Method for culturing microorganisms and method for producing polyhydroxyalkanoic acid

Abstract

Microorganisms belonging to the genus Cupriavidus are cultured in a culture solution containing an oil or a fatty acid, and before or during culturing, at least one type of hydroxy fatty acid selected from the group consisting of 3-hydroxydodecanoic acid, 3-hydroxytetradecanoic acid, 2-hydroxyhexadecanoic acid, 2-hydroxyoctadecaneoic acid, and 12-hydroxy octadecenoic acid, or an oil containing the hydroxy fatty acid as a constituent fatty acid, are added to the culture solution. The microorganisms may be microorganisms capable of producing a polyhydroxyalkanoic acid, and the polyhydroxyalkanoic acid produced by the microorganisms may be recovered.

Description

Method for culturing microorganisms and method for producing polyhydroxyalkanoic acid

[0001] This invention relates to a method for culturing microorganisms, and a method for producing polyhydroxyalkanoic acid (hereinafter also referred to as PHA) by culturing microorganisms.

[0002] Generally, in the fermentation industry, useful substances are produced by cultivating aerobic microorganisms. The culture medium used for cultivating such useful substances is a liquid culture solution containing nutrients that promote the growth and proliferation of the microorganisms. Furthermore, aeration and stirring of the culture solution are performed to ensure proper microbial cultivation.

[0003] However, this aeration and stirring of the culture medium results in the inclusion of air bubbles in the culture medium. These bubbles, generated during cultivation, are often difficult to break due to the action of secretions from microorganisms such as proteins, microbial decomposition products, and carbon sources and decomposition products of nutrients such as carbon sources. This leads to a decrease in the liquid specific gravity, resulting in reduced production volume and increased costs due to a decrease in the culture medium filling rate of the culture tank. Furthermore, in some cases, this can lead to the leakage of microbial cells and a resulting decrease in production yield. Therefore, technologies to suppress air bubbles in fermentation production are needed.

[0004] A common method for suppressing bubbles in fermentation production is to add antifoaming agents such as polyalkylene glycol to the culture medium.

[0005] Furthermore, Patent Document 1 discloses a method for suppressing bubbles in a PHA production culture medium by using high-concentration oxygen to reduce the amount of air permeating into the culture medium and increase the liquid specific gravity.

[0006] In the aforementioned prior art, substances added during fermentation production cannot be assimilated by microorganisms and therefore cannot be removed, leading to a decrease in wastewater treatment rate in the wastewater treatment process, and in some cases, contamination of the product. This posed problems for industrial use. Furthermore, the use of high-concentration oxygen increases costs. Thus, challenges remain in foam suppression technology for fermentation production, and further technological development is necessary.

[0007] Although not a technology related to microbial culture, Non-Patent Literature 1 reports that in beer, the higher the content of trihydroxyoctadecenoic acid (including 9,10,13-trihydroxy-11-octadecenoic acid and 9,12,13-trihydroxy-10-octadecenoic acid), the worse the foam retention of the beer after it is poured into a glass.

[0008] Japanese Patent Publication No. 2024-118674

[0009] Yabunouchi, Nippon Tatsuzukyo Kaishishi, 1980, Vol. 75, No. 4, p. 273-276

[0010] In view of the above situation, the present invention aims to provide a method for culturing microorganisms that enables microbial cultivation while suppressing bubbles in the culture medium without using antifoaming agents such as polyalkylene glycol or gases containing high concentrations of oxygen, and a method for producing PHA that enables fermentation production of PHA.

[0011] As a result of diligent research to solve the aforementioned problems, the inventors have discovered that when culturing microorganisms of the genus Cupriavidus, adding a specific hydroxy fatty acid, or an oil containing such a hydroxy fatty acid as a constituent fatty acid, to a culture medium containing oil or fatty acids can suppress bubbles in the culture medium, thus completing the present invention.

