KR102549752B1 - Method for extracting lipid from microalgae - Google Patents

Abstract

The present invention relates to a method for extracting lipids from microalgae, and to a method for efficiently extracting lipids from microalgae using a mixed solvent of ethanol and ethyl acetate after weakening the cell membrane of microalgae under high temperature and high pressure conditions. it's about The method is economical because it does not use a large amount of highly toxic chloroform and does not include a drying step. In addition, the method of the present invention can extract lipids in high yield.

Description

Method for extracting lipids from microalgae {METHOD FOR EXTRACTING LIPID FROM MICROALGAE}

The present invention relates to a method for extracting lipids from microalgae, and to a method for efficiently extracting lipids from microalgae using an organic solvent after weakening cell membranes of microalgae under high temperature and high pressure conditions.

Among various fuels obtained from crude oil, diesel oil has good fuel efficiency, is economical, and generates a small amount of carbon dioxide, but generates a large amount of air pollutants after combustion. Therefore, in order to solve this problem, research on biodiesel is being conducted as an alternative fuel that has properties similar to those of diesel oil, is economical, and can prevent air pollution. On the other hand, biodiesel is produced by transesterification of alcohol with triglyceride, which is the main component of vegetable oils such as rapeseed oil, soybean oil, sunflower oil, and palm oil.

However, as the use of biodiesel increases rapidly, the price of vegetable oil soars, and destruction of land resources is concerned. Therefore, there is a need to develop a method for producing biodiesel using inedible raw materials.

Microalgae, a typical non-edible raw material, is a unicellular organism that grows photosynthetically using water, carbon dioxide and sunlight, and is also called phytoplankton. Microalgae fixate carbon dioxide with high efficiency and grow rapidly. In addition, the microalgae (58,700 ~ 97,790 ℓ / ha) have higher oil productivity per unit area than soybeans (446 ~ 635 ℓ / ha) or oil palm (5,336 ~ 5,950 ℓ / ha), so they are non-edible for the production of biodiesel. It is attracting attention as a raw material.

Microalgae have a very high water content, and the dry weight of the harvested microalgae is less than about 1%. However, in the lipid extraction method using a solvent, the high water content of microalgae increases the amount of solvent used. Therefore, in order to extract lipids from microalgae, a step of removing moisture is required, and this process lowers the economic feasibility of producing biodiesel.

In this regard, Korean Patent Registration No. 10-1502355 discloses a method for extracting lipids using a supercritical carbon dioxide extraction method and exhibiting high yields of lipids and fatty acid methyl esters.

On the other hand, the present inventors completed the present invention by effectively extracting lipids from microalgae treated with high temperature and high pressure using an organic solvent while studying a process for efficiently extracting lipids from microalgae.

Republic of Korea Patent No. 10-1502355

An object of the present invention is to provide a method for efficiently extracting lipids from microalgae.

Another object of the present invention is to provide a system for efficiently extracting lipids from microalgae.

In order to achieve the above object, the present invention comprises the steps of heating the microalgae-containing solution under high pressure; And it provides a method for extracting lipids from microalgae, comprising the step of extracting lipids by adding a mixed solvent of ethyl acetate and alcohol to the heated microalgae-containing solution.

In addition, the present invention is a first step of heating the microalgae-containing solution under high pressure; A second step of obtaining a solvent layer by adding a mixed solvent of ethyl acetate and alcohol to the heated microalgae-containing solution; And it provides a system for extracting lipids from microalgae, including a third step of obtaining lipids from the solvent layer.

Furthermore, the present invention provides a first step of heating the microalgae-containing solution under high pressure; A second step of separating the solvent layer and pellets by solid-liquid separation of the heated microalgae-containing solution; A third step of extracting lipids by adding ethyl acetate to the solvent layer; A fourth step of extracting lipids by adding a mixed solvent of ethyl acetate and alcohol to the pellets; And it provides a system for extracting lipids from microalgae, including a fifth step of obtaining the lipids extracted in the third and fourth steps.

The method of extracting lipids from microalgae of the present invention is economical because it does not use a large amount of highly toxic chloroform and does not include a drying step. In addition, the method of the present invention can extract lipids in high yield by weakening the cell walls of microalgae under conditions of high temperature and high pressure and then extracting lipids using an organic solvent.

