KR101766956B1 - Method for producing esters from microbial fermentation using high-pressure carbon dioxide gas - Google Patents

Abstract

The present invention relates to a method for producing an ester from a microbial fermentation process using a high-pressure carbon dioxide gas, and more specifically, to an esterification process in which an organic acid and / or an alcohol is produced by microbial organic acid fermentation, Wherein the esterification reaction is carried out under a pressure of from 5 bar to 50 bar of carbon dioxide.
According to the present invention, there is no fear of causing problems such as degradation of enzyme catalytic activity since an acid catalyst is not added, and since no additional alkaline substance is added for pH control, no problem of salt accumulation is caused, An ester compound can be produced.

Description

TECHNICAL FIELD The present invention relates to a method for producing an ester from a microbial fermentation process using a high-pressure carbon dioxide gas,

The present invention relates to a method for producing an ester from a microbial fermentation process using high pressure carbon dioxide gas in a method for producing an ester by fermentation reaction of an organic acid producing microbial strain.

Conventional microbial fermentation reactions are used to produce various organic acid or alcohol-based compounds. For example, various organic acids such as acetic acid, propionic acid, butyric acid, and succinic acid can be produced through microbial fermentation reaction, and various alcohol compounds such as ethanol and butanol can also be produced through a microbial fermentation reaction. The resulting organic acids or alcohol compounds can be used as platform chemicals in a variety of chemical processes and can be used to synthesize materials with higher value through subsequent catalytic processes.

On the other hand, the term "organic acid fermentation by microorganisms" refers to a process for producing various organic acids or alcohols by incomplete oxidation of carbon compounds through a fermentation reaction with microorganisms. The organic acids or alcohols produced by the fermentation reaction are organic solvents Is separated from the microbial culture liquid by extraction using used or separation using an ion exchange resin.

The separated organic acids or alcohols may then be converted into various biofuels and biochemical raw materials including alcohols, carboxylic acids, ethers and esters. For example, in the following Reaction Schemes 1 to 4, production by microbial fermentation Butyric acid or hexanoic acid reacts with an alcohol to form the ester dialkyl acid:

<Reaction Scheme 1>

<Reaction Scheme 2>

<Reaction Scheme 3>

<Reaction Scheme 4>

At this time, the reactions are very sensitive to the pH conditions and the pH of the reaction solution should generally be kept low since the organic acid should not be ionized for the esterification reaction. Therefore, conventionally, an esterification reaction of an organic acid and an alcohol is generally carried out by adding an acid catalyst such as hydrochloric acid, sulfuric acid or the like as a reaction catalyst.

For example, Korean Patent Laid-Open Publication No. 10-2009-0129619 discloses a method for producing biodiesel from a raw material channel containing free fatty acid. In this method, Amberlyst-15, Amberlyst BD20, WO ₃ / ZrO _{In the} presence of an acid catalyst such as WO ₃ / Al ₂ O ₃ , WO ₃ / SiO ₂ , SO ₄ ^{2 -} / ZrO ₂ , SO ₄ ^{2 -} / Al ₂ O ₃ , SO ₄ ^{2 -} / SiO ₂ , And esterifying the raw oil containing free fatty acid and an alcohol.

In addition, Korean Patent Publication No. 10-1436428 discloses a method and an apparatus for producing biodiesel from sewage sludge. In this method, an alcohol and an acid catalyst are mixed with sewage sludge, and a transesterification reaction is carried out And a method for producing biodiesel comprising the steps of:

However, in the process of producing an organic acid or alcohol by the microbial fermentation reaction, when the acid catalyst is added as described above, the shape of the enzyme protein is changed by the ionic strength of the added acid catalyst, Leading to a problem of ultimately stopping the fermentation reaction. As the concentration of the organic acid in the fermentation reaction solution increases, the pH of the fermentation reaction solution is lowered, and the lowered pH is further increased by the fermentation reaction of microorganisms . &Lt; / RTI &gt;

Accordingly, a means for returning the lowered pH to a pH suitable for the fermentation reaction as the reaction progresses is required. To adjust the pH, a substance such as an alkali salt is added to artificially raise the pH of the lowered fermentation reaction solution , The added alkali salt reacts with the acid catalyst in the fermentation reaction liquid to produce a salt, and the salt thus formed is accumulated in the fermentation reaction liquid, and the volume of the fermentation reaction solution is also increased.

