WO2020036216A1

WO2020036216A1 - Method for culturing heterotrophic microalga using palm oil mill effluent (pome), and method for producing dha

Info

Publication number: WO2020036216A1
Application number: PCT/JP2019/032054
Authority: WO
Inventors: 清志多田; 信渡邉; 吉田　昌樹; 順子伊藤; 敏秀中島; ゴータママイケル
Original assignee: ＭｏＢｉｏｌ株式会社
Priority date: 2018-08-16
Filing date: 2019-08-15
Publication date: 2020-02-20
Also published as: JP6884451B2; MY188642A; JPWO2020036216A1

Abstract

The present invention addresses the problem of providing: a culture method whereby it becomes possible to proliferate a microalga with high efficiency using a POME discharged in a palm oil production process; and a method for producing a high-value-added valuable substance from a microalga with high efficiency. By using a POME hydrolysate, it becomes possible to proliferate a heterotrophic microalga capable of producing a ω-3 polyunsaturated fatty acid such as docosahexaenoic acid (DHA) satisfactorily with high efficiency, and it also becomes possible to improve the productivity of DHA.

Description

Method for culturing heterotrophic microalgae using palm oil mill effluent (POME) and method for producing DHA

The present invention provides a method for culturing heterotrophic microalgae and a method for producing DHA, using a palm oil mill effluent (Palm Oil Mill Effluent: POME) as a medium.

国 In countries such as Indonesia and Malaysia where the production of palm oil extracted from oil palm fruits is a key industry, palm oil factory effluent (hereinafter referred to as POME) is a major environmental and economic problem. For example, in Indonesia, about 455,000 t / day of POME is released from a palm oil manufacturing plant into a lagoon and is subjected to oxidation pond treatment → anaerobic treatment → aerobic treatment. In this process, fermentation gas causes odor, damage to insects and pests, water pollution, and the like, and methane gas generated by anaerobic treatment is released to the atmosphere. Methane has a very large global warming potential, 21 times that of carbon dioxide, and has a negative impact on global warming. As described above, POME causes emission of warming substances and water pollution, causing widespread and serious environmental problems such as global warming, deterioration of living environment, and adverse effects on wildlife. For example, since the palm oil production industry is a key industry in Indonesia, Malaysia, etc., solving the environmental problems of POME is crucial for the sustainable development of the national society where the palm oil industries, such as Indonesia and Malaysia, are thriving. Is an important issue.

As a method for solving the environmental problem of POME, a method is known in which methane gas is generated in an anaerobic state as described above, and the methane gas is collected and biogas power is generated (Patent Documents 1 and 2). However, since the amount of biogas power generation is not so high, profitability is poor and most of them are not feasible as a business. Therefore, there is still a strong need for a new method of using POME locally.

Conventionally, microalgae have been proliferated, and lipids or proteins, vitamins, and other valuable materials used as raw materials for fuels, feeds, health foods, and the like have been produced and stored in the cells of the microalgae. It is used for fuel and food. In particular, production and storage of high value-added valuables such as ω-3 polyunsaturated fatty acids have attracted attention as a component that can be immediately industrialized.

増殖 The growth of microalgae is also used in wastewater treatment such as purification of sewage and industrial wastewater (Patent Literature 3) and exhaust gas treatment for absorbing and utilizing carbon dioxide (Patent Literature 4). However, if the microalgae do not proliferate efficiently, the amount of valuables stored in the cells will not always be sufficient, and there is a risk that effective utilization of the valuables will be difficult. It has been reported that microalgae such as chlorella proliferate using POME (Non-Patent Documents 1-5). However, microalgae are low in algal biomass productivity and lipid productivity and poor in POME utilization efficiency. The production method of high value-added valuables from has not been established.

: Republished 2017-026370 : Patent No. 6049937 : Japanese Patent Laid-Open No. 05-301097 : JP 2010-022331 A

In view of the above-mentioned problems, the present invention provides a culture method capable of efficiently growing microalgae using POME discharged from a palm oil production process, and efficiently producing high value-added valuables from microalgae. It is an object to provide a production method.

The present inventors have conducted intensive studies in order to improve the utilization efficiency of POME. Heterotrophic production of ω-3 polyunsaturated fatty acids such as DHA (docosahexaenoic acid) by using POME hydrolyzate has been studied. The present inventors have found that microalgae are efficiently and favorably proliferated, and that the production amount of DHA is improved, and completed the present invention.

The present invention relates to a culture method and a DHA production method capable of efficiently growing heterotrophic microalgae producing ω-3 polyunsaturated fatty acids such as DHA (docosahexaenoic acid) by using a POME hydrolyzate. provide.

