KR101727292B1 - Producing Method of Biodiesel Using Acid Oil Derived From Soapstock and Enzymes - Google Patents

Abstract

More particularly, the present invention relates to a process for producing biodiesel, which comprises preparing an acid oil from a soapstock produced as a pollutant in the process of producing rice bran oil, And a method for producing biodiesel by an esterification reaction. According to the production method of the present invention, biodiesel can be obtained at a high yield.

Description

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method for producing biodiesel using an acid oil derived from soap,

The present invention relates to a process for producing biodiesel using acid oils derived from soap fractions and enzymes, and more particularly, to a process for producing biodiesel from acid soap from soapstock produced as a by-product in the process of producing rice bran oil. ), And subjecting the acid oil to an esterification reaction in the presence of an enzyme to produce biodiesel.

As the global petroleum reserves decrease and the reduction of greenhouse gas emissions caused by the use of fossil fuels becomes mandatory, the importance of biodiesel development as an alternative fuel is increasing. Biodiesel is a non-toxic, biodegradable and renewable fuel energy with the advantage of low greenhouse gas emissions such as carbon monoxide, carbon dioxide and sulfuric acid gas.

Biodiesel is a monoalkyl ester of long chain fatty acids and short chain alcohols in vegetable oils and animal fats. Generally, the biodiesel synthesis reaction uses a chemical catalyst or an enzyme catalyst. The chemical catalytic reaction uses sodium hydroxide, potassium hydroxide, or sulfuric acid, and has a merit that a time and an initial cost are small in the production of biodiesel, There is a way. However, there is a disadvantage in that it takes a great deal of cost for treating the wastewater generated in the neutralization reaction and the salt removal process, which must be performed after the reaction, and that glycerol can not be recovered.

On the other hand, when an enzyme is used as a catalyst in the production of biodiesel, there is a disadvantage in that the reaction time is relatively long and the enzyme is expensive. However, since the catalyst and the reaction product are easily separated from each other, There is an advantage that recovery is easy. In addition, since the enzyme reaction takes place under relatively mild conditions, there is no need to use additional energy, and the advantage of preventing soap formation and increasing the biodiesel yield can be obtained.

Currently, refined oil is used mainly for research on biodiesel production. Refining oil costs account for about 70% of biodiesel production cost. Because refined oil is too expensive to be used as raw material for biodiesel, There is a problem that the economy is poor. In addition, since most refined oils are produced from crops, prices fluctuate more or less depending on crops of the crops.

Therefore, there is a high expectation for a production method that uses an enzyme in a biodiesel production method, and increases the reaction efficiency thereof and lowers the production cost to make it economical.

Korean Patent Publication No. 10-2010-0029458 Korean Patent Publication No. 10-2013-0008672 Korean Patent Publication No. 10-2013-0099537

Lara Pizarro, A.V., Park, E.Y. 2003. Lipase-catalyzed production of biodiesel fuel from vegetable oils contained in waste activated bleaching earth. Process Biochemistry, 38 (7), 1077-1082. Ma, F., Hanna, M.A. 1999. Biodiesel production: a review. Bioresource Technology, 70 (1), 1-15.

The present invention relates to a process for producing an acid oil from a soapstock derived from a microbial oil and using the acidic oil and an alcohol having 1 to 4 carbon atoms as a substrate, It is an object of the present invention to provide a method for producing diesel.

It is another object of the present invention to provide a condition for obtaining biodiesel from the biodiesel production method.

It is another object of the present invention to improve the economical efficiency and efficiency of the biodiesel production process by reusing the enzyme used in the biodiesel production process.

In order to achieve the above object,

(1) preparing an acid oil from a soapstock derived from a rice bran oil; And

(2) the above-mentioned substrate is made of the acidic oil and the alcohol having 1 to 4 carbon atoms, and the substrate is composed of Novozym 435, Lipozyme RM IM and Lipozyme TL IM Wherein the esterification reaction is carried out by reacting at least one kind of enzyme selected from the group consisting of:

The biodiesel production method of the present invention can obtain biodiesel at a high yield under optimum conditions.

Further, the biodiesel production method of the present invention can reuse the enzyme, thereby increasing the efficiency and economical efficiency.

