KR101990211B1 - A method for producing hexanoic acid using waste biomass and Megasphaera hexanoica strain - Google Patents

Abstract

The present invention relates to a method for producing hexanoic acid by culturing a pulmonary biomass mixed with food waste and anaerobic digestion sludge, and then inoculating and culturing megasparrahexanoic acid strain in the culture.
According to the present invention, since pulmonary biomass is used without artificial medium, the production rate and productivity of hexanoic acid can be drastically improved without additional anaerobic treatment and sterilization treatment, and production cost of hexanoic acid can be remarkably reduced have.

Description

FIELD OF THE INVENTION [0001] The present invention relates to a method for producing hexanoic acid by using pulmonary biomass and megasparrahexanoic acid,

The present invention relates to a method for producing hexanoic acid by culturing a pulmonary biomass mixed with food waste and anaerobic digestion sludge, and then inoculating and culturing megasparrahexanoic acid strain in the culture.

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 on ethanol as a C2 material and butanol as a C4 material as a transportation fuel, but aviation fuel requires 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 H ₂ , butyric acid, and 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 / m 2 by oil culture of immobilized megasphaera elsdenii in an incubator containing amberlite IRA-400, L to 19 g / L, which is 30%. In Korean Patent No. 10-1316732, a method of increasing the production amount of hexanoic acid by using Megaspera Elsevier Dennis NCIMB 702410 and extracting fermentation has been disclosed However, since the production amount is not sufficient, it is not enough to apply to the production process of biofuel.

In addition, in the above-mentioned conventional techniques, acetic acid and butyric acid should be supplied from outside of the production of hexanoic acid by using an artificial medium, yeast extract, minerals and the like should be added to increase production, There is a problem that the production method is cumbersome and the production cost is high because additional processes are required. In addition, when hexanoic acid is produced using pulmonary biomass without using an artificial medium, the production speed is slow and the production amount is not sufficient, so that it is difficult to put into practice.

KR 10-1316732

Roddick, F.A. and M.L. Britz, Journal of Chemical Technology and Biotechnology, 1997 69 (3) 383-391.

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 of producing hexanoic acid at a high yield using lung biomass and megasparrahexanoic acid strains without using an artificial medium .

In order to solve the above problems,

(a) mixing food waste and anaerobic digestion sludge and culturing under predetermined temperature and anaerobic conditions to produce a primary culture medium in which acetic acid and butyric acid are produced;

(b) inoculating and culturing Megasphaera hexanoica strain (KCCM11835P) in the primary culture to produce a secondary culture; And

(c) recovering the hexanoic acid from the secondary culture.

According to the present invention, the anaerobic digestion sludge may be thawed after being frozen for more than 7 days at -50 to -100 ° C.

According to the present invention, there is provided a method for the production of a culture medium, comprising the steps (a) and (b) of: (i) carrying out subculturing under anaerobic conditions by mixing the primary culture medium of step (a) with food wastes .

At this time, step (i) may be performed when the concentration of butyric acid contained in the primary culture liquid of step (a) is 6 g / L or more and the concentration of acetic acid is 2 g / L or more.

According to the present invention, the predetermined temperature of the step (a) may be 20 to 40 ° C.

According to the present invention, the concentration of the food waste may be 50 to 150 g / L.

According to the present invention, the concentration of the anaerobic digestion sludge may be 30 to 70 mL / L.

According to the present invention, the step (b) may be performed under the condition that 10 to 30 g / L of fructose is added to the primary culture.

According to the present invention, the Megasphaera hexanoica strain (KCCM11835P) inoculated into the primary culture in step (b) may be a yeast culture.

At this time, the enrichment culture is carried out in the presence of 8 to 12 g / L of yeast extract, 1 to 3 g / L of K ₂ HPO ₄ , 30 to 50 mL / L of salt solution, 10 to 30 g / L, 1 to 5 g / L of sodium acetate and 0.1 to 1 g / L of cysteine-HCl.