That is, the method for culturing heterotrophic microalgae according to the present invention for solving the above-mentioned problems is characterized in that a POME hydrolyzate obtained by hydrolyzing a POME containing a squeezed juice obtained by squeezing oil from palm fruit in a palm oil production process is used. , Wherein microalgae producing high value-added components are heterotrophically cultured.

において The method for culturing microalgae according to the present invention is characterized in that a POME hydrolyzate is supplied.

Accordingly, the present application provides the following inventions.
(1) A method for culturing heterotrophic microalgae using a medium containing a POME hydrolyzate.
(2) The culture method according to (1), wherein the POME hydrolyzate is a POME hydrolyzate obtained by a chemical, physical, and / or biological treatment.
(3) The culture method according to (2), wherein the POME hydrolyzate obtained by the chemical treatment is an acid or alkali POME hydrolyzate.
(4) The culture method according to (3), wherein the acid is sulfuric acid, hydrochloric acid, and / or phosphoric acid.
(5) The culture method according to (3), wherein the alkali is sodium hydroxide, potassium hydroxide, and / or ammonia.
(6) The culture method according to (2), wherein the POME hydrolyzate obtained by the physical treatment is a POME hydrolyzate obtained by heat and / or pressure.
(7) The culture method according to (2), wherein the POME hydrolyzate obtained by the biological treatment is a POME hydrolyzate obtained by peptidase or amylase.
(8) The culture method according to any one of (1) to (7), wherein the heterotrophic microalgae is an algae that produces ω-3 polyunsaturated fatty acids.
(9) The culture method according to (8), wherein the ω-3 polyunsaturated fatty acid is DHA.
(10) The culture method according to any one of (1) to (9), wherein the algae is a strain of algae belonging to Thraustochytriales.
(11) The culture method according to (10), wherein the Thraustochytriales is a species belonging to the genus Aurantiochytrium.
(12) The culture method according to (11), wherein the algae of the genus Aurantiochytrium are culture strains belonging to Aurantiochytrium limacinum.
(13) The culture method according to (11), wherein the algae of the genus Aurantiochytrium are culture strains belonging to Aurantiochytrium mangrovei.
(14) The culture according to (12), wherein the cultured strain of Aurantiochytrium limacinum is any one selected from 4W-1b strain, Aurantiochytrium limacinum SR-21 strain, and Aurantiochytrium limacinum NIES3737 strain. The culture method as described above.
(15) The culture method according to (13), wherein the cultured strain of Aurantiochytrium mangrovey is an Aurantiochytrium mangrovey strain 18W-13a.
(16) The culture method according to any one of (1) to (15), wherein the POME hydrolyzate has a solid content removed by centrifugation and / or filtration.
(17) The culture method according to any one of (1) to (16), wherein the medium further contains a sugar component.
(18) The culture according to (17), wherein the sugar component is one or more sugars selected from glucose, galactose, fructose, maltose, sucrose, lactose, oligosaccharides, sugar alcohols such as glycerol, and the like. Method.
(19) The culture method according to (17) or (18), wherein the total amount of the sugar component is 10 to 30 g / medium L.
(20) The culture method according to any one of (1) to (19), wherein the medium further includes a mineral component.
(21) The mineral component is a sulfate such as potassium sulfate, magnesium sulfate, iron sulfate, ammonium sulfate, copper sulfate, nickel sulfate or zinc sulfate; a phosphate such as potassium phosphate; a carbonate such as calcium carbonate; Chlorides such as manganese chloride, sodium chloride and calcium chloride; selenites and molybdates such as sodium molybdate and sodium selenite; halides such as potassium bromide and potassium iodide; natural seawater salts; The culture method according to (20), which is a single salt or a plurality of salts selected from artificial seawater salts.
(22) The culture method according to (20) or (21), wherein the total amount of the mineral components is 5 to 35 g / medium L.
(23) DHA production, comprising collecting heterotrophic microalgae cultured by the culture method according to any one of (1) to (22), and extracting DHA from the collected alga. Method.

According to the present invention, DME can be produced as a valuable resource by efficiently culturing heterotrophic microalgae while effectively utilizing POME.

Surprisingly, when the medium containing POME hydrolyzate is used, the Aurantiochytrium perimacinum 4W-1b strain grows at a higher growth rate than when the medium containing only POME non-hydrolyzate is used. To achieve a high concentration of DHA production.

FIG. 1 shows the Aurantiochi using a medium to which 50% POME sulfuric acid and a hydrolyzate by heating, a 50% POME hydrolyzate by heating, and a 50% POME (control) without hydrolysis were added. The algal cell concentration of Thorium perimacinum 4W-1b strain is shown (OD660). FIG. 2 shows the Aurantiochi using a medium to which 50% POME sulfuric acid and a hydrolyzate obtained by heating, a 50% POME hydrolyzate obtained by heating, and a 50% POME (control) not subjected to hydrolysis were added. 3 shows the total lipid concentration and DHA concentration of Thorium limacinum 4W-1b strain.

