Method for manufacturing microorganisms and use of the same

Abstract

To provide a method for manufacturing microorganisms in which foaming of a culture solution can be suppressed while lipids in the culture solution is micronized.SOLUTION: A method for manufacturing microorganisms includes: a filling step of filling a microorganism culture solution into a container equipped with an agitator; a dispersion step of spraying lipids into the culture solution together with gas by using a two-fluid nozzle and dispersing the lipids into the culture solution; and an agitation step of agitating the culture solution by agitation blades having a predetermined structure fixed to the agitator.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] The present invention relates to a method for culturing a microorganism and its use. [Background technology]

[0002] In recent years, against the backdrop of growing awareness of environmental issues, food issues, health and safety, and a growing preference for natural or natural products, the significance and importance of microbial cultivation and the production of substances using microorganisms (fermentation production, bioconversion, etc.) has been increasing.

[0003] Generally, in the cultivation of microorganisms and the production of substances by microorganisms, a carbon source (carbon source for cultivation, fermentation, etc.) that the microorganisms can utilize is essential. Typical carbon sources for microbial cultivation include lipids (e.g., fatty acids, oils and fats).

[0004] It is known that in the cultivation of microorganisms and the production of substances by microorganisms, the efficiency of cultivation and substance production can be improved by increasing the specific surface area of ​​lipids, which serve as carbon sources in the culture solution. For example, Patent Document 1 describes a method for improving the efficiency of microbial cultivation by vigorously stirring lipids in the culture solution to form fine particles. [Prior art documents] [Patent documents]

[0005] [Patent Document 1] International Publication No. WO2021 / 106462 Summary of the Invention [Problem to be solved by the invention]

[0006] Although the above-mentioned conventional techniques are excellent, the present inventors have newly discovered that when microparticulating lipids, the fatty acid masses generated during the lipid decomposition process are excessively microparticulated due to the shear force generated by stirring, and these microparticulated fatty acids become surface active components, causing excessive foaming in the culture solution. Furthermore, the inventors have also discovered that the foaming causes the liquid level of the culture solution to rise, making it necessary to scale up the culture tank, which results in a problem of reduced efficiency in the production of substances by microorganisms.

[0007] In view of the above circumstances, one aspect of the present invention is to provide a method for culturing microorganisms that can reduce the size of lipids in the culture medium while suppressing foaming of the culture medium. [Means for solving the problem]

[0008] As a result of intensive research aimed at solving the above problems, the present inventors have discovered that (1) spraying lipids into a microbial culture solution using a two-fluid nozzle and (2) stirring the culture solution using a stirring blade having a specific structure can reduce the size of lipids in the culture solution while suppressing foaming of the culture solution, and have completed the present invention. That is, one aspect of the present invention includes the following configuration.

[0009] [1] A method for culturing microorganisms, comprising: a filling step of filling a container equipped with a stirrer with a culture solution containing the microorganisms; a dispersion step of using a two-fluid nozzle to submerse lipids together with a gas into the culture solution, thereby dispersing the lipids in the culture solution; and a stirring step of stirring the culture solution with a stirring blade fixed to the stirrer, wherein the stirring blade comprises a fixed part fixed to the rotating shaft of the stirrer, a disk part provided on the fixed part and perpendicular to the rotating shaft, and a plurality of blade body parts arranged radially along the radial direction of the disk part, with a pair of upper and lower blades forming a gap between the opposing surfaces of the upper and lower blades, the gap being inclined so as to widen in the rotation direction of the stirring blade, and intersecting with the outer periphery of the disk part.

[0010] [2] The method for culturing microorganisms according to [1], wherein the ratio (d / D) of the blade diameter d of the stirring blade to the inner diameter D of the vessel is 0.35 to 0.5.

[0011] [3] The method for culturing microorganisms according to [1] or [2], wherein the lipids have a solubility in water of 1% or less.

[0012] [4] The method for culturing a microorganism according to any one of [1] to [3], wherein the lipids are at least one type of lipid selected from the group consisting of oils and fats derived from palm oil, oils and fats derived from plants and animals, edible oils and fats, and waste edible oil.

[0013] [5] The method for culturing a microorganism according to any one of [1] to [4], wherein the microorganism is a microorganism that produces polyhydroxyalkanoates.

[0014] [6] A method for producing a polyhydroxyalkanoate, which comprises, as one step, the method for culturing a microorganism according to any one of [1] to [5]. [Effects of the Invention]

[0015] According to one aspect of the present invention, a method for culturing a microorganism can be provided that can reduce the size of lipids in a culture solution while suppressing foaming of the culture solution. [Brief explanation of the drawings]

[0016] [Figure 1] FIG. 1 is a diagram showing a schematic configuration of a culture device used in one embodiment of the present invention. [Figure 2] 1A and 1B are a top view (left) and a side view (right) of an agitating impeller used in one embodiment of the present invention. [Figure 3] 1 shows a top view (left) and a side view (right) of the stirring impeller used in the comparative example. [Figure 4] FIG. 1 is a graph showing the transition of the foam surface height of the culture medium during culture in Examples and Comparative Examples. DETAILED DESCRIPTION OF THE INVENTION

[0017] One embodiment of the present invention will be described below, but the present invention is not limited thereto. The present invention is not limited to the respective configurations described below, and various modifications are possible within the scope of the claims. Furthermore, embodiments or examples obtained by combining the technical means disclosed in different embodiments or examples are also included in the technical scope of the present invention. Furthermore, new technical features can be formed by combining the technical means disclosed in each embodiment. All academic literature and patent documents described in this specification are incorporated herein by reference. Furthermore, unless otherwise specified in this specification, the term "A to B" representing a numerical range means "greater than or equal to A (including and greater than A) and less than or equal to B (including and less than B)."

[0018] 1. Microbial Cultivation Method A method for culturing microorganisms according to one embodiment of the present invention (hereinafter sometimes referred to as the "main culture method") comprises: a filling step of filling a container equipped with a stirrer with a culture solution containing microorganisms; a dispersion step of using a two-fluid nozzle to submersely spray lipids together with a gas into the culture solution, thereby dispersing the lipids in the culture solution; and a stirring step of stirring the culture solution with a stirring blade fixed to the stirrer, wherein the stirring blade comprises a fixed portion fixed to the rotating shaft of the stirrer; a disk portion provided on the fixed portion and perpendicular to the rotating shaft; and a plurality of blade body portions arranged radially along the radial direction of the disk portion, each of which has a pair of upper and lower blades that form a gap between the opposing surfaces of the upper and lower blades, the gap being inclined so as to widen in the rotation direction of the stirring blade, and which intersects with the outer periphery of the disk portion.