INDUSTRIAL APPLICABILITY According to the present invention, it is possible to produce hexanoic acid without the need to supply acetic acid and butyric acid outside, which uses pulverized biomass without an artificial medium, dramatically improves production speed and productivity of hexanoic acid, Significant savings can be achieved.

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is a view showing a process of a production method of hexanoic acid according to the present invention. FIG.
FIG. 2 is a graph showing the results of analyzing the microbial community change according to the freezing time of the anaerobic digestion sludge.
FIG. 3 is a graph showing the results of measuring the content of organic acids produced when the anaerobic digestion sludge not frozen according to Example 2-1 was mixed with food waste and cultured. FIG.
FIG. 4 is a graph showing the results of measuring the content of organic acids produced when the anaerobic digestion sludge thawed after freezing for 7 days according to Example 2-2 was mixed with food waste and cultured.
FIG. 5 is a graph showing the results of measurement of the content of organic acids produced when the anaerobic digested sludge thawed after freezing for 15 days according to Example 2-3 was mixed with food waste and cultured.
FIG. 6 is a graph showing the results of measuring the content of organic acids produced when the anaerobic digested sludge thawed after freezing for 32 days according to Example 2-4 was mixed with food waste and cultured. FIG.
FIG. 7 is a graph showing the results of measuring the content of organic acids produced during subculture by mixing anaerobic digested sludge thawed after freezing for 7 days according to Example 3 with food waste, mixing the mixed culture with food waste, .
FIG. 8 is a graph showing the yield of hexanoic acid produced when a megasporea hexaenoic acid strain (KCCM11835P) was inoculated into a mixed culture of food waste and anaerobic digestion sludge according to Example 4.
9 is a graph showing the results of inoculation of megasporea hexanoic acid strain (KCCM11835P) after adding fructose to a mixed culture of food waste and anaerobic digestion sludge according to Example 5, Fig.

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

Conventionally, a method for producing hexanoic acid which is a precursor of hexanol, which is a biofuel, has been disclosed. However, in general, acetic acid and butyric acid must be supplied from the outside of the production of hexanoic acid using an artificial medium. In order to increase the production amount, yeast extract, minerals and the like have to be added, and anaerobic treatment and sterilization process are required, resulting in cumbersome production method and high production cost. In addition, when hexanoic acid is produced using pulmonary biomass without using an artificial medium, there is a problem that the production speed is slow and the production amount is insufficient, making it difficult to put into practical use.

Accordingly, the present invention provides a method for producing hexanoic acid which can remarkably improve the productivity of hexanoic acid without significantly adding an external organic acid such as acetic acid or butyric acid, and can significantly reduce the production cost of hexanoic acid.

Therefore,

(a) mixing food waste and anaerobic digestion sludge and culturing under predetermined temperature and anaerobic conditions to produce a primary culture medium in which acetic acid and butyric acid are produced;

(b) inoculating and culturing Megasphaera hexanoica strain (KCCM11835P) in the primary culture to produce a secondary culture; And

(c) recovering the hexanoic acid from the secondary culture.

Specifically, in the step (a), food waste and anaerobic digestion sludge are mixed and cultured under predetermined temperature and anaerobic conditions to produce a primary culture medium in which acetic acid and butyric acid are produced. Megasphaera hexanoica strain (KCCM11835P) used in the present invention is characterized by producing hexanoic acid by consuming acetic acid and butyric acid. Acetic acid and butyric acid produced in step (a) are hexanoic acid It is a factor that has a great influence on Iksan production.

Therefore, there is a need to establish a condition capable of improving the production yield of acetic acid and butyric acid through step (a). For this, the anaerobic digestion sludge is preferably subjected to pretreatment such as heating, freezing, alkali or acid treatment, and it is more preferable to use a sludge subjected to thawing after freezing. When the pretreatment process such as the thawing process after the freezing is performed, bacteria that do not form spores, such as bacteria, archaea, and methanogenic bacteria, are inhibited and spore-forming bacteria that produce acetic acid and butyric acid from the organic matter are activated . In other words, the spore microorganisms such as methane-producing bacteria are suppressed and the microorganisms existing in the anaerobic digestion sludge are changed such that the spore-forming microorganisms producing acetic acid and butyric acid are activated. Especially, If thawed after more than one day, microbial communities suitable for the production of acetic acid and butyric acid are formed, and the production of acetic acid and butyric acid can be greatly improved when mixed with food waste.