In general, palm oil production processes include steam treatment for inactivating palm fruit clusters (Fresh Fruit Bunch: FFB), separation of palm fruit and palm empty clusters (Empty Fruit Bunch: EFB), and fruit parts from palm fruit ( It goes through a pretreatment process of separation of mesoca) and seed part. The process proceeds to the extraction and separation and purification of crude palm oil from the fruit, producing high-purity palm oil. POME in the present invention means used steam water, a mixed effluent of oil juice and unrecovered oil, and / or unrecovered oil obtained by centrifuging the above-mentioned mixed liquid. This refers to the discharged liquid that has been removed or recovered (temperature is 70-90 ° C), and the discharged liquid that is discharged after being cooled and then subjected to oxidation pond treatment → anaerobic treatment → aerobic treatment. POME is rich in carbohydrates, organic acids, vitamins, amino acids, peptides, proteins, minerals, etc., derived from raw materials. POME used for algae culture may be before or after the oxidation pond treatment, anaerobic treatment, or aerobic treatment. In some embodiments, it is preferable to use POME before performing these oxidation pond treatment, anaerobic treatment, and aerobic treatment.

@POME contains various solids derived from raw materials. When culturing the DHA-producing algae, a POME hydrolyzate may be prepared using POME containing a solid substance and used directly in the medium. Alternatively, POME obtained by hydrolyzing and removing solid matter by a known means such as centrifugation or filtration may be used. Alternatively, the solid content may be removed from the POME by a known means such as centrifugation or filtration, and the solid content may be hydrolyzed and added to the supernatant POME for use in the medium. However, the POME hydrolyzate may or may not contain solids such as non-hydrolyzed solids, and is not limited.

POME hydrolyzate can be prepared by any means. For example, chemical treatment with an acid such as sulfuric acid, hydrochloric acid or phosphoric acid, or an alkali such as sodium hydroxide, potassium hydroxide, or ammonia; physical treatment with heat, pressure, or the like; biological treatment with an enzyme such as peptidase or amylase Examples include, but are not limited to, hydrolysis such as treatment. In the present application, the term “peptidase” refers to an enzyme that catalyzes a hydrolysis reaction of a peptide bond, is synonymous with “protease”, and is used interchangeably.

The hydrolysis may be performed by arbitrarily combining chemical treatment, physical treatment, biological treatment and the like. For example, POME may be hydrolyzed by combining a chemical treatment with an acid or an alkali and a physical treatment such as heating. For example, in one embodiment, a medium containing sulfuric acid and / or heated POME hydrolyzate is used. When POME is hydrolyzed by an acid or alkali chemical treatment, the concentration of the acid or alkali to be used is not limited, and examples thereof include 1%, 2%, 3%, 5%, 10%, and the like. In certain embodiments, the concentration of the acid or alkali may be in the range of 0.1-10%, 0.3-8%, 0.5-5%, 1-3%, etc. by volume. When POME is hydrolyzed by heating, the heating temperature is not limited, but for example, at least 40 to 374 ° C, 40 to 300 ° C, 45 to 250 ° C, 50 to 200 ° C, 60 to 180 ° C, 70 to 170 ° C, 80 to 80 ° C. To 160 ° C, 90 to 150 ° C, 100 to 140 ° C, 110 to 130 ° C, 115 to 125 ° C, and the like. When POME is hydrolyzed by pressure, the pressure to be added is not limited, but for example, at least 1.0 atm to 5.0 atm, 1.5 atm to 4.0 atm, 2.0 atm to 3.0 atm, 5.0 atm to 50 atm, 50 atm To 100 atm, 100 to 150 atm, 150 to 200 atm, 200 to 218.0 atm, and the like. When the hydrolysis is carried out by a biological treatment with an enzyme such as a peptidase, at a concentration of 100 to 1000 Unit, 300 to 800 Unit, 400 to 700 Unit, for example, 60 to 20 ° C., 50 to 30 ° C., 40 to The reaction may be performed at a temperature at which the enzyme is activated, such as 35 ° C. or 37 ° C. The processing time for hydrolyzing POME is not limited, but is 1 to 4 weeks, 1 to 7 days, 1 to 24 hours, 20 to 100 minutes, 30 to 90 minutes, 40 to 80 minutes, 50 to 70 minutes, 55 Up to 65 minutes. For example, it can be arbitrarily set according to the hydrolysis treatment, such as setting a longer time for the biological treatment. When the above processes are combined, these processes may be performed simultaneously or in any order. However, the conditions such as the method, order, temperature, and time of the hydrolysis treatment are arbitrary as long as POME can be hydrolyzed, and are not limited to the above ranges.

