KR20130028823A - Methods for preparing biodiesel using animal cell culture waste - Google Patents

Abstract

PURPOSE: A method of manufacturing biodiesel is provided to manufacture biodiesel using cultured animal cell waste without a separate waste treatment process. CONSTITUTION: A method of manufacturing biodiesel from cultured animal cell salvage comprises a first extracting step of adding a polar organic solvent to the collected wet cultured animal cell salvage and stirring to extract lipid; a crude lipid yielding step of layer separating the supernatant and cell pellets and concentrating the supernatant to manufacture concentrate; a second extracting step of adding acid/alcohol solution to yielded crude lipid, refluxing and extracting with a non-polar organic solvent; and a separating step of collecting the non-polar solvent layer and concentrating under the reduced pressure to separating biodiesel. The animal cell is the CHO (Chinese hamster ovary) cell, the BHK (baby hamster kidney) cell, the HEK (human embryonic kidney) cell, and the NS 0 (myeloma cell line) cell. The animal cell is the CHO cell. The polar organic solvent is methanol, ethanol, isopropyl alcohol, acetone or methanol/chloroform solvent mixture. The polar organic solvent is applied at 1-10 times of volume to the volume of the cultured animal cell salvage. The mixing ratio of the methanol/chloroform mixed solution is 1:4 to 4:1. The acid/alcohol solution in the second extracting step is sulfuric acid/methanol solution, sulfuric acid/ethanol solution, anhydrous hydrochloride/methanol solution, anhydrous hydrochloride/ethanol solution, acetyl chloride/methanol, and acetyl chloride/ethanol solution. The acid/alcohol solution in the second extracting step has 1-5 % (w/v) of concentration. The acid/alcohol solution in the second extracting step is applied to 0.2-20 times of volume (v/w) to the crude lipid.

Description

Method for preparing biodiesel using cultured animal cell waste {Methods for preparing biodiesel using animal cell culture waste}

The present invention relates to a method for producing biodiesel, and more particularly, to a method for producing biodiesel using culture animal cell waste.

In general, biodiesel, which is one of the important sustainable production energy, is produced using fatty acid glycerol ester as alkyl ester of fatty acid, and most fatty acid glycerol ester is an edible oil which is the most important food resource for human survival. When used as a raw material of diesel, it can cause a shortage of food resources and a sharp increase in the price of food resources.

Since the wastes generated by human production are the main cause of the destruction of the environment, the utilization of wastes is necessary for the continued survival of humankind. In particular, the technology for producing and reusing energy from these wastes is attracting attention because it can solve the problems of the production of biodiesel using the food resources described above and prevent environmental destruction.

On the other hand, animal cells such as CHO cells are widely used for the production of expensive pharmaceuticals such as therapeutic antibodies, and the quality of the products is excellent. As waste has a high organic content, it rapidly raises the demand for biological oxygen at disposal and consumes a lot of energy when incinerated for disposal. It is being discarded. Therefore, there is a need for the development of a method for recycling the remaining cell waste used for protein production.

However, cultured animal cell waste is a living cell that is recovered wet, and is composed of lipids, carbohydrates, proteins, nucleic acids, other trace elements, and large amounts of water, and is thought to be recyclable through the use of the cellular components themselves. Since the development of the method and the method of use is not made, it is not recycled and is disposed of entirely.

The present invention is to solve a number of problems, including the above problems, it is an object of the present invention to provide a method for producing biodiesel using a culture animal cell waste. However, these problems are exemplary and do not limit the scope of the present invention.

According to one aspect of the invention, the first extraction step of extracting the lipid by stirring after adding a polar organic solvent to the recovered recovered cultured animal cells; After separating the supernatant and the cell precipitate, the supernatant is obtained by concentrating the supernatant to prepare a concentrate. A secondary extraction step of adding an acid / alcohol solution to the obtained crude oil and heating it under reflux to extract it with a nonpolar organic solvent; And recovering the non-polar organic solvent layer and concentrating under reduced pressure to separate the biodiesel.

At this time, the culture animal cells may be used as CHO (Chinese hamster ovary) cells, BHK (baby hamster kidney) cells, HEK (human embryo kidney) cells, NS0 (myeloma cell line) cells, preferably CHO cells Can be used.

The polar organic solvent may be chloroform-methanol mixed solvent, isopropyl alcohol, ethanol, methanol or acetone.

The polar organic solvent may be added in a volume of 1 to 10 times the volume of the culture animal cell harvest, more preferably 4 to 7 times. The mixing ratio of the methanol / chloroform mixed solution in the polar organic solvent may be 1: 4 to 4: 1, preferably 1: 1 to 3: 1.