Korean Patent Publication No. 10-2009-0129619 Korean Registered Patent No. 10-1436428

SUMMARY OF THE INVENTION Accordingly, the present invention has been made to solve the above problems of the prior art, and it is an object of the present invention to provide a method for producing an ester by producing an organic acid or alcohol by microbial organic acid fermentation, To provide a method for producing an ester which can maintain the pH condition favorable to the reaction to produce an ester with excellent efficiency, and further, there is no pH adjusting agent to be added separately, thereby enabling cost reduction and no problem of salt accumulation in the reaction solution do.

In order to solve the above problems,

a) culturing an organic acid producing microorganism strain in a carbon source medium to produce an organic acid; And

b) adding an alcohol to the culture medium of step a) to perform an esterification reaction between the organic acid and the added alcohol in the culture solution,

Characterized in that the esterification reaction in step b) is carried out under a pressure of 5 bar to 50 bar of carbon dioxide.

Further, in order to solve the above problems,

a) culturing an alcohol producing microorganism strain in a carbon source medium to produce an alcohol; And

b) adding an organic acid to the culture medium of step a) to perform an esterification reaction between the alcohol in the culture medium and the added organic acid,

Characterized in that the esterification reaction in step b) is carried out under a pressure of 5 bar to 50 bar of carbon dioxide.

Further, in order to solve the above problems,

a) culturing an organic acid and an alcohol-producing microorganism strain in a carbon source medium to produce an organic acid and an alcohol; And

b) performing an esterification reaction of the organic acid and the alcohol produced in the step a)

Characterized in that the esterification reaction in step b) is carried out under a pressure of 5 bar to 50 bar of carbon dioxide.

Further, in order to solve the above problems,

a) co-culturing an organic acid-producing microorganism strain and an alcohol-producing microorganism strain in a carbon source medium to produce an organic acid and an alcohol;

b) performing an esterification reaction of the organic acid and the alcohol produced in the step a)

Characterized in that the esterification reaction in step b) is carried out under a pressure of 5 bar to 50 bar of carbon dioxide.

According to an embodiment of the present invention, the esterification reaction in step b) may be carried out in a culture solution of the microorganism strain at a pH of 3.0 to 4.0.

According to another embodiment of the present invention, after the esterification reaction in the step b) is completed, the pH of the culture medium of the microorganism strain may be restored to 5.0 to 6.0 by performing a carbon dioxide degassing process on the culture solution of the microorganism strain have.

According to another embodiment of the present invention, it is possible to filter out the strain of step a) before performing step b), and to perform the step b) only for the filtered culture.

According to another embodiment of the present invention, the organic acid producing microorganism strain is Clostridium tyrobutyricum), Clostridium butyric rikum (Clostridium butyricum), Clostridium Cluj berry (Clostridium 　 kluyveri ), caproic acid produsense galactitol riborans ( Caproiciproducens galactitolivorans , Megasphaera &lt; RTI ID = 0.0 &gt; hexanoica , and Megasphaera elsdenii ; and at least one strain selected from the group consisting of: The alcohol-producing microbial strain may be Saccharomyces cerevisiae cerevisiae ); The organic acid and the alcohol-producing microorganism strain is Clostridium acetonitrile butyronitrile rikum (Clostridium acetobutyricum) and Clostridium Bay that linkage (Clostridium beijerinckii ). &lt; / RTI &gt;

According to another embodiment of the present invention, the esterification reaction of step b) may be carried out at a temperature of 40 ° C to 60 ° C for 1 hour to 4 hours.

According to another embodiment of the present invention, the carbon dioxide may be carbon dioxide produced from the microorganism strain culture of step a).