Next, between step (a) and step (b), (i) a step of subculturing under anaerobic conditions by mixing the primary culture of step (a) with food wastes . Specifically, in the subculture, a part of the primary culture liquid produced through the step (a) is mixed with food waste, cultured under anaerobic conditions, and a part of the culture liquid is mixed with food waste and cultured under anaerobic conditions Lt; / RTI > In this case, the subculture is for maximizing the production of acetic acid and butyric acid by igniting the microbial community producing acetic acid and butyric acid. In the course of culturing a part of the previous culture with food waste, the yield of acetic acid and butyric acid is high It is preferable to proceed to the next cultivation process at the time point. Through this process, it is possible to continuously produce a culture liquid in which acetic acid and butyric acid are produced in a high yield, and consequently to maximize the production amount of hexanoic acid produced by megasparrahexanoic acid. More specifically, the step (i) is most preferably performed when the concentration of butyric acid contained in the primary culture liquid of step (a) is 6 g / L or more and the concentration of acetic acid is 2 g / L or more.

In addition, it is preferable that the step (a) is performed under a predetermined temperature and anaerobic condition. At this time, the predetermined temperature may be a middle temperature of 20 to 40 ° C. Among the various strains contained in the anaerobic digestion sludge under the temperature condition, acetic acid and butyric acid producing bacteria suitable for the medium temperature condition are activated to produce acetic acid and butyric acid The production of acetic acid and butyric acid is maximized near room temperature of about 25 ° C.

In addition, the concentration of the food waste mixed in the step (a) and the step (i) is preferably 50 to 150 g / L. If the concentration of the food garbage is below the lower limit, there is a problem that sufficient carbon is not provided for the growth of the strains present in the anaerobic digestion sludge. If the concentration exceeds the upper limit, the concentration of the salt contained in the food garbage is large, There is a problem.

The concentration of the anaerobic digestion sludge mixed in the step (a) is preferably 30 to 70 mL / L. When the concentration of the anaerobic digestion sludge is less than the lower limit, there is a problem that acetic acid and butyric acid producing bacteria take a long time to ignite rightward and the initial production rate of acetic acid and butyric acid decreases. If the concentration exceeds the upper limit, There is a problem that production bacteria may be ignited.

In addition, in order to prevent the production efficiency of hexanoic acid from being lowered due to the existence of various strains in the primary culture solution, it is preferable that, in step (b), before the inoculation into the primary culture liquid, megasparrahexanoyl It is preferable that the Megasphaera hexanoica strain (KCCM11835P) undergoes a multiplication culturing process under predetermined conditions. Specifically, the enrichment culture is carried out in the presence of 8 to 12 g / L of yeast extract, 1 to 3 g / L of K ₂ HPO ₄ , 30 to 50 mL / L of Salt solution, 10 to 30 g of fructose / L, 1 to 5 g / L of sodium acetate and 0.1 to 1 g / L of cysteine-HCl.

In order to improve the production of hexanoic acid by megasparrahexanoic acid in the step (b), 10 to 30 g / L of fructose is preferably added to the primary culture medium . At this time, if the content of fructose is less than the lower limit, the effect of improving hexanoic acid production is insignificant, and if the content exceeds the upper limit, the effect of further improving the production is not exhibited.

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  One. Mega Spasera Hexanoika  Isolation of strain

In order to isolate microorganisms producing hexanoic acid, small intestine containing gastric juice was purchased and processed anaerobically in the wuxi market in Majang-dong, Seoul, Korea. The anaerobically treated gastric juice was inoculated into Reinforced Clostridia medium (difco) containing 5 g / L hexanoic acid and incubated at 37 ° C for 3 days. This enrichment culture was subcultured several times in the same medium. The cultures were cultured in Reinforced Clostridia medium (difco) medium to form colonies.