1 is a graph showing the yield of biodiesel according to the type of enzyme.
2 is a graph showing the yield of biodiesel according to molar ratio between substrates when Novozym 435 (Novozym 435) alone is used.
FIG. 3 is a graph showing the yield of biodiesel according to molar ratio between substrates when Lipozyme RM IM alone is used.
FIG. 4 is a graph showing the yield of biodiesel according to molar ratio between substrates when lipozyme TL IM (Lipozyme TL IM) alone is used.
5 is a graph showing the yield of biodiesel according to the reaction temperature when Novozym 435 (Novozym 435) alone is used.
6 is a graph showing the yield of biodiesel according to the reaction temperature when Lipozyme RM IM is used alone.
FIG. 7 is a graph showing the yield of biodiesel according to the reaction temperature when lipozyme TL IM (Lipozyme TL IM) alone is used.
8 is a graph showing the yield of biodiesel according to the amount of enzyme used when Novozym 435 (Novozym 435) alone is used.
FIG. 9 is a graph showing the yield of biodiesel according to the amount of enzyme used when Lipozyme RM IM alone is used.
10 is a graph showing the yield of biodiesel according to the amount of enzyme used when lipozyme TL IM (Lipozyme TL IM) alone is used.
11 is a graph showing the yield of biodiesel after primary esterification with Novozym 435 (Novozym 435) followed by secondary esterification with Lipozyme TL IM. At this time, the first ester reaction time in the two-step reaction was varied, and the results were shown. 5 minutes (●), 15 minutes (○), 30 minutes (▼), 60 minutes (△). ■ and □ indicate a single enzyme reaction of Novozym 435, Lipozyme TL IM, respectively.
12 is a graph showing the yield of biodiesel after primary esterification with Lipozyme RM IM and subsequent secondary esterification with Lipozyme TL IM. At this time, the first ester reaction time in the two-step reaction was varied, and the results were shown. 5 minutes (●), 15 minutes (○), 30 minutes (▼), 60 minutes (△). ■ and □ represent the single enzyme reaction of Lipozyme RM IM and Lipozyme TL IM, respectively.
13 is a schematic diagram showing a continuous reactor in which glycerol is not removed.
14 is a schematic view showing a continuous reactor for removing glycerol.
15 is a graph showing the yield of biodiesel according to the moisture content of the substrate when using liposomes TL IM (Lipozyme TL IM) in a continuous reactor.
16 is a graph showing the yield of biodiesel according to the reaction temperature when using liposomes TL IM (Lipozyme TL IM) in a continuous type reactor.
17 is a graph showing the yield of biodiesel according to the molar ratio between the substrates when the liposomes TL IM (Lipozyme TL IM) is used in a continuous reactor.
18 is a graph showing the yield of biodiesel according to the number of times of using the enzyme when the liposome TL IM is used in a continuous type reactor, b is the case where glycerol produced during the reaction is removed, and a is not removed .

Hereinafter, the present invention will be described in more detail.

The present invention relates to a method for producing biodiesel,

(1) preparing an acid oil from a soapstock derived from a rice bran oil; And

(2) the above-mentioned substrate is made of the acidic oil and the alcohol having 1 to 4 carbon atoms, and the substrate is composed of Novozym 435, Lipozyme RM IM and Lipozyme TL IM And a step of reacting the enzyme with at least one enzyme selected from the group consisting of an esterification reaction and an esterification reaction.

The method for producing biodiesel according to the present invention uses an acid oil made from soapstock derived from rice bran oil as a substrate to prevent environmental pollution and reduce manufacturing cost. The soapstock derived from the rice bran oil is a by-product produced during the rice bran oil refining process.

The acid oil obtained from soya oil derived soapstock according to the present invention is composed of free fatty acids (39-79%), acylglycerol (18-30%), pigments and other hydrophilic substances, It is a raw material suitable for manufacturing.

In the step (1), the acid oil may be prepared by adding isopropanol, hexane, sulfuric acid, and water together with the soap-derived soap component and stirring.

More specifically, the acid oil was prepared by stirring the soya bean oil derived from ungreased oil and hexane with an impeller mixer, adding sulfuric acid and distilled water, transferring the reaction product to a separating funnel, adding hexane and washing with distilled water three or four times, 5 or more, the hexane layer is collected in a round flask while passing through anhydrous sodium sulfate, and the hexane is evaporated using a vacuum rotary evaporator.