[0012] In other words, the present invention relates to a method for culturing microorganisms, comprising the steps of culturing a microorganism of the genus Cupriavidus in a culture medium containing oil or fatty acids, and adding to the culture medium, before or during cultivation, at least one hydroxy fatty acid selected from the group consisting of 3-hydroxydodecanoic acid, 3-hydroxytetradecanoic acid, 2-hydroxyhexadecanoic acid, 2-hydroxyoctadecanoic acid, and 12-hydroxyoctadecenoic acid, or an oil having the hydroxy fatty acid as a constituent fatty acid. The present invention also relates to a method for producing polyhydroxyalkanoic acid, comprising the steps of: culturing a microorganism of the genus Cupriavidus having the ability to produce polyhydroxyalkanoic acid in a culture medium containing oil or fatty acids; adding at least one hydroxy fatty acid selected from the group consisting of 3-hydroxydodecanoic acid, 3-hydroxytetradecanoic acid, 2-hydroxyhexadecanoic acid, 2-hydroxyoctadecanoic acid, and 12-hydroxyoctadecenoic acid, or an oil having the hydroxy fatty acid as a constituent fatty acid, to the culture medium before or during cultivation; and recovering the polyhydroxyalkanoic acid produced by the microorganism.

[0013] According to the present invention, it is possible to provide a method for culturing microorganisms that enables microbial cultivation while suppressing bubbles in the culture medium without using antifoaming agents such as polyalkylene glycol or gases containing high concentrations of oxygen, and a method for producing PHA that enables fermentation production of PHA.

[0014] According to the method for culturing microorganisms or producing PHA described herein, since bubbles in the culture medium are suppressed, it is possible to reduce the amount of culture medium that can be filled into the culture tank and reduce risks such as culture medium overflow, making it possible to carry out stable cultivation and produce PHA.

[0015] Embodiments of the present invention will be described in detail below. However, the present invention is not limited to the embodiments described below, and various modifications are possible within the scope defined in the claims. Furthermore, the configurations described below can be combined in any way, and such combinations may also constitute an embodiment of the present invention.

[0016] This disclosure relates to a method for culturing microorganisms, comprising the step of culturing microorganisms in a culture medium containing oils or fatty acids, and to a method for producing PHA, comprising the step of culturing microorganisms capable of producing PHA (hereinafter also referred to as PHA-producing microorganisms) in a culture medium containing oils or fatty acids.

[0017] The microorganisms usable in the culture method relating to this disclosure are microorganisms of the genus Cupriavidus. These microorganisms may be wild-type strains, mutant strains obtained by artificially mutating wild-type strains, or transformed strains obtained by genetically modifying them using genetic engineering techniques.

[0018] The PHA-producing microorganisms usable in the PHA production method described herein are microorganisms of the genus Cupriavidus. These microorganisms may be wild-type strains that inherently possess the PHA synthase gene, mutant strains obtained by artificially mutating such wild-type strains, or transformed strains into which an exotic PHA synthase gene has been introduced by genetic engineering techniques.

[0019] The host of the aforementioned microorganism, or the transformed microorganism, is not particularly limited as long as it belongs to the genus Capriavidus, but is preferably Capriavidus necator.

[0020] (PHA) The type of PHA produced by the PHA-producing microorganisms according to this disclosure is not particularly limited as long as it is a PHA that the microorganism can produce, but preferred are homopolymers of one monomer selected from 3-hydroxyalkanoates having 4 to 16 carbon atoms, copolymers of two or more monomers selected from 3-hydroxyalkanoates having 4 to 16 carbon atoms, copolymers of one monomer selected from 3-hydroxyalkanoates having 4 to 16 carbon atoms and other hydroxyalkanoates (e.g., 2-hydroxyalkanoates, 4-hydroxyalkanoates, 5-hydroxyalkanoates, 6-hydroxyalkanoates, etc. having 4 to 16 carbon atoms), and copolymers of two or more monomers selected from 3-hydroxyalkanoates having 4 to 16 carbon atoms and other hydroxyalkanoates.

[0021] Particularly preferred PHA is a homopolymer of 3-hydroxyalkanoic acid having 4 carbon atoms, or a copolymer containing 3-hydroxyalkanoic acid having 4 carbon atoms. Examples 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) (abbreviation: P3HB3HH); P(3HB-co-4HB), a copolymer of 3HB and 4-hydroxybutyric acid (abbreviation: 4HB); and PHA containing lactic acid (abbreviation: LA) as a constituent, such as the copolymer P(LA-co-3HB) of 3HB and LA. Among these, P3HB3HH is preferred from the viewpoint of having a wide range of applications as a polymer.

[0022] The type of PHA to be produced can be appropriately selected depending on the purpose, based on the type of PHA synthase gene possessed by the microorganism used or introduced separately, the type of metabolic pathway genes involved in the synthesis, culture conditions, and the like.