According to another embodiment of the present invention, the organic acid is at least one organic acid selected from the group consisting of acetic acid, propionic acid, butyric acid, succinic acid, hexanoic acid, and mixtures thereof, and the alcohol is ethanol, propanol, butanol, hexanol and mixtures thereof And at least one alcohol selected from the group consisting of

According to another embodiment of the present invention, the esterification reaction is carried out in the presence of zeolite, heteropoly acids, silica-alumina, Nafion-H, para-toluenesulfonic acid (p -toluenesulfonic acid), SO4 ^2- / ZrO ₂ , SO 4 ^2- / TiO ₂ -La ₂ O _3, and mixtures thereof.

According to another embodiment of the present invention, the esterification reaction can be catalyzed by at least one enzyme catalyst selected from the group consisting of esterase and lipase.

According to another embodiment of the present invention, the esterification reaction can be performed by an immobilized enzyme in which an enzyme catalyst is immobilized on a polymer resin carrier.

According to another embodiment of the present invention, the ester produced may be extracted using at least one extraction solvent selected from the group consisting of dodecane, tetradecane, hexadecane, and mixtures thereof.

According to the present invention, there is no fear of causing problems such as degradation of enzyme catalytic activity since an acid catalyst is not added, and since no additional alkaline substance is added for pH control, no problem of salt accumulation is caused, Esters can be produced.

BRIEF DESCRIPTION OF DRAWINGS FIG. 1 is a schematic diagram showing a system configuration for carrying out a process for producing an ester according to the present invention; FIG.
2 schematically shows the reaction in the high-pressure reactor in the process according to the invention.
FIG. 3 is a graph showing the conversion efficiency of butyric acid according to the pH when the pH of the culture liquid is adjusted using a sodium hydroxide solution for the microbial fermentation broth.
4 is a graph showing the conversion efficiency of different butyric acid in the reaction time under the carbon dioxide pressurization condition for the microorganism culture solution.

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

In accordance with the present invention, there is provided a method for producing an ester by producing an organic acid and / or an alcohol by fermentation of microorganism organic acid and then performing an esterification reaction using the same, wherein the esterification reaction is carried out under a pressure of 5 bar to 50 bar In the presence of a base.

As described above, the esterification reaction is carried out by reacting an organic acid with an alcohol. In the present invention, there are four possible types of the esterification reaction. That is, i) the organic acid producing microorganism strain is cultured to produce an organic acid, and then alcohol is added to the produced organic acid; ii) culturing an alcohol-producing microbial strain to produce an alcohol, and then adding an organic acid to the produced alcohol; iii) culturing a microorganism strain producing both an organic acid and an alcohol to produce an organic acid and an alcohol, and then reacting the produced organic acid with an alcohol; Or iv) a method in which an organic acid producing microorganism strain and an alcohol producing microorganism strain are co-cultured to produce an organic acid and an alcohol, respectively, and then the produced organic acid is reacted with an alcohol.

Particularly, in the present invention, the esterification reaction is carried out under a pressurization condition of 5 bar to 50 bar, so that the carbon dioxide gas pressurized in the pressure range is brought into gas-liquid contact with the reaction solution in the reactor, When the carbon dioxide gas is dissolved into the solution, the reaction solution is kept acidic by the generated carbonate ion. Since the pH of the reaction solution is maintained at 3 to 4 by the carbon dioxide pressurization, the esterification reaction of the organic acid and the alcohol within the pH range can be performed with excellent efficiency. In addition, the carbon dioxide gas required for the carbon dioxide pressurization process can supply external carbon dioxide gas through a separate carbon dioxide storage device, but it is also possible to recycle the carbon dioxide gas generated by culturing the microorganism strain.

1, there is shown a system block diagram for carrying out the method for producing an ester according to the present invention. Referring to FIG. 1, in the incubator 110, an organic acid producing microorganism strain, an alcohol producing microorganism strain, And an organic acid producing microorganism strain are cultured, and this microorganism culture solution is conveyed to the filter 120. [ In the filter 120, only the microorganisms are filtered through a membrane process or the like, the filtered microorganisms are returned to the incubator 110, and the cultured microbes are transported to the culture medium reservoir 130. Then, the culture solution stored in the culture solution reservoir 130 is transferred to the high-pressure reactor 140 for performing the esterification reaction, and the carbon dioxide gas is supplied to the high-pressure reactor 140 under pressure. The ester produced in the high-pressure reactor 140 is extracted from the organic solvent for extraction, which is separately supplied from the organic solvent storage unit 150, and is taken out to the outside, and then recovered through a distillation process or the like.

