KR20220046971A - Menufacturing method for organic acid using methanotrophs - Google Patents

Abstract

A method for manufacturing an organic acid according to an embodiment of the present invention includes: a preparation step of preparing methylotrophic bacillus; a repeated culturing step of culturing 30 or more times by introducing methylotrophic bacillus into a medium; and an organic acid production step of producing an organic acid by reacting the methylotrophic bacillus that have undergone the repeated culturing step in a reaction vessel containing methane.

Description

Manufacturing method of organic acid using methanogenic bacteria

One embodiment of the present invention relates to a method for producing an organic acid using a methanogen. Specifically, an embodiment of the present invention relates to a method for producing an organic acid using the methanogen, which improves the gas consumption rate of the methanogen and at the same time increases the production of the organic acid.

Methane gas is the main component of natural gas and shale gas and is known as a causative agent of the greenhouse effect. Unlike liquid hydrocarbons represented by petroleum, methane by itself is a gas, so it is difficult to convert it into a high value-added thing and use it as a fuel is also difficult. Accordingly, efforts have been made to convert methane gas into useful compounds.

Methanotrophic bacteria are microorganisms that can grow on methane as the sole energy source, and were first isolated by Sohngen in 1906. After that, by taxonomic study, it was classified into 25 types according to the type of cell, the cell in the stationary phase, or the structure of the inner cell membrane. These methane-oxidizing bacteria contain an enzyme called methane monooxygenases (MMO), and can easily convert methane to methanol at room temperature and under normal pressure. Methane is oxidized to methanol by methane monooxygenase, and then methanol is oxidized to formaldehyde or formic acid by methanol dehydrogenase (MDH) to carbon dioxide. The biosynthesis of carbon compounds from methane proceeds by the ribulose monophosphate cycle (RuMP cycle) and the serine cycle, and in general, type I methane-oxidizing bacteria perform the ribulose monophosphate cycle. , it is known that type II methanoxidizing bacteria synthesize biomass using the serine cycle (Biocatalytic Conversion of Methane to Methanol as a Key Step for Development of Methane-Based Biorefineries J. Microbiol. Biotechnol. In Yeub Hwang et al (2014) ), 24(12), 1597-605). Due to the physiological characteristics of these methane-oxidizing bacteria, efforts to reduce harmful methane gas by converting methane into organic compounds using methane-oxidizing bacteria and to utilize it as an energy source have been continued.

On the other hand, succinic acid, which is known to have high solubility among carbon compounds, is an organic acid having colorless columnar or tubular crystals, and is one of the major organic acids constituting the TCA cycle and is a type of carboxylic acid. Succinic acid can be used in various fields such as food, agriculture, and polymer industry, and it is judged to be a compound with an international market value of about 15 billion dollars, and a method for producing succinic acid using a biological process is continuously studied.

An embodiment of the present invention provides a method for producing an organic acid using a methanogen. Specifically, an embodiment of the present invention provides a method for producing an organic acid using the methanogen, which improves the gas consumption rate of the methanogen and at the same time increases the production of the organic acid.

A method for producing an organic acid according to an embodiment of the present invention includes a preparation step of preparing methanogenic bacteria; Repeated culturing step of culturing 30 or more times by introducing methanogenic bacteria into the medium; and an organic acid production step of producing an organic acid by reacting the methanogenic bacteria that have undergone the repeated culture step in a reaction vessel containing methane.

Methanobacteria are genus Methylomonas, genus Methylobacter, genus Methylococcus , genus Methylomicrobium , genus Methylosphaera , genus methylocaldum ( Methylocaldum ), genus methyloglobus ( Methyloglobus ), genus methylosarcina , genus methylopropundus ( Methyloprofundus ), genus methylothulmus ( Methylothermus ), genus methylohalobius ( Methylohalobius ) , genus Methylogaea , genus Methylomarinum , genus Methylovulum, genus Methylomarinovum , genus Methylorubrum , genus Methyloparacoccus ( Methyloparacoccus ), genus Methylosinus , genus Methylocystis , genus Methylocella , genus Methylocapsa, genus Methylofurula , genus Methylacidiphyllum ( Methylacidiphilum ) and methylacidimicrobium genus ( Methylacidimicrobium ) It may include one or more of.