However, the method for producing the acid oil is not limited thereto, and the method for producing the acid oil from the un-oil-derived soap powder can be applied to anyone skilled in the art.

In the step (2), biodiesel can be obtained by adding an enzyme to the substrate using the acid oil and the alcohol having 1 to 4 carbon atoms as a substrate to proceed the esterification reaction .

The enzyme may be at least one selected from the group consisting of Novozym 435 (Novozym 435), Lipozyme RM IM, and Lipozyme TL IM.

As a result of an active screening of various enzymes for obtaining biodiesel optimally, the three enzymes were found to be significantly higher in basic conditions than those of enzymes derived from other strains (derived from Lipase AYS-Candida rugosa and Lipase PS-Pseudomonas fluorescence) In the present invention, the three enzymes were selected as the enzymes used for producing biodiesel.

The esterification reaction of step (2) of the present invention may be a batch or a continuous process.

The type of enzyme used, the molar ratio between the substrates, the reaction temperature, and the amount of enzyme used are very important factors in the esterification reaction of the substrate using the enzyme.

When each of the above three enzymes is used alone, reaction conditions for optimally obtaining biodiesel through the esterification reaction are as follows.

When Nobobizam 435 (Novozym 435) is used alone, the esterification reaction conditions are such that the molar ratio of the acid oil used as the substrate and the alcohol having 1 to 4 carbon atoms is 1: 4 to 1: 6, preferably 1: 5. When the molar ratio between the substrates is less than 1: 4, the yield of biodiesel decreases. When the molar ratio exceeds 1: 6, there is no distinct difference from the case where the molar ratio is 1: 5.

The reaction temperature is preferably 20 to 60 ° C. When the reaction temperature is lower than 20 ° C, the yield of biodiesel decreases. When the reaction temperature exceeds 60 ° C, Novozym 435 enzyme may be degraded.

The Novozym 435 enzyme is preferably used in an amount of 5 to 15% by weight based on the total weight of the substrate. When the content is less than 5% by weight, the yield of biodiesel is reduced. When the content is more than 15% by weight, the difference is not remarkably different from that when the content is more than 15% by weight.

When the liposomes RM IM are used alone, the esterification reaction conditions are such that the molar ratio of the acidic oil used as the substrate and the alcohol having 1 to 4 carbon atoms is 1: 2 to 1: 6, 1: 5. When the molar ratio between the substrates is less than 1: 2, the yield of biodiesel decreases. When the molar ratio exceeds 1: 6, there is no distinct difference from the case where the molar ratio is 1: 6.

The reaction temperature is preferably 10 to 50 占 폚. If the reaction temperature is lower than 10 DEG C or higher than 50 DEG C, the yield of biodiesel decreases.

In addition, the lipozyme RM IM enzyme is preferably used in an amount of 2.5 to 15% by weight based on the total weight of the substrate. When the content is less than 2.5% by weight, the yield of biodiesel is reduced. When the content is more than 15% by weight, the difference is not remarkably different from that when the content is more than 15% by weight.

When the liposomes TL IM is used alone, the esterification reaction conditions are such that the molar ratio of the acid oil used as the substrate and the alcohol having 1 to 4 carbon atoms is 1: 2 to 1: 6, 1: 5. When the molar ratio between the substrates is less than 1: 2, the yield of biodiesel decreases. When the molar ratio exceeds 1: 6, there is no distinct difference from the case where the molar ratio is 1: 6.

The reaction temperature is preferably 10 to 45 ° C. If the reaction temperature is below 10 ° C or above 45 ° C, the yield of biodiesel decreases.

In addition, the lipozyme TL IM enzyme is preferably used in an amount of 10 to 15% by weight based on the total weight of the substrate. When the content is less than 10% by weight, the yield of biodiesel is decreased. When the content is more than 15% by weight, the difference is not remarkably different from that when the content is more than 15% by weight.

The numerical ranges of the molar ratio between the substrates, the reaction temperature and the amount of enzyme used when the above three enzymes are used alone do not limit the scope of the present invention but are included in the equivalent range of the present invention.

