JP6709484B1 - Method for culturing heterotrophic microalgae using palm oil factory effluent (POME) and method for producing DHA - Google Patents

Abstract

An object of the present invention is to provide a culture method capable of efficiently growing microalgae using POME discharged from a palm oil manufacturing process, and a method of efficiently producing high value-added valuables from microalgae. That is. By culturing heterotrophic microalgae that produce ω-3 polyunsaturated fatty acids such as DHA (docosahexaenoic acid) using POME, these heterotrophic microalgae can be efficiently propagated and DHA at a high concentration. Produced.

Description

The present invention provides a method for culturing heterotrophic microalgae and a method for producing DHA, which uses palm oil mill effluent (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 the palm oil manufacturing plant into the lagoon, where oxidation pond treatment → anaerobic treatment → aerobic treatment is performed. During this process, fermentation gas causes foul odors, harmful insects and pests, water pollution, and methane gas generated by anaerobic treatment is released to the atmosphere. The global warming potential of methane is 21 times as large as that of carbon dioxide, which has an adverse effect on global warming. As described above, POME causes global warming, deterioration of living environment, adverse effects on wildlife, and other widespread and serious environmental problems by causing emission of warming substances and water pollution. 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 the national society in which the palm oil industry, such as Indonesia, Malaysia, etc., is thriving. Has become a problem.

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

Conventionally, microalgae are grown, lipids, which are raw materials for fuels, feeds, health foods, etc., or valuable materials such as proteins and vitamins are produced and stored in the cells of the microalgae, and the stored valuable materials are used. Are used as fuel and food. In particular, the production and storage of high value-added valuables such as ω-3 polyunsaturated fatty acids have been attracting attention as ingredients that can be industrialized immediately.

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 proliferate efficiently, the amount of valuables stored in the cells will not necessarily be sufficient, which may make it difficult to effectively use the valuables. It has been reported that microalgae such as Chlorella proliferate using POME (Non-patent documents 1-5), but microalgae are poor in algal biomass productivity and lipid productivity and poor in POME utilization efficiency. Has not established a production method for high value-added valuables.

Republished 2017-026370 bulletin Japanese Patent No. 6049937 JP 05-301097 A JP 2010-022331 A

HABIB et. al. “Growth and Nutritional Values of Moinamicrura 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 Biotechnoloty 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 and the like, the present invention efficiently cultivates high value-added valuables from a culture method and microalgae that can efficiently grow microalgae using POME discharged from the palm oil manufacturing process. It is an object to provide a method for producing.

The present inventor has conducted diligent studies to solve the above-mentioned problems, and as a result, using POME, ω-such as DHA (docosahexaenoic acid) with or without an organic carbon source such as sugar and a mineral component as required. 3 By culturing heterotrophic microalgae that produce polyunsaturated fatty acids, it was found that these heterotrophic microalgae are efficiently proliferated and DHA is produced at a high concentration, thus completing the present invention. I made it happen.

That is, the method for cultivating a heterotrophic microalgae according to the present invention for solving the above-mentioned problems is a high value-added component using POME containing squeezed juice squeezing oil from palm fruit in the palm oil production process. It is characterized by heterotrophic culture of microalgae that produce.

The method for culturing microalgae according to the present invention is characterized in that palm fruit is squeezed to supply POME containing squeezed juice produced after the production of palm oil as a component.