The polar organic solvent used in the crude oil obtaining step may be methanol or ethanol, and may be added in a volume of 1 to 10 times that of the concentrate.

The acid / alcohol solution of the second extraction step may be sulfuric acid / methanol solution, sulfuric acid / ethanol solution anhydrous hydrochloric acid / methanol, anhydrous hydrochloric acid / ethanol, acetyl chloride / methanol and acetyl chloride / ethanol, and the concentration is 1-5% ( w / v), and may be added in a volume of 0.2 to 20 times (v / w) relative to the crude oil. Heat reflux in the secondary extraction step may be carried out at a temperature of 50 to 75 ℃, may be carried out for 2 hours to 10 hours preferably 5 hours to 8 hours. In the second extraction step, the nonpolar organic solvent may be normal hexane, normal heptane, cyclohexane, diethyl ether, or a mixed solvent of at least two or more thereof, and may have a volume of 2 to 20 times (v / v) relative to the acid / alcohol solution. Can be added.

The cultured animal cell precipitate produced and produced by biodiesel may be a dry organic matter that is easily composted after drying.

Generally, when extracting lipids, raw materials are used in a dried state, but cultured animal cell wastes recovered in the purification process after cultivation are recovered in a wet, viscous liquid soup state, and for drying, it consumes a large amount of energy such as freeze drying and spray drying. It is necessary to process. Therefore, the present inventors have attempted to develop a method of directly extracting lipids from animal cell waste in a wet state using organic solvents by using a feature that lipids are transferred to organic solvents. When extracting lipids by directly adding an organic solvent to the recovered product, it was confirmed that lipids can be extracted according to the type of organic solvent. Recovered wet cultured animal cell wastes typically contain 80-85% water, and the cells are powdered according to the type of organic solvent used when stirring and extracting organic solvents directly to wet cultured animal cell wastes. It was confirmed that the lipid can be extracted in high yield.

When polar organic solvents such as chloroform-methanol mixed solvent, isopropyl alcohol, ethanol, methanol and acetone were added at a volume of 1 to 10 times the volume of wet animal cells, no gel was formed and lipids were extracted in high yield. The residue can be easily made into a solid component so that the extracted crude oil can be used as a raw material for biodiesel production, and the remaining residue can be recovered for easy drying and used for secondary utilization. The extracted crude oil is a large amount of side reaction saponification reaction when heated under the base bio-diesel conversion method of base / methanol solvent, making it difficult to produce biodiesel, and 0.2 times volume, which is the conventional method even when using the acid catalyst sulfuric acid / methanol method. When methanol was used, it was confirmed that only a part of biodiesel was decomposed into fatty acids. To overcome this problem, heating reflux was performed within 10 hours using sulfuric acid / methanol of 10 times or more, and normal hexane and normal By confirming that evaporation of the solvent after extraction, such as heptane, cyclohexane, diethyl ether, or a mixed solvent of any two or more thereof, can be effectively converted to biodiesel when extracted with a nonpolar organic solvent having advantageous low temperature volatility. The invention has been completed (see FIGS. 1 and 2).

According to one embodiment of the present invention made as described above, it is possible to produce biodiesel that is renewable energy from animal cell waste that has been discarded until now, by producing biodiesel without a separate waste treatment process, environmental improvement and energy acquisition The degreasing animal cell sediment remaining after the manufacture of biodiesel is also easy to dry, making it a dry powder that is easy to store and transport, which makes it easier to reuse and recycles as compost to prevent environmental pollution and reuse. Economic effects can be achieved. Of course, the scope of the present invention is not limited by these effects.

1 is a photograph of a test tube containing biodiesel prepared from CHO cell waste using the method according to an embodiment of the present invention.
2 is a gas chromatogram of biodiesel prepared from CHO cell waste using the method according to one embodiment of the present invention:
A: myristic acid; B: palmitic acid; C: stearic acid;
D: oleic acid; E: 10-octadecanoic acid F: nonadecanoic acid (internal standard);
G: linoleic acid; H: linolenic acid; And I: arachidonic acid.

Hereinafter, the present invention will be described in detail with reference to Examples and Experimental Examples. However, the following Examples and Experimental Examples are merely illustrative of the present invention, and the present invention is not limited to the following Examples and Experimental Examples.