1 is a schematic diagram showing a process for extracting lipids from microalgae according to the present invention.
Figure 2 is a schematic diagram showing the process of extracting lipids from the solid-liquid separated microalgae according to the present invention.

Hereinafter, the present invention will be described in detail.

The present invention comprises the steps of heating a microalgae-containing solution under high pressure; And it provides a method for extracting lipids from microalgae, comprising the step of extracting lipids by adding a mixed solvent of ethyl acetate and alcohol to the heated microalgae-containing solution.

As used herein, the term "microalgae" is a photosynthetic single-celled plant and refers to algae with a size of 50 μm or less. The cultured microalgae may include a culture medium, and the harvested microalgae may include seawater or freshwater depending on the habitat of the microalgae. The microalgae may be cultured or harvested. At this time, the dry weight of microalgae can be adjusted to 20% by weight by concentrating or diluting the cultured or harvested microalgae-containing solution. The concentration or dilution may be appropriately performed by a person skilled in the art according to the dry weight of cultured or harvested microalgae.

The microalgae may be marine microalgae or freshwater microalgae. The marine microalgae are from the group consisting of Nitzsia sp., Spirulina sp., Dunaliela sp., Nanochloropsis sp., and combinations thereof. It can be any one selected. Meanwhile, the freshwater microalgae may be any one selected from the group consisting of Chlorella sp., Scenedesmus sp., and combinations thereof. In one embodiment of the present invention, the microalgae Nannochloropsis Oceania ( Nannochloropsis oceania ) It may be.

In the method for extracting lipids from microalgae, lipids may be extracted from a solution containing microalgae having a moisture content of 80% by weight or more. The water content may be 80 to 99% by weight, 85 to 97% by weight or 90 to 95% by weight. At this time, the dry weight of microalgae in the microalgae-containing solution may be 20% by weight or less.

The high pressure may be 5 to 50 bar. In addition, the heating may be performed at a temperature of 160 to 260 °C, 180 to 260 °C, 200 to 260 °C, 220 to 260 °C or 240 to 260 °C. In one embodiment of the present invention, the heating may be performed at a temperature of about 160, 180, 200, 220, 240 or 260 °C. In addition, the heating may be performed for 10 seconds to 40 minutes, 5 minutes to 20 minutes, 30 seconds to 10 minutes, or 10 seconds to 1 minute. The heating time may be reduced as the reaction temperature increases. In one embodiment of the present invention, the heating may be performed for 30 minutes in a batch reaction. Since microalgae have cell walls of different compositions and structures depending on the species, the heating temperature and time may vary depending on the type of microalgae.

In the method according to the present invention, lipids can be extracted by adding a mixed solvent of ethyl acetate and alcohol to the heated microalgae-containing solution. The mixed solvent may be a solvent in which ethyl acetate and alcohol are mixed at a volume ratio of 1:1 to 5:1, 1.5:1 to 4:1, or 1.8:1 to 3:1. In one embodiment of the present invention, the mixed solvent may be a mixed solvent of ethyl acetate and alcohol in a volume ratio of 2:1. In this case, the alcohol may be a C _1-4 lower alcohol, and the lower alcohol may be any one selected from the group consisting of ethanol, methanol _, and mixtures thereof.

The lipid extraction method of the present invention using the solvent can extract both neutral lipids and polar lipids. The neutral lipid may include monoacylglycerol, diacylglycerol, triacylglycerol or free fatty acid. Meanwhile, the polar lipid may include phospholipid or glycolipid.

The method according to the present invention may further include a solid-liquid separation step after heating. The solid-liquid separation step may be performed through any one selected from the group consisting of centrifugation, decanting, filtration, and combinations thereof. Lipids can be extracted by adding ethyl acetate or a mixed solvent of ethyl acetate and alcohol to each of the supernatant and the pellet obtained through solid-liquid separation. The organic solvent may be a mixed solvent of ethyl acetate and alcohol.

