanoic acids having 4 to 16 carbon atoms and another hydroxyalkanoic acid (e.g., 4-hydroxyalkanoic acids having 4 to 16 carbon atoms, lactic acid, etc.), or a copolymer of two or more monomers selected from 3-hydroxyalkanoic acids having 4 to 16 carbon atoms. Specific examples include, but are not limited to, P(3HB), a homopolymer of 3-hydroxybutyric acid (abbreviation: 3HB), P(3HB-co-3HV) (abbreviation: PHBV) copolymer of 3HB and 3-hydroxyvaleric acid (abbreviation: 3HV), P(3HB-co-3HH) (abbreviation: PHBH) copolymer of 3HB and 3-hydroxyhexanoic acid (abbreviation: 3HH), P(3HB-co-4HB) copolymer of 3HB and 4-hydroxybutyric acid (abbreviation: 4HB), and PHA containing lactic acid (abbreviation: LA) as a constituent, such as P(LA-co-3HB) copolymer of 3HB and LA.

[0016] From the viewpoint of a wide range of applications as a polymer, PHA containing at least 3-hydroxybutyrate units is preferred, and a homopolymer of 3-hydroxybutyrate units or a copolymer of 3-hydroxybutyrate units and other hydroxyalkanoate units is more preferred. Among the copolymers, PHBV and PHBH are more preferred, and PHBH is particularly preferred.

[0017] The type of PHA produced can be appropriately selected depending on the type of PHA synthase gene possessed by the microorganism used or introduced separately, the type of metabolic gene involved in the synthesis, the culture conditions, etc.

[0018] The PHA-producing microorganism is not particularly limited as long as it is a microorganism capable of producing PHA, and may be a microorganism found in nature, or a mutant or transformant. Specifically, the following bacteria are included: the genus Cupriavidus such as Cupriavidus necator; the genus Alcaligenes such as Alcaligenes latas; the genus Pseudomonas such as Pseudomonas putida, Pseudomonas fluorescens, Pseudomonas aeruginosa, Pseudomonas resinovorans, and Pseudomonas oleovorans; the genus Bacillus such as Bacillus megaterium; the genus Azotobacter; the genus Nocardia; Aeromonas caviae; and Aeromonas hydrophila. Examples of such organisms include the genera Aeromonas, Ralstonia, Wautersia, and Comamonas, such as Aeromonas hydrophila, Ralstonia, Wautersia, and Comamonas (Microbiological Reviews, pages 450-472, 1990). Biological cells artificially modified to produce PHA by introducing a PHA synthase genera using genetic engineering techniques can also be used. For example, gram-negative bacteria such as Esherichia, gram-positive bacteria such as Bacillus, yeasts such as Saccharomyces, Yarrowia, and Candida, and cells of higher organisms such as plants can also be used. Bacteria are preferred because they are capable of accumulating large amounts of PHA, and bacteria belonging to the genus Capillavidus are particularly preferred.

[0019] The PHA synthase gene to be introduced by transformation is not particularly limited, and examples include PHA synthase genes derived from Aeromonas viridis, Aeromonas hydrophila, Pseuromonas SP 61-3, Cupriavidus necator, and variants thereof. The variants refer to nucleotide sequences encoding PHA synthases having an amino acid sequence in which one or more amino acid residues have been deleted, added, inserted, or substituted.

[0020] PHA can be accumulated in the cells by culturing a PHA-producing microorganism in the presence of a carbon source. Fat or oil is used as the carbon source. However, carbon sources other than fat or oil may be used in combination with fat or oil.

[0021] The oils and fats include triglycerides, which are ester compounds of constituent fatty acids and glycerin. The constituent fatty acids may include unsaturated fatty acids having one or more carbon-carbon unsaturated bonds and / or saturated fatty acids having no carbon-carbon unsaturated bonds. The oils and fats that can be used include, but are not limited to, animal oils and fats, vegetable oils and fats, mixtures thereof, interesterified oils, and fractionated oils. Specific examples of vegetable oils and fats include rapeseed oil, sunflower oil, soybean oil, olive oil, corn oil, palm oil, palm kernel oil, cottonseed oil, sesame oil, nut oil, jatropha oil, and rice bran oil. Specific examples of animal oils and fats include lard. These can be used alone or in combination.

[0022] The constituent fatty acids of the oils and fats include short-chain fatty acids having 2 to 4 carbon atoms, medium-chain fatty acids having 5 to 12 carbon atoms, and long-chain fatty acids having 12 or more carbon atoms. Of these, oils and fats containing at least three constituent fatty acids selected from the group consisting of lauric acid, myristic acid, myristoleic acid, pentadecanoic acid, palmitic acid, margaric acid, stearic acid, oleic acid, linoleic acid, erucic acid, and linolenic acid are preferred. Furthermore, oils and fats containing at least two constituent fatty acids selected from the group consisting of palmitoleic acid, heptadecanoic acid, oleic acid, linoleic acid, linolenic acid, and erucic acid are particularly preferred.