Example 1 Preparation of Wet CHO Cell Waste

CHO cells were cultured in an animal cell incubator using serum free medium ProCHO5 (Lonza, 12-766Q). Specifically, the medium made by adding 4 mM glutamine to ProCHO5 was sterile filtered and injected with 1 L (working volume) into a 3 L Applikon continuous agitated cell incubator, followed by adding 4 mM glutamine to ProCHO5 in a 125 ml Erlenmeyer flask. CHO DUKX-B11 cells incubated for 3 days at 37 ° C. with 5% CO ₂ were inoculated with cells cultured in 6 20 ml flasks at a cell concentration of 3 × 10 ⁵ cells / ml in the incubator. Cultivation was started and the cultured cells were cultured for 8 days at 100 rpm, DO 40%, 37 ℃. As a result, the maximum cell concentration reached 3.76 × 10 ⁶ cells / ml on the 4th day of culture.

Aseptically filter medium made by adding 4 mM glutamine to ProCHO5 and inject 14 L into a 20 L continuous agitated cell culture, inoculate 1 L of the cultured CHO DUKX-B11 cells at 100 rpm, DO 40%, 37 Incubated for 8 days.

The culture was then centrifuged at 1500 rpm for 3 minutes to give 320 ml of wet CHO cell waste in a viscous liquid phase.

Example 2: Extraction of Lipids and Preparation of Skim CHO Cells

Into each 20 ml of CHO cell waste, 100 ml of normal hexane, chloroform, acetone, methanol, ethanol, isopropyl alcohol, methanol / chloroform 2: 1 mixed solvent was added and stirred at room temperature overnight using a magnetic stirrer to extract lipids. Next, the supernatant and cell residues were separated by centrifugation at 3,000 rpm for 10 minutes. The supernatant and the cell precipitate were separated, and the supernatant was concentrated in a water bath at 50 ° C. using a reduced pressure rotary evaporator, and evaporated again by adding 20 ml methanol to 1-2 g of the concentrated residue, and the cell residue was obtained by hot air dryer. Dried overnight at 40 ° C. to obtain dry CHO cell residue (Table 1).

Yield of Crude and Dried CHO Cell Residues by Extracted Organic Solvents Organic solvent Crude oil (g) Degreasing CHO Cells (g) Degreasing
CHO Cell Status Methanol / chloroform
2: 1 (v / v) 0.87 2.69 powder Isopropyl Alcohol 0.80 2.84 Powder + lump ethanol 0.87 2.88 Lump Methanol 0.74 2.65 Lump Acetone 0.60 3.07 Lump chloroform ND ND ND Normal hexane ND ND ND ND: not measurable

As a result, when a polar organic solvent such as methanol / chloroform mixed solvent, isopropyl alcohol, ethanol, methanol, acetone was used, crude lipid was separated and degreasing CHO cells from which lipid components were removed were separated. On the other hand, when extracted with chloroform or normal hexane, which is a non-polar organic solvent, the emulsification phenomenon was intensified and the supernatant was not separated, so that only 20 ml of the extraction solvent was separated and concentrated and used for the biodiesel conversion reaction.

Example 3: Conversion of Biodiesel

10% volume (v / w) of 3% (W / v) sulfuric acid / methanol solution was added to each crude oil extracted in Example 2, and the reaction was performed by heating at 65 ° C. for 5 hours in a sealed state, and then using 3% (W / v) 10 mg of sulfuric acid / methanol solution, 0.3 mg naphthalene / normal heptane solution was added, vigorously stirred with a vortex mixer for 1 minute, and the upper layer was taken and analyzed by gas chromatography to determine the amount of fatty acid methyl produced. 2). Naphthalene is an internal standard in gas chromatography analysis, and normal heptane was used as a solvent.

Production of Biodiesel by Extraction Solvents Organic solvent Fatty acid methyl
Production amount (g) of biodiesel Biodiesel conversion per dry cell (%) Methanol / chloroform
2: 1 (v / v) 0.26 7.2 Isopropyl Alcohol 0.16 4.4 ethanol 0.14 3.9 Methanol 0.09 2.5 Acetone 0.10 2.8 chloroform 0.04 ^{1 *} 1.3 Normal hexane 0.01 ^{2 *} 0.3 ^{1 *, 2 *} : Extracts only 20 ml of extract, 5 times the value after concentration

As a result, it was found that the methanol / chloroform mixed solvent, isopropyl alcohol and ethanol produced high amounts of fatty acid methyl, and the biodiesel conversion per dry cell was also excellent. In the case of methanol and acetone, the yield was slightly lower than those of the three polar organic solvents, but fatty acid methyl was produced. On the other hand, in the case of non-polar organic solvents, chloroform and normal hexane, which failed to extract crude oil, the amount of fatty acid methyl produced was extremely low.