FIG. 2 schematically shows the reaction inside the high-pressure reactor 140. For example, reaction of synthesizing butylbutyrate, which is an ester, by using butyric acid as an organic acid and butanol as an alcohol is shown. Referring to FIG. 2, butyric acid and butanol contained in the culture liquid produce water and butyl butyrate through an esterification reaction. At this time, butyric acid and butanol, which are raw materials, are difficult to dissolve in the extraction organic solvent exhibiting hydrophobicity, but butylbutyrate, which is the result of the esterification reaction, is easily extracted from the culture medium to the organic solvent since it exhibits hydrophobicity. Therefore, when the hydrophilic culture liquid and the hydrophobic organic solvent form the lower liquid layer and the upper liquid layer in the high-pressure reactor 140 and only the upper liquid layer is separated from the high-pressure reactor 140, the target ester . The esterification reaction can be carried out at a temperature of 40 ° C to 60 ° C for 1 hour to 4 hours. Examples of the catalyst for facilitating the esterification reaction include zeolite, heteropoly acids, Silica-alumina, Nafion-H, p-toluenesulfonic acid, SO4 ^{2 -} / ZrO ₂ , SO 4 ^2- / TiO ₂ --La ₂ O ₃ and mixtures thereof A chemical catalyst selected from the group consisting of: Or an enzyme catalyst selected from the group consisting of esterase and lipase can be used. Preferably, in the present invention, an immobilized enzyme in which an enzyme catalyst such as esterase or lipase is immobilized on a polymer resin carrier or the like can be used. For example, an immobilized enzyme such as Novozym 435 or Lipozyme TLIM can be used for the reaction. In this case, since the immobilized enzyme does not sink into the culture medium of the lower layer in the reactor 140, it remains in the high-pressure reactor 140 without circulating through the culture liquid, and catalyzes the esterification reaction.

On the other hand, when the method according to the present invention is performed using an immobilized enzyme, there is an advantage that the target can be extracted in the form of an ester simultaneously with the organic acid fermentation, but the reaction time may be lengthened or the efficiency may be lowered. However, these disadvantages can be overcome to some extent by optimizing process conditions and increasing the amount of enzyme to improve the initial reaction rate. In addition, when a chemical catalyst is used, a high initial reaction rate and excellent efficiency can be expected. However, since the chemical catalyst may inhibit the microbial activity, it can not be used in a form mixed with the microbial fermentation broth. It is not possible to extract the object. For example, commercially available chemical catalysts include Amberlyst 131, DBSA and Nafion NR-50.

As described above, extraction solvents suitable for extracting an objective ester exhibiting sufficient hydrophobicity include, but are not limited to, dodecane, tetradecane, hexadecane, and mixtures thereof. Particularly, the hydrophobic extraction solvents to be used in the present invention are characterized by i) having a very high hydrophobicity, omitting a separate water separation process, ii) having no toxicity to the microorganism, and iii) having a property that the reactivity is small and stable In addition, iv) when extracting esters such as dialkyl acid, the distribution coefficient is very large, so that a large amount of the objective substance can be extracted using a small amount of extraction solvent.

In the above-mentioned process, butylaurate as an organic acid and butanol as an alcohol are used to synthesize butylbutyrate ester. However, organic acids usable in the present invention include, but are not limited to, acetic acid, propionic acid , Butyric acid, succinic acid, hexanoic acid, and mixtures thereof. Examples of alcohols include, but are not limited to, alcohols selected from the group consisting of ethanol, propanol, butanol, hexanol, and mixtures thereof And the like. For example, when hexanoic acid is used as an organic acid and hexanol is used as an alcohol, hexylhexanoate is used as an ester, hexanoic acid is used as an organic acid, and butylhexanoate is used as an ester when butanol is used as an alcohol. Butyric acid is used as an organic acid, and hexyl butyrate is used when hexanol is used as an alcohol. As a matter of course, those skilled in the art can easily recognize that various ester compounds can be produced from various organic acids and alcohols.