For example, in some embodiments, 50% by volume POME is hydrolyzed by heating at 121 ° C. for 60 minutes, or by adding 1%, 2%, 3% by volume sulfuric acid, or 1%, 2%, After adding sulfuric acid of 3% or the like, a culture medium may be prepared by performing a hydrolysis treatment by heating at 121 ° C. for 60 minutes.

In the present invention, the medium containing the POME hydrolyzate may be sterilized by known means such as filtration sterilization, autoclave sterilization, boiling sterilization, and radiation sterilization, and may be sterilized by known means such as sodium hypochlorite or ozone treatment. May be sterilized.

において In the present invention, “using a medium containing a POME hydrolyzate” does not exclude the use of a non-POME hydrolyzate as long as the POME hydrolyzate is used in the medium. For example, a medium containing only POME non-hydrolyzate without POME hydrolyzate is prepared and cultured for a certain period of time, for example, 12 hours, 24 hours, 36 hours, 48 hours, and 60 hours, and then the POME hydrolyzate is added. The culture may be continued for a certain period of time, for example, 12 hours, 24 hours, 36 hours, 48 hours, and 60 hours, or vice versa. Alternatively, some or all of the POME in the medium may be hydrolyzed. The ratio of the POME hydrolyzate to the POME non-hydrolysate in the medium containing the POME hydrolyzate is arbitrary. For example, the ratio of the POME hydrolyzate to the total amount of the POME (the total of the POME hydrolyzate and the POME non-hydrolysate) is The percentage of the degradant may be at least 1%, at least 10%, at least 20%, at least 25%, at least 50%, at least 75%, at least 80%, 100% (by volume). In some embodiments, the proportion of POME hydrolyzate to the total amount of POME may be in the range of 1-100%, 10-90%, 20-80%, 25-75% (% by volume), and the like. In the present invention, the content of POME in the medium of the heterotrophic microalgae (the total amount of POME including the POME hydrolyzate and the POME non-hydrolyzate) is not limited, but, for example, at least 1%, at least in the medium. It may be at a concentration of 10%, at least 25%, at least 50%, at least 75%, 100% (% by volume). In certain embodiments, the content of POME in the medium (total POME) may be in the range of 1-100%, 10-90%, 20-80%, 25-75% (% by volume), and the like. .

In the present invention, by adding various additives to the medium containing the POME hydrolyzate, the medium may be prepared to have a composition suitable for culturing DHA-producing algae. Examples of the additive include saccharides, organic acids, inorganic acids, organic bases, inorganic bases, vitamins, amino acids, peptides, proteins, and minerals (including natural seawater, artificial seawater, and the like). In some embodiments, it may be preferable to add sugars and / or minerals. In some embodiments, it may be preferable not to add minerals. If no mineral is added, there are advantages in that metal corrosion of the apparatus can be prevented, wastewater treatment and discharge after culturing are easy, and the cost of the culture medium is kept low. Further, if necessary, the pH can be appropriately adjusted by adding an appropriate acid or base. The suitable pH of the medium depends on the type of DHA-producing algae to be cultured, and may be adjusted to, for example, pH 6 to pH 7.

The mineral component can be, but is not limited to, chemically defined inorganic salts, such as alkali and alkaline earth metal salts, and salts of other metals. Such inorganic salts include, for example, potassium sulfate, magnesium sulfate, iron sulfate, ammonium sulfate, or sulfates such as copper sulfate, nickel sulfate, and zinc sulfate; phosphates such as potassium phosphate; carbonates such as calcium carbonate; Chlorides such as cobalt chloride, manganese chloride, sodium chloride and calcium chloride; alkali metal oxides; selenites and molybdates such as sodium molybdate and sodium selenite; halogens such as potassium bromide or potassium iodide Or an artificial seawater salt such as Red @ Sea @ Salt (Red Sea Salt). The salts may be added in a total amount of 0.01 to 5.0, 5 to 40, 5 to 35, 10 to 30, or 10 to 25% (g / medium L). For example, in one embodiment, the salts are added in a total amount of 19.4% (19.4 g / L of medium).

In certain embodiments, no additional mineral components, eg, salts such as chlorides, are added to the medium. In such embodiments, the medium does not contain additional mineral components other than those originally contained in the POME hydrolyzate. When a DHA-producing algae such as Thraustochytriales is cultured without adding a mineral component to the medium of the present invention using a POME hydrolyzate, DHA productivity may increase. In the case of a medium that does not contain additional mineral components other than that originally contained in POME or the like, the concentration of minerals in such a medium may be, for example, a total of 6.0%, 5.0%, 4.0% in terms of NaCl amount. ‰, 3.0 ‰, 2.0 ‰, less than 1.0 ‰ (g / medium L), total less than 3.0 ‰, 2.0 ‰, 1.0 ‰ (g / medium L) in terms of Cl amount Can be

In one embodiment, the growth of the heterotrophic microalgae may be promoted by including a saccharide together with the POME hydrolyzate in the culture solution. Saccharides are a carbon source of heterotrophic microalgae, but when POME hydrolyzate does not contain sufficient saccharides available for microalgae, or promote growth of heterotrophic microalgae If desired, saccharides that can be used in any form by microalgae may be added to the culture solution.