Therefore, the present application provides the following inventions.
(1) A method for culturing heterotrophic microalgae using a medium containing POME.
(2) The culture method according to (1), wherein the heterotrophic microalgae are algae that produce ω-3 polyunsaturated fatty acids.
(3) The culture method according to (2), wherein the ω-3 polyunsaturated fatty acid is DHA.
(4) The culture method according to any one of (1) to (3), wherein the alga is a strain of alga belonging to Thraustochytriales.
(5) The culture method according to (4), wherein the Thraustochytrids is a species belonging to the genus Aurantiochytrium.
(6) The culture method according to (5), wherein the alga of the genus Aurantiochytrium is a culture strain belonging to Aurantiochytrium limacinum.
(7) The culture method according to (5), wherein the alga of the genus Aurantiochytrium is a culture strain belonging to Aurantiochytrium mangrovei.
(8) The culture strain of the aurantiochytrium limacinum is any strain selected from the 4W-1b strain, the aurantiochytrium limacinum SR-21 strain, and the aurantiochytrium limacinum NIES3737 strain (6) The described culturing method.
(9) The culture method according to (7), wherein the culture strain of the aurantiochytrium mangrove bay is the aurantiochytrium mangrove bay 18W-13a strain.
(10) The culture method according to any one of (1) to (9), wherein POME is clarified by centrifugation and/or filtration.
(11) The culture method according to any one of (1) to (10), wherein the medium further contains a sugar component.
(12) The culture according to (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. Method.
(13) The culture method according to (11) or (12), wherein the total amount of the sugar component added is 10 to 30 g/medium L.
(14) The culture method according to any one of (1) to (13), wherein the medium further contains a mineral component.
(15) The mineral component is potassium sulfate, magnesium sulfate, iron sulfate, ammonium sulfate, or a sulfate salt such as copper sulfate, nickel sulfate, or zinc sulfate; a phosphate salt such as potassium phosphate; a carbonate salt such as calcium carbonate; Chlorides such as cobalt, manganese chloride, sodium chloride and calcium chloride; alkali metal oxides; selenites and molybdates such as sodium molybdate and sodium selenite; halides such as potassium bromide or potassium iodide. The method for culturing according to (14), which is a single or a plurality of salts selected from natural seawater salts; and artificial seawater salts.
(16) The culture method according to (14) or (15), wherein the total amount of the mineral components added is 5 to 35 g/medium L.
(17) A method for producing DHA, which comprises collecting heterotrophic microalgae cultured by the culture method according to any one of (1) to (16) and extracting DHA from the collected algae. .

According to the present invention, it is possible to effectively use POME, efficiently culture heterotrophic microalgae, and produce DHA as a valuable resource.

Surprisingly, when the medium supplemented with POME was used, DHA-producing algae such as aurantiochytrium limacinum 4W-1b strain were satisfactorily grown at a high growth rate not seen in conventional microalgae, and a high concentration was obtained. DHA production achieved.

FIG. 1 shows the change over time in the concentration of algae in the aurantiochytrium limacinum 4W-1b strain when an artificial seawater salt-added medium adjusted so that the addition rate of the POME supernatant is 10 to 50%. Shown (OD660). FIG. 2 shows total lipids (left figure) of the aurantiochytrium limacinum 4W-1b strain when an artificial seawater salt-added medium adjusted so that the addition rate of the POME supernatant is 10 to 50% and The production of DHA (right panel) is shown. FIG. 3 shows the aurunch when using a medium in which glucose and artificial seawater salts are added to a POME supernatant liquid (100%) and a dilution liquid adjusted so that the addition ratio of the POME supernatant liquid is 10 to 75%. The time-dependent change of the algal body concentration of Ochthorium lymacinum 4W-1b strain is shown (OD660). FIG. 4 shows POME supernatant (100%) and POME when a medium prepared by adding glucose and artificial seawater salts to a diluent adjusted to have an addition rate of POME supernatant of 10 to 75% is used. The correlation of the addition rate of a clear fluid and the proliferation of the aurantiochytrium limacinum 4W-1b strain is shown. FIG. 5 shows the aurunch in the case of using a medium in which glucose and artificial seawater salts are added to a POME supernatant liquid (100%) and a dilution liquid adjusted so that the addition ratio of the POME supernatant liquid is 10 to 75%. 3 shows total lipids and DHA production of Ochthorium limacinum 4W-1b strain. FIG. 6 shows that glucose and 0.00%, 0.38%, 0.75%, 1.50%, 3.00% (wt%) of Red Sea salt were added to the POME supernatant (100%). 2 shows the changes over time (OD660) in the algal concentration of the aurantiochytrium limacinum 4W-1b strain and the DHA concentration when the culture medium was used. FIG. 7 shows the total lipid and DHA production of various strains of the genus Aurantiochytrium when using a medium in which glucose and artificial seawater salts were added to POME supernatant (100%).