Therefore, in the present invention, although not limited thereto, the organic acid producing microbial strain may be Clostridium tyrobutyricum, Clostridium &lt; RTI ID = 0.0 &gt; butyricum , Clostridium 　 kluyveri), kapeuroyi Procedures Two sense galactose Tito ribonucleic lance (Caproiciproducens galactitolivorans), Mega Spanish la-hexahydro noise car (Megasphaera hexanoica) and Mega Spanish la Els Denny (one or more strains selected from the group consisting of Megasphaera elsdenii), the alcohol production Microbial strains include Saccharomyces cerevisiae cerevisiae ), and the organic acid and alcohol producing microorganism strains include Clostridium acetobutyricum) and Clostridium Bay that linkage (Clostridium beijerinckii ). &lt; / RTI &gt;

Referring again to FIG. 1, the culture medium from which the ester produced by the above-described organic solvent has been extracted is transferred from the high-pressure reactor 140 to the carbon dioxide degassing vessel 160. In the carbon dioxide degassing vessel 160, for example, the carbon dioxide dissolved in the culture liquid is degassed by applying a pressure of 100 mmHg to 760 mmHg and a temperature condition of 20 占 폚 to 30 占 폚 to the culture liquid dissolved in the gas- . Thus, the pH of the fermentation broth in which carbon dioxide has been degassed can be restored to a level of about 5.0 to 6.0.

Taken together, the method for producing an ester according to the present invention can produce the target ester compound with excellent efficiency by the above-mentioned process.

EXAMPLES Hereinafter, the present invention will be described in more detail with reference to the following examples. However, the following examples are intended to assist the understanding of the present invention and should not be construed as limiting the scope of the present invention.

Example

Comparative Example

Butyric acid and butanol were added at the same concentration of 0.03 M to the microbial fermentation broth at a sugar concentration of 20 g / L, and the pH of the culture broth was adjusted to 2.0, 3.0, 4.0 and 4.5, respectively, using sodium hydroxide solution. 5.0 and 6.0, respectively. 5 ml of each culture solution and 5 ml of tetradecane as an extraction solvent were added at 40 ° C and normal pressure, and 10 mg of novozym 435 immobilized with lipase was added thereto. The mixture was stirred at a constant temperature of 150 rpm using an incubator equipped with a stirring function. Stirring was carried out for 4 hours, after which it was possible to separate the culture medium and the extraction solvent without further oil separation. Before and after the experiment, concentrations of butyric acid and butylbutyrate in the culture broth and extraction solvent were analyzed. The ester conversion and extraction efficiency was calculated by the formula (number of moles of butylbutyrate in extraction solvent after extraction) / (number of moles of butyrate before extraction) × 100 The results are shown in Fig. The GC-FID analysis confirmed that the produced ester was recovered by 99% or more of the extraction solvent. No detectable amount of ester (ppm) or more remained in the culture solution.

Referring to FIG. 3, it can be seen that the conversion efficiency of butyric acid increases as the pH is lowered, and the conversion efficiency is maximum at the pH 2 to 3 level.

Example

In a 500 mL anaerobic fermentor, 100 mL of culture medium (sugar concentration 20 g / L) and clostridium tirobutiricum Tyrobutyricum ) were inoculated. The culture conditions were maintained at 37 ° C for 20 hours. After the fermentation, the microorganisms were separated from the fermentation broth and the concentration of butyric acid was measured to be 4.8 g / L in the fermentation broth filtered with microorganisms, and the final pH was 6.0. Butanol was not separately obtained by fermentation and was added separately as much as the concentration of butyric acid.