[0023] In this embodiment, two types of fats and oils are used as carbon sources, each having a different content of unsaturated fatty acids in its constituent fatty acids. The two types of fats and oils are classified as fats and oils A and B, and are defined as follows: Oil and fat A: refers to the total of oils and fats used until the accumulation of poly(3-hydroxyalkanoate) in the poly(3-hydroxyalkanoate)-producing microorganism reaches 16% by weight, and the average content of unsaturated fatty acids, which are the constituent fatty acids, in the entire oil and fat A is 25% by weight or more but less than 75% by weight. Oil / fat B: The content of unsaturated fatty acids, which are constituent fatty acids in oil / fat B, is higher than the average content of unsaturated fatty acids in oil / fat A as a whole.

[0024] The content of unsaturated fatty acids is the ratio of the weight of unsaturated fatty acids to the total weight of fatty acids constituting the oil or fat, and can be calculated by measuring the weight of the constituting fatty acids. The measurement method involves saponifying the oil or fat with a strong alkali to form free fatty acids, and then methyl-esterifying the carboxyl groups of the fatty acids to increase volatility, and then separating them by gas chromatography to identify saturated fatty acids and unsaturated fatty acids.

[0025] (Oil A) Oil and fat A refers to the total of oils and fats used until the amount of poly(3-hydroxyalkanoate) accumulated in the poly(3-hydroxyalkanoate)-producing microorganism reaches 16% by weight, and the average content of unsaturated fatty acids in the total oil and fat A is within the range of 25% by weight or more and less than 75% by weight. The average content of unsaturated fatty acids is preferably 30% by weight or more and 70% by weight or less, more preferably 40% by weight or more and 65% by weight or less, and even more preferably 50% by weight or more and 60% by weight or less.

[0026] The average palmitic acid content of fat / oil A is preferably 20% by weight or more and 65% by weight or less, more preferably 25% by weight or more and 60% by weight or less, and even more preferably 30% by weight or more and 55% by weight or less, based on the total weight of fat / oil A.

[0027] As long as the average content ratio of unsaturated fatty acids is satisfied, fat A may be composed of one type of available fat or oil, such as vegetable fat or oil, or may be composed of two or more types of available fat or oil. Examples of fats and oils that fall solely under fat and oil A include palm oil and lard.

[0028] When fat / oil A is composed of two or more types of fats / oils, the fat / oil A as a whole only needs to satisfy the above-mentioned average content ratio of unsaturated fatty acids. The content ratio of unsaturated fatty acids in each fat / oil constituting fat / oil A is not particularly limited, and does not have to be within the range of 25% by weight or more and less than 75% by weight. Furthermore, when fat / oil A is composed of two or more types of fats / oils, the two or more types of fats / oils may be mixed and added to the medium, or may be added to the medium simultaneously or sequentially without being mixed.

[0029] In this embodiment, "cultivation of a poly(3-hydroxyalkanoate)-producing microorganism" refers to the final stage, "main culture," which is carried out for the purpose of causing the poly(3-hydroxyalkanoate)-producing microorganism to accumulate poly(3-hydroxyalkanoate) at a high concentration. "Preculture" and "seed culture," which are carried out before "main culture," are not included in "culture" in this embodiment. Therefore, the carbon sources used in "preculture" and "seed culture" are not included in "oil / fat A."

[0030] (Oil B) Fat B is a carbon source that is used after the amount of poly(3-hydroxyalkanoate) accumulated in the poly(3-hydroxyalkanoate)-producing microorganism exceeds 16% by weight, and refers to fat B in which the content of unsaturated fatty acids is higher than the average content of unsaturated fatty acids in all of fat A. There are no particular limitations on the difference between the content (% by weight) of unsaturated fatty acids in fat B and the average content (% by weight) of unsaturated fatty acids in all of fat A, but from the viewpoint of better achieving the effects of using fat A and fat B in combination, it is preferably 5% by weight or more, more preferably 10% by weight or more, and even more preferably 20% by weight or more.

[0031] The content of unsaturated fatty acids in oil B is defined relative to the average content of unsaturated fatty acids in the entire specific oil A used in combination therewith, and the specific numerical value is not limited. The specific numerical value of the content of unsaturated fatty acids in oil B is not particularly limited as long as it is higher than the average content of unsaturated fatty acids in the entire specific oil A used in combination therewith, and may be 75% by weight or more, or may be within the range of 25% by weight or more and less than 75% by weight. For example, as shown in Example 8 described below, oil A having an unsaturated fatty acid content of 58% by weight can be used with oil B having an unsaturated fatty acid content of 66% by weight.