In addition, the present invention is a first step of heating the microalgae-containing solution under high pressure; A second step of obtaining a solvent layer by adding a mixed solvent of ethyl acetate and alcohol to the heated microalgae-containing solution; And it provides a system for extracting lipids from microalgae, including a third step of extracting lipids from the solvent layer.

The first step is a step of heating the microalgae-containing solution under high pressure. The microalgae-containing solution of the first step may be concentrated or filtered so that the water content in the solution is 80% by weight or more after culturing or harvesting the microalgae. The microalgae may be heated to a temperature of 160 to 260° C. under a high pressure of 5 to 50 bar while being introduced into the heat exchanger.

The second process is a process of obtaining a solvent layer by adding a mixed solvent of ethyl acetate and alcohol to the microalgae-containing solution heated in the first process. In this case, the mixed solvent may be a mixture of ethyl acetate and alcohol at a volume ratio of 1:1 to 5:1. A solution containing microalgae to which a mixed solvent of alcohol and ethyl acetate is added may be separated into a solvent layer and an aqueous solution layer (water and precipitate). Lipids to be extracted may be included in the solvent layer, and substances such as amino acids, proteins, sugars, carbohydrates, and nucleic acids, which are remnants of microalgae, may be included in the aqueous layer.

The third step is a step of obtaining lipids from the obtained solvent layer. For example, lipids can be obtained by purging the solvent layer with nitrogen to remove the solvent. After obtaining the lipid through the above process, the mixed solvent of ethyl acetate and alcohol may be separated and used again in the second process.

After the solid-liquid separation of the precipitate layer separated in the second step by using decantation, filtration, centrifugation, etc., a part of ethyl acetate and ethanol may be recovered from the supernatant. On the other hand, the mixed solvent of ethyl acetate and ethanol partially dissolved in the precipitate layer can be separated through distillation after removing the pellet. The separated mixed solvent may be used again in the second process.

In addition, the present invention is a first step of heating the microalgae-containing solution under high pressure; A second step of separating the solvent layer and pellets by solid-liquid separation of the heated microalgae-containing solution; A third step of extracting lipids by adding ethyl acetate to the solvent layer; A fourth step of extracting lipids by adding a mixed solvent of ethyl acetate and alcohol to the pellets; And it provides a system for extracting lipids from microalgae, including a fifth step of obtaining the lipids extracted in the third and fourth steps.

The first step is a step of heating the microalgae-containing solution under high pressure. The microalgae-containing solution of the first step may be concentrated or filtered so that the water content in the solution is 80% by weight or more after culturing or harvesting the microalgae. The microalgae may be heated to a temperature of 160 to 260° C. under a high pressure of 5 to 50 bar while being introduced into the heat exchanger.

The second process is a process of separating the solvent layer and pellets by solid-liquid separation of the heated microalgae-containing solution. The solid-liquid separation may be performed using any one method selected from the group consisting of decantation, filtration, centrifugation, and combinations thereof. At this time, the solvent layer may include a small amount of lipid leaked out of the microalgae in the first step.

The third step is a step of extracting a small amount of lipid leaked out of the microalgae by adding ethyl acetate to the separated solvent layer. The ethyl acetate can be separated after extracting lipids and used again in the third process.

The fourth process is a process of extracting lipids by adding a mixed solvent of ethyl acetate and alcohol to the separated pellets. In this case, the mixed solvent may be a mixture of ethyl acetate and ethanol at a volume ratio of 1:1 to 5:1. After extracting lipids through the above process, the mixed solvent of ethyl acetate and ethanol is separated and can be used again in the fourth process.

The fifth step is a step of obtaining the lipids extracted in the third and fourth steps. For example, lipids can be obtained by removing the solvent by purging the ethyl acetate extract of the third step and the mixed solvent extract of ethyl acetate and alcohol of the fourth step, respectively, with nitrogen.

Hereinafter, the present invention will be described in detail by the following examples. However, the following examples are only for illustrating the present invention, and the present invention is not limited thereto.

comparative example One. hydrothermal Confirmation of lipid extraction content in case of no pretreatment

Using the Bligh & Dyer method, lipids were extracted from the microalgae Nannochloropsis Oceania ( Nannochloropsis oceania ).