Examples of sugars include, but are not limited to, one or more sugars selected from glucose, galactose, fructose, maltose, sucrose, lactose, oligosaccharides, and sugar alcohols such as glycerol. The saccharide may be added in a total amount of 5 to 40, 10 to 30, and 15 to 25 (g / medium L). For example, in one embodiment, the saccharide is added in a total amount of 20% (20 g / medium L).

The microalgae used in the present invention may be an algae that produces ω-3 polyunsaturated fatty acids such as DHA (hereinafter referred to as DHA-producing algae). Examples of DHA-producing algae include organisms belonging to Thraustochytriales, such as Aurantiochytrium, Schizochytrium, Thraustochytrium, Parietichytrium, and Parietichytrium. Organisms belonging to the genus Ulkenia or organisms belonging to the genus Oblongichytrium are exemplified.
Organisms belonging to the genus Aurantiochytrium include, for example, Aurantiochytrium limacinum, Aurantiochytrium mangrovei, and the like.
Examples of organisms belonging to the genus Schizochytrium include Schizochytrium aggregatum. For example, strains such as Schizochytrium sp. Maku-1 can be used.
Organisms belonging to the genus Thraustochytrium include, for example, Thraustochytrium aureum, Thraustochytrium pachydermum, Thraustochytrium aggregatum, and the like.
Organisms belonging to the genus Parietichytrium include, for example, Parietichytrium sarkarianum. For example, strains such as Parietichytrium sp. 6F-10b can be used.
Examples of the organism belonging to the genus Ulkenia include Ulkenia visurgensis and Ulkenia profunda. For example, strains such as Ulkenia sp. Yonez 6-9 can be used.
Examples of the organism belonging to the genus Oblongichytrium include Oblongichytrium multirudimentale, Oblongichytrium minutum, and the like. For example, strains such as Oblongichytrium sp. H9 can be used.
Particularly preferred are algae of the genus Aurantiochytrium, such as Aurantiochytrium Limasinum 4W-1b, Aurantiochytrium Limasinum SR-21, and Aurantiochytrium Limasinum NIES3737, and the like. Limasinum strains and Aurantiochytrium mangrovey strains such as Aurantiochytrium mangrovey strain 18W-13a can be used.

The cultivation of algae producing DHA or the like in the present invention is carried out by inoculating the algae on a medium containing the POME hydrolyzate prepared as described above and culturing them according to a standard method. Alternatively, as described above, culture may be performed for a certain period of time in a medium containing only the POME non-hydrolysate, and then culture may be continued for a certain period of time in a medium containing the POME hydrolysate, or vice versa. The culture conditions depend on the type of algae producing DHA or the like to be cultured, and the temperature is 5 to 40 ° C, preferably 10 to 35 ° C, particularly preferably 20 to 25 ° C, more preferably 25 ° C ± 1 ° C. Culture can be performed for 1 to 10 days, preferably 2 to 8 days, for example, 3 to 7 days, for example, 4 to 5 days, and can be performed by aeration-agitation culture, shaking culture, or static culture.

The production algae such as DHA used in the present invention may be cultured in a culture device having an appropriate cell culture means. "Cell culturing means" means a means having all functions for culturing cells, for example, a culturing tank, and the culturing tank includes a stirring device, a vibration device, a temperature control device, a pH control device, and a turbidity device. It may have one or more devices selected from a degree measuring device, a light control device, a device for measuring the concentration of a specific gas such as O ₂ and CO ₂ and a pressure measuring device. The culture tank may be the same tank as the concentration / separation tank, or may be a separate tank from the concentration / separation tank. When it is a separate tank from the concentration / separation tank, the tank may be connected by an appropriate means, for example, a flow path or the like.

The culture method is not limited as long as a medium containing the POME hydrolyzate is used. For example, after growing using a medium containing a POME hydrolyzate by a chemical treatment with sulfuric acid or the like and a physical treatment such as heating, the PME hydrolyzate containing only the physical treatment is subjected to the chemical treatment. Proliferation may be performed using a medium that does not contain a substance such as sulfuric acid used in the above. Alternatively, after growing using a medium containing POME hydrolyzate by chemical treatment, use a medium containing POME hydrolyzate by physical treatment only but not containing the substance used in chemical treatment. Propagation may be performed. Alternatively, after growing using a medium containing POME hydrolyzate by chemical treatment, physical treatment, biological treatment, etc., growth is performed using a medium containing no POME hydrolyzate. And vice versa.