[0023] (PHA Synthase Gene) The PHA synthase (PhaC) gene possessed by the PHA-producing microorganism according to the present disclosure may be inherently possessed by the host, or may be an exogenous gene. The PHA synthase gene is not particularly limited, and examples include PHA synthase genes derived from Aeromonas caviae, Aeromonas hydrophila, Pseudomonas sp. 61-3, or Cupriavidus necator; chimeric PHA synthase genes obtained by combining two or more of the aforementioned PHA synthase genes; and genes encoding proteins consisting of an amino acid sequence that exhibits 90% or more sequence identity with the amino acid sequence of any of the aforementioned PHA synthases. The sequence identity is preferably 95% or more, more preferably 97% or more, and even more preferably 99% or more. The number of PHA synthase genes possessed by the transformed microorganism according to the present disclosure may be one, or may be plural. When a plurality of PHA synthase genes are possessed, they may be the same gene or different genes.

[0024] (Culture of Microorganisms) In the method for culturing a microorganism or the method for producing PHA according to the present disclosure, a microorganism or a PHA-producing microorganism is cultured in a culture solution containing a carbon source. When a PHA-producing microorganism is cultured, PHA can be accumulated in the cells of the microorganism. As a method for culturing a microorganism or a PHA-producing microorganism, a conventional microbial culture method can be followed, and the culture may be performed in a culture solution containing a carbon source. There are no particular limitations on the composition of the culture solution other than oils and fats or fatty acids, the method of adding the carbon source, culture scale, aeration and stirring conditions, culture temperature, culture time, and the like.

[0025] At least oil or fat or fatty acid is used as a carbon source during culture. In conventional microbial culture using oil or fat or fatty acid as a carbon source, the problem that bubbles in the culture solution are difficult to suppress even when an antifoaming agent such as polyalkylene glycol is added to the culture solution has been remarkable. In contrast, according to the method for culturing a microorganism or the method for producing PHA according to the present disclosure, despite using oil or fat or fatty acid as a carbon source, it is possible to culture the microorganism while suppressing bubbles in the culture solution and produce PHA.

[0026] The type of oil or fat used as a carbon source is not particularly limited, and examples thereof include oils and fats such as palm oil, palm kernel oil (including palm olein, palm double olein, and palm kernel oil olein, which are low-melting fractions obtained by fractionating these oils), corn oil, coconut oil, olive oil, soybean oil, rapeseed oil, and jatropha oil, fractionated oils thereof, and refined by-products thereof. A part or all of the oil or fat may be degraded oil. The type of fatty acid used as a carbon source is not particularly limited, and examples thereof include lauric acid, oleic acid, stearic acid, palmitic acid, and myristic acid.

[0027] The oil or fat or fatty acid used as a carbon source may contain the specific hydroxy fatty acid of the present disclosure or an oil or fat having the hydroxy fatty acid as a constituent fatty acid, or may not contain the specific hydroxy fatty acid of the present disclosure or an oil or fat having the hydroxy fatty acid as a constituent fatty acid.

[0028] When using oils or fatty acids as a carbon source, it is preferable that the concentration of oils or fatty acids in the culture medium be 10% by weight or less, from the viewpoint of suppressing foaming. It may also be 5% by weight or less, 3% by weight or less, or 1% by weight or less. The lower limit can be set appropriately considering the growth rate of microorganisms and the PHA production rate, but for example, it may be 0.1% by weight or more, or 0.5% by weight or more.

[0029] The method of adding a carbon source containing oil or fatty acids to the culture medium is not particularly limited, and methods include adding the carbon source all at once, adding it in installments, or adding it continuously, intermittently, or intermittently (continuous addition, intermittent addition, or intermittent addition), but it is preferable to add the carbon source continuously or intermittently.

[0030] The amount of carbon source to be added, whether continuously or intermittently, is not particularly limited, but it is desirable to add the carbon source while taking care to ensure that the carbon source concentration in the culture medium remains within the range described above.

[0031] In the method for culturing microorganisms or producing PHA according to this disclosure, at least one hydroxy fatty acid selected from the group consisting of 3-hydroxydodecanoic acid, 3-hydroxytetradecanoic acid, 2-hydroxyhexadecanoic acid, 2-hydroxyoctadecanoic acid, and 12-hydroxyoctadecenoic acid is added to the culture medium before or during the cultivation of the microorganism or PHA-producing microorganism. Only one type of hydroxy fatty acid may be added, or two or more types may be added.

