JP6884451B2 - Heterotrophic microalgae culture method and DHA production method using palm oil factory effluent (POME) - Google Patents

Description

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

In countries such as Indonesia and Malaysia where palm oil production extracted from oil palm fruits is a key industry, palm oil factory effluent (hereinafter referred to as POME) has become a major environmental and economic problem. For example, in Indonesia, about 455,000 tons / day of POME is discharged from a palm oil manufacturing plant into the lagoon and is treated by an oxide pond, anaerobic treatment, and aerobic treatment. In this process, the fermentation gas causes foul odors, damage to pests and vermin, water pollution, etc., and methane gas generated by anaerobic treatment is released into the atmosphere. The global warming potential of methane is 21 times higher than that of carbon dioxide, which has an adverse effect on global warming. In this way, POME causes the release of warming substances and water pollution, causing widespread and serious environmental problems such as global warming, deterioration of the 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 important for the sustainable development of a national society in which the palm oil industry is thriving, such as Indonesia and Malaysia. It has become an issue.

As a solution to the environmental problems of POME, since methane gas is generated in an anaerobic state as described above, a method of recovering these and performing biogas power generation is known (Patent Documents 1 and 2). However, since the amount of biogas power generation is not so high, it is not profitable and most of it cannot be established as a business. Therefore, there is still a strong need for new ways to use POME locally.

Conventionally, microalgae are grown to produce and store lipids that are raw materials for fuels, feeds, health foods, etc., or valuable resources such as proteins and vitamins in the cells of microalgae, and the stored valuable resources are used. It is used for fuel and food. In particular, the production and storage of high value-added valuables such as ω-3 polyunsaturated fatty acids are attracting attention as components that can be immediately industrialized.

This growth of microalgae is also used for wastewater treatment such as purification of sewage and industrial wastewater (Patent Document 3) and exhaust gas treatment for the purpose of absorbing and utilizing carbon dioxide (Patent Document 4). However, if the microalgae do not grow efficiently, the amount of valuable resources stored in the cells will not always be sufficient, which may make effective use of the valuable resources difficult. It has been reported that microalgae such as chlorella grow using POME (Non-Patent Document 1-5), but since algae biomass productivity and lipid productivity are low and POME utilization efficiency is poor, microalgae No method for producing high value-added valuables from Japan has been established.

: Republished 2017-0263 70 : Patent No. 6049937 : Japanese Patent Application Laid-Open No. 05-301097 : Japanese Patent Application Laid-Open No. 2010-022331

HABIB et. Al. “Growth and Nutritional Values of Moina micrura Fed on Chlorella vulgaris Grown in Digested Palm Oil Mill Effluent”, Asian Fisheries Science, 16, 107-119, 2003 Putri et. Al. “Investigation of Microalgae for High Lipid Content using Palm Oil Mill Effluent (Pome) as Carbon Source”, 2011 International Conference on Environment and Industrial Innovation, IPCBEE, 12, 85-89, 2011 Nwuche et. Al. “Use of Palm Oil Mill Effluent as Medium for Cultivation of Chlorella sorokiniana”, British Biotechnology Journal, 4 (3): 305-316, 2014 Nur et. Al. “Enhancement of Chlorella vulgaris Biomass Cultivated in POME Medium as Biofuel Feedstock under Mixotrophic Conditions”, Journal of Engineering and Technology Sciences, 47 (5), 487-497, 2015 Cheah et. Al. “Enhancing biomass and lipid productions of microalgae in palm oil mill effluent using carbon and nutrient supplementation”, Energy Conversion and Management, 164, 188-197, 2018

In view of the above problems, the present invention is a culturing method capable of efficiently growing microalgae using POME discharged from the palm oil manufacturing process, and efficiently producing high value-added valuables from microalgae. The challenge is to provide a method of production.

The present inventor has conducted diligent studies to improve the utilization efficiency of POME, and found that the use of POME hydrolyzate produces ω-3 polyunsaturated fatty acids such as DHA (docosahexaenoic acid). The present invention has been completed by discovering that microalgae can be efficiently and satisfactorily grown and that the amount of DHA produced is improved.

The present invention provides a culture method and a DHA production method capable of efficiently growing heterotrophic microalgae that produce 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 problems is a POME hydrolyzate obtained by hydrolyzing POME containing squeezed juice obtained by squeezing oil from palm fruit in the process of producing palm oil. It is characterized in that microalgae producing high value-added components are heterotrophically cultured using.