[0032] In a preferred embodiment, the content of unsaturated fatty acids in fat / oil B is preferably 60% by weight or more and 98% by weight or less, more preferably 65% ​​by weight or more and 96% by weight or less, even more preferably 70% by weight or more and 95% by weight or less, and particularly preferably 75% by weight or more and 94% by weight or less.

[0033] Oil / fat B may be composed of one type of available oil / fat, such as vegetable oil, or may be composed of two or more types of available oil / fat, so long as the content of unsaturated fatty acids in the oil / fat B is higher than the average content of unsaturated fatty acids in the entire oil / fat A. An example of an oil / fat that falls under oil / fat B is rapeseed oil. Furthermore, oil / fat B may be discarded edible oil or the like, so long as it satisfies the above requirements.

[0034] Studies by the present inventors have revealed that when fat B, which has a relatively high content of unsaturated fatty acids, is used alone as a carbon source to culture a PHA-producing microorganism, the PHA production rate slows down. However, in this embodiment, a good PHA production rate can be achieved by using fats and oils A and B in combination under specific conditions. This allows fats and oils B to be effectively used as a carbon source for PHA-producing microorganisms. From the viewpoint of effective use of fats and oils B, the higher the proportion of fats and oils B used, the better. Specifically, the amount of fats and oils B used relative to the total amount of fats and oils A and B used in the entire culture is 10% by weight or more, and may be 40% by weight or more, 60% by weight or more, or even 80% by weight or more. The upper limit of the amount of fats and oils B used is not particularly limited, but is preferably 97% by weight or less, more preferably 95% by weight or less, and even more preferably 90% by weight or less. Even when fats and oils B are used at such a high proportion, a high PHA production rate equivalent to that achieved when fats and oils A are used alone can be achieved.

[0035] The amount of fat A used relative to the total amount of fat A and fat B used throughout the culture is not particularly limited, but from the viewpoint of achieving an improvement in the PHA production rate, it is preferably 3% by weight or more, more preferably 5% by weight or more, and even more preferably 10% by weight or more. The upper limit of the amount of fat A used is 90% by weight or less, and may be 60% by weight or less, 40% by weight or less, or 20% by weight or less.

[0036] (Use of carbon source) In this embodiment, in culturing a PHA-producing microorganism, fat A having a relatively low content of unsaturated fatty acids is used as a carbon source at least until the amount of accumulated poly(3-hydroxyalkanoate) reaches 16% by weight. After that, once the amount of accumulated poly(3-hydroxyalkanoate) exceeds 16% by weight, fat B having a relatively high content of unsaturated fatty acids is used as a carbon source.

[0037] In this way, by using oil A in the early stages of cultivation and then oil B, a high level of PHA production rate can be achieved even when using oil B, which slows the PHA production rate when used alone. The mechanism behind this is unknown, but it is presumed that in the early stages of cultivation, the growth of PHA-producing microorganisms takes priority over PHA accumulation, and that oil A, which contains a relatively low proportion of unsaturated fatty acids, is more suitable as a carbon source during this cell growth stage, but once growth has progressed to a certain extent and PHA accumulation takes priority, the effect of the type of carbon source is reduced and oil B also becomes effective as a carbon source.

[0038] After the PHA accumulation amount exceeds 16% by weight, it is preferable to continue using an oil or fat having the same unsaturated fatty acid content as oil or fat A for a certain period of time, but at some point, change to oil or fat B, which has a relatively higher unsaturated fatty acid content. According to a preferred embodiment, the carbon source used after the PHA accumulation amount in the PHA-producing microorganism exceeds 16% by weight and becomes less than 85% by weight is preferably oil or fat B. This allows oil or fat B, which slows the PHA production rate when used alone, to be effectively used as a carbon source when culturing the PHA-producing microorganism. The PHA accumulation amount in the PHA-producing microorganism is preferably 20% by weight to 80% by weight, more preferably 25% by weight to 50% by weight, even more preferably 25% by weight to 45% by weight, and particularly preferably 30% by weight to 40% by weight.

[0039] The amount of PHA accumulated in PHA-producing microorganisms can be calculated by measuring the weight of dried cells obtained by recovering a certain amount from the culture medium, mixing it with an organic solvent to remove oils and fats, washing it with water, and drying it, and then measuring the weight of PHA recovered from the same amount of culture medium, using the following formula. PHA accumulation (%) = [PHA weight (g) recovered from a certain amount of culture medium] / [dry cell weight (g) obtained from a certain amount of culture medium] × 100

[0040] In a preferred aspect of this embodiment, it is preferable to start the culture in the presence of fat / oil A, change the type of carbon source during the culture (before the amount of PHA accumulated in the PHA-producing microorganism reaches the final level), continue the culture in the presence of fat / oil B, and terminate the culture when the amount of PHA accumulated in the PHA-producing microorganism reaches the final level. In this way, by using fat / oil A as the carbon source in the early stage of the culture and then using fat / oil B as the carbon source during the culture, a high level of PHA production rate can be easily achieved despite using fat / oil B as the carbon source, which slows the PHA production rate when used alone.