[0019] According to this culture method, it is possible to reduce the size of lipids in the culture solution while suppressing foaming of the culture solution. Therefore, according to this culture method, (1) lipids can be reduced in size, allowing microorganisms to efficiently assimilate the lipids. As a result, substance production by microorganisms can be carried out efficiently. Furthermore, (2) in conventional methods, it was necessary to fill the culture tank (culture device) with an amount of culture solution obtained by subtracting the volume of the foaming solution from the capacity of the culture tank (culture device) in order to take into account foaming due to the surfactant action of fatty acids derived from lipids. However, according to this culture method, foaming of the culture solution is suppressed, so a larger amount of culture solution can be filled in the culture tank, resulting in more efficient substance production by microorganisms.

[0020] <1-1.Culture device> First, an example of a culture apparatus capable of carrying out each step in the present culture method will be described in detail using FIG. 1, but the culture apparatus for carrying out each step of the present culture method is not limited to this embodiment.

[0021] As shown in Figure 1, the culture apparatus 100 comprises a container 1 filled with a culture solution 2, a stirrer 3 having a rotating shaft 31 inserted into the container 1 and an agitator blade 32 and a drive unit 33 connected to the rotating shaft 31, a two-fluid nozzle 4 installed on the side of the container 1 and having a liquid supply port (liquid injection hole) 41 and a gas supply port (gas injection hole) 42, an aeration ring sparger 5, an exhaust line 6, a probe-type video microscope 7, a tubing pump 8 and an oil tank 9 connected to the liquid supply port 41 of the two-fluid nozzle 4, and a flow control valve 10 connected to the gas supply port 42 of the two-fluid nozzle 4 and having a pressure gauge 11 attached thereto.

[0022] (container) The container 1 is filled with a culture solution 2 containing microorganisms, and thereby accommodates the culture solution 2 and functions as a culture tank where the microorganisms are cultured.

[0023] (mixer) The agitator 3 includes a rotating shaft 31 inserted into the container 1, and an agitator blade 32 and a drive unit 33 connected to the rotating shaft 31. In the agitator 3, when the drive unit 33 is driven, the rotating shaft 31 rotates in a rotational direction, and the agitator blade 32 connected to the rotating shaft 31 rotates in the rotational direction in conjunction with the rotation of the rotating shaft 31, thereby agitating the culture solution filled in the container 1.

[0024] Mixing blade The specific configuration of the agitating blade 32 will be explained in more detail using Figure 2. The left diagram of Figure 2 is a top view of the agitating blade 32, and the right diagram of Figure 2 is a side view of the agitating blade 32, showing the state where it is fixed to the rotating shaft 32. The arrow in the figure indicates the rotation direction of the agitating blade 32.

[0025] As shown in Figure 2, the agitator blade 32 comprises a fixed portion 321 fixed to the rotating shaft 31, a disk portion 322 provided on the fixed portion 321 and perpendicular to the rotating shaft, and a plurality of blade body portions 325 arranged radially along the radial direction of the disk portion 322, each of which is a pair of upper and lower blades 323a and 323b, forming a gap 324 between the opposing surfaces of the upper blade 323a and the lower blade 323b, and which are inclined so that the gap 324 widens in the rotational direction of the agitator blade 32 and intersects with the outer periphery of the disk portion 322.

[0026] As described above, the impeller 32 has a blade body 325 with an inclined gap 324, and therefore can perform stirring with a lower shear force than a conventional impeller with a blade body without a gap (for example, a flat turbine). As a result, the impeller 32 can prevent fatty acid clumps generated in the process of decomposing lipids in the culture solution 2 from being excessively broken down into smaller particles due to stirring, and as a result, the culture solution 2 can be stirred while preventing foaming of the culture solution 2, and lipid droplets 43 can be broken down into smaller particles.

[0027] Furthermore, by providing the disc portion 322 as described above, the stirring blade 32 can shorten the radial length of the blade body portion 325. As a result, stirring can be performed with a lower driving power.

[0028] The blade diameter d of the impeller 32 is not particularly limited and can be selected appropriately to match the inner diameter D of the vessel 1. Specifically, the ratio to the inner diameter D of the vessel 1 (d / D) is preferably 0.35 to 0.5, more preferably 0.37 to 0.45, and even more preferably 0.39 to 0.40. If the ratio (d / D) of the blade diameter d of the impeller 32 to the inner diameter D of the vessel 1 is within the above range, in other words, if the ratio (d / D) of the blade diameter d of the impeller 32 to the inner diameter D of the vessel 1 is within the above range, it is possible to minimize the shearing effect on the fatty acid masses in the culture solution while promoting the reduction of the size of bubbles in the culture solution.

[0029] The shapes of the upper blade 323a and the lower blade 323b of the stirring blade 32 are not particularly limited, and may be flat as shown in Figures 2 and 3, or may have an arc-shaped cross section or a blade-shaped cross section (both not shown), etc.

[0030] As described above, gap 324 is formed at an angle, and therefore has a shape that opens in the rotation direction. In this specification, the wider gap (on the rotation direction side) of gap 324 is referred to as the first gap, and the narrower gap (on the opposite side to the rotation direction) is referred to as the second gap.

[0031] The number of blade bodies 325 in the stirring blade 32 is not particularly limited as long as it is two or more, but is preferably 2 to 10, and more preferably 4 to 8. Furthermore, the blade bodies 325 are preferably provided on the outer periphery of the disk portion 322 so that the blade bodies 325 are spaced equally apart.

[0032] The material of the stirring blades 32 is not particularly limited, but stainless steel, ceramic, or titanium is preferable because it can suppress the generation of corrosion products such as rust and prevent such products from contaminating the culture solution.

[0033] In Figure 1, an example is shown in which the agitator 3 has two agitator blades 32, but the number of agitator blades 32 that the agitator 3 has is not particularly limited to this and may be one, or three or more.However, it is preferable that the number of agitator blades 32 that the agitator 3 has is two, as this can further suppress foaming of the culture solution.

[0034] (two-fluid nozzle) The two-fluid nozzle 4 has a liquid supply port (liquid ejection port) 41 for ejecting any gas, and a gas supply port 42 for ejecting any gas, which is arranged independently from the gas ejection port. In this specification, the term "two-fluid nozzle" refers to a nozzle that can simultaneously eject any gas and any liquid from separate supply ports, as described above.

[0035] Lipids are supplied in liquid form from an oil tank 9 via a tubing pump 8 to a liquid supply port 41, and the lipids are sprayed into the culture solution 2 via a liquid supply port 4. A gas is supplied to a gas supply port 42 via a flow rate control valve 10, and the gas is sprayed into the culture solution 2 via the gas supply port 42 simultaneously with the lipids sprayed from the liquid supply port 4. The gas sprayed from the gas supply port 42 applies a shear force to the lipids 43 sprayed from the liquid supply port 4, causing them to break down, thereby enabling the lipid droplets 43 to be dispersed as fine droplets in the culture solution 2.