The method for culturing the microalgae according to the present invention is characterized by supplying a POME hydrolyzate.

Therefore, 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 chemical, physical, and / or biological treatment.
(3) The culture method according to (2), wherein the POME hydrolyzate by the chemical treatment is a POME hydrolyzate by an acid or an alkali.
(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 by the physical treatment is a POME hydrolyzate by heat and / or pressure.
(7) The culture method according to (2), wherein the POME hydrolyzate by the biological treatment is a POME hydrolyzate by peptidase or amylase.
(8) The culture method according to any one of (1) to (7), wherein the heterotrophic microalgae are algae that produce 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 are strains of algae belonging to Chytrids (Thraustochytriales).
(11) The culture method according to (10), wherein the chytrids are species belonging to the genus Aurantiochytrium.
(12) The culture method according to (11), wherein the algae of the genus Aurantiochytrium is a culture strain belonging to Aurantiochytrium limacinum.
(13) The culture method according to (11), wherein the algae of the genus Aurantiochytrium is a culture strain belonging to Aurantiochytrium mangrovei.
(14) The culture strain of Aurantiochytrium lymacinum is any of the strains selected from 4W-1b strain, Aurantiochytrium lymacinum SR-21 strain, or Aurantiochytrium lymacinum NIES3737 strain (12). The culture method described.
(15) The culture method according to (13), wherein the culture strain of Aurantiochytrium manglow bay is Aurantiochytrium manglow bay 18W-13a strain.
(16) The culture method according to any one of (1) to (15), wherein the POME hydrolyzate has its 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 added is 10 to 30 g / medium L.
(20) The culture method according to any one of (1) to (19), wherein the medium further contains a mineral component.
(21) The mineral component is a sulfate such as potassium sulfate, magnesium sulfate, iron sulfate, ammonium sulfate, copper sulfate, nickel sulfate, zinc sulfate; a phosphate such as potassium phosphate; a carbonate such as calcium carbonate; cobalt chloride. Chlorides such as manganese chloride, sodium chloride, calcium chloride; selenates and molybdates such as sodium molybdate, sodium selenate; halides such as potassium bromide, potassium iodide; 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 added is 5 to 35 g / medium L.
(23) DHA production including recovery of heterotrophic microalgae cultured by the culture method according to any one of (1) to (22) and extraction of DHA from the recovered algae. Method.

According to the present invention, POME can be effectively used, and DHA can be produced as a valuable resource by efficiently culturing heterotrophic microalgae.

Surprisingly, Aurantiochytrium lymasinum 4W-1b strain grows better when using a medium containing POME hydrolyzate than when using a medium containing only POME non-hydrolyzate. And achieved high concentration DHA production.

FIG. 1 shows Aurantiochytrium when a medium containing 50% POME sulfuric acid and a hydrolyzate by heating, a hydrolyzate by heating 50% POME, and 50% POME (control) without hydrolysis was used. The algae concentration of the trillium lymasinum 4W-1b strain is shown (OD660). FIG. 2 shows Aurantiochytrium when a medium containing 50% POME sulfuric acid and a hydrolyzate by heating, a hydrolyzate by heating 50% POME, and 50% POME (control) without hydrolysis was used. The total lipid concentration and DHA concentration of the thorium lymasinum 4W-1b strain are shown.

In general, the palm oil manufacturing process involves steaming for inactivating the Fresh Fruit Bunch (FFB), separating the Palm Fruit from the Empty Fruit Bunch (EFB), and the fruit portion from the palm fruit (EFB). It goes through a pretreatment process of separating the seeds from the mesoca). Crudo palm oil extraction from the fruit part and separation and refining proceed to produce high-purity palm oil. POME in the present invention refers to used steam water, a mixed drainage liquid of oil squeezed juice and unrecovered oil, and / or unrecovered oil produced by centrifuging the above mixed liquid, which is generated in the manufacturing process of this palm oil. The effluents that have been removed or recovered (temperature is 70-90 ° C), and the effluents that are discharged after cooling through oxidation pond treatment → anaerobic treatment → aerobic treatment. POME is rich in sugars, organic acids, vitamins, amino acids, peptides, proteins, minerals and the like derived from raw materials. The POME used for culturing algae may be before or after the oxide pond treatment, the anaerobic treatment, or the aerobic treatment. In certain embodiments, it is preferable to use POME before these pond treatments, anaerobic treatments, and aerobic treatments.