[0032] Alternatively, instead of the hydroxy fatty acid, oils and fats containing the hydroxy fatty acid as a constituent fatty acid may be added. Castor oil is a specific example of such an oil. Furthermore, this oil may be added together with the hydroxy fatty acid.

[0033] The amount of hydroxy fatty acid or oil added can be appropriately set considering the effects of the invention, but it is preferable to add an amount such that the concentration of hydroxy fatty acid in the culture medium increases by 2 ppm to 30,000 ppm. When oil is added, the increase in the concentration of hydroxy fatty acid is calculated from the amount of oil added and the proportion of hydroxy fatty acid contained in the oil. Specific methods will be shown in the examples below.

[0034] By adding the hydroxy fatty acid or the oil / fat such that the concentration of the hydroxy fatty acid increases by 2 ppm or more, the foam-suppressing effect of the hydroxy fatty acid can be achieved. The foam-suppressing effect improves as the amount of hydroxy fatty acid or the oil / fat added increases, so the lower limit of the increase in concentration is preferably 10 ppm or more, more preferably 100 ppm or more, even more preferably 200 ppm or more, particularly preferably 500 ppm or more, and most preferably 800 ppm or more.

[0035] Furthermore, adding an amount of the hydroxy fatty acid or oil such that the increase in concentration exceeds 30,000 ppm is undesirable from a cost standpoint. The upper limit of the increase in concentration may be 10,000 ppm or less, 5,000 ppm or less, 2,000 ppm or less, or 1,200 ppm or less.

[0036] In particular, if the added hydroxy fatty acid is at least one selected from the group consisting of 3-hydroxydodecanoic acid, 3-hydroxytetradecanoic acid, 2-hydroxyhexadecanoic acid, and 2-hydroxyoctadecanoic acid, the upper limit of the increase in concentration may be 10,000 ppm or less. Also, if the added hydroxy fatty acid is 12-hydroxyoctadecenoic acid, or if the aforementioned oil or fat is added, the upper limit of the increase in concentration may be 30,000 ppm or less.

[0037] The aforementioned increase in concentration refers to the increase in the total concentration of hydroxy fatty acids when two or more types of hydroxy fatty acids are added, or when both hydroxy fatty acids and oils and fats are added.

[0038] The timing of adding hydroxy fatty acids or oils to the culture medium is not particularly limited. Methods include adding hydroxy fatty acids or oils to the culture medium before starting the culture, or adding hydroxy fatty acids or oils to the culture medium while the culture is in progress. These methods may be combined as appropriate.

[0039] The method of adding hydroxy fatty acids or oils to the culture medium is not particularly limited, and examples include adding the hydroxy fatty acids or oils all at once, adding them in installments, or adding them continuously, intermittently, or intermittently (continuous addition, intermittent addition, or intermittent addition). However, it is preferable to add the hydroxy fatty acids or oils continuously or intermittently. The hydroxy fatty acids added may be one of the five types of hydroxy fatty acids mentioned above, or two or more types. In addition, both hydroxy fatty acids and oils may be added.

[0040] Furthermore, since the amount of bubbles generated is small in the early stages of cultivation, there is little need to add hydroxy fatty acids or oils to the culture medium. Therefore, the addition of hydroxy fatty acids or oils may be started after a certain amount of time has passed since the start of cultivation and a large amount of bubbles have begun to be generated.

[0041] In the method for culturing microorganisms or producing PHA in this disclosure, it is preferable to cultivate the microorganisms using a culture medium containing the above-mentioned carbon source, a nitrogen source which is a nutrient source other than a carbon source, inorganic salts, and other organic nutrients. Examples of nitrogen sources, though not limited to those listed below, include ammonia; ammonium salts such as ammonium chloride, ammonium sulfate, and ammonium phosphate; peptone, meat extract, yeast extract, etc. Examples of inorganic salts include potassium dihydrogen phosphate, disodium hydrogen phosphate, magnesium phosphate, magnesium sulfate, and sodium chloride. Examples of other organic nutrients include amino acids such as glycine, alanine, serine, threonine, and proline, and vitamins such as vitamin B1, vitamin B12, and vitamin C.