[0041] In the preferred embodiment, the timing of changing the carbon source from fat A to fat B is not particularly limited and can be determined appropriately depending on the amount of PHA accumulated in the PHA-producing microorganism and the proportion of fat B used, but may be, for example, a time point when the amount of PHA accumulated in the PHA-producing microorganism is more than 16% but less than 85% by weight, preferably 20% to 80% by weight, more preferably 25% to 50% by weight, even more preferably 30% to 45% by weight, and particularly preferably 30% to 40% by weight. By changing the type of carbon source at such a time point, a high PHA production rate can be achieved while increasing the amount of fat B used.

[0042] The amount of PHA accumulated in the PHA-producing microorganism at the end of the culture is not particularly limited and may be determined appropriately, but is preferably 80% by weight or more, more preferably 90% by weight or more.

[0043] The method of adding fat A or fat B to a medium containing a PHA-producing microorganism may be either a lump addition or a continuous addition, but continuous addition is preferred. That is, the PHA-producing microorganism is preferably cultured by continuously adding fat A and / or fat B to a medium containing the PHA-producing microorganism. Here, "continuous addition" includes not only a mode in which fat A and / or fat B are added continuously without interruption over time, but also a mode in which fat A and / or fat B are added intermittently with temporary rest periods between additions.

[0044] In one specific embodiment of continuous addition, it is preferable to continuously add and disperse fat A in a medium containing a PHA-producing microorganism while culturing, and then change the type of carbon source and continue culturing while continuously adding and dispersing fat B.

[0045] (Culture medium) The medium used for culturing PHA-producing microorganisms may be any liquid medium containing nutrients that contribute to the growth and proliferation of the microorganisms. Preferably, the PHA-producing microorganisms are mixed with a liquid containing the above-mentioned carbon sources as well as nitrogen sources other than the carbon sources, inorganic salts, and other organic nutrient sources, and dispersed by stirring, shaking, etc.

[0046] Examples of nitrogen sources include ammonium salts such as ammonia, ammonium chloride, ammonium sulfate, and ammonium phosphate, as well as 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.

[0047] A culture medium containing such nutrient sources, a carbon source, and a PHA-producing microorganism are dispersed in a container to obtain a culture solution. The culture conditions can be those of a conventional microbial culture method, except for the carbon source and its addition method, and there are no particular limitations on the culture scale, aeration and agitation conditions, culture temperature, culture pH, culture time, etc.

[0048] (PHA recovery) After culturing for an appropriate period of time to allow PHA to accumulate in the cells, PHA can be recovered from the cells using a well-known method. The recovery method is not particularly limited, but can be carried out, for example, by the following method. For example, after culturing is completed, the cells are separated from the culture medium using a centrifuge or the like, washed with distilled water, methanol, or the like, and dried. PHA is extracted from these dried cells using an organic solvent such as chloroform. Cell components are removed from this PHA-containing solution by filtration or the like, and a poor solvent such as methanol or hexane is added to the filtrate to precipitate PHA. Furthermore, the supernatant is removed by filtration or centrifugation, and the PHA can be recovered by drying.

[0049] In another example, bacterial cells are separated from the culture medium using a centrifuge or the like, and then washed with distilled water, methanol, etc. Subsequently, the washed sample is mixed with a sodium lauryl sulfate (SDS) solution, the cell membrane is disrupted by ultrasonication, the bacterial components and PHA are separated using a centrifuge or the like, and the PHA can be recovered by drying the PHA.

[0050] According to this embodiment, it is possible to produce PHA at a good production rate while using fats and oils having a relatively high content of unsaturated fatty acids, which slows down the PHA production rate when used alone. [Example]

[0051] The present invention will be described in more detail below with reference to examples, but the present invention is not limited to these examples.

[0052] (Oils and fats used) For the oils and fats 1 to 11 used in the following Examples, Comparative Examples, and Reference Examples, the content ratio of fatty acids constituting each oil and fat, the total content ratio of saturated fatty acids, and the total content ratio of unsaturated fatty acids are shown in Table 1. Oil and fat 1 is palm oil, oil and fat 3 is rapeseed oil, and oil and fat 10 is lard.