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

The POME hydrolyzate can be prepared by any means. For example, chemical treatment with acids such as sulfuric acid, hydrochloric acid, phosphoric acid, alkalis such as sodium hydroxide, potassium hydroxide, or ammonia; physical treatment with heat, pressure, etc .; biological treatment with enzymes such as peptidase, amylase, etc. Hydrolysis of treatment and the like can be mentioned, but the present invention is not limited to these. In the present application, the term "peptidase" refers to an enzyme that catalyzes the hydrolysis reaction of a peptide bond, and is synonymous with "protease" and is used interchangeably.

Hydrolysis may be carried out by any combination of 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 with heating or the like. For example, in some embodiments, a medium containing sulfuric acid and / or a heated POME hydrolyzate is used. When POME is hydrolyzed by chemical treatment with an acid or an alkali, the concentration of the acid or alkali used is not limited, and examples thereof include volumes of 1%, 2%, 3%, 5% and 10%. In certain embodiments, the acid or alkali concentration may be in the range of 0.1-10%, 0.3-8%, 0.5-5%, 1-3% and the like by volume. When the 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. Examples thereof include ~ 160 ° C., 90 to 150 ° C., 100 to 140 ° C., 110 to 130 ° C., 115 to 125 ° C. and the like. When the POME is hydrolyzed by pressure, the pressure applied 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. Examples thereof include ~ 100 atm, 100 atm to 150 atm, 150 atm to 200 atm, 200 atm to 218.0 atm, and the like. When hydrolyzed by biological treatment with an enzyme such as peptidase, the concentration is 100 to 1000 Unit, 300 to 800 Unit, 400 to 700 Unit, etc., for example, 60 to 20 ° C, 50 to 30 ° C, 40 to It may be carried out at a temperature such as 35 ° C., 37 ° C., etc. at which the enzyme is activated. The treatment time for hydrolyzing POME is not limited, but 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. ~ 65 minutes and the like can be mentioned. For example, the time of the biological treatment can be set to be longer, which can be arbitrarily set according to the hydrolysis treatment. When the above processes are combined, these processes may be performed at the same time or in any order. However, 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 range.

For example, in one embodiment, 50% volume POME is hydrolyzed by heating at 121 ° C. for 60 minutes, or by adding 1%, 2%, 3% sulfuric acid, or 1%, 2% volume. A medium may be prepared by adding 3% or more sulfuric acid and then hydrolyzing 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 known means such as sodium hypochlorite and ozone treatment. It may be sterilized with.

In the present invention, "using a medium containing a POME hydrolyzate" does not exclude the use of a POME non-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, 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, 60 hours, or vice versa. Alternatively, some or all of POME in the medium may be hydrolyzed. The ratio of POME hydrolyzate to POME non-hydrolyzate in the medium containing POME hydrolyzate is arbitrary, for example, POME water addition to the total amount of POME (total of POME hydrolyzate and POME non-hydrolyzate). The proportion of degradation products may be at least 1%, at least 10%, at least 20%, at least 25%, at least 50%, at least 75%, at least 80%, 100% (volume%). In certain embodiments, the ratio of the POME hydrolyzate to the total amount of POME may be in the range of 1-100%, 10-90%, 20-80%, 25-75% (volume%) and the like. Further, in the present invention, the content of POME in the medium of the heterotrophic microalgae (total amount of POME including POME hydrolyzate and non-POME hydrolyzate) is not limited, but for example, at least 1% in the medium, at least. The concentration may be 10%, at least 25%, at least 50%, at least 75%, 100% (volume%). In certain embodiments, the content of POME in the medium (total amount of POME) may be in the range of 1-100%, 10-90%, 20-80%, 25-75% (volume%) and the like. ..

In the present invention, by adding various additives to a medium containing a POME hydrolyzate, the medium may be prepared so as to have a composition suitable for culturing DHA-producing algae. Examples of the additive include sugars, organic acids, inorganic acids, organic bases, inorganic bases, vitamins, amino acids, peptides, proteins and minerals (including natural seawater and artificial seawater). 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 minerals are added, there are advantages that metal corrosion of the device can be prevented, wastewater treatment and discharge after culturing are facilitated, and medium cost 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.