In the present invention, the esterification reaction in the step (2) may be carried out by using a different enzyme to conduct the first esterification reaction and then the second esterification reaction.

Novozymes 435 and Lipozyme RM IM enzymes have high activity and residual activity, which can be widely used for catalytic reactions using enzymes. However, since both enzymes are expensive, commercial production of biodiesel using the above two enzymes may cause a rise in cost. Therefore, the present invention aims to reduce the production cost by using lipozyme TL IM as a substitute enzyme which is at least 10 times and at most 20 times lower than the two enzymes, so that it can be applied in real industry.

Accordingly, in the present invention, the first esterification reaction is performed using at least one enzyme selected from the group consisting of Novozym 435 (Novozym 435) and Lipozyme RM IM as enzymes in step (2) A step of filtering and removing the enzyme, and a step of performing a secondary esterification reaction by adding Lipozyme TL IM as an enzyme to the biodiesel.

In other words, we tried to control the reaction rate by reacting two different enzymes step by step.

Specifically, the first esterification reaction was carried out for a relatively short time (5, 15, 30, 60 minutes) with Novozyme 435 or Lipozyme RM IM having high enzyme activity, and the liposomal TL IM was used for the remaining 24 hours To proceed the secondary esterification reaction.

The primary esterification reaction is carried out for 5 to 60 minutes, preferably for 15 minutes. If the primary esterification reaction time is less than 5 minutes, the reaction time may be too short, which may lower the initial reaction rate. If the primary esterification reaction time exceeds 60 minutes, the primary esterification reaction time is not different from 60 minutes or less. Is lowered.

When Novozym 435 is used alone as the enzyme in the primary esterification reaction, the molar ratio of the acid oil used as the substrate and the alcohol having 1 to 4 carbon atoms is 1: 4 to 1: 6, and the reaction temperature is 20 to 60 ° C., and Novozym 435 is used in an amount of 5 to 15% by weight based on the total weight of the substrate.

When the liposome RM IM is used alone as the enzyme in the primary esterification reaction, the molar ratio of the acid oil used as the substrate and the alcohol having 1 to 4 carbon atoms is 1: 2 to 1: 6, 50 < 0 > C, and lipozyme RM IM is used in an amount of 2.5 to 15% by weight based on the total weight of the substrate.

After the first esterification reaction, the enzyme is removed by filtration, and liposome TL IM is added to proceed the secondary esterification reaction to produce biodiesel. At this time, the conditions of use of the lipozyme TL IM are the same as or similar to those of the single use.

This method is advantageous in that the initial reaction rate is faster and the maximum yield is higher than the reaction using liposomal TL IM alone. In this way, the biodiesel with the maximum yield similar to the reaction using the expensive enzyme alone can be obtained in a shorter time. In addition, since an expensive enzyme is used for a short time, there is an advantage that the number of times of enzyme reuse can be increased.

Also, the present invention can provide a method for producing biodiesel by reacting the substrate and the enzyme in a packed-bed reactor (PBR) in the step (2).

The molar ratio of the acid oil used as the substrate and the alcohol having 1 to 4 carbon atoms is 1: 3 to 1: 6, preferably 1: 5, as a condition for performing the esterification reaction in the continuous reactor of the substrate and the enzyme . When the molar ratio between the substrates is less than 1: 3, the yield of biodiesel decreases. When the molar ratio exceeds 1: 6, there is no distinct difference from the case where the molar ratio is 1: 6.

The reaction temperature is preferably 0 to 35 占 폚. When the reaction temperature exceeds 35 DEG C, the yield of biodiesel decreases.

In addition, the esterification reaction may proceed in an environment where moisture is added, wherein the water content is preferably 1 to 6 wt% based on the total weight of the substrate. When the content is less than 1% by weight, the yield of biodiesel is reduced. When the content is more than 6% by weight, the difference is not remarkably different from that when the content is more than 6% by weight.

Since the above method uses a continuous reactor, the activity of the lipozyme TL IM enzyme can be maintained and the period of use can be extended by removing glycerol by injecting an alcohol having 1 to 4 carbon atoms per reaction period.

Therefore, by adding the step of removing glycerol in the step (2), the period of use of the enzyme can be extended.