[0042] The culture is preferably carried out while aerating the culture medium. The aeration may be performed throughout the entire culture period or at a certain stage of the culture. Furthermore, the aeration may be performed continuously, intermittently, or intermittently, and is preferably performed continuously or intermittently, and more preferably continuously. Continuous means continuing without interruption over time, intermittent means continuing with temporary interruptions, and intermittent means repeating aeration and interruptions at regular intervals.

[0043] The gas used for ventilation is not particularly limited; any gas containing oxygen will suffice, and air can be suitably used.

[0044] While there are no particular limitations on the method of aerating the culture medium, it is preferable to aerate it in a way that generates bubbles in the culture medium, for example, by providing a gas outlet in the culture medium. Furthermore, it is preferable to stir the culture medium in order to improve the amount of oxygen supplied to the culture medium. This allows the bubbles in the culture medium to be made finer by the shear force of the stirring blades.

[0045] In the method for producing PHA according to this disclosure, PHA can be accumulated in the bacterial cells by culturing for an appropriate period of time, and then the PHA can be recovered from the bacterial cells using a well-known method. The recovery method is not particularly limited, but for example, after culturing is complete, the bacterial cells can be separated from the culture medium using a centrifuge or separation membrane, dried, and then PHA can be extracted from the dried bacterial cells using an organic solvent such as chloroform. Cellular components can be removed from this PHA-containing organic solvent solution by filtration, and a poor solvent such as methanol or hexane can be added to the filtrate to precipitate the PHA. The supernatant can then be 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 the PHA particles can be separated from the aqueous phase by filtration or centrifugation and recovered by drying.

[0046] As another example, bacterial cells can be separated from the culture medium using a centrifuge or similar device, and then washed with distilled water, ethanol, methanol, etc. Subsequently, the washed sample is mixed with a sodium lauryl sulfate (SDS) solution, the cell membrane is destroyed by ultrasonic disruption, and the bacterial components and PHA are separated using a centrifuge or similar device. The PHA can then be recovered by drying.

[0047] The following items list preferred embodiments of the present disclosure, but the present invention is not limited to these items. [Item 1] A method for culturing a microorganism of the genus Cupriavidus in a culture medium containing oil or fatty acids, and adding to the culture medium, before or during cultivation, at least one hydroxy fatty acid selected from the group consisting of 3-hydroxydodecanoic acid, 3-hydroxytetradecanoic acid, 2-hydroxyhexadecanoic acid, 2-hydroxyoctadecanoic acid, and 12-hydroxyoctadecenoic acid, or an oil having the hydroxy fatty acid as a constituent fatty acid. [Item 2] The method for culturing a microorganism according to Item 1, wherein the amount of the hydroxy fatty acid or the oil added is such that the concentration of the hydroxy fatty acid in the culture medium increases by 2 ppm or more and 30,000 ppm or less. [Item 3] The method for culturing a microorganism according to Item 1 or 2, wherein the microorganism is Cupriavidus necator. [Item 4] A method for producing polyhydroxyalkanoic acid, comprising the steps of: culturing a microorganism of the genus Cupriavidus having the ability to produce polyhydroxyalkanoic acid in a culture medium containing oil or fatty acids; adding at least one hydroxy fatty acid selected from the group consisting of 3-hydroxydodecanoic acid, 3-hydroxytetradecanoic acid, 2-hydroxyhexadecanoic acid, 2-hydroxyoctadecanoic acid, and 12-hydroxyoctadecenoic acid, or an oil having the hydroxy fatty acid as a constituent fatty acid, to the culture medium before or during cultivation; and recovering the polyhydroxyalkanoic acid produced by the microorganism. [Item 5] The method for producing polyhydroxyalkanoic acid according to Item 4, wherein the amount of the hydroxy fatty acid or the oil added is such that the concentration of the hydroxy fatty acid in the culture medium increases by 2 ppm to 30,000 ppm. [Item 6] The method for producing polyhydroxyalkanoic acid according to Item 4 or 5, wherein the microorganism is Cupriavidus necator.

[0048] The present invention will be described in more detail below with reference to examples. However, the present invention is not limited to these examples.

[0049] (PHA Production by PHA-producing Microbial Strains) Culture studies were conducted using the Cupriavidus necator H16 strain under the following conditions.

[0050] (Medium) The composition of the seed medium was 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, 0.15 w / v% KH ２ PO ４ , (pH 6.8).