First, 50 ml of isopropanol was added to 5 ml of Nannochloropsis Oceania having a moisture content of 93.3% (dry weight 6.7%) and heated at 80° C. for 5 minutes. 25 ml of chloroform was added thereto and reacted at 4°C for one day. The reactant was centrifuged for 10 minutes at 25° C. and 3,000 rpm to obtain a first extract, and 25 ml of chloroform was additionally added thereto to perform a second extraction. After separating the layers by adding 50 ml of 0.9% (w/v) KCl solution to the secondary extract, the separated chloroform layer was mixed with the primary extract. The mixture was purged with nitrogen to remove the solvent and the total lipid was obtained. The obtained lipid was subjected to transesterification using sulfuric acid and methanol, and the content of fatty acids was analyzed by gas chromatography (GC).

As a result, 26.2 mg of fatty acids were extracted from 355 mg of dry weight of microalgae (7.8%).

Comparative Example 2. Confirmation of Lipid Extraction Content in the Case of Hot Water Pretreatment

Conditions and methods identical to those of Comparative Example 1, except that 100 ml of Nannochloropsis Oceania having a moisture content of 93.3% (dry weight 6.7%) was pretreated with hot water at a temperature of 160° C. for 30 minutes under a condition of 6 bar. Lipids were extracted and the content of extracted fatty acids was analyzed.

As a result, 26.1 mg of fatty acid was extracted from 335 mg of dry weight of microalgae (7.8%).

Example One. hydrothermal Confirmation of lipid extraction content according to pretreatment temperature

A hydrothermal pretreatment process was added to the lipid extraction process from Nannochloropsis Oceania, and lipid extraction contents were compared according to the hotwater pretreatment temperature.

First, 100 ml of Nannochloropsis Oceania having a moisture content of 93.3% (dry weight 6.7%) was heated at 160 ° C / 6 bar, 180 ° C / 10 bar, 200 ° C / 16 bar, 220 ° C / 23 bar, 240 ° C / 34 bar Lipids were extracted under the same conditions and methods as in Comparative Example 1, and the content of the extracted fatty acids was analyzed, except that hot water pretreatment was performed for 30 minutes under a bar or 250 ° C / 40 bar conditions. The calculated fatty acid content was calculated as a percentage based on the fatty acid content extracted in Comparative Example 1 and is shown in Table 1 below.

Comparative Example 1 160℃ 180℃ 200℃ 220℃ 240℃ fatty acid
content(%) 100 114.5 86.3 69.1 28.2 14.9 note tea production tea production tea production tea production

As a result, in the case of hot water pretreatment at 160 ° C., the extracted fatty acid content increased by 14.5% compared to Comparative Example 1. On the other hand, in the case of hot water pretreatment at 180 ° C. or higher, the fatty acid content decreased. The above result seemed to be due to the binding of lipids to the generated solid content difference (char).

Example 2. Confirmation of lipid content extracted with a mixed solvent of ethyl acetate and ethanol

First, 5 ml of a mixed solvent in which ethyl acetate and ethanol were mixed at a volume ratio of 2:1 was added to 5 ml of Nanochloropsis Oceania pretreated with hot water under the same conditions and method as in Comparative Example 2. After reacting at 85 °C for 10 minutes, centrifugation was performed at 25 °C and 3,000 rpm for 10 minutes to obtain a supernatant. The obtained supernatant was purged with nitrogen to remove the solvent, and the total lipid was extracted. The extracted lipids were transesterified using sulfuric acid and methanol, and the content of fatty acids was analyzed by GC method. The calculated fatty acid content was calculated as a percentage based on the fatty acid content extracted in Comparative Example 2 and is shown in Table 2 below.

Pre-treatment of hot water Fatty acid content (%) Comparative Example 2 ○ 100 Example 2 ○ 107.3

As shown in Table 2, in the case of extraction with a mixed solvent of ethyl acetate and ethanol after pretreatment with hot water, the extracted fatty acid content increased by 7.3% compared to Comparative Example 2.

Example 3. After solid-liquid separation, check the lipid content extracted according to the type of organic solvent

Nannochloropsis Oceania pretreated with hot water under the same conditions and method as in Comparative Example 2 was subjected to solid-liquid separation to obtain pellets and supernatants, respectively, and lipids were extracted from each of them.

