KR20160131236A - A method for producing hexanoic acid using Megasphaera hexanoica strain - Google Patents
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Abstract
Description
The present invention relates to a method for producing hexanoic acid using megasperahexanoic acid.
Currently, 86% of world energy demand depends on fossil fuels such as petroleum, coal, and natural gas. The use of a wide range of fossil fuels can cause severe energy and resource shortages due to environmental climate change and depletion of fossil resources, and efforts to produce fuels from renewable resources are increasing to address them.
Bio energy, solar power, wind power, intelligence, and tidal power generation are emerging as alternative energy sources currently available. Among them, bio-energy is highly utilized as transportation fuel, and the emission of environmental pollutants is less than 30% Due to its ability to reduce up to 90%, it has attracted worldwide attention. Bio-energy is a sustainable source of energy made from biomass in the natural world. It is produced from plants, agriculture and environmental waste such as corn, sugarcane, and waste cellulose, There is an effect of reducing waste by using various organic wastes without increasing carbon dioxide (CO 2 ). It also does not contain heavy metals and other harmful substances. In the case of liquid biofuels, it can be mixed with existing automotive liquid fuels. As a biofuel derived from such renewable vegetable raw materials, a lot of research has already been carried out as a fuel for transporting ethanol, which is a C2 material, and butanol, a C4 material, but aviation fuels require a material having a higher carbon number It is necessary to develop new biofuels.
On the other hand, hexanoic acid is a linear fatty acid having 6 carbons which is used to make perfumes, drugs, lubricating greases, rubbers, dyes, etc., and can be converted into hexanol through a simple catalytic reaction. Hexanol can be used as gasoline, diesel and jet fuel through esterification, and it is necessary to develop a technology to produce hexanoic acid as a precursor of biofuels.
The production of hexanoic acid by microbial fermentation is one of the most advanced microorganisms for the production of hexanoic acid using the anaerobic bacterium Clostridium kluyveri (1942). The strain can produce H2, butyric acid, hexanoic acid using ethanol, acetate or succinate. As the acetic acid is overproduced, hexanoic acid is mainly produced. In these relationships, it has been found that butyric acid is used as an intermediate in the synthesis of hexanoic acid.
In order to increase hexanoic acid production, rodick et al. Reported that the yield of hexanoic acid production was increased to 6 g /
SUMMARY OF THE INVENTION The present invention has been made in view of the above problems, and it is an object of the present invention to provide a method for producing hexanoic acid at a high yield by improving productivity.
In order to solve the above problems,
Culturing a culture of Megasphaera hexanoica strain (KFCC11466P) in a culture medium; And recovering the hexanoic acid from the culture liquid.
According to an embodiment of the present invention, the culture liquid may further include acetate and butyrate. At this time, the concentration of the acetate may be 7 to 9 g / L, and the concentration of the butyrate may be 13 to 15 g / L.
In addition, the culture solution may be supplemented with fructose, yeast extract, tryptophan, peptone, beef extract, K 2 HPO 4 , Tween 80, cysteine-HCl x H 2 O, resazurin, salt solution, distilled water, .
According to another embodiment of the present invention, the culture solution may further contain 6 to 18 g / L of yeast extract.
According to another embodiment of the present invention, the culture may further contain 5.5 to 10.5 g / L of yeast extract and 2.3 to 10.5 g / L of peptone. At this time, the concentration of the yeast extract may be 8.5 to 9.5 g / L, and the concentration of the peptone may be 8.5 to 9.5 g / L.
The culture may also contain fructose, K 2 HPO 4 , cysteine-HCl x H 2 O, resazurin, salt solution, distilled water, vitamin K1 solution or a mixture thereof.
According to another embodiment of the present invention, the pH may be maintained between 6 and 6.5 during the culturing and the pH may be maintained between 4 and 5 during the recovery of the hexanoic acid after the cultivation.
Further, in order to solve the above problems,
Culturing a culture of Megasphaera hexanoica strain (KFCC11466P) in a culture medium; Adding a mixed solvent of oleyl alcohol and alanine 336 to the culture medium during the culturing; And removing the mixed solvent to obtain a hexanoic acid in a mixed solvent.
According to an embodiment of the present invention, the volume ratio of the oleyl alcohol and the alumin 336 may be 9: 1 to 6: 3.