Mineral components may be, but are not limited to, chemically defined inorganic salts such as alkaline and alkaline earth metal salts, as well as salts of other metals. Examples of such inorganic salts include 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, calcium chloride; alkali metal oxides; selenates and molybdates such as sodium molybdate and sodium selenate; halogens such as potassium bromide or potassium iodide Examples include single or multiple salts selected from the compounds; natural seawater salts; and artificial seawater salts such as Red Sea Salt; The salts may be added in a total amount of 0.01-5.0, 5-40, 5-35, 10-30, or 10-25 ‰ (g / medium L). For example, in certain embodiments, the salts are added in a total amount of 19.4 ‰ (19.4 g / medium L).

In certain embodiments, no additional mineral components, such as salts such as chlorides, are added to the medium. In such an embodiment, the medium contains no additional mineral components other than those originally contained in the POME hydrolyzate. DHA productivity may increase when DHA-producing algae such as Thraustochytriales are cultivated in the medium of the present invention using a POME hydrolyzate without adding mineral components. In the case of a medium containing no additional mineral components other than those originally contained in POME or the like, the mineral concentrations in such a medium are, for example, 6.0 ‰, 5.0 ‰, 4.0 in total in terms of NaCl amount. Less than ‰, 3.0 ‰, 2.0 ‰, 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 certain embodiments, the inclusion of sugars in the culture medium along with the POME hydrolyzate may promote the growth of the heterotrophic microalgae. Sugars are a carbon source for heterotrophic microalgae, but when the POME hydrolyzate does not contain sufficient sugars that can be used by microalgae, or promotes the growth of the heterotrophic microalgae. If desired, sugars that can be used by microalgae in some form may be added to the culture solution.

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

The microalgae used in the present invention may be algae that produce ω-3 polyunsaturated fatty acids such as DHA (hereinafter referred to as DHA or the like-producing algae). Examples of DHA-producing algae include Aurantiochytrium, Schizochytrium, Thraustochytrium, Parietichytrium, which are organisms belonging to Thraustochytriales. Organisms belonging to the genus Ulkenia or organisms belonging to the genus Aurantiochytrium can be mentioned.
Examples of organisms belonging to the genus Aurantiochytrium include Aurantiochytrium limacinum and Aurantiochytrium mangrovei.
Examples of organisms belonging to the genus Schizochytrium include Schizochytrium aggregatum. For example, strains such as Schizochytrium sp. Maku-1 can be used.
Examples of organisms belonging to the genus Thraustochytrium include Thraustochytrium aureum, Thraustochytrium pachydermum, and Thraustochytrium aggregatum.
Examples of organisms belonging to the genus Parietichytrium include 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 organisms belonging to the genus Oblongichytrium include Oblongichytrium multirudimentale and Oblongichytrium minutum. For example, strains such as Oblongichytrium sp. H9 can be used.
Particularly preferred are Aurantiochytrium algae, such as Aurantiochytrium Limasinum 4W-1b strain, Aurantiochytrium Limasinum SR-21 strain, and Aurantiochytrium Limasinum NIES3737 strain. A strain of Limasinum and a strain of Aurantiochytrium manglow bay such as Aurantiochytrium manglow bay 18W-13a strain can be used.

Culturing of DHA or the like-producing algae in the present invention is carried out by sowing the algae in a medium containing the POME hydrolyzate prepared as described above and culturing according to a conventional method. Alternatively, as described above, the culture may be carried out in a medium containing only the POME non-hydrolyzate for a certain period of time, and then continued in the medium containing the POME hydrolyzate for a certain period of time, or vice versa. The culture conditions depend on the type of DHA-producing algae to be cultured, and the temperature is usually 5 to 40 ° C., preferably 10 to 35 ° C., particularly preferably 20 to 25 ° C., more preferably 25 ° C. ± 1 ° C. The culture can be carried out 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 carried out by aeration stirring culture, shaking culture or static culture.

The DHA-producing algae used in the present invention may be cultured in a culture apparatus having a suitable cell culture means. The "cell culture means" means a means having all functions for culturing cells, for example, a culture tank, and the culture tank is a stirrer, a vibration device, a temperature control device, a pH control device, and a turbidity. degree measuring apparatus, the light control device, O _2, CO may have one or a plurality of devices is selected from the specific gas concentration measuring device and a pressure measuring device ₂ or the like. The culture tank may be the same as the concentration / separation tank, or may be a separate tank from the concentration / separation tank. When the tank is different from the concentration / separation tank, it may be connected by an appropriate means such as a flow path.