The alcohol having 1 to 4 carbon atoms used in the above-described biodiesel production method is preferably a straight chain, more preferably ethanol. If the carbon number of the alcohol is more than 4, the produced biodiesel can be in a gel state at a low temperature.

Hereinafter, embodiments of the present invention will be described in detail with reference to the drawings. However, the following examples are illustrative of the present invention and are not intended to limit the scope of the present invention.

Manufacturing example  One. Rice oil  origin Soap powder  Used Acid  Manufacture of oil

In a beaker, 100 g of soap, 300 mL of isopropanol, and 300 mL of hexane are stirred with an impeller mixer. 10 mL of sulfuric acid and 10 mL of distilled water are added, the reaction product is transferred to a separatory funnel, and 700 mL of hexane is added. The pH of the solution was adjusted to 5 or higher. The hexane layer was collected in a round flask while passing through anhydrous sodium sulfate and the hexane was evaporated with a rotary vacuum evaporator to prepare an acid oil.

Example  1. Screening of Enzymes Optimized for Biodiesel Production

3 g of a mixture of an acid oil (1.80 g, 6.52 mmol) and a 99% ethanol aqueous solution (1.20 g, 26.09 mmol) as a substrate was placed in a 25 mL scourcap Erlenmeyer flask to test the amount of biodiesel synthesis according to the type of immobilized lipase, 0.3 g (10% by weight) of AYS (Lipase AYS), Lipase PS (Lipase PS), Novozym 435 (Novozym 435), Lipozyme RM IM and Lipozyme TL IM And stirred at a reaction temperature of 30 DEG C at a speed of 300 rpm. The reaction was started and samples were taken at predetermined times, analyzed by gas chromatography, and ethanol was evaporated with nitrogen to measure the acid value.

Sample analysis

20 μL of the biodiesel (fatty acid ester) synthesized after the reaction and the remaining fatty acid, monoglyceride, diglyceride and triglyceride were dissolved in 1 mL of chloroform in a sample collected at regular intervals and analyzed by gas chromatography (Model 3800; Varian, Palo Alto , CA, USA). A flame ionization detector (FID) was used as the detector and a fused silica capillary column (DB-1ht, 15 m x 0.25 mm i.d. X 0.15 μm film thickness, J & W Scientific, Folsom, CA, USA) was used. A 1 μL sample was injected through an autosampler in split mode (split ratio 50: 1), the temperature of the column was maintained at 120 ° C. for 3 minutes, then increased to 370 ° C. at a rate of 20 ° C./minute and maintained at 370 ° C. for 5 minutes It was programmed. The carrier gas was flowed at 1.5 mL / min using helium. Both the injector and the detector were maintained at 370 ° C.

Acid value measurements were performed by the AOCS Cde 3d-63 method.

As a result, the yield of the biodiesel was found to be about 20% or less, while that of Novozym 435, Lipozyme RM (Lipase A RM) and Lipasex RM (Lipase PS) IM) and Lipozyme TL IM (TM) enzyme showed a yield of biodiesel of 80% or more (Fig. 1).

Therefore, three enzymes of Novozym 435 (Novozym 435), RM IM (Lipozyme RM IM) and Lipozyme TL IM (IM) were determined to be suitable enzymes for producing biodiesel, and the three enzymes Was used to prepare biodiesel, and the yield was measured.

Example  2. When manufacturing biodiesel Interstitial Molar ratio  optimization

In order to test the biodiesel synthesis amount according to the molar ratio between substrates, 3 g of a mixture of the acid oil and the 99% ethanol aqueous solution prepared in Preparation Example 1 as a substrate was changed from a molar ratio range of 1: 2 to 1: 6, And the mixture was stirred at a reaction temperature of 30 DEG C at a rate of 300 rpm together with 0.15 g (5% by weight) of Lipozyme RM IM and Lipozyme TL IM. The reaction was started and samples were taken at predetermined times, analyzed by gas chromatography, and ethanol was blown with nitrogen to measure the acid value.

The biodiesel (fatty acid ester) synthesized after the reaction and remaining fatty acid, monoglyceride, diglyceride, triglyceride and acid value were measured in the same manner as in Example 1 above.