[0036] The two-fluid nozzle 4 may be either an external mixing type two-fluid nozzle in which the liquid and gas are sprayed from separate supply ports and mixed outside the nozzle, or an internal mixing type two-fluid nozzle in which the liquid and gas are mixed inside the nozzle and sprayed in a mixed state outside the nozzle. However, since it is possible to spray finer lipid droplets 43 into the liquid, the external mixing type two-fluid nozzle 4 is preferable.

[0037] The two-fluid nozzle 4 is installed so that the liquid supply port 41 and the gas supply port 42 are located below the liquid surface of the culture solution 2, in other words, so that the liquid supply port 41 and the gas supply port 42 are located in the culture solution 2. By installing the two-fluid nozzle 4 in this manner, it becomes possible to spray lipids from the two-fluid nozzle 4 into the liquid.

[0038] The diameter of the liquid supply port 41 in the two-fluid nozzle 4 is not particularly limited, but is preferably 0.5 to 6.0 mm, and more preferably 0.6 to 5.5 mm, since this has the advantage that the supplied liquid is likely to be subjected to uniform shear from the gas discharged from the gas supply port 42. The diameter of the gas supply port 42 is also not particularly limited, but is preferably 1.0 to 10.0 mm, and more preferably 1.5 to 9.0 mm, since this makes it possible to apply uniform shear to the liquid discharged from the liquid supply port 41. Note that the diameters of the liquid supply port 41 and the gas supply port 42 in this specification refer to the diameters of the supply ports.

[0039] The material of the two-fluid nozzle 4 (in other words, the liquid supply port 41 and the gas supply port 42) is not particularly limited, but is preferably stainless steel, ceramic, or titanium, for example, because they have excellent stability in the culture solution 2.

[0040] The spray pattern of the two-fluid nozzle 4 is not particularly limited and can be any spray pattern such as a circular, hollow cone, full cone, or fan shape, but a circular shape is preferred because it allows for a uniform droplet size distribution.

[0041] Although FIG. 1 shows an example of an embodiment having only one two-fluid nozzle 4, the number of two-fluid nozzles 4 in the culture device 100 is not particularly limited, and may be two or more.

[0042] (Other configurations) When culturing aerobic microorganisms in the culture device 100, aeration air 51 can be supplied into the culture solution 2 through the aeration ring sparger 5. The supplied air 51 is ultimately discharged to the outside of the culture device 100 through the exhaust line 6. In addition, the probe-type video microscope 7 makes it possible to observe and photograph lipid droplets 43 in the culture solution 2.

[0043] <1-2. Filling process> Next, each step that may be included in the present culture method will be described in detail. The present culture method includes a filling step of filling a culture solution containing a microorganism into a container equipped with a stirrer.

[0044] (culture solution containing microorganisms) The culture medium containing the microorganisms to be filled into the container in the filling step is a culture medium containing the cells of the microorganisms to be cultured, and is intended to be a solution in which the above-mentioned cells and each component necessary for microbial culture (e.g., proteins, sugars, minerals, buffers, antibiotics, growth factors, other additives, etc.) are dispersed or dissolved in water as a solvent.

[0045] The microorganisms to be cultured in the culture medium are not particularly limited, and various microorganisms (e.g., eubacteria, archaea, eukaryotes, etc.) that use known lipids as a nutrient source (carbon source) can be cultured. Among these, microorganisms capable of producing various substances such as bioethanol, bioplastics, and biodiesel are preferred, and in particular, microorganisms capable of producing polyhydroxyalkanoates, which are a type of biodegradable bioplastic that can be used in a variety of applications.

[0046] The temperature of the culture solution containing the microorganisms filled into the container in the filling step is not particularly limited, but is preferably a temperature appropriate for culturing the microorganisms, and more preferably a temperature appropriate for culturing the microorganisms and higher than the melting point of the lipids dispersed in the culture solution in the subsequent dispersion step. Controlling the culture solution to such a temperature makes it possible to disperse the lipids in a liquid state in the culture solution, allowing the microorganisms to efficiently assimilate the lipids. As a result, microbial growth and substance production can be efficiently carried out. The temperature of the culture solution can be appropriately set by those skilled in the art to satisfy the above conditions depending on the type of microorganism to be cultured and the type of lipids to be supplied, but is preferably, for example, 10 to 40°C, more preferably 15 to 37°C, and even more preferably 20 to 35°C.

[0047] (Container and agitator) Regarding the specific aspects of the container into which the culture solution containing the microorganism is filled in the filling process and the agitator provided in the container, the description of the container 2 and the agitator 3 related to the culture apparatus 100 detailed in the above section <1-1. Culture apparatus> is to be used as appropriate.

[0048] <1-3. Dispersion process> This culture method includes a dispersion step in which lipids are dispersed in the culture solution by submerged spraying of lipids together with a gas into the culture solution using a two-fluid nozzle. By carrying out this dispersion step, lipids, which serve as a carbon source for microorganisms, can be dispersed in the culture solution as fine droplets, thereby enabling efficient microbial culture and substance production.

[0049] (two-fluid nozzle) For specific aspects of the two-fluid nozzle in the dispersion step, the description of the two-fluid nozzle 4 related to the culture apparatus 100 detailed in the above section <1-1. Culture apparatus> will be appropriately cited.

[0050] (lipids) As used herein, "lipids" refers to fatty acids or compounds that produce fatty acids upon hydrolysis. More specifically, "lipids" include fatty acids, oils and fats, waxes, phosphoglycerides, sphingolipids, steryl esters, and complexes of fatty acids with proteins or sugars. While any of the above lipids can be used as the lipids used in the dispersion step of this culture method, oils and fats are preferred because they can maintain a stable dispersion state in an aqueous dispersion medium for a long period of time.

[0051] ·Fatty acids As used herein, the term "fatty acids" refers to monocarboxylic acids consisting of a long-chain hydrocarbon and a carboxyl group. Fatty acids include saturated fatty acids with no unsaturated bonds and unsaturated fatty acids with one or more unsaturated bonds.

[0052] Fatty acids that can be used in the culture method include fatty acids, fatty acid salts such as fatty acid sodium, fatty acid potassium, fatty acid calcium, and fatty acid magnesium, fatty acid esters such as triglycerides, diglycerides, and monoglycerides, etc. More specifically, preferred examples of lipids having a solubility in water at 25°C of 1% or less include palm olein-containing oils and fats, soybean oil, and grain oils such as rapeseed oil.