The fatty acid methyl content was calculated by the method described in the following experimental example.

Experimental Example 1: Content analysis of biodiesel

A 0.3 m mm inner diameter 15 m capillary column coated with 5% phenylpolydimethylsiloxane crosslinked (DH-5HT) filler 0.1 μm thick was used on a Younglin M600D gas chromatography instrument. After raising 15 ℃ per minute to 180 ℃ and then to 7 ℃ per minute to 230 ℃ and then to 30 ℃ per minute to 380 ℃ and then maintained at 380 ℃ for 10 minutes. Helium gas was flowed into the mobile phase by 3 ml per minute, the inlet temperature was 300 ° C, the detector was used a flame ionization detector (FID) and 380 ° C. The sample was injected with 1 μl.

Naphthalene was dissolved in normal heptane at 0.3 mg / ml as an internal standard and biodiesel (purity 99.5% KS M 2413 method) prepared by soybean oil was used as a reference standard. The amount of biodiesel was quantified by the sum of the area of peaks between 7 minutes and 12 minutes, which is the detection time of biodiesel, and the formula used for the quantification is as follows.

[∑A] _std : Total area of peak from RT7 to RT12 in the control soybean oil biodiesel assay

A _Istd: Area of internal standard peak in control soybean oil biodiesel analysis

m _std: Sample weight (g) taken from control soybean oil biodiesel

V _std : Volume of control standard soybean oil biodiesel dissolved naphthalene solution (ml)

[∑A] _sample : Total area of the peak from RT7 to RT12 of the analysis sample.

A _Isample : area of naphthalene (internal standard) of analytical sample

V _sample : Volume of extracted normal hexane of analytical sample (ml)

Example 4 Preparation of Biodiesel from Wet CHO Cell Waste

After adding 750 ml of methanol / chloroform = 2: 1 (v / v) solution to 150 ml of wet CHO cell waste, the mixture was stirred overnight at room temperature with a magnetic stirrer and centrifuged at 1500 rpm for 10 minutes to obtain a supernatant. Meanwhile, 100 ml of methanol / chloroform = 2: 1 (v / v) solution was added to the precipitated cell residue, and then resuspended. The mixture was centrifuged at 1500 rpm for 10 minutes to obtain a supernatant, which was combined with the supernatant obtained above under reduced pressure. It was concentrated by heating with a water bath. 100 ml methanol was added to the concentrated residue, suspended, and concentrated under reduced pressure to obtain 6.7 g of crude fat. The supernatant was separated and the remaining precipitate was collected and dried overnight at 40 ° C. in a hot air dryer to obtain 20.2 g of degreased CHO cell powder.

67 ml of 3% sulfuric acid / methanol solution was added to 6.7 g of the crude fat, which was heated under reflux for 5 hours, followed by extraction with 670 ml of normal hexane twice. The layers of normal hexane were collected and washed with 300 ml of purified water. The concentrated residue was washed with 1 ml distilled water, and then concentrated under reduced pressure at 1 mmHg high vacuum to obtain 1.4 g of light yellow biodiesel transparent liquid (FIG. 1).

The ratio of fatty acids constituting the obtained biodiesel was as shown in Table 3 of the GC-MS analysis (Fig. 2), and the cloud point was 8 ° C.

 Fatty Acid Distribution of CHO Cell-Derived Biodiesel Fatty Acid Methyl Ester percentage(%) Relative distribution Remarks Myristic acid 1.36 0.09 Saturated fatty acid Palmitic acid 14.58 One Saturated fatty acid 7-hexadecanoic acid 2.20 0.15 Unsaturated fatty acid Palistoleic acid 5.67 0.39 Unsaturated fatty acid Stearic acid 7.17 0.49 Saturated fatty acid Elide acid 0.98 0.07 Unsaturated fatty acid Oleic acid 50.42 3.45 Unsaturated fatty acid 10-octadecanoic acid 16.60 1.13 Unsaturated fatty acid Linoleic acid 0.48 0.03 Unsaturated fatty acid Linolenic acid 0.41 0.03 Unsaturated fatty acid Arachidonic acid 0.13 0.01 Saturated fatty acid sum 100 Saturated fatty acid 23.24 1.59 Monounsaturated fatty acids 75.86 5.187 Polyunsaturated fatty acid 0.89 0.061

As shown in Table 3, the biodiesel extracted from CHO cells has an oleic acid content of more than 50%, an unsaturated fatty acid content of more than 75%, a relatively low cloud point, and is suitable as a fuel for transportation internal combustion engines. It can be seen.