The novozyme 435 enzyme showed optimum conditions for obtaining biodiesel at a molar ratio of acid oil and ethanol of 1: 4 to 1: 6 (Fig. 2), and the lipozyme RM IM and lipozyme TL IM enzyme were dissolved in acid oil and ethanol Showed optimum conditions for obtaining biodiesel at a molar ratio of 1: 2 to 1: 6 (FIGS. 3 and 4).

Example  3. Optimization of temperature during biodiesel production

3 g of a mixture of the acid oil prepared in Preparation Example 1 (1.64 g, 5.94 mmols) and a 99% ethanol aqueous solution (1.36 g, 29.57 mmols) as a substrate was placed in a 25 mL scourcap Erlenmeyer flask And the mixture was stirred at a speed of 300 rpm with 0.15 g (5% by weight) of Novozyme 435, Lipozyme RM IM and Ripozyme TL IM. The reaction temperature was 10 ° C to 60 ° C at 10 ° C intervals. The reaction was started and samples were taken at predetermined times, analyzed by gas chromatography, and ethanol was blown with nitrogen to measure the acid value.

The biodiesel (fatty acid ester) synthesized after the reaction and remaining fatty acid, monoglyceride, diglyceride, triglyceride and acid value were measured in the same manner as in Example 1.

The Novozym 435 enzyme showed optimal conditions for obtaining biodiesel at a temperature of 20 to 60 ° C (FIG. 5), and the lipozyme RM IM enzyme showed optimal conditions for obtaining biodiesel at a temperature of 10 to 50 ° C 6), the lipozyme TL IM enzyme showed optimum conditions for obtaining biodiesel at a temperature of 10 to 45 ° C (FIG. 7).

Example  4. When manufacturing biodiesel Enzymatic  optimization

3 g of a mixture of the acid oil (1.64 g, 5.94 mmols) prepared in Preparation Example 1 and 99% ethanol aqueous solution (1.36 g, 29.57 mmols) as a substrate was added to a 25 mL scourcap Erlenmeyer flask And the mixture was stirred at a speed of 300 rpm with 0.15 g (5% by weight) of Novozyme 435, Lipozyme RM IM and Ripozyme TL IM. The reaction temperature was 40 ° C for Novozyme 435 and 30 ° C for Lipozyme RM IM and Lipozyme TL IM. The amount of the enzyme was 0.075 g (2.5 wt%), 0.15 g (5 wt%), 0.3 g (10 wt%), and 0.45 g (15 wt%). The reaction was started and samples were taken at predetermined times, analyzed by gas chromatography, and ethanol was blown with nitrogen to measure the acid value.

The biodiesel (fatty acid ester) synthesized after the reaction and remaining fatty acid, monoglyceride, diglyceride, triglyceride and acid value were measured in the same manner as in Example 1.

The novozyme 435 enzyme showed optimum conditions for obtaining biodiesel at 5 to 15 wt% (Fig. 8), the liposome RM IM enzyme showed optimal conditions for obtaining biodiesel at 2.5 to 15 wt% (Fig. 9) , The lipozyme TL IM enzyme showed optimum conditions for obtaining biodiesel at 10 to 15 wt% (Fig. 10).

Example  5. Two steps with two enzymes Esterification  Biodiesel Synthesis via Reaction

3 g of a mixture of the acid oil (1.64 g, 5.94 mmols) prepared in Preparation Example 1 and 99% ethanol aqueous solution (1.36 g, 29.57 mmols) was added to a 25 mL scallop In the Erlenmeyer flask, Novozyme 435 or Lipozyme RM IM was used for the first step, and the enzyme used in the first step for the second step was removed by filtration through a 0.45 μL nylon microfilter. Then, Lipozyme TL IM was added thereto, Lt; / RTI > The reaction temperature and enzyme amount for each enzyme type were 0.3 g (10 wt%) at 40 ° C for Novozyme 435, 0.15 g (5 wt%) at 30 ° C for liposome RM IM, 30 Lt; 0 > C (10 wt%). The reaction was started and samples were taken at predetermined times, analyzed by gas chromatography, and ethanol was blown with nitrogen to measure the acid value.

The biodiesel (fatty acid ester) synthesized after the reaction and remaining fatty acid, monoglyceride, diglyceride, triglyceride and acid value were measured in the same manner as in Example 1.