[0051] The composition of the preculture medium was 1.1 w / v% Na ２ HPO ４ ・12H ２ O, 0.19 w / v% KH ２ PO ４ , 1.29 w / v% (NH ４ ) ２ SO ４ , 0.1 w / 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 ３ ・6H ２ O, 1 w / v% CaCl ２ ・2H ２ O, 0.02 w / v% CoCl ２ ・6H ２ O, 0.016 w / v% CuSO ４ ・5H ２ O, 0.012 w / v% NiCl ２ ・6H ２ O dissolved in 0.1 N hydrochloric acid).

[0052] The composition of the PHA production medium was 0.385 w / v% Na ２ HPO ４ ・12H ２ O, 0.067 w / v% KH ２ PO ４ , 0.291 w / v% (NH ４ ) ２ SO ４ , 0.1 w / v% MgSO ４7H ２ O, 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 dissolved in water)

[0053] (PHA Production Culture) PHA production culture was performed as follows. First, a glycerol stock (50 μl) of the Curiavidus necator H16 strain was inoculated into a seed medium (10 ml) and cultured for 24 hours to perform seed culture. Next, the seed culture solution was inoculated at 1.0 v / v% into a 3 L jar fermenter (Marubishi Bioengin MDL-300 model) containing 1.8 L of pre-culture medium. The operating conditions were a culture temperature of 30°C, a stirring speed of 500 rpm, and an aeration rate of 1.8 L / min. The culture was performed for 28 hours while controlling the pH between 6.7 and 6.8 to perform pre-culture. A 14% ammonium hydroxide aqueous solution was used for pH control.

[0054] Next, the pre-culture solution was inoculated at 5.0 v / v% into a 5 L jar fermenter (Marubishi Bioengin MDS-U50 model) containing 2.5 L of PHA production medium. The operating conditions were a culture temperature of 33°C, a stirring speed of 420 rpm, and an aeration rate of 2.1 L / min, with the pH controlled between 6.7 and 6.8. A 25% ammonium hydroxide aqueous solution was used for pH control. A carbon source was added intermittently. Palm olein oil was used as the carbon source. Culturing was continued until the ratio of PHA accumulation to dry cell mass reached 80% or more.

[0055] (Evaluation Example 1) 50 mL of the culture medium was placed in a 200 mL graduated cylinder, and then 2-hydroxyhexadecanoic acid (2-C16) was added in an amount ranging from 1 to 500 mg as shown in Table 1. The graduated cylinder was then shaken back and forth 10 times by hand to create foam. After foaming, the mixture was left to stand for 5 minutes, and the foam suppression was evaluated by measuring the height of the foam surface at that time. A lower foam surface indicates better foam suppression. The foam suppression was also evaluated in the same manner for a culture medium without the addition of 2-hydroxyhexadecanoic acid (2-C16). The measurement results are shown in Table 1.

[0056]

[0057] Table 1 shows the following: In each case where 2-hydroxyhexadecanoic acid (2-C16) was added to the culture medium, the bubble level was significantly lower compared to the case where it was not added. Furthermore, the bubble level decreased as the amount of 2-C16 added increased. From this, it can be said that 2-C16 has an effect of suppressing bubbles in the culture medium.

[0058] (Evaluation Example 2) Under the same conditions as in Evaluation Example 1, 5 mg of 3-hydroxydodecanoic acid (3-C12), 3-hydroxytetradecanoic acid (3-C14), or 2-hydroxyoctadecanoic acid (2-C18) was added to 50 mL of the culture medium, and the foam-suppressing properties were evaluated. The measurement results are shown in Table 2.

[0059]

[0060] Table 2 shows the following: In each case where 3-hydroxydodecanoic acid (3-C12), 3-hydroxytetradecanoic acid (3-C14), or 2-hydroxyoctadecanoic acid (2-C18) was added to the culture medium, the bubble level was lower compared to the case where no additive was added, similar to the case where 2-hydroxyhexadecanoic acid (2-C16) was added. From this, it can be said that not only 2-C16, but also hydroxy fatty acids such as 3-C12, 3-C14, and 2-C18 have the effect of suppressing bubbles in the culture medium.

[0061] (Evaluation Example 3) Under the same conditions as in Evaluation Example 1, 5 mg, 100 mg, 625 mg, or 6250 mg of 12-hydroxyoctadecenoic acid (12-C18) was added to 50 mL of the culture medium, and the foam-suppressing properties were evaluated. The measurement results are shown in Table 3.