[0053] ·Oils and fats As used herein, the term "fats and oils" refers to compounds in which one or more hydroxyl groups of glycerol are ester-bonded to a fatty acid. Fats and oils include monoacylglycerin, in which one hydroxyl group of glycerol is ester-bonded to a fatty acid; diacylglycerin, in which two hydroxyl groups of glycerol are ester-bonded to a fatty acid; and triacylglycerin, in which three hydroxyl groups of glycerol are ester-bonded to a fatty acid. In other words, fats and oils are mixtures of monoacylglycerin, diacylglycerin, and triacylglycerin. Furthermore, the fatty acids ester-bonded to glycerol may be the same type, or different types may be mixed. Triacylglycerin is also sometimes referred to as neutral fat.

[0054] The oils and fats that can be used in this culture method are not particularly limited, but oils and fats derived from palm oil, oils and fats derived from plants or animals (other than palm), edible oils and fats, and waste edible oil, etc., can be suitably used.

[0055] Specific examples of fats and oils derived from palm oil (sometimes simply referred to as "palm oil") include crude palm oil, crude palm kernel oil, palm oil, palm kernel oil, palm olein, palm double olein, palm kernel olein, PFAD (palm oil fatty acid distillate), PKFAD (palm kernel oil fatty acid distillate), POME (Palm Oil Mill Effluent, a waste liquid discharged in the process of obtaining crude palm oil from oil palm fruits), and EFB juice (Empty Fruit Bunch Juice, a by-product obtained in the process of obtaining empty fruit bunch pellets from empty fruit bunches of oil palm), as well as crudely refined oils, fractionated oils, hardened oils, and interesterified oils of these fats and oils. These fats and oils may be used alone or in combination of two or more. Among these, palm fractionated oils such as palm olein and palm double olein, which are low-melting point fractions obtained by fractionating palm oil, and palm kernel fractionated oils such as palm kernel oil olein, which is a low-melting point fraction obtained by fractionating palm kernel oil, are preferred because they are easily stabilized in a droplet state in the culture medium.

[0056] Specific examples of oils and fats derived from plants or animals (other than palm) include beef tallow, lard, milk fat, fish oil, soybean oil, rapeseed oil, sunflower oil, olive oil, sesame oil, canola oil, peanut oil, almond oil, grapeseed oil, avocado oil, tung oil, corn oil, cottonseed oil, rice oil, linseed oil, camellia oil, perilla oil, safflower oil, palm oil, shea butter, sal fat, illipe butter, cocoa butter, jatropha oil, and oils and fats derived from algae, as well as crudely refined oils, fractionated oils, hardened oils, and interesterified oils of these oils, etc. As the oils and fats derived from plants or animals, one type of these may be used alone, or two or more types may be used in combination.

[0057] As the edible oils and fats, among the above-mentioned vegetable or animal-derived oils and fats, those for edible use, such as rapeseed oil, soybean oil, olive oil, corn oil, linseed oil, sesame oil, perilla oil, peanut oil, almond oil, grapeseed oil, and avocado oil, can be suitably used, and as the waste edible oil, these edible oils and fats after use for cooking or the like can be suitably used.

[0058] Physical properties of lipids The solubility of the lipids used in this culture method in water at 25°C is not particularly limited, but since this tends to stabilize the oil droplet size in the culture medium, it is preferably 1% or less, more preferably less than 1%, even more preferably 0.5% or less, and even more preferably 0.2% or less.

[0059] The melting point of the lipids used in the present emulsion production method is not particularly limited, and for example, lipids of 120°C or lower can be used. However, since the lipids can exist in a liquid state in the culture medium and are easily micronized, it is preferable that the temperature be lower than the temperature of the culture medium in which the lipids are dispersed. Specifically, the melting point of the lipids used in the present culture method is preferably 30°C or lower, more preferably 25°C or lower, and even more preferably 15°C or lower. Furthermore, the lower limit of the melting point of the lipids is not particularly limited, and may be, for example, -10°C or higher, or -20°C or higher.

[0060] Methods for measuring the melting point and solubility of lipids In this specification, the melting point of lipids can be measured by heating the target lipids to a temperature at which they are all liquid, then gradually lowering the temperature and measuring the temperature at which all of the lipids solidify. The solubility of lipids in water can be calculated as the amount of lipids dissolved by preparing an aqueous solution in which the target lipids are saturated at room temperature and measuring the absorbance of the aqueous solution.

[0061] (in-liquid spray) In this specification, "submerged spraying" refers to spraying gas and liquid from a two-fluid nozzle into a liquid (a culture solution for microorganisms in this culture method) with the gas injection hole and liquid injection hole of the two-fluid nozzle immersed in the liquid.

[0062] ·gas In the dispersion step, the gas ejected from the two-fluid nozzle is not particularly limited, and examples thereof include air (compressed air), oxygen, nitrogen, water vapor, and mixtures thereof. In particular, when the microorganisms to be cultured are aerobic, the gas ejected from the nozzle can be used to supply oxygen to the microorganisms, so air or oxygen is preferably used. Conversely, when the microorganisms to be cultured are anaerobic, an oxygen-free gas such as nitrogen can be preferably used to prevent an increase in the oxygen concentration in the culture solution. The temperature of the gas is also not particularly limited, and may be, for example, room temperature (specifically, 20 to 25°C). However, in order to prevent solidification of lipids in the two-fluid nozzle, the temperature is preferably at least 10°C below the melting point of the lipids to be ejected.

[0063] ·liquid In the dispersion step, the liquid ejected from the two-fluid nozzle is liquid lipids. Regarding lipids, the description in the above section (Lipids) is used as appropriate.

[0064] In the dispersion step, when lipids having a melting point higher than room temperature (solid at room temperature) are used as the liquid to be sprayed, it is preferable to heat the two-fluid nozzle to melt the lipids and spray them in a liquid state.

[0065] In the dispersion step, the temperature to which the two-fluid nozzle is heated is not particularly limited as long as it is a temperature at which the lipids to be ejected melt, but it may be, for example, 50° C. or higher, or 60° C. or higher, or 70° C. or higher. The upper limit of the temperature is also not particularly limited, but from the viewpoint of suppressing adverse effects on the microorganisms to be cultured, it is preferably 100° C. or lower, preferably 90° C. or higher, and more preferably 80° C. or lower.

[0066] Gas linear velocity The linear velocity (vg) of the gas ejected from the two-fluid nozzle in the dispersion step is not particularly limited, but is preferably 100 m / sec or more. By setting the linear velocity of the gas in the dispersion step to 100 m / sec or more, a stronger shear force can be applied to the lipids ejected from the liquid supply port, making it possible to supply finer droplets of the lipids into the aqueous dispersion medium.

[0067] In this specification, the gas linear velocity is a value calculated by the following formula. Gas linear velocity (vg) [m / sec] = gas flow rate [m 3 / sec] / area of ​​gas supply port of two-fluid nozzle [m 2 ].