The culture method is not limited as long as a medium containing a POME hydrolyzate is used. For example, after proliferation using a medium containing a POME hydrolyzate by a chemical treatment such as sulfuric acid and a physical treatment such as heating, a chemical treatment containing a POME hydrolyzate only by a physical treatment is performed. The growth may be carried out using a medium containing no substance such as sulfuric acid used in 1. Alternatively, after growth using a medium containing POME hydrolyzate by chemical treatment, use a medium containing POME hydrolyzate only by physical treatment but not containing the substance used in the chemical treatment. May be propagated. Alternatively, the growth is carried out using a medium containing a POME hydrolyzate by chemical treatment, physical treatment, biological treatment, etc., and then the growth is carried out using a medium containing no POME hydrolyzate. And vice versa.

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

The DHA produced by the DHA-producing algae of the present invention can be extracted and analyzed by a method known to those skilled in the art. For example, when DHA-producing algae are cultured and propagated as described above and DHA-producing algae are recovered from the obtained culture solution, they can be recovered by an existing method such as centrifugation or filtration. The recovered pellets of DHA-producing algae are dried by freeze-drying or drying by heating, or the wet algae that are not dried after concentrating the DHA-producing algae culture are used in the DHA extraction step. May be good.

DHA can be extracted from the obtained dried algae or wet algae. The extraction method is not particularly limited, and known methods such as organic solvent extraction, squeezing, and supercritical extraction can be used. 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 a mixture of two or more liquids of a polar solvent and a non-polar solvent. Liquids can also be used. The algae may be crushed before extraction, and the algae may be crushed by a known method such as chemical crushing with an alkali or acid, mechanical crushing with a homogenizer, ultrasonic waves, a bead mill, or biological crushing with an enzyme. You may. After the above extraction, the obtained extract may be used to concentrate and purify DHA by a method known to those skilled in the art. For example, column chromatography using silica gel or acidic clay, high-speed liquid chromatography, liquid distribution, urea addition. It may be concentrated and purified by using a known method such as a method.

The DHA or the like-producing algae may contain at least 5%, at least 15%, at least 20%, at least 25%, and at least 30% (% by weight) of DHA per 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 lymasinum 4W-1b strain in a medium in which glucose and salts are added to 50% POME hydrolyzate, production of total lipid and DHA (seed algae)
In the following medium, Aurantiochytrium lymasinum 4W-1b strain, which belongs to the algae, provided by the University of Tsukuba, was used in GTY medium (1 L of 1/2 diluted seawater containing 20 g of glucose, 5 g of yeast extract, and 10 g of tryptone) for 3 days. It was cultivated and used as a seed alga.

High-temperature POME provided by a palm oil factory in Liau, Indonesia in April 2018 was quickly cooled in a refrigerator, stored for 3 days, and then hydrolyzed by heat treatment at 121 ° C for 60 minutes. Add sulfuric acid to 1% and hydrolyze by the same heat treatment (at 121 ° C for 60 minutes), and not hydrolyze at all. Adjust the pH to 7.0 using NaOH for each. _{A total of 19.4 g / L of salts (15.5 g / L of Na 2} ) so that the hydrolyzed supernatant obtained by centrifugation at 4000 rpm (2,700 g) for 15 minutes becomes 50% (volume%). _{SO 4, 0.69g / L CaCl 2} · 2H 2 O _{in, 5.74g / L MgSO 4 · 7H} 2 O in artificial seawater containing glucose _KH 2 PO ₄₎ and 20 g / L of 0.59 g / L Diluted with is used as a medium.

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

A growth curve was obtained by collecting 1 ml of the culture solution during culturing at a predetermined time point and measuring the optical turbidity at 660 nm with an ultraviolet-visible absorbance meter. Cultures were performed for up to 60 hours in all cultures until growth reached a plateau. After completion of the culture, 50 to 100 mL of the culture solution was collected and centrifuged at 3900 rpm for 15 minutes with a high-speed cooling centrifuge. Then, the supernatant was discarded, the pellets were washed with distilled water, and centrifugation was performed again. Then, the sample was frozen at −80 ° C., and these were dried in a freeze dryer to obtain dried algae. The weight of dried algae was measured with a microbalance, and the amount of algae per 1 L of the culture solution was calculated.