5 ml of the culture solution and 5 ml of tetradecane as an extraction solvent were put into a high-pressure reactor according to the present invention, and carbon dioxide was supplied to each of them to apply a pressure of 50 bar. The mixture was stirred at a constant temperature of 150 rpm using an incubator equipped with a stirring function. The agitation was carried out for 1 hour, 2 hours and 4 hours respectively. After that, it was possible to separate the culture medium and the extraction solvent without any oil separation.

The concentrations of butyric acid and butylbutyrate in the culture broth and extraction solvent were analyzed before and after the experiment. The conversion of ester and the extraction efficiency thereof were found to be (moles of butylbutyrate in extraction solvent after extraction) / (moles of butyric acid before extraction) The results are shown in Fig. For comparison, FIG. 4 also shows the results of reaction under the same conditions as described above, but under normal pressure for 2 hours.

Referring to FIG. 4, it can be seen that the conversion efficiency of butyric acid under the carbon dioxide high-pressure condition is significantly improved compared to the case where the reaction is carried out under atmospheric pressure without any pH adjustment.

Claims (15)

a) culturing an organic acid producing microorganism strain in a carbon source medium to produce an organic acid; And
b) adding an alcohol to the culture medium of step a) to perform an esterification reaction between the organic acid and the added alcohol in the culture solution,
The esterification reaction of step b) is carried out under a pressure of 5 bar to 50 bar of carbon dioxide,
The step b) is carried out in the reactor by passing the culture medium of the strain through a filter to filter out the strain of step a) and supplying the filtered culture medium to the reactor before performing the step b)
The strains thus filtered are transported into an incubator containing the carbon source medium,
Wherein the culture broth of the strains strained with the strains is supplied to a reservoir arranged on a line connecting the strainer and the reactor and temporarily stored before performing the step b) Wherein the microorganism fermentation step comprises supplying the culture broth of the strain so as to maintain 3.0 to 4.0. delete delete delete delete The method according to claim 1, wherein after the completion of the esterification reaction in step b), the pH of the culture solution of the microorganism strain is restored to 5.0 to 6.0 by performing a carbon dioxide degassing process on the culture solution of the microorganism strain A method for producing an ester from a microbial fermentation process. delete The microorganism producing microorganism according to claim 1, wherein the microorganism producing microorganism is selected from the group consisting of Clostridium tyrobutyricum, Clostridium butyricum , Clostridium kluyveri , Wherein the microorganism is at least one strain selected from the group consisting of Caproiciproducens galactitolivorans , Megasphaera hexanoica , and Megasphaera elsdenii . The process for producing an ester from a microbial fermentation process according to claim 1, wherein the esterification reaction in step b) is carried out at a temperature of 40 ° C to 60 ° C for 1 hour to 4 hours. The method for producing an ester from a microbial fermentation process according to claim 1, wherein the carbon dioxide is carbon dioxide produced from a microbial strain culture in step a). The method of claim 1, wherein the organic acid is at least one organic acid selected from the group consisting of acetic acid, propionic acid, butyric acid, succinic acid, hexanoic acid, and mixtures thereof, and the alcohol is selected from the group consisting of ethanol, propanol, butanol, &Lt; / RTI &gt; wherein said at least one alcohol is selected from at least one selected from the group consisting of alcohols. The method of claim 1, wherein the esterification reaction is carried out in the presence of a catalyst selected from the group consisting of zeolite, heteropoly acids, silica-alumina, Nafion-H, p-toluenesulfonic acid, , SO4 ^2- / ZrO ₂ , SO 4 ^2- / TiO ₂ -La ₂ O _3, and mixtures thereof. The method for producing an ester from a microbial fermentation process according to claim 1, The process for producing an ester from a microorganism fermentation process according to claim 1, wherein the esterification reaction is catalyzed by at least one enzyme catalyst selected from the group consisting of esterase and lipase. The method for producing an ester from a microbial fermentation process according to claim 1, wherein the esterification reaction is carried out with an enzyme-immobilized enzyme immobilized on a polymer resin carrier. The process according to claim 1, wherein the ester produced is extracted using an extraction solvent selected from the group consisting of dodecane, tetradecane, hexadecane, and mixtures thereof.

Images