Each of the selected colonies was inoculated into a Reinforced Clostridia medium (difco) liquid medium and cultured at 37 ° C for 3 days. Then, hexanoic acid production was measured by gas chromatography (GC). As a result, microorganisms producing the greatest amount of hexanoic acid were isolated. The separated microorganisms were deposited on April 28, 2016 with the deposit number KCCM11835P to the Korea Microorganism Conservation Center, a microorganism depository organization under the Budapest Treaty.

Example  2. Anaerobic Digestion Sludge  Changes in microbial community and measurement of acetic acid and butyric acid production according to freezing conditions

First, the microbial community change according to the freezing conditions of the anaerobic digestion sludge was observed, and the results are shown in FIG.

Specifically, 50 mL / L of anaerobic digestion sludge was thawed at room temperature after freezing for 7 days at -80 ° C. (Example 2-1), thawing at room temperature after freezing for 15 days at -80 ° C. (Example 2-3), microbial communities were observed at the family level after freezing at 80 ° C for 32 days, thawing at room temperature (Example 2-4), and mixing with food gut at 150 g / L for 5 days.

As a result, it was confirmed that Ruminococcaceae has a low proportion of 1.81% in microbial communities, while Veillonellaceae has a greatest proportion in 22.51% of unfreezed sludge . However, in the case of more than 7 days of freezing , it was confirmed that Ruminococcaceae , Lachnospiraceae , Lactobacillaceae , Clostridiaceae and Bifidobacteriaceae were distributed evenly in the microbial community.

From the 7th day after freezing, it was confirmed that the microbial community of anaerobic digestion sludge changed to form a microbial community suitable for the production of acetic acid and butyric acid.

Next, 50 mL / L of the anaerobic digestion sludge according to Examples 2-1 to 2-4 was mixed with 150 g / L of food waste (total amount 200 mL, pH 6.2-7.0), stirred at room temperature and at 150 rpm The anaerobic cultivation was carried out under the condition that the organic acid production was measured according to the incubation time, and the results are shown in FIG. 3 to FIG. 6.

As a result of measurement, the production rate and production of acetic acid tended to increase with the lapse of incubation time in both of the case where the freeze pretreatment process and the freeze pretreatment process were performed, while the production rate and production amount of butyric acid It was confirmed that the improvement was rapid in one case.

Example  3. Pass  Measurement of organic acid production by culture

In accordance with Example 2-2, 50 mL / L of anaerobic digested sludge thawed at room temperature after freezing at -80 ° C. for 7 days was mixed with 150 g / L of food waste (total amount 200 mL, pH 6.2-7.0) And 150 rpm under the agitation condition, 10 ml of the culture solution was mixed with 150 g / L of food waste and cultured, and the change of organic acid production during the culture period was measured. 7.

As a result of the measurement, it was confirmed that when the subculture according to the present invention was carried out, the microbial community producing acetic acid and butyric acid was ignited, and the culture medium with high production of acetic acid and butyric acid during the culture period could be continuously produced. In order to ignite the acetic acid and butyric acid-producing microbial communities, subculture was performed at the point where the production of butyric acid and acetic acid rapidly increased (when the concentration of butyric acid was 6 g / L or more and the concentration of acetic acid was 2 g / L or more) It was confirmed that it is more desirable to

Example  4. Food waste and anaerobic Digester Sludge  In the mixed fermentation broth Mega Spasera Hexanoika ( Megasphaera hexanoica ) Strain KCCM11835P ), And cultured Sano Iksan  Production quantity measurement

In accordance with Example 2-2, 50 mL / L of anaerobic digested sludge thawed at room temperature after freezing at -80 ° C. for 7 days was mixed with 150 g / L of food waste (total amount 200 mL, pH 6.2-7.0) And 150 rpm, and the amount of hexanoic acid produced during inoculation and culture of Megasphaera hexanoica strain (KCCM11835P) was measured in the primary culture medium in which acetic acid and butyric acid were produced. 8.