Generally, the palm oil production process includes steam treatment for inactivating palm fruit bunch (FFB), separation of palm fruit and palm empty bunch (EFB), and fruit part from palm fruit ( It goes through a pretreatment step of separating meso-ca) and seeds. The crude palm oil is extracted from the fruit part and then separated and refined to produce high-purity palm oil. The POME in the present invention, this path - occurring in the manufacturing process of Muoiru, unrecovered oil and used steam water, by centrifugation a mixture of a mixed effluent residual liquid containing unrecovered oil after squeezing oil The effluent that has been removed or collected (the temperature is 70-90°C), and the effluent that is discharged after being subjected to oxidation pond treatment → anaerobic treatment → aerobic treatment after cooling. POME contains abundant sugars, organic acids, vitamins, amino acids, peptides, proteins, minerals, etc. derived from raw materials. The POME used for culturing the alga of the present invention may be one before or after the oxidation pond treatment, anaerobic treatment, or aerobic treatment. In one embodiment, it is preferable to use POME before the oxidation pond treatment, the anaerobic treatment, and the aerobic treatment.

POME contains various solids derived from raw materials. In culturing the DHA-producing alga, POME may or may not contain a solid matter, and is not limited. When the solid matter in POME is removed, a clear POME obtained by removing by a known means such as centrifugation or filtration may be used as the medium.

In the present invention, POME is sterilized by known means such as filter sterilization, autoclave sterilization, boiling sterilization, and radiation sterilization, but may be sterilized by known means such as sodium hypochlorite and ozone treatment.

In the present invention, although the content of POME in the heterotrophic microalgal medium is not limited, for example, the concentration of 1%, at least 10%, at least 25%, at least 50%, 75%, 100% (volume %) And may be contained in the culture medium.

In the present invention, various additives may be added to the medium containing POME so that the medium has 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 minerals are not added, there are advantages that metal corrosion of the device can be prevented, wastewater treatment and discharge after culture can be facilitated, and culture medium cost can be kept low. Further, if necessary, the pH can be adjusted appropriately by adding an appropriate acid or base. The suitable pH of the medium depends on the type of DHA-producing alga to be cultivated, and may be adjusted to pH 6 to pH 7, for example.

Mineral components may include, but are not limited to, chemically defined inorganic salts such as alkali and alkaline earth metal salts, and 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 and calcium chloride; alkali metal oxides; selenites and molybdates such as sodium molybdate and sodium selenite; halogens such as potassium bromide or potassium iodide. Compounds; natural seawater salts; and artificial seawater salts 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 16.7‰ (16.7 g/L medium).

In certain embodiments, no additional mineral components, such as salts such as chloride, are added to the medium. In such an embodiment, the medium contains no additional mineral components other than those originally contained in POME and the like. Cultivation of DHA-producing algae such as Thraustochytriales without adding mineral components to the medium of the present invention using POME may increase DHA productivity. In the case of a medium that does not contain an additional mineral component other than that originally contained in POME or the like, the concentration of minerals in the medium is, 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), less than 3.0 ‰, 2.0 ‰, less than 1.0 ‰ (g/medium L) in terms of Cl amount. Can be

In certain embodiments, the inclusion of saccharides with POME in the culture may promote growth of the heterotrophic microalgae. The saccharide is a carbon source of the heterotrophic microalgae, but when the POME stock solution does not sufficiently contain saccharides that can be used by the microalgae, or when it is desired to promote the growth of the heterotrophic microalgae. In some cases, saccharides that can be used by microalgae may be added to the culture solution in any form.

The saccharides include, but are not limited to, one or more saccharides selected from glucose, galactose, fructose, maltose, sucrose, lactose, oligosaccharides, sugar alcohols such as glycerol, and the like. The saccharides 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 saccharides are added in a total amount of 20‰ (20 g/L of medium).