According to another embodiment of the present invention, the volume ratio of the culture medium to the mixed solvent may be 1: 0.5 to 1: 3.
According to another embodiment of the present invention, 2-6 M of butyric acid may be added as a pH adjusting agent during the culturing. At this time, the pH may be maintained at 5.5 to 6.5 during the culturing.
According to another embodiment of the present invention, fructose and nitrogen source may be periodically added to the culture medium during the culturing. At this time, the nitrogen source may be yeast extract, peptone, or a mixture thereof. Also, the concentration of the introduced fructose may be 20 to 60 g / L, and the concentration of the introduced nitrogen source may be 5 to 20 g / L.
According to another embodiment of the present invention, during the culturing, concentrated fructose and nitrogen source may be continuously added to the culture solution. At this time, the fructose and the nitrogen source may be introduced at a rate of 0.1 to 3 ml / hr.
According to another embodiment of the present invention, the culture liquid may include acetate, butyrate, hexanoic acid, or a mixture thereof.
By using the hexanoic acid production method according to the present invention, the productivity of hexanoic acid can be remarkably improved.
1 is a graph showing the yield of hexanoic acid according to acetate and butyrate concentrations contained in the medium.
2 is a graph showing the parameters (acetate, butyrate, and pH) and their concentrations for applying the reaction surface method.
Fig. 3 shows the experimental design of the reaction surface analysis method and the yield of hexanoic acid.
FIG. 4 shows the effect of each parameter (acetate, butyrate, and pH) on the concentration of hexanoic acid obtained through the reaction surface analysis by a statistical method.
FIG. 5 is a diagram showing a prediction model for maximum hexanoic acid production through analysis and modeling of importance of each parameter (acetate, butyrate, and pH) according to the reaction surface analysis method.
Figure 6 is a graph showing the yield of actual hexanoic acid under the conditions of each parameter (acetate, butyrate, and pH) derived through the prediction model.
FIG. 7 shows the effect of each parameter (yeast extract and beef extract) on the concentration of hexanoic acid obtained through the reaction surface analysis method through a statistical method.
FIG. 8 shows the effect of each parameter (yeast extract and peptone) on the concentration of hexanoic acid obtained through the reaction surface analysis method through statistical methods.
FIG. 9 is a graph showing the production of hexanoic acid and cell growth according to the concentration of yeast extract. FIG.
10 is a graph showing the extraction efficiency of hexanoic acid according to pH after culturing the strain.
FIG. 11 is a graph showing the productivity of hexanoic acid during fermentation with the volume ratio of the culture medium and mixed solvent being 1: 1.
FIG. 12 is a graph showing the productivity of hexanoic acid during fermentation by adding 3 M butyric acid as a pH adjusting agent at a volume ratio of 1: 1 of the culture medium to the mixed solvent.
13 shows the productivity of hexanoic acid during fermentation by adding 3 M of butyric acid as a pH regulator with the volume ratio of the culture medium and mixed solvent being 2: 1, periodically feeding fructose and nitrogen source, Fig.
14 is a reaction process diagram for continuously supplying a concentrated nitrogen source and a carbon source at the time of extraction fermentation.
15 shows the productivity of hexanoic acid when subjected to extraction fermentation by continuously adding 3 M butyric acid as a pH adjusting agent and a fructose and nitrogen source continuously at a volume ratio of 2: 1 for the culture medium and mixed solvent Fig.
Hereinafter, the present invention will be described in more detail.
Conventionally, a method of producing hexanoic acid which is a precursor of hexanol, which is a biofuel, has been disclosed. However, since the production amount thereof is not sufficient, in the present invention, productivity of megasperahexanoic acid strain And to provide a method for producing greatly improved hexanoic acid.
The method for producing hexanoic acid according to the present invention comprises the steps of: inoculating and culturing a culture solution of Megasphaera hexanoica strain (KFCC11466P); And recovering the hexanoic acid from the culture.
At this time, it is preferable that the culture medium further contains acetate and butyrate as a basic medium. As can be seen from the results of the following examples, acetate and butyrate have an important influence on promotion of cell growth and production of hexanoic acid, and in particular, acetate has been found to be the most influential factor for production of hexanoic acid. Therefore, as can be seen from the results of the following examples, it is preferable that the concentration of the acetate is 7 to 9 g / L and the concentration of the butyrate is 13 to 15 g / L in order to greatly increase the production amount of hexanoic acid desirable.