[0068] ·Liquid linear velocity (VF) The linear velocity (vf) of the liquid ejected from the two-fluid nozzle in the dispersion process is not particularly limited and can be set appropriately according to the concentration of lipids suitable for culturing microorganisms, etc., and may be, for example, 0.5 m / sec or more, or 1.0 m / sec or more.

[0069] In this specification, the liquid linear velocity (vf) is a value calculated by the following formula. Liquid linear velocity (vf) [m / sec] = liquid flow rate [m 3 / sec] / area of ​​the liquid supply port of the two-fluid nozzle [m 2 ].

[0070] ·Linear velocity ratio (vg / vf) In the dispersion step, sufficient shear force can be applied to the lipids ejected from the liquid supply port, making it possible to disperse finer lipid droplets into the culture solution. Therefore, it is preferable to adjust the gas linear velocity (vg) and the liquid linear velocity (vf) so that the linear velocity ratio (vg / vf) between the gas linear velocity (vg) and the liquid linear velocity (vf) is 50 to 2000. From the above viewpoint, the linear velocity ratio (vg / vf) is more preferably 55 to 1500, more preferably 60 to 1000, and even more preferably 65 to 600.

[0071] In this specification, the linear velocity ratio (vg / vf) is a value calculated by the following formula. Linear velocity ratio (vg / vf) = gas linear velocity (vg) / liquid linear velocity (vf).

[0072] <1-4. Mixing process> This culture method includes a stirring step of stirring the culture solution filled with a microbial culture solution using a stirring blade fixed to a stirrer provided in a container. The stirring step can also be said to be a step of culturing the microorganisms while stirring the microbial culture solution.

[0073] In this culture method, the dispersion step and the stirring step may be carried out separately or simultaneously, but preferably, the dispersion step and the stirring step are carried out simultaneously. That is, in this production method, it is preferable to carry out a stirring operation using a stirring blade fixed to a stirrer in parallel with the spraying of lipids into the liquid from the two-fluid nozzle.

[0074] (mixing blade) In the stirring step, a stirring blade having a predetermined shape is used to stir the microbial culture solution. For specific aspects of the stirring blade used in the stirring step, the description of the stirrer 3 and the stirring blade 32 related to the culture apparatus 100 detailed in the above section <1-1. Culture apparatus> is appropriately cited.

[0075] In the stirring step, by using stirring blades having the above-mentioned predetermined shape, the lipids dispersed in the culture solution in the dispersion step can be further refined and foaming due to the surfactant action of fatty acids derived from the lipids can be suppressed, allowing a larger amount of culture solution to be filled into the culture tank. As a result, this culture method can more efficiently culture microorganisms and produce substances than conventional culture methods.

[0076] (Number of stirring blades) The number of stirring blades used in the stirring process is not particularly limited and may be one or two or more, but it is preferable that the number of stirring blades 32 provided in the stirrer 3 is two or more, as this can further suppress foaming of the culture solution, and two is more preferable from the viewpoint of decomposing excess fatty acid blocks and suppressing foaming of the culture solution.

[0077] (Stirring speed of the impeller) The stirring speed of the impeller in the stirring step is not particularly limited, but a higher stirring speed, i.e., a higher shear rate applied to bubbles in the culture solution, has the advantage of improving the dispersibility of the bubbles (making the bubbles finer), while keeping the stirring speed at a certain speed or less can prevent excessive fineness of fatty acid clumps in the culture solution. From the above viewpoints, the stirring speed of the impeller in the stirring step is preferably 400 to 500 rpm, more preferably 450 to 500 rpm, and even more preferably 480 to 500 rpm.

[0078] (Average particle size of lipids) In the stirring step, the volume average particle size (D50) of the lipids dispersed in the culture solution is not particularly limited, but is preferably 1 to 45 μm, more preferably 5 to 35 μm, and even more preferably 10 to 30 μm. If the volume average particle size of the lipids during culture is within the above range, contact between the lipids and the microorganisms in the culture solution and assimilation of the lipids by the microorganisms are efficiently carried out, thereby enabling efficient microbial culture and substance production.

[0079] Because of the above advantages, in this culture method, it is preferable to carry out stirring so that the volume average particle diameter of the lipid droplets in the culture solution falls within the above range. The volume average particle diameter of the lipid droplets in the culture solution can be controlled by adjusting the linear velocity of the gas and liquid sprayed into the liquid in the dispersion step, and the shape, size, and stirring speed of the stirring blades used in the stirring step.

[0080] (Method for measuring particle size of lipids) The volume-average particle diameter (D50) of lipids dispersed in a culture medium can be calculated, for example, by photographing the culture medium in question with a probe-type video microscope (e.g., PVM (registered trademark) V819 manufactured by METTLER TOLEDO), measuring the particle diameters of at least 100 or more lipid droplets captured in the photographed image, calculating the volume of each droplet from the obtained particle diameters, and accumulating these to obtain the particle diameter at 50% of the cumulative volume.

[0081] 2. Method for producing polyhydroxyalkanoate According to this production method, as described above, it is possible to efficiently culture microorganisms and produce substances using microorganisms. Therefore, this production method is suitable for use in methods for producing substances using microorganisms, particularly in the production of polyhydroxyalkanoates, which are a type of biodegradable bioplastic that can be used in a variety of applications. That is, in one embodiment of the present invention, there is provided a method for producing polyhydroxyalkanoates, which includes this culture method as one step. Hereinafter, the "method for producing polyhydroxyalkanoates according to one embodiment of the present invention" may be referred to as the "method for producing polyhydroxyalkanoates."

[0082] According to this method for producing polyhydroxyalkanoate, contact between lipids serving as carbon sources and microorganisms in the microbial culture solution and assimilation of lipids by the microorganisms can be efficiently carried out, and foaming of the culture solution is suppressed, making it possible to produce polyhydroxyalkanoate more efficiently than conventional methods for producing polyhydroxyalkanoate.

[0083] The method for producing the present polyhydroxyalkanoate can be carried out in the same manner as known methods for producing substances using microorganisms, except that it includes the method for producing the present emulsion as one step, in other words, culturing microorganisms using the present culture method.