[0062]

[0063] Table 3 shows the following: In the example where 12-hydroxyoctadecenoic acid (12-C18) was added to the culture medium, the bubble level was lower compared to the example where no additive was added, similar to the example where 2-hydroxyhexadecanoic acid (2-C16) was added. From this, it can be said that not only 2-C16 but also 12-C18 has the effect of suppressing bubbles in the culture medium.

[0064] (Evaluation Example 4) Under the same conditions as in Evaluation Example 1, 5 mg, 100 mg, 700 mg, or 2100 mg of castor oil containing 12-hydroxyoctadecenoic acid as a constituent fatty acid was added to 50 mL of the culture medium, and the foam-suppressing properties were evaluated. The measurement results are shown in Table 4.

[0065] Since the castor oil used in the example contains 80% 12-hydroxyoctadecenoic acid (12-C18), the increase in the concentration of hydroxy fatty acids in the culture medium (ppm) was calculated using the following formula: Amount of castor oil added (mg) × 80% ÷ 0.05 (L) = Increase in the concentration of hydroxy fatty acids (ppm)

[0066] The 12-C18 content in castor oil was estimated as follows. The main hydroxy fatty acid in castor oil is 12-C18, and this fatty acid has one hydroxyl group per molecule. On the other hand, since triglycerides, the main component of castor oil, are composed of three molecules of 12-C18, their theoretical hydroxyl value was calculated to be 180.3 mgKOH / g using the following formula: Theoretical hydroxyl value = 56100 × 3 / 933.43 (molecular weight of 12-C18) The 12-C18 content in castor oil can be estimated according to the following formula by dividing the hydroxyl value of castor oil by the theoretical hydroxyl value. The 12-C18 content in castor oil (weight %) = hydroxyl value (mgKOH / g) / 180.3 × 100. Since the hydroxyl value of the castor oil used was 145 mgKOH / g, the 12-C18 content in the castor oil was calculated to be 80%.

[0067]

[0068] Table 4 shows the following: In the example where castor oil was added to the culture medium, the foam surface height was lower compared to the example where no castor oil was added, similar to the example where 2-hydroxyhexadecanoic acid (2-C16) was added. From this, it can be said that not only 2-C16 but also castor oil has the effect of suppressing bubbles in the culture medium.

Claims

1. A method for culturing microorganisms, comprising the steps of culturing a microorganism of the genus Cupriavidus in a culture medium containing oil or fatty acids, and adding to the culture medium, before or during cultivation, at least one hydroxy fatty acid selected from the group consisting of 3-hydroxydodecanoic acid, 3-hydroxytetradecanoic acid, 2-hydroxyhexadecanoic acid, 2-hydroxyoctadecanoic acid, and 12-hydroxyoctadecenoic acid, or an oil having the hydroxy fatty acid as a constituent fatty acid.

2. The method for culturing microorganisms according to claim 1, wherein the amount of hydroxy fatty acid or oil added is such that the concentration of the hydroxy fatty acid in the culture medium increases by 2 ppm to 30,000 ppm.

3. The method for culturing a microorganism according to claim 1 or 2, wherein the microorganism is Cupriavidus necator.

4. A method for producing polyhydroxyalkanoic acid, comprising the steps of: culturing a microorganism of the genus Cupriavidus having the ability to produce polyhydroxyalkanoic acid in a culture medium containing oil or fatty acids; adding at least one hydroxy fatty acid selected from the group consisting of 3-hydroxydodecanoic acid, 3-hydroxytetradecanoic acid, 2-hydroxyhexadecanoic acid, 2-hydroxyoctadecanoic acid, and 12-hydroxyoctadecenoic acid, or an oil having the hydroxy fatty acid as a constituent fatty acid, to the culture medium before or during cultivation; and recovering the polyhydroxyalkanoic acid produced by the microorganism.

5. The method for producing polyhydroxyalkanoic acid according to claim 4, wherein the amount of hydroxy fatty acid or oil added is such that the concentration of the hydroxy fatty acid in the culture medium increases by 2 ppm to 30,000 ppm.

6. The method for producing polyhydroxyalkanoic acid according to claim 4 or 5, wherein the microorganism is Cupriavidus necator.

Images