As a result of the above measurement, esterification reaction was carried out twice as compared with the case where biodiesel was produced by one esterification reaction using Novozyme 435, Lipozyme RM IM and Lipozyme TL IM alone, (Fig. 11 and Fig. 12). ≪ tb >< TABLE >

Example  6. Biodiesel Production Using Continuous Reactor

The esterification of acid oil and ethanol using lipase as a catalyst was carried out in a small packed-bed reactor (PBR) (Fig. 13). The reaction was injected at a specific rate by an electronic syringe pump (Model 200; KD Scientific, New Hope, Pa., USA). Before starting the reaction, the substrate was passed through at a rate of 3 minutes (350 μl / min) for 5 times. After the reaction was started, each product was obtained after passing through 3 times.

Sample analysis

10, 20, 30 minutes, 1, 2, 4, and 8 hours, respectively. After the reaction, 10 μL of the product was dissolved in 1 mL of chloroform and analyzed by gas chromatography (Model 3800; Varian, Palo Alto, Calif., USA). The columns were DB-1ht column (15 m x 0.25 mm i.d .; J & W Scientific, Folsom, CA, USA) and flame ionization detector was used as the detector. The temperature of the column was programmed to be maintained at 120 ° C for 3 minutes and then ramped up to 370 ° C at a rate of 20 ° C / min and then held at 370 ° C for 3 minutes. The carrier gas was flowed at 1.5 mL / min using helium. Both the injector and the detector were maintained at 370 ° C.

Acid value measurements were performed by the AOCS Cd 3d-63 method.

Moisture content of the substrate was measured by a Karl-Fisher moisture analyzer (Model 870 KF Titrino plus; Metrohm AG; Herisau, Switzerland).

Example  7. Optimization of water content in the production of biodiesel using continuous reactors

The continuous esterification reaction according to the method of Example 6 was carried out. In order to test the biodiesel synthesis amount according to the water content, a mixture of the acid oil prepared in Preparation Example 1 and the 99% ethanol aqueous solution at a molar ratio of 1: 5 as a substrate was placed in a continuous reactor having 0.5 g of Lipozyme TL IM And reacted at 30 ° C. The water content was measured to be 1 to 6 wt% based on the weight of the substrate.

The reaction rate and the maximum yield of biodiesel increased with increasing water content. However, when the water content was 4 wt% or more, there was no significant difference in reaction rate and maximum yield. Thus, the optimum moisture content was 4 wt%, and the yield of biodiesel at this time was 90% (Fig. 15).

Example  8. Optimization of reaction temperature in the production of biodiesel using continuous reactor

The continuous esterification reaction according to the method of Example 6 was carried out. In order to test the biodiesel synthesis amount according to the reaction temperature, a mixture of the acidic oil prepared in Preparation Example 1 and 99% ethanol aqueous solution as a substrate in a molar ratio of 1: 5 was added to a continuous reactor And reacted at 0 to 30 ° C, respectively. At this time, the water content is 4% by weight based on the substrate.

The reaction rate and the biodiesel yield were almost the same at the reaction temperature of 10 to 30 ° C. Also, even at the reaction temperature of 0 ° C, the initial reaction rate was only slightly slower, and the maximum biodiesel yield was not significantly different.

It was confirmed that the activity of liposomes TL IM was resistant to high temperatures in the acid hydrolysis or interesterification, and that the activity of the enzyme decreased sharply with increasing temperature in the presence of ethanol . The optimal temperature was selected as the room temperature of 20 ° C in consideration of the energy economy and the effect of denaturing the enzyme (FIG. 16).

Example  9. When producing biodiesel using a continuous reactor Interstitial  Optimization of molar ratio

The continuous esterification reaction according to the method of Example 6 was carried out. In order to test the synthesis amount of biodiesel according to the molar ratio between substrates, 3 g of a mixture of the acid oil and the 99% ethanol aqueous solution prepared in Preparation Example 1 as a substrate was changed from a molar ratio range of 1: 3 to 1: 6, Was injected into a continuous reactor loaded with 0.5 g of TL IM. At this time, the water content was 4% by weight based on the substrate and the temperature was 20 ° C.