[0084] (Polyhydroxyalkanoate) As used herein, "polyhydroxyalkanoate" refers to a polymer having a 3-hydroxyalkanoate unit as a constituent unit (monomer unit). As used herein, "3-hydroxyalkanoate" may also be referred to as "3HA." Specific examples of polyhydroxyalkanoates include poly(3-hydroxybutyrate) (P3HB), poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) (P3HB3HH), poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (P3HB3HV), poly(3-hydroxybutyrate-co-4-hydroxybutyrate) (P3HB4HB), poly(3-hydroxybutyrate-co-3-hydroxyoctanoate) (P3HB3HO), poly(3-hydroxybutyrate-co-3-hydroxyoctadecanoate) (P3HB3HOD), poly(3-hydroxybutyrate-co-3-hydroxydecanoate) (P3HB3HD), and poly(3-hydroxybutyrate-co-3-hydroxyvalerate-co-3-hydroxyhexanoate) (P3HB3HV3HH). Among these, P3HB, P3HB3HH, P3HB3HV, and P3HB4HB are preferred because they can be easily produced industrially.

[0085] Furthermore, by changing the composition ratio of the repeating units, it is possible to change the melting point and degree of crystallinity, and as a result, it is possible to change physical properties such as Young's modulus and heat resistance, and it is possible to impart physical properties between those of polypropylene and polyethylene.In addition, as described above, it is easy to produce industrially and is a physically useful plastic, so P3HB3HH, a copolymer of 3-hydroxybutyric acid and 3-hydroxyhexanoic acid, is more preferred as the polyhydroxyalkanoate produced by this polyhydroxyalkanoate production method.

[0086] (Polyhydroxyalkanoate-producing bacteria) In the method for producing polyhydroxyalkanoate, a polyhydroxyalkanoate-producing bacterium, which is a type of microorganism, is cultured in the presence of lipids as a carbon source to produce polyhydroxyalkanoate. In other words, in the method for producing polyhydroxyalkanoate, a polyhydroxyalkanoate-producing bacterium is cultured in the culture step (i.e., the main culture method).

[0087] The polyhydroxyalkanoate-producing bacterium is not particularly limited as long as it is a microorganism capable of producing polyhydroxyalkanoate intracellularly. For example, microorganisms isolated from nature and deposited in a depository institution for strains (e.g., IFO, ATCC, etc.), or mutants and transformants prepared from such microorganisms, can be used. Specific examples of polyhydroxyalkanoate-producing bacteria include Bacillus megaterium, which was first discovered in 1925 and produces P3HB, a type of polyhydroxyalkanoate. Other examples include naturally occurring microorganisms such as Cupriavidus necator (formerly classified as Alcaligenes eutrophus and Ralstonia eutropha) and Alcaligenes latus. Examples of bacteria that produce copolymers of hydroxybutyrate, a type of polyhydroxyalkanoate, with other hydroxyalkanoates include Aeromonas caviae, which produces P3HB3HV and P3HB3HH, and Alcaligenes eutrophus, which produces P3HB4HB. In particular, with regard to P3HB3HH, Alcaligenes eutrophus AC32 (FERM BP-6038) (T. Fukui, Y. Doi, J. Bacteriol., 179, pp. 4821-4830 (1997)), into which genes encoding PHA synthases have been introduced, is a more preferred example of a polyhydroxyalkanoate-producing bacteria. In addition to the above, genetically modified microorganisms into which various polyhydroxyalkanoate synthesis-related genes have been introduced can also be used as polyhydroxyalkanoate-producing bacteria, depending on the composition, size, etc. of the polyhydroxyalkanoate to be produced.

[0088] (Recovery process) The method for producing a polyhydroxyalkanoate preferably includes a recovery step of recovering the polyhydroxyalkanoate from the cells of the polyhydroxyalkanoate-producing bacteria.

[0089] In the recovery step, the method for recovering polyhydroxyalkanoate from the polyhydroxyalkanoate-producing bacteria is not particularly limited. For example, polyhydroxyalkanoate can be recovered by a method including the following steps: Inactivation process: A process for inactivating polyhydroxyalkanoate-producing bacteria Alkali treatment process: A process of adding an alkaline aqueous solution to a culture solution containing polyhydroxyalkanoate-producing bacteria. Enzyme treatment step: a step of adding an enzyme to a culture solution containing a polyhydroxyalkanoate-producing microorganism to enzymatically treat the microorganism. Oxidation treatment process: A process of adding an oxidizing agent to a culture solution containing polyhydroxyalkanoate-producing bacteria. - Surfactant addition process: A process of adding a surfactant to a culture solution containing polyhydroxyalkanoate-producing bacteria. Centrifugation step: A step of centrifuging the aqueous suspension obtained in the above step. Membrane filtration process: A process of membrane filtration of the aqueous suspension obtained in the above process. Drying step: A step of drying the aqueous suspension or filter cake obtained in the above step to obtain a polyhydroxyalkanoate powder.

[0090] The present invention is not limited to the above-described embodiments, and various modifications are possible within the scope of the claims. Embodiments obtained by appropriately combining the technical means disclosed in different embodiments are also included in the technical scope of the present invention. [Example]

[0091] The present invention will be explained in more detail with reference to the following examples and comparative examples, but the present invention is not limited to these examples, and examples obtained by appropriately combining the technical means disclosed in each example are also included in the scope of the present invention.Unless otherwise specified, each operation below was carried out under conditions of 23°C and a relative humidity of 60%.

[0092] [Measurement method] (Volume average particle size of lipids (D50)) The volume-average particle diameter (D50) of lipids was calculated by photographing lipid droplets in a liquid using a probe-type video microscope (PVM (registered trademark) V819, manufactured by METTLER TOLEDO), measuring the particle diameters of at least 100 lipid droplets captured in the photographed image, calculating the volume of each droplet from the obtained particle diameters, and accumulating these to obtain the particle diameter at 50% of the cumulative volume.

[0093] Example 1 (Miniaturization test) An oil dispersion was prepared using a production apparatus (production apparatus A) equipped with a vessel equipped with a stirrer and a two-fluid nozzle, as shown in Figure 1. Specifically, 80 L of water (25°C) was placed in a 200 L vessel, and liquid palm oil (a fat with a solubility in water at 25°C of 0.2% and a melting point of 30°C) was sprayed into the vessel at a linear velocity of 1.41 m / sec from the two-fluid nozzle, simultaneously with room-temperature air at a linear velocity of 106 m / sec, until the amount of palm oil reached 1.0 wt% relative to the liquid volume in the vessel. The two-fluid nozzle used was an external-mixing two-fluid spray nozzle (two-fluid nozzle A, liquid supply diameter 1.0 mm, gas supply diameter 2.0 mm, manufactured by Spraying Systems) with a circular spray pattern and stainless steel nozzle material. Simultaneously with the spraying of palm oil into the vessel, the stirrer was started, and the water (and palm oil) in the vessel was stirred at a stirring speed of 500 rpm using the stirring blades attached to the stirrer, as shown in Figure 2. The impeller used (Impeller 1, manufactured by Satake Multimix) had a blade diameter of 187.5 mm and consisted of six uniformly spaced blades, each consisting of an upper and lower blade with a first gap of 30 mm, a second gap of 10 mm, and a blade width of 20 mm. Two impellers were fixed to a rotating shaft located in the center of the vessel. The lower impeller was located 100 mm above the bottom of the vessel, and the upper impeller was fixed 260 mm above the lower impeller. The temperature of the culture solution during stirring was 34°C. This procedure resulted in a dispersion of palm oil in water.