The present invention further provides a DHA production method, wherein DHA-producing algae cultured by the above-described culture method are collected using a medium containing a POME hydrolyzate, and DHA is extracted from the collected DHA-producing algae. The DHA production method of the present invention is characterized in that DHA or the like-producing algae is produced by culturing DHA or the like-producing algae using a medium containing a POME hydrolyzate.

ＤDHA produced by the DHA-producing algae of the present invention can be extracted and analyzed by methods known to those skilled in the art. For example, when a DHA-producing algae is cultured and grown as described above, and the DHA-producing algae is recovered from the obtained culture solution, the DHA-producing algae can be recovered by an existing method such as centrifugation or filtration. The recovered DHA-producing algae pellets are dried by freeze-drying or drying by heating or the like, or the DHA-producing algal culture is concentrated, and then the undried wet alga is used for the DHA extraction step. Is also good.

ＤDHA can be extracted from the obtained dried algal cells or wet algal cells. The extraction method is not particularly limited, and can be performed by a known method such as extraction with an organic solvent, squeezing, or supercritical extraction. Examples of the organic solvent used in the organic solvent extraction method include hexane, acetone, chloroform, methanol, ethanol, diethyl ether and the like, and may be used alone or as a mixture of two or more liquids of a polar solvent and a nonpolar solvent. A liquid can also be used. Algae may be crushed before extraction, but is not limited to, chemical crushing with an alkali or acid, homogenizer or ultrasonic, mechanical crushing with a bead mill, biological crushing with an enzyme, etc., performed by known methods. May be. After the above-mentioned extraction, the obtained extract may be subjected to concentration and purification of DHA by a method known to those skilled in the art, for example, column chromatography using silica gel or acid clay, high-performance liquid chromatography, liquid-liquid distribution, urea addition. Concentration and purification may be performed using a known method such as a method.

産生 The DHA-producing algae may contain at least 5%, at least 15%, at least 20%, at least 25%, at least 30% (% by weight) of DHA based on its dry weight.

Next, the present invention will be described in more detail with reference to examples, but the present invention is not limited thereto.
(Example 1) Growth of Aurantiochytrium Limasinum 4W-1b strain in a medium in which glucose and salts were added to 50% POME hydrolyzate, production of total lipids and DHA (seed algae)
Aurantiochytrium limacinum 4W-1b strain belonging to Thraustochytriales provided by the University of Tsukuba in a GTY medium (containing 1 g of 1/2 diluted seawater, 20 g of glucose, 5 g of yeast extract, and 10 g of tryptone) in the following medium for 3 days It was cultured and used as a seed algae.

A high-temperature POME provided from a palm oil plant in Riau, Indonesia in April 2018 is immediately cooled in a refrigerator, stored for 3 days, and then hydrolyzed by heating at 121 ° C. for 60 minutes. Sulfuric acid was added so that the concentration became 1%, and hydrolysis was performed by the same heat treatment (121 ° C. for 60 minutes), and the hydrolysis was not performed at all, and the pH was adjusted to 7.0 with NaOH for each. The hydrolyzed supernatant obtained by centrifugation at 4000 rpm (2,700 g) for 15 minutes was subjected to a total of 19.4 g / L salts (15.5 g / L Na ₂ _{_{_{SO 4, CaCl 2 · 2H 2}}} O of _{_{0.69g / L, MgSO 4 · 7H}} 2 O of 5.74g / L, _KH 2 PO ₄ of 0.59 g / L) and the 20 g / L Those diluted in artificial seawater containing glucose was used as a medium.

２００ 200 ml of each of the above media was poured into a 500 ml Erlenmeyer flask, and sterilized in an autoclave at 120 ° C. for 20 minutes. After cooling the medium, the seed algae of Aurantiochytrium perimacinum 4W-1b strain was washed with sterilized seawater, and then seeded on the medium. The mouth of the flask was sealed with a silicon stopper, and cultured at 25 ° C. with reciprocal shaking (100 strokes / min).

１ 1 ml of the culture solution during the culture was collected at a predetermined time point, and the optical turbidity at 660 nm was measured with an ultraviolet-visible absorptiometer to obtain a growth curve. Cultures were performed for up to 60 hours until growth reached a plateau in all cultures. After the completion of the culture, 50 to 100 mL of the culture solution was collected and centrifuged at 3900 rpm for 15 minutes using a high-speed cooling centrifuge. Thereafter, the supernatant was discarded, the pellet was washed with distilled water, and centrifuged again. Thereafter, the sample was frozen at −80 ° C. and dried with a freeze dryer to obtain a dried alga body. The weight of the dried algal cells was measured with a microbalance, and the amount of algal cells per liter of the culture solution was calculated.