In addition, the above culture medium contains, as a basic medium component, fructose, yeast extract, tryptophan, peptone, beef extract, K 2 HPO 4 ,
Further, as can be seen from the results of the following examples, it is preferable that the culture medium further contains 6 to 18 g / L of yeast extract in the basic medium for increasing the production amount of hexanoic acid.
Further, as can be seen from the results of the following examples, in order to increase the production amount of hexanoic acid, the culture medium contained 5.5 to 10.5 g / L of yeast extract and 2.3 to 10.5 g / L of peptone in the basic medium It is more preferable that the concentration of the yeast extract is 8.5 to 9.5 g / L and the concentration of the peptone is 8.5 to 9.5 g / L.
The culture medium may also contain fructose, K 2 HPO 4 , cysteine-HCl x H 2 O, resazurin, salt solution, distilled water, vitamin K1 solution or a mixture thereof as a basic medium component . For example, mPYF medium having the composition shown in the following Table 2 can be used as a basic medium.
Further, as can be seen from the results of the following examples, it is preferable to maintain the pH at 6 to 6.5 during the culture of the strain, and it is preferable to maintain the pH at 4 to 5 during the recovery of the hexanoic acid after the cultivation Do.
The method for producing hexanoic acid according to the present invention comprises the steps of inoculating and culturing a culture solution of Megasphaera hexanoica strain (KFCC11466P); Adding a mixed solvent of oleyl alcohol and alanine 336 to the culture medium during the culturing; And removing the mixed solvent to obtain hexanoic acid in the mixed solvent.
The mixed solvent can be added to the culture solution when the growth of the strain enters the logarithmic growth phase. It is most preferable that the mixed solvent is added to the Megasphaera hexanoica strain (KFCC11466P) according to the present invention at the time of culturing for about 10-14 hours. In addition, the volume ratio of the oleyl alcohol and the alanine 336 contained in the mixed solvent is preferably 9: 1 to 6: 3. If the amount of ALAMIN 336 is too much outside the above range, toxicity of the microorganism may be induced and the growth may be inhibited.
It is preferable that the volume ratio of the culture medium and the mixed solvent is 1: 0.5 to 1: 3. As can be seen from the results of the following examples, it is more preferable that 1: 1 in case of batch culture and 1: 2 in case of oil culture Do.
It is most preferable that the pH is maintained in the range of 5.5 to 6.5 during the incubation period of the present invention. When the hexanoic acid is extracted by adding the mixed solvent according to the method of the present invention, the pH of the culture solution is continuously increased, In the present invention, it is preferable to add 2-6 M of butyric acid as a controlling agent capable of preventing the pH from rising through pH buffering, and the butyric acid acts as an electron acceptor As can be seen from the results of the following examples, the production amount of hexanoic acid can be further increased.
During the culturing, the production amount of hexanoic acid can be further increased by feeding fructose (carbon source) and nitrogen source to the culture liquid periodically and culturing the oil. At this time, the nitrogen source is most preferably yeast extract, peptone or a mixture thereof. Also, the concentration of the introduced fructose may be 20 to 60 g / L, and the concentration of the introduced nitrogen source may be 5 to 20 g / L. When the fructose and nitrogen source are added at an optimum concentration, the yield of hexanoic acid can be increased. If the concentration is less than the lower limit, the effect is insignificant. If the upper limit is exceeded, the yield is not increased any more.
In addition, in the present invention, productivity of hexanoic acid can be further improved by continuously feeding fructose and nitrogen source concentrated at a high concentration into the culture solution during the culturing. The reason for continuously injecting the fructose and nitrogen sources at a high concentration is to mitigate the impact on the growth of bacteria caused by the simultaneous introduction of the carbon source fructose and the nitrogen source, It is preferable to be charged at a rate of 3 ml / hr. If the amount is less than the lower limit, the effect of improving the productivity of hexanoic acid is insignificant, and if it exceeds the upper limit, the effect of alleviating the impact on the growth of the bacteria can be reduced.