[0094] When the oil droplets in the obtained palm oil dispersion were measured using a probe-type video microscope (PVM (registered trademark) V819, manufactured by METTLER TOLEDO), the volume median diameter (D50) was found to be 29 μm, indicating that the palm oil droplets had been sufficiently refined. Furthermore, even after stirring was stopped, the droplets did not coalesce and remained in a stably refined state.

[0095] (microbial culture) ·Seed culture First, a glycerol stock (50 μL) of the KNK-631 strain, a polyhydroxyalkanoate-producing bacterium, was inoculated into a seed medium (10 mL) and cultured at 30° C. for 24 hours to perform seed culture.

[0096] The seed 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, with a pH of 6.8.

[0097] ·Preculture The seed culture obtained by the above procedure was inoculated at 1.0 v / v% into a 10 L jar fermenter (Marubishi Bioengine MDL-300 model) containing 3.5 L of pre-culture medium. The operating conditions of the jar fermenter were a culture temperature of 30°C, an agitation speed of 400 rpm, and an aeration rate of 4.0 L / min, and the pre-culture was carried out by culturing for 24 hours while controlling the pH at 6.5. A 25% aqueous ammonium hydroxide solution was used to control the pH.

[0098] 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, 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 oil was added as a carbon source at a concentration of 10 g / L.

[0099] ·Main culture The preculture solution obtained by the above procedure was inoculated at 1.0 v / v% into the container of the aforementioned production equipment A, which was pre-filled with the main culture medium. Melted palm oil heated to 50°C was sprayed into the culture solution using a two-fluid nozzle A equipped on the production equipment A, while controlling the palm oil concentration in the culture solution to 1.0 v / v%. During the cultivation, a phosphoric acid solution was sprayed into the solution at a constant rate to initiate microbial cultivation. During cultivation, the impeller was rotated at a speed of 500 rpm to continuously agitate the culture solution. Other operating conditions for the production equipment A were a cultivation temperature of 34°C, an aeration rate of 60 L / min, and pH controlled at 6.5. A 25% ammonium hydroxide solution was used to control pH. The main cultivation was continued for 28 hours, and the ratio of fatty acid concentration (FFA) to the total concentration (Total-C) of palm oil and the carbon components produced by its decomposition (FFA / Total-C) remaining in the culture solution was measured at the end of the cultivation. During the main culture, the height of the foam surface in the culture medium was measured over time as a percentage of the height of the foam surface relative to the height of the vessel. The results are shown in Table 1 and Figure 4.

[0100] The composition of the culture medium was 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).

[0101] Example 2 (Miniaturization test) Instead of impeller 1, a culture apparatus (culture apparatus B) having the same structure as the above-mentioned manufacturing apparatus A was used, except that a stirring impeller (stirring impeller 2, manufactured by Satake Multimix) with a blade diameter of 200.0 mm and six blade bodies consisting of upper and lower blades uniformly arranged, with a first gap of 30 mm, a second gap of 10 mm, and a blade width of 20 mm, was fixed to the rotating shaft.A dispersion in which palm oil was dispersed in water was obtained by the same operation as in Example 1, except that manufacturing apparatus B was used.

[0102] When the oil droplets in the obtained palm oil dispersion were measured using a probe-type video microscope, the volume median diameter (D50) was found to be 29 μm, indicating that the palm oil droplets were sufficiently micronized, similar to Example 1. Furthermore, even after stirring was stopped, the droplets did not coalesce and maintained a stably micronized state.

[0103] (Main culture) The polyhydroxyalkanoate-producing strain KNK-631 was cultured in the same manner as in Example 1, except that the above-mentioned culture apparatus B was used as the culture apparatus for the main culture (i.e., agitator impeller 2 was used as the agitator impeller). At the end of the culture, the ratio (FFA / Total C) of the fatty acid concentration (FFA) to the concentration (Total-C) of palm oil remaining in the culture broth and carbon components produced by its decomposition was measured. Furthermore, during the main culture, the foam surface height of the culture broth was continuously measured as the ratio of the foam surface height to the height of the vessel. The results are shown in Table 1 and Figure 4.

[0104] Example 3 (Miniaturization test) A culture apparatus (culture apparatus B) having the same structure as the above-mentioned manufacturing apparatus A, except that instead of the lower one of the two stirring blades 1, stirring blade 2 was fixed to the rotating shaft, was used, and a dispersion liquid in which palm oil was dispersed in water was obtained by the same operation as in Example 1, except that manufacturing apparatus B was used.

[0105] When the oil droplets in the obtained palm oil dispersion were measured using a probe-type video microscope, the volume median diameter (D50) was found to be 29 μm, indicating that the palm oil droplets were sufficiently finely divided, similar to Examples 1 and 2. Furthermore, even after stirring was stopped, the droplets did not coalesce and remained in a stably finely divided state.

[0106] (Main culture) The polyhydroxyalkanoate-producing strain KNK-631 was cultured in the same manner as in Example 1, except that the above-mentioned culture apparatus C was used as the culture apparatus for the main culture (i.e., agitator blade 1 was fixed to the upper side of the rotating shaft and agitator blade 2 was fixed to the lower side). The ratio (FFA / Total C) of the fatty acid concentration (FFA) to the concentration (Total-C) of palm oil and carbon components produced by its decomposition remaining in the culture solution at the end of the culture was measured. Furthermore, during the main culture, the foam surface height of the culture solution was continuously measured as the ratio of the foam surface height to the height of the container. The results are shown in Table 1 and Figure 4.

[0107] Comparative Example 1 (Miniaturization test) An attempt was made to prepare a dispersion in which palm oil was dispersed in water by the same procedure as in Example 1, except that a culture apparatus (culture apparatus D) having the same structure as the above-mentioned manufacturing apparatus A was used, except that six flat disc turbine blades (6FT) having the structure shown in Figure 3 were fixed to the rotating shaft instead of the stirring blade 1, and no gas was supplied from the two-fluid nozzle (i.e., only palm oil was supplied directly).

[0108] When the oil droplets in the resulting palm oil dispersion were measured using a probe-type video microscope, the volume median diameter (D50) was found to be 45 μm. Furthermore, the droplets coalesced immediately after stirring was stopped, and the finely divided state could not be maintained.