[0053] [Table 1]

[0054] (Calculation method for PHA accumulation) The amount of PHA accumulated (wt%) was calculated by measuring the weight of dried cells obtained by mixing a certain amount of culture medium with an organic solvent, washing with water, and drying, and the weight of PHA recovered from the same amount of culture medium, using the following formula. PHA accumulation amount (wt %)=[PHA weight (g) obtained in each Example, Comparative Example, or Reference Example] / [dry cell weight (g) in each Example, Comparative Example, or Reference Example]×100

[0055] (Calculation method for PHA productivity) PHA productivity (%) was calculated using the following formula as the ratio of the weight (g) of PHA obtained per liter of culture solution in each Example, Comparative Example, or Reference Example to the weight (g) of PHA obtained per liter of culture solution in Reference Example 1 or Reference Example 5, in which PHA was produced using only Oil / Fat 1. Note that the reference example used was selected from Reference Example 1 or 5, which used the same PHA-producing microorganism. PHA productivity (%)=[PHA weight (g) obtained in each Example, Comparative Example, or Reference Example] / [PHA weight (g) obtained in Reference Example 1 or Reference Example 5]×100

[0056] (Comparative Examples 1 to 7 and Reference Examples 1 to 4) Using the KNK-005 strain (see U.S. Patent No. 7,384,766) as a PHA-producing microorganism, (1) preculture, (2) seed culture, and (3) main culture were carried out in the following order by the methods described below. In the main culture, each of the oils shown in Table 2 was used alone as the oil or carbon source.

[0057] (1) Preculture First, 20 μL of a glycerol stock of the KNK-005 strain was inoculated into 20 mL of a preculture medium and cultured at 30° C. for 18 hours. The pre-culture medium consisted of 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).

[0058] (2) Seed culture The resulting preculture solution was inoculated at 1.0 v / v% into a 3 L jar fermenter (Marubishi Bioengine MDL-8C) containing 1.8 L of seed culture medium. The operating conditions were a culture temperature of 30°C, an agitation speed of 500 rpm, and an aeration rate of 1.8 L / min. Seed culture was carried out for 24 hours while controlling the pH between 6.5 and 6.6. A 14% aqueous ammonium hydroxide solution was used to control the pH. The seed 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.

[0059] (3) Main culture The resulting seed culture solution was inoculated at 5.0 v / v% into a 5 L jar fermenter (Bioneer-Neo, manufactured by Marubishi Bioengine) containing 2.5 L of main culture medium. The operating conditions were a culture temperature of 34°C, an agitation speed of 600 rpm, and an aeration rate of 6.0 L / min, and the pH was controlled between 6.5 and 6.6. A 25% aqueous ammonium hydroxide solution was used to control the pH. The main culture was carried out while intermittently adding each of the fats and oils shown in Table 2 as a carbon source during the culture period. The main culture was carried out for 48 hours, and after completion of the culture, a certain amount of the culture medium was collected, washed with distilled water and methanol, and then vacuum-dried, and the dry bacterial weight was measured. After washing the bacterial cells as described above, the bacterial components were dissolved using SDS, and the PHA and bacterial components were separated by ultrasonic disruption. Only the PHA was collected, and the amount of PHA accumulated was measured. Based on this, PHA productivity was calculated and is shown in Table 2. The culture medium contained 0.385 w / v% Na2HPO4·12H2O, 0.067 w / v% KH2PO4, 0.291 w / v% (NH4)2SO4, 0.1 w / v% MgSO4·7H2O, 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), and 0.05 w / v% BIOSPUREX 200K (antifoaming agent: manufactured by Cognis Japan).

[0060] [Table 2]

[0061] The following can be seen from Table 2: The fats and oils used in Comparative Examples 1 to 7 had a high content of unsaturated fatty acids, and when these fats and oils were used alone as carbon sources, PHA productivity was low at less than 80%. On the other hand, the fats and oils used in Reference Examples 1 to 4 had a low content of unsaturated fatty acids at less than 75% by weight, and when these fats and oils were used alone as carbon sources, PHA productivity was good at 80% or more.

[0062] Example 1 (1) Pre-culture, (2) Seed culture, and (3) Main culture were sequentially carried out under the same conditions as Comparative Examples 1 to 7 and Reference Examples 1 to 4, except for the following points. In the main culture, the first fat (fat 1) was first intermittently added as a carbon source while the main culture was initiated. When the amount of PHA accumulated in the microorganism reached 15% by weight, the carbon source was switched to the second fat (fat 2), and the main culture was continued while the second fat was intermittently added. The culture was terminated 48 hours after the start of the main culture. Table 3 shows the proportions of the first fat and the second fat in the carbon source used in the main culture, and the calculated PHA productivity values. In this example, the entire amount of fat 1 and the small amount of fat 2 used until the PHA accumulation reached 15% to 16% by weight correspond to fat A. At this time, the average content of unsaturated fatty acids in fat A is about 58%. Furthermore, fat 2 corresponds to fat B.