The microalgae used in the present invention may be, in particular, algae that produce ω-3 polyunsaturated fatty acids such as DHA (hereinafter referred to as DHA-producing algae). Examples of algae that produce DHA include aurantichytrium (Aurantiochytrium), Schizochytrium, Thraustochytrium, and Parietichytrium which are organisms belonging to the Thraustochytriales. Examples include organisms belonging to the genus Ulkenia, or organisms belonging to the genus Oblongichytrium.
Examples of organisms belonging to the genus Aurantiochytrium include Aurantiochytrium limacinum and Aurantiochytrium mangrovei.
Examples of the organisms belonging to the genus Schizochytorium include Schizochytrium aggregatum. For example, strains such as Schizochytrium sp. Maku-1 can be used.
Examples of the organisms belonging to the genus Thraustochytrium include Thraustochytrium aureum, Thraustochytrium pachydermum, Thraustochytrium aggregatum, and the like.
Examples of the organisms belonging to the genus Parietichytrium include Parietichytrium sarkarianum. For example, strains such as Parietichytrium sp. 6F-10b can be used.
Examples of the organisms belonging to the genus Ulkenia include Ulkenia visurgensis, Ulkenia profunda, and the like. For example, strains such as Ulkenia sp. Yonez 6-9 can be used.
Examples of the 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 algae of the genus Aurantiochytrium, for example, Aurantiochytrium limacinum 4W-1b strain, aurantiochytrium limacinum SR-21 strain, and aurantiochytrium such as aurantiochytrium limacinum NIES3737 strain. Limacinum strains, as well as aurantiochitrium mangrovei strains such as 18W-13a strain, and the like can be used.

Cultivation of algae producing DHA or the like in the present invention is carried out by inoculating the algae in a medium containing POME prepared as described above and culturing according to a standard method. The culture conditions depend on the type of algae such as DHA to be cultivated, 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. The culture can be performed for 1 to 10 days, preferably 3 to 7 days, for example 4 to 5 days, and can be performed by aeration and agitation culture, shaking culture or static culture.

The DHA-producing algae used in the present invention may be cultivated in a culturing device having an appropriate cell culturing means. The “cell culture means” means a means having all the functions for culturing cells, and is, for example, a culture tank, and the culture 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 temperature measuring device, a light control device, a specific gas concentration measuring device 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 a tank different from the concentration/separation tank. When it is a tank different from the concentration/separation tank, they may be connected by an appropriate means such as a flow path.

The present invention further provides a method for producing DHA, which collects DHA-producing algae cultivated by the above-mentioned culturing method using a medium containing POME, and extracts DHA from the collected DHA-producing algae. The method for producing DHA of the present invention is characterized by culturing DHA-producing algae using a medium containing POME to cause the DHA-producing algae to produce DHA.

DHA produced by algae such as DHA of the present invention can be extracted and analyzed by methods known to those skilled in the art. For example, when the DHA-producing alga is cultured and proliferated as described above and the DHA-producing alga is recovered from the obtained culture solution, it can be recovered by an existing method such as centrifugation or filtration. The collected pellets of DHA-producing algae are dried by freeze-drying or drying by heating, etc., or the wet Algae bodies which have not been dried after the DHA-producing algae culture is concentrated are used in the DHA extraction step. Good.

DHA can be extracted from the obtained dry algal cells or wet algal cells. The extraction method is not particularly limited, and known methods such as organic solvent extraction, compression, and supercritical extraction can be used. Examples of the organic solvent used in the organic solvent extraction method include hexane, acetone, chloroform, methanol, ethanol, and diethyl ether, which may be used alone or a mixture of two or more liquids such as a polar solvent and a nonpolar solvent. A liquid can also be used. The algal cells may be crushed before extraction, including, but not limited to, chemical crushing with alkali or acid, mechanical crushing with homogenizer or ultrasonic wave, bead mill, biological crushing with enzymes, etc. by known methods. May be. After the above extraction, the obtained extract may be concentrated and purified 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 method, etc. May be concentrated and purified using a known method.

The DHA-producing alga may contain at least 5%, at least 15%, at least 20%, at least 25%, at least 30% (wt%) 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 limacinum 4W-1b strain, production of total lipids and DHA (seed algae) in an artificial seawater salt-added medium adjusted so that the addition rate of the POME supernatant is 10 to 50% )
In the following medium, the aurantiochytrium limacinum 4W-1b strain belonging to the Thraustochytrids provided by the University of Tsukuba was GTY medium (containing 20 g of glucose, 5 g of yeast extract and 10 g of tryptone in 1 L of 1/2 diluted seawater) for 4 days. It was cultured and used as a seed alga.