[0109] As shown in Figure 3, the six flat disc turbine blades 400 used in Comparative Example 1 comprise a fixed portion 401 fixed to a rotating shaft, a disk portion 402 provided on the fixed portion 401 and perpendicular to the rotating shaft, and a plurality of blade body portions 403 each consisting of a single flat plate arranged radially along the radial direction of the disk portion 402 and intersecting the outer periphery of the disk portion 402.

[0110] (Main culture) The polyhydroxyalkanoate-producing strain KNK-631 was cultured in the same manner as in Example 1, except that the culture apparatus used for the main culture was the above-mentioned culture apparatus D (i.e., a 6FT stirring blade) and gas was not supplied from the two-fluid nozzle. At the end of the culture, the ratio of the fatty acid concentration (FFA) to the total concentration (Total-C) of palm oil and the carbon components produced by its decomposition remaining in the culture solution (FFA / Total-C) was measured. During the main culture, the foam surface height of the culture solution was continuously measured as the ratio of the foam surface height to the height of the vessel. The results are shown in Table 1 and Figure 4.

[0111] Comparative Example 2 (Miniaturization test) A dispersion liquid in which palm oil was dispersed in water was prepared in the same manner as in Example 1, except that the culture apparatus D was used instead of the culture apparatus A.

[0112] When the oil droplets in the obtained palm oil dispersion were measured using a probe-type video microscope, the volume median diameter (D50) was found to be 29 μm, indicating that the palm oil droplets were sufficiently finely divided, similar to Examples 1 to 3. Furthermore, even after stirring was stopped, the droplets did not coalesce and maintained a stable finely divided state.

[0113] (Main culture) The polyhydroxyalkanoate-producing strain KNK-631 was cultured in the same manner as in Example 1, except that the above-mentioned culture apparatus D was used as the culture apparatus for the main culture (i.e., 6FT stirring blades were used). The ratio (FFA / Total C) of the fatty acid concentration (FFA) to the concentration (Total-C) of palm oil and carbon components produced by its decomposition remaining in the culture solution at the end of the culture was measured. Furthermore, during the main culture, the foam surface height of the culture solution was continuously measured as the ratio of the foam surface height to the height of the vessel. The results are shown in Table 1 and Figure 4.

[0114] Comparative Example 3 (Miniaturization test) An attempt was made to prepare a dispersion in which palm oil was dispersed in water by the same procedure as in Example 1, except that no gas was supplied from the two-fluid nozzle (only palm oil was supplied directly).

[0115] When the oil droplets in the resulting palm oil dispersion were measured using a probe-type video microscope, the volume median diameter (D50) was found to be 58 μm. Furthermore, the droplets coalesced immediately after stirring was stopped, and the finely divided state could not be maintained.

[0116] (Main culture) The polyhydroxyalkanoate-producing strain KNK-631 was cultured in the same manner as in Example 1, except that gas was not supplied from the two-fluid nozzle. The ratio (FFA / Total C) of the fatty acid concentration (FFA) to the total concentration (Total-C) of palm oil and carbon components produced by its decomposition remaining in the culture solution at the end of the culture was measured. Furthermore, during the main culture, the foam surface height of the culture solution was continuously measured as the ratio of the foam surface height to the height of the vessel. The results are shown in Table 1 and Figure 4.

[0117] [Table 1]

[0118] 〔summary〕 As is clear from Table 1, it was shown that supplying lipids (palm oil) into the culture solution by submerged spraying using a two-fluid nozzle makes it possible to disperse the lipids in the culture solution as fine droplets with a sufficiently small median volume diameter, and that finely dispersing lipids in the culture solution increases the FFA / Total C. Note that a higher FFA / Total C indicates that lipids have been metabolized and broken down into fatty acids, i.e., that the microorganisms are assimilating lipids more efficiently.

[0119] Furthermore, as is clear from Figure 4, stirring using the impeller having the structure shown in Figure 2 can suppress the rise in the liquid level of the culture solution, i.e., it can suppress foaming of the culture solution, compared to when the commonly used impeller having the structure shown in Figure 3 is used.

[0120] From the above, it has been shown that this production method (1) makes it possible to finely disperse lipids, allowing microorganisms to efficiently assimilate lipids, and further (2) suppresses foaming of the culture medium due to the surfactant action of fatty acids derived from lipids, thereby enabling more efficient substance production by microorganisms. [Industrial Applicability]

[0121] According to one aspect of the present invention, there is provided a method for culturing a microorganism, which can micronize lipids in a culture solution while suppressing foaming of the culture solution. By utilizing one aspect of the present invention, it is possible to efficiently culture the microorganism and produce a substance by the microorganism. [Explanation of symbols]

[0122] 1 container 2 Culture solution 3. Mixer 4. Two-fluid nozzle 5 Ventilation ring sparger 6 Exhaust line 7. Probe-type video microscope 8 Flow control valve 9. Oil Tank 10. Flow control valve 11 Pressure gauge 31 Rotation axis 32 Mixing blade 33 Drive unit 41 Liquid supply port 42 Gas supply port 43 Lipid droplets 51 Ventilated Air 100 Spray culture device 321 Fixed part 322 Disc Section 323a upper wing 323b lower wing 324 Gap 325 Wing body

Claims

1. A method for culturing a microorganism, comprising: a filling step of filling the culture solution containing the microorganism into a container equipped with a stirrer; a dispersing step of dispersing the lipids in the culture solution by submerged spraying of the lipids together with a gas into the culture solution using a two-fluid nozzle; a stirring step of stirring the culture solution with a stirring blade fixed to the stirrer, The stirring blade is a fixing portion fixed to the rotating shaft of the agitator; a disk portion provided on the fixed portion and perpendicular to the rotation axis; A method for culturing microorganisms, comprising a pair of upper and lower blades arranged along the radial direction of the disk portion, forming a gap between the opposing surfaces of the upper and lower blades, the gap being inclined so as to widen in the rotation direction of the agitator blade, and comprising a plurality of blade body portions arranged radially so as to intersect with the outer periphery of the disk portion.

2. 2. The method for culturing microorganisms according to claim 1, wherein the ratio (d / D) of the blade diameter d of the stirring blade to the inner diameter D of the container is 0.35 to 0.

5.

3. 2. The method for culturing microorganisms according to claim 1, wherein the lipids have a solubility in water of 1% or less.

4. 2. The method for culturing microorganisms according to claim 1, wherein the lipids are at least one type of lipid selected from the group consisting of oils and fats derived from palm oil, oils and fats derived from plants and animals, edible oils and fats, and waste edible oil.

5. 2. The method for culturing a microorganism according to claim 1, wherein the microorganism is a microorganism that produces polyhydroxyalkanoates.

6. A method for producing a polyhydroxyalkanoate, which comprises, as one step, the method for culturing a microorganism according to any one of claims 1 to 5.

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