When the molar ratio was 1: 4 to 1: 6, there was no significant difference in the reaction rate and the biodiesel yield. However, in the case of the molar ratio of 1: 3, the yield of biodiesel was no longer increased after 30 minutes of reaction time, and the equilibrium was reached at a low level of biodiesel yield. Therefore, the optimum molar ratio of the reaction was 1: 4, and the maximum yield was 93% (FIG. 17).

Example  10. Measurement of enzyme activity at optimal conditions of biodiesel production using continuous reactor

The activity of the enzyme was measured from the biodiesel yield in the optimum conditions (moisture content 4% by weight, reaction temperature 20 ° C, molar ratio of acid oil and ethanol 1: 4, residence time 4 hours) measured in Examples 7 to 9 . To investigate the effect of glycerol on the inhibition of enzyme activity, the reaction was divided into a reactor which does not remove glycerol (FIG. 13) and a reactor which removes glycerol produced during the reaction at regular intervals (FIG. 14) The yield of diesel was compared. Ethanol was used as a solvent to remove glycerol, and 10 mL of ethanol was passed through every 12th reaction.

As a result of no ethanol injection (curve 18a), the yield of biodiesel decreased steadily as the reaction was repeated. In particular, yields of 80% or more were obtained until 19th day, but then decreased rapidly thereafter, yielding 34% in 49th day. As a result of the injection of ethanol (curve 18b), the biodiesel yield gradually decreased as the reaction was repeated. Therefore, it was confirmed that the removal of glycerol produced during the reaction from the liposome TL IM having a hydrophilic resin was effective in maintaining the enzyme activity and prolonging the use period.

Claims (12)

(1) preparing an acid oil from a soapstock derived from a rice bran oil; And
(2) the above-mentioned substrate is made of the acidic oil and the alcohol having 1 to 4 carbon atoms, and the substrate is composed of Novozym 435, Lipozyme RM IM and Lipozyme TL IM Wherein the esterification reaction is carried out by reacting at least one enzyme selected from the group consisting of
When Novozym 435 is used as the enzyme of step (2), the esterification reaction conditions are as follows: the molar ratio of the acid oil and the alcohol having 1 to 4 carbon atoms is 1: 4 to 1: 6, the reaction temperature is 40 to 60 ° C , Novozyme 435 is used in an amount of 10 to 15% by weight based on the total weight of the substrate,
When the liposome RM IM is used as the enzyme of the step (2), the esterification reaction is carried out under the conditions that the molar ratio of the acid oil and the alcohol having 1 to 4 carbon atoms is 1: 2 to 1: 6, And 5 to 15% by weight of liposome RM IM is used relative to the total mass of the substrate,
When the liposome TL IM is used as the enzyme of the step (2), the esterification reaction conditions are as follows: the molar ratio of the acid oil and the alcohol having 1 to 4 carbon atoms is 1: 4 to 1: 6, , And 10 to 15% by weight of liposomal TLIM is used relative to the total weight of the substrate. [Claim 2] The method according to claim 1, wherein the acid oil is prepared by reacting unsalted soap, isopropanol, hexane, sulfuric acid and water. delete delete delete The method according to claim 1, wherein in the step (2), the enzyme is subjected to a primary esterification reaction using at least one selected from the group consisting of Novozym 435 (Novozym 435) and Lipozyme RM IM, Wherein the enzyme is filtered and removed, and a second esterification reaction is carried out by adding Lipozyme TL IM as an enzyme. [Claim 7] The method according to claim 6, wherein the primary esterification reaction is carried out for 5 to 60 minutes. [3] The method according to claim 1, wherein in step (2), the substrate and the enzyme are reacted in a packed-bed reactor (PBR) to perform an esterification reaction. [8] The method according to claim 8, wherein the esterification reaction conditions in the continuous reactor of step (2) are as follows: the molar ratio of the acid oil and the alcohol having 1 to 4 carbon atoms is 1: 3 to 1: 6, Wherein the water content is 1 to 6% by weight based on the total weight of the substrate. [Claim 9] The method according to claim 8, further comprising the step of removing glycerol produced in the step (2). [Claim 9] The method according to claim 8, wherein the enzyme is lipozyme TL IM. The method for producing biodiesel according to claim 1, wherein the alcohol having 1 to 4 carbon atoms is ethanol.

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