The high-temperature POME provided by the factory in Riau, Indonesia in April 2018 was immediately cooled in the refrigerator and stored for 3 days, after which it was centrifuged at 5000 rpm for 15 minutes, and the supernatant liquid (hereinafter POME supernatant (Referred to as liquid). A medium containing 16.7 g/L of Red Sea salt (artificial seawater salts) was prepared so that the addition ratio of the POME supernatant was 10%, 25%, and 50% (volume %).

200 ml of each medium 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 alga of Aurantiochytrium limacinum 4W-1b strain was washed with sterilized seawater and then seeded in the medium. The mouth of the flask was sealed with a silicon stopper and cultured at 25° C. with reciprocal shaking (100 strokes/minute).

A growth curve was obtained by collecting 1 ml of the culture solution during the culture at a predetermined time point and measuring the optical turbidity at 660 nm with an ultraviolet-visible absorption spectrometer. Culturing was carried out for up to 48 hours until growth reached a plateau in all cultures. 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 pellet was washed with distilled water, and centrifuged again. Then, 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 by a microbalance to calculate the amount of algal cells per 1 L of the culture solution.

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

The above results are shown in FIGS.
FIG. 1 shows the time-dependent changes in the algal cell concentration of aurantiochytrium limacinum 4W-1b cultured in 10%, 25%, and 50% series of POME supernatant solutions (OD660). The strain grew in all the addition rate mediums, and the higher the POME concentration (addition rate), the higher the growth of the strain.
FIG. 2 shows the total lipid and DHA production after 48 hours of culture. Lipids and DHA were produced in all the addition ratio media, and the higher the POME concentration (addition ratio), the higher the lipid and DHA production of the strain.

Table 1 shows the growth amount (g/L), algal cell productivity (mg/L/day), lipid content (%), and total amount after the strain was grown for 48 hours in each addition rate medium of Example 1. Table summarizing lipid amount (mg/L), lipid productivity (mg/L/day), DHA content (%), DHA production amount (mg/L), and DHA productivity (mg/L/day) Indicates. 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, which are converted per day. The lipid content and the DHA content each represent the ratio to the amount of algal bodies (% by weight).

The higher the POME concentration (addition rate), the higher the alga body growth rate/productivity, total lipid and DHA production rate/productivity, and especially the alga body productivity and lipid productivity are 1760 mg/L/day and 395 mg, respectively. It was as high as /L/day.

Table 2 shows a comparison between the prior art in which other microalgae were cultured using POME and the present invention, in terms of alga body growth amount/productivity and total lipid production amount/productivity.

From Table 2, the algal cell productivity of the conventional microalga is 2.9 to 1,040 (mg/L/day), while the algal cell productivity of the present invention is 50% POME, without glucose addition. Even when the medium was used, it was 1,760 (mg/L/day), which was as high as 1.7 to 600 times. Further, the total lipid productivity of the conventional microalgae is 0.63 (mg/L/day) to 100.9 (mg/L/day), whereas in the present invention, aurantiochitrium limacinum 4W- The algal cell productivity when 1b was used was as high as 3.9 to 626 times even when the medium containing 50% POME and glucose was not used. As shown below, these effects became more prominent when 100% POME was used and a medium supplemented with 20 g/L glucose was used. Furthermore, all the microalgae of the prior art accumulate lipids containing saturated fatty acids or monovalent unsaturated fatty acids as main components, and from this, valuable ω-3 polyunsaturated fatty acids such as DHA are collected. Hard to get.