Before the inoculation, the Megasphaera hexanoica strain (KCCM11835P) was added with 10 g / L of yeast extract, ₂ g / L of K ₂ HPO ₄ , 40 mL / L of salt solution , 20 g / L of fructose, 3 g / L of sodium acetate and 0.5 g / L of cysteine-HCl.

As shown in FIG. 8, when the method of the present invention was followed, the production of hexanoic acid was found to increase from the early stage of the culture of Megasphaera hexanoica strain (KCCM11835P), and the maximum yield was about 3.5 g / L and the productivity was excellent.

Example  5. Food waste and anaerobic Digester Sludge  In the mixed fermentation broth Fructose  Add Mega Spasera Hexanoika ( Megasphaera hexanoica ) Strain (KCCM11835P) was inoculated and cultured Hexanoic acid  Production quantity measurement

In accordance with Example 2-2, 50 mL / L of anaerobic digested sludge thawed at room temperature after freezing at -80 ° C. for 7 days was mixed with 150 g / L of food waste (total amount 200 mL, pH 6.2-7.0) And 150 rpm, and 20 g / L of fructose was added to the primary culture medium in which acetic acid and butyric acid were produced. Then, Megasphaera hexanoica strain (KCCM11835P) was inoculated and cultured with hexa The yield of No Ike acid was measured and the results are shown in Fig.

Before the inoculation, the Megasphaera hexanoica strain (KCCM11835P) was added with 10 g / L of yeast extract, ₂ g / L of K ₂ HPO ₄ , 40 mL / L of salt solution , 20 g / L of fructose, 3 g / L of sodium acetate and 0.5 g / L of cysteine-HCl.

As shown in FIG. 9, it was confirmed that the production of hexanoic acid from the initial stage of culture when the megaspaira hexanoica strain (KCCM11835P) was cultured after the addition of fructose according to the method of the present invention, Was about 8.5 g / L, indicating that the productivity was remarkably improved.

Korea Microorganism Conservation Center (overseas) KCCM11835P 20160428

Claims (10)

(a) mixing food waste and anaerobic digestion sludge and culturing under predetermined temperature and anaerobic conditions to produce a primary culture medium in which acetic acid and butyric acid are produced;
(b) inoculating and culturing Megasphaera hexanoica strain deposited with the deposit number KCCM11835P in the primary culture to produce a secondary culture; And
(c) recovering the hexanoic acid from the secondary culture,
The anaerobic digestion sludge was thawed after being frozen for more than 7 days at -50 to -100 ° C,
Between step (a) and step (b): (i) performing subculturing under anaerobic conditions by mixing the primary culture of step (a) with food waste,
Wherein the step (i) is carried out when the concentration of butyric acid contained in the primary culture liquid of step (a) is 6 g / L or more and the concentration of acetic acid is 2 g / L or more . delete delete delete The method according to claim 1,
Wherein the predetermined temperature of step (a) is 20 to 40 占 폚. The method according to claim 1,
Wherein the food waste has a concentration of 50 to 150 g / L. The method according to claim 1,
Wherein the concentration of the anaerobic digestion sludge is 30 to 70 mL / L. The method according to claim 1,
Wherein the step (b) is performed under the condition that 10 to 30 g / L of fructose is added to the primary culture. The method according to claim 1,
Wherein the strain of Megasphaera hexanoica deposited with the deposit number KCCM11835P inoculated into the primary culture in step (b) is cultivated in an enriched manner. 10. The method of claim 9,
The enrichment culture is carried out in the presence of 8 to 12 g / L yeast extract, 1 to 3 g / L K ₂ HPO ₄ , 30 to 50 mL / L salt solution, 10 to 30 g / L fructose, 1 to 5 g / L of sodium acetate and 0.1 to 1 g / L of cysteine-HCl in a culture medium containing hexanoic acid.

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