The algae were recovered from the above 10 mL of the algae culture solution by centrifugation. The obtained algal pellets were ultrasonically crushed, 20 mL of a chloroform / methanol (volume ratio 2: 1) mixed solution was added, and the mixture was well mixed. 4 mL of a 0.9% aqueous sodium chloride solution was added, mixed well, and after centrifugation, the chloroform layer was collected by filtering with a filter paper. The filtrate was concentrated to dryness, and the amount of lipid was measured. 0.2 mg of tricosan methyl ester was added to the lipid as an internal standard substance, 0.5 mL of 0.5 M NaOH methanol solution was added, and a saponification reaction was carried out at 100 ° C. for 9 minutes, followed by 14% boron trifluoride. 0.7 mL of methanol solution was added, and the methyl esterification reaction was carried out at 100 ° C. for 7 minutes. 3 mL of saturated brine and 3 mL of hexane were added to the sample, mixed well, and after centrifugation, the hexane layer was measured for DHA content by GC (gas chromatography) analysis.

The above results are shown in FIGS. 1 and 2.
FIG. 1 shows a medium obtained by hydrolyzing POME having a volume of 50% by a sulfuric acid treatment having a volume of 1% and a heat treatment at 121 ° C., a medium obtained by hydrolyzing only a heat treatment at 121 ° C. The time course of the algae concentration of Aurantiochytrium lymasinum 4W-1b cultured in% POME medium (hereinafter referred to as control medium) is shown (OD660). The strain grew on all media, but the hydrolyzed medium showed clearly better growth than the control medium. Furthermore, the medium hydrolyzed with sulfuric acid and heat showed better growth than the medium hydrolyzed with POME only by heating.
FIG. 2 shows the total lipid and DHA production after 48 hours of culturing. Lipids and DHA were produced in all media, but in terms of lipid production, 1% sulfuric acid and hydrolyzed medium by heating were about 1.3 times higher than those of the control. Regarding the amount of DHA produced, the hydrolyzed medium produced by 1% sulfuric acid and heating and the hydrolyzed medium produced only by heating were clearly increased from the control.
Table 1 shows the growth amount (g / L), algae productivity (mg / L / day), total lipid content (%), and total lipid amount (mg / L) after growing the strain in each medium for 48 hours. A table summarizing L), lipid productivity (mg / L / day), DHA content (%), DHA production (mg / L), and DHA productivity (mg / L / day) is shown. Productivity is a value obtained by converting the amount of algae, the total amount of lipids, and the amount of DHA after 48 hours of growth, respectively, per day. The total lipid content and the DHA content indicate the ratio to the algae mass (% by weight), respectively.

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

(Example 2) Growth of Aurantiochytrium lymasinum 4W-1b strain in a medium prepared using 100% POME hydrolyzed by various methods, production of total lipid and DHA Various hydrolysis conditions were set. The growth of Aurantiochytrium lymasinum 4W-1b strain, total lipid and DHA production were investigated.
As the seed strain, the same Aurantiochytrium lymasinum 4W-1b strain as in Example 1 was used. For POME, high-temperature POME provided from a palm oil factory in North Sumatra, Indonesia was quickly cooled and stored frozen, and thawed at the start of the experiment.
The POME hydrolyzate is hydrolyzed by treating the thawed POME under the conditions shown in Table 2 below (Experiment Nos. 2 to 4 in Table 2), and the pH is adjusted to 7.0 with an acid or alkali. A hydrolyzed POME supernatant (volume 100%) obtained by centrifugation at 4000 rpm for 15 minutes was used. As a control (POME non-hydrolyzate), only filtration is performed with a filter (0.45 μm) without any hydrolysis (Experiment No. 1 in Table 2), and after adjusting to pH 7.0 in the same manner, centrifugation at 4000 rpm for 15 minutes. The non-hydrolyzed POME supernatant (volume 100%) obtained by separation was used.
For culturing, a POME supernatant (volume 100%: POME non-hydrolyzed product) obtained by centrifuging the thawed POME at 4000 rpm for 15 minutes was used as an initial medium, and a 500 ml volume triangle was used as in Example 1. Pour 200 ml into a flask, sterilize at 121 ° C for 20 minutes in an autoclave, cool, then seed the seed strain in a medium, seal the mouth of the flask with a silico stopper, and perform reciprocating shaking culture (100 strokes / minute) at 25 ° C to 36. After time culturing, 50 ml of the above various POME hydrolysates or controls were added to the culture, and the culture was continued.