藻 Algal cells were recovered from 10 mL of the algal culture by centrifugation. The obtained alga body pellet was crushed by ultrasonic waves, and 20 mL of a mixed solution of chloroform / methanol (2: 1 by volume) was added, followed by thorough mixing. 4 mL of a 0.9% aqueous sodium chloride solution was added and mixed well, and after centrifugation, the chloroform layer was collected by filtration with filter paper. The filtrate was concentrated to dryness and the amount of lipid was measured. To the lipid, 0.2 mg of trichosane methyl ester was added as an internal standard substance, 0.5 mL of 0.5 M NaOH methanol solution was added, and a saponification reaction was performed at 100 ° C. for 9 minutes, followed by 14% boron trifluoride. 0.7 mL of a methanol solution was added, and a methyl esterification reaction was performed at 100 ° C. for 7 minutes. To the sample, 3 mL of saturated saline and 3 mL of hexane were added and mixed well. After centrifugation, the DHA content of the hexane layer was measured by GC (gas chromatography) analysis.

The above results are shown in FIGS.
FIG. 1 shows a medium obtained by hydrolyzing 50% by volume of POME by a 1% by volume sulfuric acid treatment and a heat treatment at 121 ° C., a medium hydrolyzed only by a heat treatment at 121 ° C., and a volume 50% without hydrolysis as a control. The change with time of the algal cell concentration of Aurantiochytrium Limasinum 4W-1b cultured in a% POME medium (hereinafter referred to as a control medium) is shown (OD660). Although the strain grew on all media, the hydrolyzed media showed significantly better growth than the control media. Furthermore, the culture medium hydrolyzed by sulfuric acid and heating showed better growth than the culture medium obtained by hydrolyzing POME only by heating.
FIG. 2 shows the total lipid and DHA production after 48 hours of culture. Lipids and DHA were produced in all media, but the amount of lipid production was about 1.3 times higher in the hydrolysis medium with 1% sulfuric acid and heating than in the control. Regarding the amount of DHA produced, the hydrolysis medium with 1% sulfuric acid and heating and the hydrolysis medium with heating alone were clearly higher than the control.
Table 1 shows the growth amount (g / L), algal productivity (mg / L / day), total lipid content (%), total lipid amount (mg / L), a table summarizing lipid productivity (mg / L / day), DHA content (%), DHA production amount (mg / L), and DHA productivity (mg / L / day). The productivity is a value obtained by converting the amount of algal bodies, the amount of total lipids, and the amount of DHA after 48 hours of growth per day. Each of the total lipid content and the DHA content indicates a ratio to the algal body content (% by weight).

Algae productivity and lipid productivity were highest in sulfuric acid + heated hydrolysis POME medium, while DHA productivity was highest in heated hydrolysis medium, followed by sulfuric acid + heated hydrolysis medium, and The control medium was the lowest.

Example 2 Growth of Aurantiochytrium Limasinum 4W-1b Strain in Medium Prepared Using 100% POME Hydrolyzed by Various Methods, Production of Total Lipids and DHA Various hydrolysis conditions were set. The growth of Aurantiochytrium Limasinum 4W-1b strain and total lipid and DHA production were examined.
As a seed strain, the same Aurantiochytrium limacinum 4W-1b strain as in Example 1 was used. As the POME, high-temperature POME provided from a palm oil factory in North Sumatra, Indonesia, which was rapidly cooled and stored frozen, was thawed at the start of the experiment and used.
The POME hydrolyzate is hydrolyzed by treating the thawed POME under the conditions shown in Table 2 below (experiment numbers 2 to 4 in Table 2), and after adjusting the pH to 7.0 with acid or alkali. The hydrolyzed POME supernatant (100% volume) obtained by centrifuging at 4000 rpm for 15 minutes was used. As a control (POME non-hydrolysate), only filtration was performed with a filter (0.45 μm) without hydrolysis at all (experiment number 1 in Table 2), and the pH was similarly adjusted to 7.0, followed by centrifugation at 4000 rpm for 15 minutes. The non-hydrolyzed POME supernatant (volume 100%) obtained by separation was used.
The culture was performed using a POME supernatant obtained by centrifuging the thawed POME at 4000 rpm for 15 minutes (volume: 100%: non-POME hydrolyzate) as an initial medium. After pouring 200 ml into the flask, sterilizing in an autoclave at 121 ° C. for 20 minutes and cooling, the seed strain was inoculated in a medium, the mouth of the flask was sealed with a silico stopper, and subjected to reciprocal shaking culture (25 strokes / 100 strokes / min) at 25 ° C. After culturing for a time, the above-mentioned various POME hydrolysates or 50 ml of control were added to the culture, and the culturing was continued.