In addition, it is preferable that the culture liquid contains acetate, butyrate, hexanoic acid or a mixture thereof as a component of the basic medium.
Hereinafter, the present invention will be described in more detail with reference to preferred embodiments and the like. It will be apparent to those skilled in the art, however, that these examples are provided for further illustrating the present invention and that the scope of the present invention is not limited thereto.
Example 1. Acetate, Butyrate And pH As a parameter. Hexanoic acid Maximum production conditions
Acetate, butyrate and pH were selected as parameters affecting the production of hexanoic acid in the culture of Megaspera hexanoica strain (KFCC11466P) of the present invention, and the optimal conditions were analyzed by the reaction surface analysis method. PYG medium having the composition described in Table 1 was used as a basic medium in strain culture.
Figure 1 is a graph showing the yield of hexanoic acid according to the concentration of acetate and butyrate, respectively. Through these results, it was confirmed that both acetate and butyetite were helpful factors for the production of hexanoic acid.
FIGS. 2 to 5 are diagrams showing the procedure for deriving the conditions for maximizing production of hexanoic acid by applying the above variables, and the results thereof. From these results, it was confirmed that acetate among the three variables had the greatest effect on the production of hexanoic acid. In addition, hexanoic acid was produced at the maximum of 8.74 g / L of acetate, 13.63 g / L of butyrate and pH of 6.24, and the amount thereof was 10.61 g / L.
The productivity of the actual hexanoic acid was evaluated under the conditions derived through the above reaction surface analysis. FIG. 6 is a graph showing the production of actual hexanoic acid under the conditions of each parameter (acetate, butyrate, and pH) derived through the prediction model. This confirmed that the maximum yield of hexanoic acid under the optimum conditions increased to 10.8 g / L.
Example 2. Maximum Production Conditions of Hexanoic Acid Derived from Response Surface Methodology with Yeast Extract and Peptone as Variables
In the case of producing hexanoic acid using conventional microorganisms, it has been experimentally conducted to derive the optimal concentration of nitrogen source, taking into account the fact that an excessive amount of nitrogen source is contained and excessive cost is incurred and adversely affects the yield of hexanoic acid .
In the present invention, yeast extract, peptone, and beef extract in the nitrogen source were selected as variables affecting the production of hexanoic acid by culturing Megasphaera hexanoica strain (KFCC11466P) Respectively. MPYF medium having the composition shown in Table 2 was used as a basic medium for strain culture.
FIG. 7 shows the results obtained by the reaction surface analysis method on the effect of yeast extract and beef extract on the production of hexanoic acid. As a result, the productivity of hexanoic acid increased as the concentration of yeast extract increased, whereas the beef extract did not improve the productivity of hexanoic acid.
Fig. 8 shows the results obtained by the reaction surface analysis method on the effect of yeast extract and peptone on the production of hexanoic acid. When yeast extract and peptone were mixed with each other, the productivity of hexanoic acid was remarkably enhanced by their synergistic effect. Especially, yeast extract and beef extract were added at a ratio of 8.75 g / L, that is, 1: 1 The maximum amount of cyclohexanoic acid produced was 10.00 g / L.
Example 3. Hexanoic acid Concentration of yeast extract for maximum production
The effect of hexanoic acid on the production of hexanoic acid was determined by the method of Example 2, and the effect of the single yeast extract on the production of hexanoic acid was measured by adding only the nitrogen source. FIG. 9 is a graph showing the production of hexanoic acid and cell growth according to the concentration of yeast extract. FIG. The yield of hexanoic acid increased with the concentration of yeast extract in the range of 6 to 18 g / L. Especially, when the concentration of yeast extract was 12 g / L, hexanoic acid was produced at the maximum, / L.
Example 4. After incubation, in the recovery step Hexanoic acid Titration to improve productivity pH
It is confirmed through Example 1 that pH is preferably maintained in the range of 5.5 to 6.5, particularly 6.0 to 6.5, when culturing the strain according to the present invention, and after culturing the strain, recovering the hexanoic acid by using an organic solvent In order to derive the optimal pH at pH 7.0. Experiments were carried out using mixed solvents in which oleic alcohol and alanine 336 were mixed at a ratio of 9: 1 under two conditions suitable for pH of 4.73 and 6.11.