60 hours after the start of culturing, 50 to 100 mL was collected in the same manner as in Example 1, and the growth amount (g / L), algae productivity (mg / L / day), total lipid content (%), and total lipid amount ( The mg / L), lipid productivity (mg / L / day), DHA content (%), DHA production (mg / L), and DHA productivity (mg / L / day) were determined.

The results are shown in Tables 3-6. Table 3 shows the effect of physical treatment (hydrolysis by heat and pressure) comparing Experiment Nos. 2 (1) and (2) with respect to Experiment No. 1. Table 4 shows the effect of chemical treatment (hydrolysis with acid) comparing Experiment Nos. 3-1 (1), (2) and (3) with respect to Experiment No. 1. Table 5 shows the effect of chemical treatment (hydrolysis with alkali) comparing Experiment Nos. 3-2 (1) and (2) with respect to Experiment No. 1. Table 6 shows the effect of biological treatment (enzymatic hydrolysis) comparing Experiment Nos. 4 (1) and (2) with respect to Experiment No. 1. In all the experiments, the production and productivity of algae, 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.

From the above results, it was shown that it is preferable to use a medium containing a POME hydrolyzate for the growth of heterotrophic microalgae such as Aurantiochytrium lymasinum 4W-1b strain and the production of lipids and DHA. It was.

Claims (17)

A method for culturing algae belonging to the genus Aurantiochytrium using a medium containing a POME hydrolyzate.
The culture method, wherein the POME hydrolyzate is a POME hydrolyzate by chemical treatment with acid or alkali, physical treatment with heat and / or pressure, and / or biological treatment with peptidase . The culture method according to claim 1 , wherein the acid is sulfuric acid, hydrochloric acid, and / or phosphoric acid. The culture method according to claim 1 , wherein the alkali is sodium hydroxide, potassium hydroxide, and / or ammonia. The culture method according to any one of claims 1 to 3 , wherein the alga belonging to the genus Aurantiochytrium is an alga that produces an ω-3 polyunsaturated fatty acid. The culture method according to claim 4 , wherein the ω-3 polyunsaturated fatty acid is DHA. The culture method according to any one of claims 1 to 5 , wherein the algae of the genus Aurantiochytrium is a culture strain belonging to Aurantiochytrium limacinum. The culture method according to any one of claims 1 to 5 , wherein the algae of the genus Aurantiochytrium is a culture strain belonging to Aurantiochytrium mangrovei. The sixth aspect of claim 6, wherein the culture strain of Aurantiochytrium lymasinum is any strain selected from the 4W-1b strain, the Aurantiochytrium lymasinum SR-21 strain, or the Aurantiochytrium lymasinum NIES3737 strain. Culture method. The culture method according to claim 7 , wherein the culture strain of Aurantiochytrium manglow bay is Aurantiochytrium manglow bay 18W-13a strain. The culture method according to any one of claims 1 to 9 , wherein the POME hydrolyzate has its solid content removed by centrifugation and / or filtration. The culture method according to any one of claims 1 to 10 , wherein the medium further contains a sugar component. The culture method according to claim 11 , 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 11 or 12 , wherein the total amount of the sugar component added is 10 to 30 g / medium L. The culture method according to any one of claims 1 to 13 , wherein the medium further contains a mineral component. The mineral components are sulfates such as potassium sulfate, magnesium sulfate, iron sulfate, ammonium sulfate, copper sulfate, nickel sulfate, zinc sulfate; phosphates such as potassium phosphate; carbonates such as calcium carbonate; cobalt chloride, manganese chloride. , Chlorides such as sodium chloride and calcium chloride; selenates and molybdates such as sodium molybdenate and sodium selenate; halides such as potassium bromide and potassium iodide; natural seawater salts; and artificial seawater salts The culture method according to claim 14 , which is a single or a plurality of salts selected from. The culture method according to claim 14 or 15 , wherein the total amount of the mineral components added is 5 to 35 g / medium L. A method for producing DHA, which comprises recovering heterotrophic microalgae cultured by the culturing method according to any one of claims 1 to 16 and extracting DHA from the recovered algae.

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