[0063] Example 2 Except for using fat 8 as the second fat in the main culture, (1) preculture, (2) seed culture, and (3) main culture were sequentially performed under the same conditions as in Example 1. Table 3 shows the proportions of the first fat and the second fat in the carbon source used in the main culture and the calculated PHA productivity values. In this example, the entire amount of fat 1 and the small amount of fat 8 used until the PHA accumulation reached 15% to 16% by weight correspond to fat A. At this time, the average content of unsaturated fatty acids in fat A is about 56%. Furthermore, fat 8 corresponds to fat B.

[0064] Examples 3 and 4 In the main culture, the first fat and the second fat shown in Table 3 were used, and the first fat was switched to the second fat when the amount of PHA accumulated in the microorganism reached 20% by weight. Except for this, (1) preculture, (2) seed culture, and (3) main culture were sequentially carried out under the same conditions as in Example 1. Table 3 shows the proportions of the first fat and the second fat in the carbon source used in the main culture and the calculated PHA productivity values. In this example, the first fat (fat 1) corresponds to fat A, and the second fat (fat 2) corresponds to fat B. The same applies to the following Examples 5 to 9.

[0065] (Examples 5 to 8) In the main culture, the first fat and the second fat shown in Table 3 were used, and the first fat was switched to the second fat when the amount of PHA accumulated in the microorganism reached 30 to 34% by weight. Except for this, (1) preculture, (2) seed culture, and (3) main culture were sequentially performed under the same conditions as in Example 1. Table 3 shows the proportions of the first fat and the second fat in the carbon source used in the main culture and the calculated PHA productivity values.

[0066] Example 9 Except for the fact that the main culture was switched from the first fat to the second fat when the amount of PHA accumulated in the microorganism reached 79% by weight, (1) preculture, (2) seed culture, and (3) main culture were sequentially performed under the same conditions as in Example 1. Table 3 shows the proportions of the first fat and the second fat in the carbon source used in the main culture and the calculated PHA productivity values.

[0067] [Table 3]

[0068] The following can be seen from Table 3. In Examples 1 to 9, main culture was initiated using a first fat or oil having an unsaturated fatty acid content of 25% by weight or more but less than 75% by weight as the carbon source, and during the main culture, the carbon source was switched to a second fat or oil having a higher unsaturated fatty acid content than the first fat or oil, and the main culture was continued. It can be seen that in all cases, PHA productivity was good, at 80% or more. In particular, in Examples 3 to 8, despite using 80% by weight or more of the second fat or oil, which has low PHA productivity when used alone, PHA productivity was extremely high, at 90% or more. Although fats and oils with a relatively high content of unsaturated fatty acids result in low PHA productivity when used alone, as shown in Comparative Examples 1 to 7 and Reference Example 4 in Table 2, it can be seen that good PHA productivity can be achieved by using them in combination with fats and oils with an unsaturated fatty acid content of 25% by weight or more but less than 75% by weight in succession.

[0069] (Comparative Example 8) (1) Pre-culture, (2) seed culture, and (3) main culture were sequentially carried out under the same conditions as in Example 1, except for the following points. The order of addition of fat 1 and fat 2 in the main culture of Example 1 was reversed, and the main culture was started while intermittently adding fat 2 as a carbon source. When the amount of PHA accumulated in the microorganism reached 30% by weight, the carbon source was switched to fat 1, and the main culture was continued while intermittently adding fat 1. The culture was terminated 48 hours after the start of the main culture. Table 4 shows the proportions of fat 1 and fat 2 in the carbon sources used in the main culture and the calculated PHA productivity values.

[0070] (Comparative Example 9) Except for the fact that the main culture was switched from oil 2 to oil 1 when the amount of PHA accumulated in the microorganism reached 80% by weight, (1) preculture, (2) seed culture, and (3) main culture were sequentially performed under the same conditions as in Comparative Example 8. Table 4 shows the proportions of oil 1 and oil 2 in the carbon source used in the main culture and the calculated PHA productivity values.

[0071] [Table 4]

[0072] Table 4 reveals the following. In Comparative Examples 8 and 9, unlike Examples 1 to 9, main culture was initiated using an oil or fat (oil 2) with a relatively high content of unsaturated fatty acids as the carbon source, and during the main culture, the carbon source was switched to an oil or fat (oil 1) with an unsaturated fatty acid content of 25% by weight or more but less than 75% by weight, and the main culture was continued. As a result, PHA productivity was low at less than 80%, and was similar to that of Comparative Examples 1 to 7. In particular, in Comparative Example 8, PHA productivity was extremely low at 76%, despite the use of 80% by weight or more of oil or fat 1, which has the highest PHA productivity when used alone. From the above, it can be seen that in order to obtain good PHA productivity, it is desirable that the carbon source used in the early stages of cultivation is not an oil or fat with a relatively high content of unsaturated fatty acids, but rather an oil or fat with an unsaturated fatty acid content of 25% by weight or more and less than 75% by weight, as in Examples 1 to 9.