10 is a graph showing the extraction efficiency of hexanoic acid according to pH after culturing the strain. As a result, it was confirmed that, compared to the extraction of about 40% of hexanoic acid at pH 6.11, the extraction of more than 95% of hexanoic acid at pH 4.73.
Example 5. Using a mixed solvent Hexanoic acid Extraction evaluation
Experiments were conducted to confirm the productivity when extracting hexanoic acid using a mixed solvent of oleic alcohol and alanine 336 while culturing Megaspera hexanoica strain (KFCC11466P) of the present invention. In the culture of the strain, medium containing 10 g / L of acetate, butyrate and hexanoic acid, respectively, was used as the basic medium.
FIG. 11 is a graph showing the productivity of hexanoic acid during fermentation with the volume ratio of the culture medium and mixed solvent being 1: 1. As shown in the figure, it was confirmed that hexanoic acid was extracted with keeping the interface without mixing the culture medium and the mixed solvent. The productivity of hexanoic acid was 0.41 g / L / hr when extracted under the above conditions And the yield of hexanoic acid was 20 g / L.
Example 6. pH With modulator Hexanoic acid Extraction evaluation
Experiments were carried out under the same conditions as in Example 5 except that 3 M butyric acid was added during the cultivation in order to examine the effect of using butyric acid as a pH regulator on productivity of hexanoic acid.
FIG. 12 is a graph showing the productivity of hexanoic acid during fermentation by adding 3 M butyric acid as a pH adjusting agent at a volume ratio of 1: 1 of the culture medium to the mixed solvent. As a result, it was confirmed that the pH was maintained at 5.99 even though hexanoic acid was extracted. Especially, productivity of hexanoic acid was 1.25 g / L / hr and production was 52 g / L.
Example 7. Fructose and When the nitrogen source is periodically added and cultured, Hexanoic acid Extraction evaluation
Experiments were carried out to confirm the productivity when extracting hexanoic acid while culturing oil by adding fructose (carbon source) and nitrogen source while culturing Megaspera hexanoica strain (KFCC11466P) of the present invention. The experiment was carried out under the same conditions as in Example 6, except that the volume ratio of the culture medium and the mixed solvent was 2: 1 during the culturing and that 40 g / L of fructose and 10 g / L of nitrogen source were added twice .
13 shows the productivity of hexanoic acid during fermentation by adding 3 M of butyric acid as a pH regulator with the volume ratio of the culture medium and mixed solvent being 2: 1, periodically feeding fructose and nitrogen source, Fig. As a result, the production rate of hexanoic acid decreased slightly to 0.27 g / L / hr as the concentration of hexanoic acid accumulated in the culture fluid increased due to oil-price cultivation. On the other hand, due to the cyclic introduction of fructose and nitrogen source, Was increased up to 140 g / L.
Example 8. Fructose and When the nitrogen source is continuously added, Hexanoic acid Extraction evaluation
In order to alleviate the impact on the growth of bacteria caused by the introduction of fructose (carbon source) and nitrogen source while culturing the Megasphaera hexanoica strain (KFCC11466P) of the present invention, fructose and nitrogen source were continuously The extraction of hexanoic acid was carried out to confirm the productivity.
14 is a reaction process diagram for continuously supplying a concentrated nitrogen source and a carbon source at the time of extraction fermentation. As shown in Fig. 14, during the cultivation of the strain, fructose concentrated at a high concentration of 700 g / L was slaughtered at a rate of 1 ml / hr using a syringe pump, and a nitrogen source concentrated at a concentration of 10 times or more at a concentration of 1 ml / hr , And the productivity of hexanoic acid in the extraction under the above conditions is shown in FIG. As a result, the maximum yield of hexanoic acid was 140.8 g / L, which was similar to that of Example 7, but the productivity of hexanoic acid was significantly improved to 0.73 g / L / hr.
Name of depository: Korea Microorganism Conservation Center (Domestic)
Accession number: KFCC11466P
Date of deposit: 20090930
Claims (20)
And recovering the hexanoic acid from the culture.
Wherein the culture liquid further comprises acetate and butyrate.
Wherein the concentration of the acetate is 7 to 9 g / L and the concentration of the butyrate is 13 to 15 g / L.