The fatty acid methyl ester content described above was analyzed according to the following experimental example.

Experimental Example 2: Fatty Acid Composition Distribution Experiment

Using the same SIMADZU GC-MS-QP2010 Plus instrument, a 0.25 mm inner diameter 100 m capillary column (Rt ^® -2560) coated with a 0.2 μm thick biscyanopropyl polysiloxane filler was used. After increasing 4 ℃ per minute up to 240 ℃ 5 minutes at 140 ℃ was maintained at 240 ℃ for 15 minutes. Helium gas was flowed into the mobile phase by 3 ml per minute, the inlet temperature was 250 ° C., the detector used a mass spectrometer at 250 ° C., and the detection voltage was 1.5 kV and the sample was injected with 1 μl.

As an internal standard, methyl methyl ester of 19 carbon atoms (C19: 0, nonadecanoic acid) was used at 0.4 mg / ml in normal heptane, and it was quantified by comparing the concentration of internal standard and fatty acid methyl ester with the peak area It was. The calculation formula used for quantification is as follows.

ΣA: Total area of fatty acid methyl ester peak

A _IS : area of nonadecanoic acid (internal standard)

A _P : Peak area of each fatty acid Methyl Ester

While the present invention has been particularly shown and described with reference to exemplary embodiments thereof, it is to be understood that the invention is not limited to the disclosed embodiments, but, on the contrary, is intended to cover various modifications and equivalent arrangements included within the spirit and scope of the appended claims. Therefore, the true technical protection scope of the present invention will be defined by the technical spirit of the appended claims.

Claims (12)

A primary extraction step of adding lipid organic solvent to the recovered wet culture animal cell harvest and stirring to extract lipids;
After separating the supernatant and the cell precipitate, the supernatant is obtained by concentrating the supernatant to prepare a concentrate.
A secondary extraction step of adding an acid / alcohol solution to the obtained crude oil and heating it under reflux to extract it with a nonpolar organic solvent; And
A method for producing biodiesel from a cultured animal cell harvest comprising a separation step of separating the biodiesel by vacuum concentration after recovering the non-polar organic solvent layer. The method of claim 1,
The animal cells are CHO (Chinese hamster ovary) cells, BHK (baby hamster kidney) cells, HEK (human embryo kidney) cells, NS0 (myeloma cell line) cells, production method of biodiesel from the culture animal cell recovery. The method of claim 2,
The animal cell is a CHO cell,
Method of producing biodiesel from cultured animal cell harvest. The method of claim 1,
The polar organic solvent is methanol, ethanol, isopropyl alcohol, acetone or methanol / chloroform mixed solvent,
Method of producing biodiesel from cultured animal cell harvest. The polar organic solvent is added in a volume of 1 to 10 times the volume of the culture animal cell harvest,
Method of producing biodiesel from cultured animal cell harvest. 5. The method of claim 4,
The mixing ratio of the methanol / chloroform mixed solution is 1: 4 to 4: 1,
Method of producing biodiesel from cultured animal cell harvest. The method of claim 1,
Acid / alcohol solution of the second extraction step is a sulfuric acid / methanol solution, sulfuric acid / ethanol solution, anhydrous hydrochloric acid / methanol solution, anhydrous hydrochloric acid / ethanol solution, acetyl chloride / methanol and acetyl / ethanol solution,
Method of producing biodiesel from cultured animal cell harvest. The method of claim 1,
Acid / alcohol solution of the secondary extraction step is 1 to 5% (w / v) concentration,
Method of producing biodiesel from cultured animal cell harvest. The method of claim 1,
The acid / alcohol solution of the second extraction step is applied to 0.2 to 20 times the volume (v / w) compared to the crude oil,
Method of producing biodiesel from cultured animal cell harvest. The method of claim 1,
Heating reflux in the secondary extraction step is carried out for 2 to 10 hours at a temperature of 50 to 75 ℃,
Method of producing biodiesel from cultured animal cell harvest. The method of claim 1,
The non-polar organic solvent is normal hexane, normal heptane, cyclohexane, diethyl ether or a mixed solvent thereof,
Method of producing biodiesel from cultured animal cell harvest. The method of claim 1,
The non-polar organic solvent is added in 2 to 20 times the volume (v / v) compared to the acid / alcohol solution,
Method of producing biodiesel from cultured animal cell harvest.

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