[0073] (Comparative Examples 10 to 12 and Reference Examples 5 to 7) Cupriavidus necator H16 strain was used as the PHA-producing microorganism, and (1) preculture, (2) seed culture, and (3) main culture were sequentially carried out under the same conditions as Comparative Examples 1 to 7 and Reference Examples 1 to 4, except that each oil or fat shown in Table 5 was used alone as the carbon source for the main culture. Table 5 shows the calculated PHA productivity values.

[0074] [Table 5]

[0075] The following can be seen from Table 5. In Comparative Examples 10 to 12, in which fats and oils with a relatively high content of unsaturated fatty acids were used alone as the carbon source, PHA productivity was low at less than 80%. On the other hand, in Reference Examples 5 to 7, in which fats and oils with a low content of unsaturated fatty acids, less than 75% by weight, were used alone as the carbon source, PHA productivity was good at 80% or more. That is, although PHA-producing microorganisms different from those used in Comparative Examples 1 to 7 and Reference Examples 1 to 4 were used in Comparative Examples 10 to 12 and Reference Examples 5 to 7, the relationship between the type of carbon source and PHA productivity showed the same tendency.

[0076] Example 10 (1) Pre-culture, (2) Seed culture, and (3) Main culture were sequentially carried out under the same conditions as Comparative Examples 10 to 12 and Reference Examples 5 to 7, except for the following points. In the main culture, the first fat (fat 1) was first intermittently added as a carbon source while the main culture was initiated. When the amount of PHA accumulated in the microorganism reached 15% by weight, the carbon source was switched to the second fat (fat 2), and the main culture was continued while the second fat was intermittently added. The culture was terminated 48 hours after the start of the main culture. Table 6 shows the proportions of the first fat and the second fat in the carbon source used in the main culture, and the calculated PHA productivity values.

[0077] Example 11 Except for using fat 8 as the second fat in the main culture, (1) preculture, (2) seed culture, and (3) main culture were sequentially performed under the same conditions as in Example 10. Table 6 shows the proportions of the first fat and the second fat in the carbon source used in the main culture and the calculated PHA productivity values.

[0078] (Examples 12 to 13) In the main culture, the first fat and the second fat shown in Table 6 were used, and the first fat was switched to the second fat when the amount of PHA accumulated in the microorganism reached 20% by weight. Except for this, (1) preculture, (2) seed culture, and (3) main culture were sequentially carried out under the same conditions as in Example 10. Table 6 shows the proportions of the first fat and the second fat in the carbon source used in the main culture and the calculated PHA productivity values.

[0079] (Examples 14 to 18) In the main culture, the first fat and the second fat shown in Table 6 were used, and the first fat was switched to the second fat when the amount of PHA accumulated in the microorganism reached 30 to 34% by weight. Except for this, (1) preculture, (2) seed culture, and (3) main culture were sequentially carried out under the same conditions as in Example 10. Table 6 shows the proportions of the first fat and the second fat in the carbon source used in the main culture and the calculated PHA productivity values.

[0080] Example 19 Except that the main culture was switched from the first fat to the second fat when the amount of PHA accumulated in the microorganism reached 82% by weight, (1) preculture, (2) seed culture, and (3) main culture were sequentially performed under the same conditions as in Example 10. Table 6 shows the proportions of the first fat and the second fat in the carbon source used in the main culture and the calculated PHA productivity values.

[0081] [Table 6]

[0082] Table 6 reveals the following. In Examples 10 to 19, as in Examples 1 to 9, main culture was initiated using a first fat or oil having an unsaturated fatty acid content of 25% by weight or more but less than 75% by weight as the carbon source, and during the main culture, the carbon source was switched to a second fat or oil having a relatively high unsaturated fatty acid content, and the main culture was continued. It can be seen that in all cases, PHA productivity was good, at 80% or more. In particular, in Examples 12 to 18, despite using 80% by weight or more of the second fat or oil, which has low PHA productivity when used alone, PHA productivity was extremely high, at 90% or more. Although fats and oils with a relatively high content of unsaturated fatty acids result in low PHA productivity when used alone, as shown in Comparative Examples 10 to 12 and Reference Example 7 in Table 5, it can be seen that good PHA productivity can be achieved by using them sequentially in combination with fats and oils with an unsaturated fatty acid content of 25% by weight or more but less than 75% by weight.