Wherein the culture medium comprises at least one selected from the group consisting of fructose, yeast extract, tryptophan, peptone, beef extract, K 2 HPO 4 , Tween 80, cysteine-HCl x H 2 O, resazurin, salt solution, distilled water, ≪ / RTI >
Wherein the culture medium further comprises a yeast extract of 6 to 18 g / L.
Wherein said culture medium further comprises 5.5 to 10.5 g / L of yeast extract and 2.3 to 10.5 g / L of peptone.
Wherein the concentration of the yeast extract is 8.5 to 9.5 g / L and the concentration of the peptone is 8.5 to 9.5 g / L.
Wherein said culture medium comprises fructose, K 2 HPO 4 , cysteine-HCl x H 2 O, resazurin, salt solution, distilled water, vitamin K1 solution or a mixture thereof .
Wherein the pH is maintained at 6 to 6.5 during the culturing and the pH is maintained at 4 to 5 during recovery of the hexanoic acid after the cultivation.
Adding a mixed solvent of oleyl alcohol and alanine 336 to the culture medium during the culturing; And
And removing the mixed solvent to obtain a hexanoic acid in a mixed solvent.
Wherein the volume ratio of the oleyl alcohol and the alumina 336 is from 9: 1 to 6: 3.
Wherein the volume ratio of the culture solution to the mixed solvent is 1: 0.5 to 1: 3.
Wherein 2-6 M of butyric acid is added as a pH regulator during the culturing.
Wherein the pH is maintained between 5.5 and 6.5 during said culturing.
The method of producing hexanoic acid according to any one of claims 1 to 5, wherein the culture solution is periodically added with fructose and a nitrogen source during the culture.
Wherein the nitrogen source is a yeast extract, peptone or a mixture thereof.
Wherein the concentration of the introduced fructose is 20 to 60 g / L, and the concentration of the introduced nitrogen source is 5 to 20 g / L.
Wherein the concentration of the introduced fructose is 20 to 60 g / L, and the concentration of the introduced nitrogen source is 5 to 20 g / L.
Wherein the fructose and the nitrogen source are introduced at a rate of 0.1 to 3 ml / hr.
Wherein the culture medium comprises acetate, butyrate, hexanoic acid or a mixture thereof.
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Application Number | Priority Date | Filing Date | Title |
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KR1020150063128A KR101745084B1 (en) | 2015-05-06 | 2015-05-06 | A method for producing hexanoic acid using Megasphaera hexanoica strain |
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KR20180059375A (en) * | 2016-11-25 | 2018-06-04 | 한양대학교 산학협력단 | A method for producing hexanoic acid using waste biomass and Megasphaera hexanoica strain |
WO2019132557A1 (en) * | 2017-12-29 | 2019-07-04 | 한양대학교 산학협력단 | Method for co-production of isovaleric acid and hexanoic acid by using megaspaera hexanoica strain |
KR20190082121A (en) * | 2017-12-29 | 2019-07-09 | 한양대학교 산학협력단 | Method for producing isovaleric acid and hexanoic acid simultaneously using Megasphaera hexanoica strain |
US10975448B2 (en) | 2018-01-23 | 2021-04-13 | Korea Institute Of Science And Technology | Clostridium sp. strain producing hexanoic acid in high yield and method for producing hexanoic acid using the same |
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KR20200015575A (en) | 2017-06-14 | 2020-02-12 | 4디 파마 리서치 리미티드 | Compositions Containing Bacterial Strains |
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Cited By (4)
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KR20180059375A (en) * | 2016-11-25 | 2018-06-04 | 한양대학교 산학협력단 | A method for producing hexanoic acid using waste biomass and Megasphaera hexanoica strain |
WO2019132557A1 (en) * | 2017-12-29 | 2019-07-04 | 한양대학교 산학협력단 | Method for co-production of isovaleric acid and hexanoic acid by using megaspaera hexanoica strain |
KR20190082121A (en) * | 2017-12-29 | 2019-07-09 | 한양대학교 산학협력단 | Method for producing isovaleric acid and hexanoic acid simultaneously using Megasphaera hexanoica strain |
US10975448B2 (en) | 2018-01-23 | 2021-04-13 | Korea Institute Of Science And Technology | Clostridium sp. strain producing hexanoic acid in high yield and method for producing hexanoic acid using the same |
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