After 60 hours from the start of the culture, 50 to 100 mL were collected in the same manner as in Example 1, and the growth amount (g / L), algal cell productivity (mg / L / day), total lipid content (%), and total lipid amount (%) were obtained. mg / L), lipid productivity (mg / L / day), DHA content (%), DHA production (mg / L), and DHA productivity (mg / L / day).

The results are shown in Tables 3 to 6. Table 3 shows the effect of physical treatments (hydrolysis by heat and pressure) comparing Run Nos. 2 (1) and (2) to Run No. 1. Table 4 shows the effect of the chemical treatment (hydrolysis with acid) comparing Experiment Nos. 3-1 (1), (2) and (3) to Experiment No. 1. Table 5 shows the effect of the chemical treatment (hydrolysis with alkali) comparing Experiment No. 3-2 (1) and (2) to Experiment No. 1. Table 6 shows the effect of biological treatment (enzymatic hydrolysis) comparing experiment numbers 4 (1) and (2) to experiment number 1. In all experiments, the production amount and productivity of alga bodies, lipids, and DHA were improved when the medium containing the POME hydrolyzate was used, as compared with the case where the control medium containing only the POME non-hydrolyzate was used.

The above results indicate that it is preferable to use a medium containing a POME hydrolyzate for the growth of heterotrophic microalgae including the Aurantiochytrium perimacinum 4W-1b strain and the production of lipid / DHA. Was.

Claims

培養 A method for culturing heterotrophic microalgae using a medium containing a POME hydrolyzate.
The culture method according to claim 1, wherein the POME hydrolyzate is a POME hydrolyzate obtained by a chemical, physical, and / or biological treatment.
The culture method according to claim 2, wherein the POME hydrolyzate obtained by the chemical treatment is an acid or alkali POME hydrolyzate.
The method according to claim 3, wherein the acid is sulfuric acid, hydrochloric acid, and / or phosphoric acid.
The method according to claim 3, wherein the alkali is sodium hydroxide, potassium hydroxide, and / or ammonia.
The culture method according to claim 2, wherein the POME hydrolyzate obtained by the physical treatment is a POME hydrolyzate obtained by heat and / or pressure.
The culture method according to claim 2, wherein the POME hydrolyzate obtained by the biological treatment is a POME hydrolyzate obtained by peptidase or amylase.
The culture method according to any one of claims 1 to 7, wherein the heterotrophic microalgae is an algae that produces ω-3 polyunsaturated fatty acids.
The culture method according to claim 8, wherein the ω-3 polyunsaturated fatty acid is DHA.
The culture method according to any one of claims 1 to 9, wherein the algae is a strain of algae belonging to Thraustochytriales.
The method according to claim 10, wherein the Thraustochytriales is a species belonging to the genus Aurantiochytrium.
The method according to claim 11, wherein the algae belonging to the genus Aurantiochytrium are culture strains belonging to Aurantiochytrium limacinum.
The culture method according to claim 11, wherein the algae belonging to the genus Aurantiochytrium are cultures belonging to Aurantiochytrium mangrovei.
13. The culture according to claim 12, wherein the cultured strain of Aurantiochytrium perimacinum is any strain selected from 4W-1b strain, Aurantiochytrium perimacinum SR-21 strain, and Aurantiochytrium perimacinum NIES3737 strain. Culture method.
The culture method according to claim 13, wherein the {Aulanthiochitrium} mangrovey strain is an Aurantiochytrium @ Mangrovey strain 18W-13a.
The culture method according to any one of claims 1 to 15, wherein the POME hydrolyzate has a solid content removed by centrifugation and / or filtration.
The culture method according to any one of claims 1 to 16, wherein the medium further contains a sugar component.
18. The culture method according to claim 17, wherein the sugar component is one or more sugars selected from glucose, galactose, fructose, maltose, sucrose, lactose, oligosaccharides, sugar alcohols such as glycerol, and the like.
The culture method according to claim 17, wherein the total amount of the sugar component is 10 to 30 g / medium L.
培養 The culture method according to any one of claims 1 to 19, wherein the medium further contains a mineral component.
The mineral components include sulfates such as potassium sulfate, magnesium sulfate, iron sulfate, ammonium sulfate, copper sulfate, nickel sulfate and zinc sulfate; phosphates such as potassium phosphate; carbonates such as calcium carbonate; cobalt chloride, manganese chloride Chlorides such as sodium chloride and calcium chloride; selenites and molybdates such as sodium molybdate and sodium selenite; halides such as potassium bromide and potassium iodide; natural seawater salts; and artificial seawater salts. The culture method according to claim 20, which is a single salt or a plurality of salts selected from the group consisting of:
The culture method according to claim 20, wherein the total amount of the mineral components is 5 to 35 g / medium L.
A method for producing DHA, comprising recovering a heterotrophic microalgae cultured by the culture method according to any one of claims 1 to 22, and extracting DHA from the recovered algae.