Example 2: POR supernatant (100%) and an aurantthiox in the case where a culture medium in which glucose and artificial seawater salts were added to a diluent adjusted so that the addition rate of the POME supernatant was 10 to 75% Growth of Thorium limacinum 4W-1b strain, production of total lipid and DHA Using the same POME and seed alga as in Example 1, 20 g/L glucose and 16. were added to the POME supernatant (100%) obtained by the same method. A 100% POME medium was prepared by adding 7 g/L of Red Sea salt. Further, the POME supernatant was diluted with distilled water so that the addition rates were 10%, 25%, 50%, and 75% (volume %), and 16.7 g/L of red sea salt and 20 g/L of glucose were added. A medium adjusted so as to contain was also prepared. These were the addition series of POME. Then, in the same manner as in Example 1 above, the growth of aurantiochytrium limacinum 4W-1b was evaluated by OD, and the dry weight of algal cells was measured to calculate the amount of algal cells per liter of culture solution. Then, the total lipid and the amount of DHA were measured.

The above results are shown in FIGS. 3, 4, and 5.
FIG. 3 shows the change over time in the algal cell concentration of the algal strain when the prepared POME addition series was used as a medium (OD660). In each of the addition series of glucose-added POME of 10%, 25% and 50% of Example 2, as compared with the POME addition rates of 10%, 25% and 50% of Example 1 performed under the condition without addition of glucose, The strain showed clearly high growth. Further, as in Example 1, the higher the POME concentration (addition rate), the higher the growth, and the highest growth was shown in the 100% POME-containing medium. FIG. 4 shows the relationship between the POME addition rate and the growth amount. It was found that the relationship between the addition rate (x) and the growth amount (y) is in a linear proportional relationship of y=4.2308x+1.64 (R ² =0.9579).
FIG. 5 shows the total lipid and DHA production after 48 hours of culture. Lipids and DHA were produced in all the addition rate mediums as in Example 1, and the higher the addition rate of POME, the higher the production amount of lipids and DHA, and the highest production was shown in the 100% POME-containing medium.

Table 3 shows the growth amount (g/L) after the strain was grown for 48 hours in each addition ratio medium of Example 2, algal cell productivity (mg/L/day), lipid content (%), and total. Table summarizing lipid amount (mg/L), lipid productivity (mg/L/day), DHA content (%), DHA production amount (mg/L), and DHA productivity (mg/L/day) Indicates. The method of obtaining each numerical value and the unit are the same as in Table 1.

In each of the addition series of glucose-added POME of 10%, 25% and 50% of Example 2, the strain showed a clearly higher growth compared to Example 1 performed under the condition without addition of glucose. As shown in Table 3, as compared with the 50% addition series of the glucose-free system of Table 1, in the 50% addition series with glucose addition, the algal cell productivity was 2,815 mg/L/day, which was double, and The lipid productivity was 1,110 mg/L/day, 2.8 times higher, and the DHA productivity was 228.5 mg/L/day, 7.7 times higher. In addition, as shown in Table 3, algal cell productivity of the strain with glucose addition at 100% POME was 4,150 mg/L/day, and lipid productivity was 1,675 mg/L/day, which was the same as in the prior art. Compared with algal cell productivity and total lipid productivity of other microalgae, the values were 4 to 1430 times higher and 16.6 to 2660 times higher, respectively.

Example 3: Time-dependent changes in proliferation and DHA concentration of aurantiochytrium limacinum 4W-1b strain in a medium in which glucose and artificial seawater salts at various concentrations were added to POME supernatant (100%) The same as in Examples 1 and 2. 20 g/L glucose and 0.00%, 0.38%, 0.75%, 1.50%, 3 were added to the POME supernatant (100%) obtained by the same method using POME and seed algae. 0.00% (wt%) Red Sea salt was added.

As in Examples 1 and 2, after sterilizing the medium and cooling, the medium was cultivated under the same culture conditions (500 mL Erlenmeyer flask (medium preparation amount 200 mL), 25° C., reciprocal shaking culture (100 strokes/minute)), and OD The growth of aurantiochytrium limacinum 4W-1b was evaluated, and the dry weight of the algal cells was measured to calculate the amount of algal cells per 1 L of the culture solution, and the DHA concentration was measured.

Results are shown in FIG. FIG. 6 shows changes in algal cell concentration (OD660) over time and DHA concentration after 48 hours of growth. The concentration of algal bodies was not significantly affected by the concentration of added mineral components, but the concentration of DHA was higher when the concentration of added mineral components was low (0.00%, 0.38%, 0.75% added) (1. 50% and 3.00% were added). That is, it can be seen that the lower the concentration of the added mineral component, the better the DHA production amount (mg/L).