[0083] (Comparative Example 13) (1) Preculture, (2) Seed culture, and (3) Main culture were sequentially performed under the same conditions as in Example 10, except for the following points. The order of addition of fat 1 and fat 2 in the main culture of Example 10 was reversed, and the main culture was started while intermittently adding fat 2 as a carbon source. When the amount of PHA accumulated in the microorganism reached 32% by weight, the carbon source was switched to fat 1, and the main culture was continued while intermittently adding fat 1. The culture was terminated 48 hours after the start of the main culture. Table 7 shows the proportions of fat 1 and fat 2 in the carbon sources used in the main culture and the calculated PHA productivity values.

[0084] (Comparative Example 14) (1) Pre-culture, (2) seed culture, and (3) main culture were sequentially performed under the same conditions as in Comparative Example 13, except that the main culture was switched from oil 2 to oil 1 when the amount of PHA accumulated in the microorganism reached 80% by weight. Table 7 shows the proportions of oil 1 and oil 2 in the carbon source used in the main culture and the calculated PHA productivity values.

[0085] [Table 7]

[0086] Table 7 reveals the following. In Comparative Examples 13 and 14, unlike Examples 10 to 19, main culture was initiated using an oil or fat (oil 2) with a relatively high content of unsaturated fatty acids as the carbon source, and during the main culture, the carbon source was switched to an oil or fat (oil 1) with an unsaturated fatty acid content of 25% by weight or more but less than 75% by weight, and the main culture was continued. As a result, PHA productivity was low at less than 80%, and was similar to that of Comparative Examples 10 to 12. In particular, in Comparative Example 13, despite the use of nearly 80% by weight of oil or fat 1, which has the highest PHA productivity when used alone, PHA productivity was extremely low at 73%. From the above, it can be seen that in order to obtain good PHA productivity, it is desirable that the carbon source used in the early stages of cultivation is not an oil or fat with a relatively high content of unsaturated fatty acids, but rather an oil or fat with an unsaturated fatty acid content of 25% by weight or more and less than 75% by weight, as in Examples 10 to 19.

Claims

1. A method for producing poly(3-hydroxyalkanoate) by culturing a poly(3-hydroxyalkanoate)-producing microorganism in the presence of a carbon source, comprising: In the culture, fats and oils A and B are used as carbon sources, The manufacturing method includes: (i) culturing the poly(3-hydroxyalkanoate)-producing microorganism in the presence of fat or oil A until the amount of poly(3-hydroxyalkanoate) accumulated in the poly(3-hydroxyalkanoate)-producing microorganism reaches more than 16% by weight and less than 85% by weight; (ii) changing the oil A to oil B and continuing to culture the poly(3-hydroxyalkanoate)-producing microorganism in the presence of oil B; The production method, wherein the amount of fat / oil B used is 10% by weight or more relative to the total amount of fat / oil A and fat / oil B used throughout the culture. Oil / fat A: Oil / fat A contains one or more types of oil / fat, and the average content of unsaturated fatty acids as constituent fatty acids in the entire oil / fat A is 25% by weight or more and less than 75% by weight. Oil / fat B: The content of unsaturated fatty acids, which are constituent fatty acids, in oil / fat B is higher than the average content of unsaturated fatty acids in oil / fat A as a whole.

2. The method according to claim 1, wherein the fat / oil B is a fat / oil having an unsaturated fatty acid content of 60% by weight or more and 98% by weight or less.

3. The method according to claim 1 or 2, wherein the amount of fat B used relative to the total amount of fat A and fat B used throughout the culture is 40% by weight or more.

4. The production method according to any one of claims 1 to 3, wherein the poly(3-hydroxyalkanoate)-producing microorganism is cultured until the amount of poly(3-hydroxyalkanoate) accumulated in the poly(3-hydroxyalkanoate)-producing microorganism reaches 80% by weight or more.

5. The method according to any one of claims 1 to 4, wherein the culture is carried out while continuously adding a fat or oil A to a medium containing the poly(3-hydroxyalkanoate)-producing microorganism, and then the culture is continued while continuously adding a fat or oil B.

6. The method according to any one of claims 1 to 5, wherein the poly(3-hydroxyalkanoate) contains at least 3-hydroxybutyrate units.

7. The method according to any one of claims 1 to 5, wherein the poly(3-hydroxyalkanoate) comprises a homopolymer of 3-hydroxybutyrate units or a copolymer of 3-hydroxybutyrate units with other hydroxyalkanoate units.

8. The method according to claim 7, wherein the other hydroxyalkanoate units are 3-hydroxyhexanoate units.

9. The method according to any one of claims 1 to 8, wherein the poly(3-hydroxyalkanoate)-producing microorganism is a bacterium.

10. The method according to claim 9, wherein the poly(3-hydroxyalkanoate)-producing microorganism is a bacterium belonging to the genus Capriavidus.

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