Specifically, 2 ml of ethyl acetate, a mixed solvent of ethyl acetate and ethanol (volume ratio 2: 1), and a mixed solvent of hexane, butyl acetate, butyl acetate and ethanol (volume ratio 2: 1) were added to the pellet, and at 85 ° C. Reacted for 10 minutes. This was repeated twice to obtain a total of 4 ml of pellet reaction. Meanwhile, 3 ml of hexane, chloroform or ethyl acetate was added to the supernatant, respectively, and reacted at room temperature for 5 minutes to obtain a supernatant reaction product. Subsequent processes were performed in the same manner and conditions as in Example 2 using the obtained pellet or the reactant in the supernatant. In addition, lipids were extracted by the Bligh & Dyer method using pellets, and the contents of the extracted lipids were compared. The calculated fatty acid content was calculated as a percentage based on the fatty acid content extracted in Comparative Example 2 and is shown in Table 3 below.

division extraction solvent Fatty acid content (%) Comparative Example 2 Bligh & Dyer 100 pellet Bligh & Dyer 77.5 ethyl acetate 41.4 Ethyl Acetate: Ethanol 83.2 nucleic acid 32.1 butyl acetate 62.3 Butyl Acetate: Ethanol 73.5 supernatant nucleic acid 2.7 chloroform 7.7 ethyl acetate 17.2

As shown in Table 3, the lipids extracted from the pellets using a mixed solvent of ethyl acetate and ethanol contained 83.2% of fatty acids based on Comparative Example 2. On the other hand, in the case of lipid extraction from the supernatant, the highest amount of 17.2% of fatty acids was extracted when ethyl acetate was used as a solvent.

Claims (15)

heating the microalgae-containing solution under high pressure; and
A method for extracting lipids from microalgae, comprising the step of extracting lipids by adding a mixed solvent of ethyl acetate and alcohol to a heated microalgae-containing solution.
The method of claim 1, wherein the microalgae-containing solution has a moisture content of 80% to 99%.
The method of claim 1, wherein the microalgae are marine microalgae or freshwater microalgae.
The method of claim 3, wherein the microalgae is Nannochloropsis Oceania ( Nannochloropsis oceania ), a method for extracting lipids from microalgae.
The method for extracting lipids from microalgae according to claim 1, wherein the high pressure is 5 to 50 bar.
The method of claim 1, wherein the heating is performed at a temperature of 160 to 260 °C.
The method of claim 1, wherein the mixed solvent is a mixture of ethyl acetate and alcohol in a volume ratio of 1:1 to 5:1.
The method of claim 7, wherein the mixed solvent is a mixture of ethyl acetate and alcohol in a volume ratio of 2:1.
The method _of claim 1, wherein the alcohol is a C _1-4 lower alcohol.
The method of claim 1, wherein the extracted lipids are neutral lipids and polar lipids.
A first step of heating the microalgae-containing solution under high pressure;
A second step of obtaining a solvent layer by adding a mixed solvent of ethyl acetate and alcohol to the heated microalgae-containing solution; and
A system for extracting lipids from microalgae, including a third step of obtaining lipids from the solvent layer.
A first step of heating the microalgae-containing solution under high pressure;
A second step of separating the solvent layer and pellets by solid-liquid separation of the heated microalgae-containing solution;
A third step of extracting lipids by adding ethyl acetate to the solvent layer;
A fourth step of extracting lipids by adding a mixed solvent of ethyl acetate and alcohol to the pellets; and
A system for extracting lipids from microalgae, comprising a fifth step of obtaining lipids extracted in the third and fourth steps.
The system for extracting lipids from microalgae according to claim 11 or 12, wherein the high pressure is 5 to 50 bar.
The system for extracting lipids from microalgae according to claim 11 or 12, wherein the heating is performed at a temperature of 160 to 260 ° C.
The system for extracting lipids from microalgae according to claim 11 or claim 12, wherein the mixed solvent is a mixture of ethyl acetate and alcohol in a volume ratio of 1:1 to 5:1.

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