The salt concentration of the POME supernatant liquid (100%) before adding the mineral component and glucose was 2230 (mg/L) in terms of Cl (chloride ion). This corresponds to 12.5% by weight seawater, and corresponds to about 0.375% by weight in terms of NaCl. That is, it can be seen that even without adding a mineral component, only the mineral component originally contained in POME is sufficient for the growth, and the DHA production amount is more preferable.

Example 4: Culture test using various strains of the genus Aurantiochytrium POME supernatant obtained by the same method as in Examples 1 to 3 except that POME provided from a factory in North Sumatra, Indonesia was used. 20 g/L glucose and 16.7 g/L Red Sea salt were added to the liquid (100%) to prepare a 100% POME medium, which was sterilized and cooled. Culture conditions similar to those of Examples 1 to 3 using various strains of the genus Aurantiochytrium shown in Table 4 below (500 mL Erlenmeyer flask (medium preparation amount 200 mL), 25° C., reciprocal shaking culture (100 strokes/minute) )), the growth of various strains was evaluated by OD, the dry weight of algal cells was measured to calculate the amount of algal cells per liter of the culture solution, and the total lipid and DHA concentration were measured.

The results are shown in FIG. 7 and Table 5. FIG. 7 shows the total lipid amount (mg/L) and DHA concentration (mg/L) of various strains of the genus Aurantiochytrium. Table 5 shows the amount of growth (g/L) after the various strains were grown for a certain time in Example 4, algal cell productivity (mg/L/day), lipid content (%), and total lipid content (mg). /L), lipid productivity (mg/L/day), DHA content (%), DHA production amount (mg/L), and DHA productivity (mg/L/day). The definition of each value is the same as in Table 1. From these results, it is understood that the algae of the genus Aurantiochytrium exhibit extremely excellent algal growth/productivity, total lipid and DHA production/productivity when the medium of the present invention is used.

Example 5: Culture test using various strains of other DHA-producing algae Cultured under the same conditions as in Example 4 except that various strains of Thraustochytrids and other DHA-producing algae as shown in Table 6 below were used. Then, the productivity of algal cells, total lipids, and DHA was measured.

The results are shown in Table 7. Table 7 shows algal cell productivity (mg/L/day), lipid productivity (mg/L/day), and DHA productivity (mg/L/day) after growing various strains in Example 5. Is a table summarizing. The definition of each value is the same as in Table 1. Since the culture time was different for each strain, only productivity was described. From these results, it can be seen that DHA-producing algae such as Thraustochytrids generally show very excellent algal cell productivity, total lipid and DHA productivity when the medium of the present invention is used.

Claims (10)

A method for culturing algae belonging to Aurantiochytrium limacinum, which uses a medium containing POME. Is any strains algae belonging to the O-lunch Oki thorium Rimashinamu is O lunch Oki thorium Rimashinamu 4W-1b strains, O-lunch Oki thorium Rimashinamu SR-21 strain, or is selected from O lunch Oki thorium Rimashinamu NIES3737 strain, wherein The culture method according to Item 1. The culture method according to claim 1 or 2, wherein POME is clarified by centrifugation and/or filtration. The culture method according to claim 1, wherein the medium further contains a sugar component. The sugar component is glucose, galactose, fructose, maltose, sucrose, lactose, oligosaccharides, and is one or more saccharides are sugar alcohols or we choose such as glycerol, the culture method of claim 4. The culture method according to claim 4 or 5, wherein the total amount of the sugar component added is 10 to 30 g/medium L. The culture method according to claim 1, 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 and manganese chloride. , sodium, chloride and calcium chloride; potassium bromide, halides such as potassium iodide; selenite, such as sodium selenite; molybdate such as sodium molybdate; alkaline metal oxides natural seawater The culture method according to claim 7, which is a single salt or a plurality of salts selected from salts ; artificial seawater salts. The culture method according to claim 7, wherein the total amount of the mineral components added is 5 to 35 g/medium L. A method for producing DHA, comprising collecting algae cultured by the culture method according to claim 1, and extracting DHA from the collected algae.

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