The methanogen may include the genus Methylomonas DH-1.

It can be cultured 40 to 60 times in the repeated culture step.

In the repeated culture step, it may be cultured in an atmosphere containing 20 to 50% by volume of methane and 20% by volume or less of oxygen.

In the repeated culture step, when methanogenic bacteria are introduced, OD600 may be added to 0.05 to 0.5.

In the repeated culture step, it can be cultured so that the OD600 is 1 to 2.5 per culture.

In the repeated culture step, it can be cultured for 1 to 5 days per culture.

In the organic acid production step, the reaction may be carried out in a reaction vessel containing 20 to 50% by volume of methane and 20% by volume or less of oxygen.

The method for producing an organic acid using methanogens according to an embodiment of the present invention stably consumes gas for a long time.

The method for producing an organic acid using methanogenic bacteria according to an embodiment of the present invention can increase the production of succinic acid and acetic acid at the same time.

1 is a graph of OD600 according to incubation time during repeated culture in Example 1.
2 is a graph showing (A) methane consumption rate before ALE, (B) oxygen consumption rate before ALE, (C) methane consumption rate after ALE, and (D) oxygen consumption rate after ALE in Experimental Example 1;
3 is a graph of (A) microbial growth curve (OD600, linear scale) (B) microbial growth curve (log scale) before and after ALE in Experimental Example 2.
4 is a graph of the organic acid production pattern in Experimental Example 3 (A) before ALE, (B) after ALE 50.

The terminology used herein is for the purpose of referring to specific embodiments only, and is not intended to limit the present invention. As used herein, the singular forms also include the plural forms unless the phrases clearly indicate the opposite. As used herein, the meaning of "comprising" specifies a particular characteristic, region, integer, step, operation, element and/or component, and the presence or absence of another characteristic, region, integer, step, operation, element and/or component It does not exclude additions.

Although not defined otherwise, all terms including technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the present invention belongs. Commonly used terms defined in the dictionary are additionally interpreted as having a meaning consistent with the related technical literature and the presently disclosed content, and unless defined, are not interpreted in an ideal or very formal meaning.

Hereinafter, embodiments of the present invention will be described in detail so that those of ordinary skill in the art can easily implement them. However, the present invention may be embodied in several different forms and is not limited to the embodiments described herein.

As used herein, the term “succinic acid” is an organic acid having a colorless columnar or tubular crystal having a chemical formula of HOOC-CH ₂ CH ₂ -COOH. Succinic acid is one of the major organic acids constituting the TCA cycle and is a type of carboxylic acid. In the TCA cycle, alpha-ketoglutaric acid is dehydrogenated and decarboxylated to form succinyl co-enzyme A (succinyl Co-A), which is then converted to succinic acid and produced. Succinic acid is oxidized to fumaric acid by succinic acid dehydrogenase. do. In addition, in the case of the glyoxylic acid cycle, succinic acid is produced in the process in which isocitrate is converted to succinic acid and glyoxylic acid by isocitrate lyase, and glyoxylic acid is converted into malic acid, oxaloacetic acid, and citric acid by the glyoxylic acid cycle. Through the process, it is converted back to isocitric acid.

As used herein, the term “methanotroph” refers to bacteria using methane as a main carbon source and energy source. ATP is obtained by completely oxidizing methane to CO ₂ using the oxidation pathway of methane. Among methylotrophs that use a compound having one carbon number, such as methane, methanol, or methylamine, as an energy source, strains that can use methane together are also considered methanogenic bacteria.

As used herein, the term “culture” refers to growth in environmental conditions artificially regulated, such as a target cell or tissue. The environmental conditions that are artificially controlled include nutrients, temperature, osmotic pressure, pH, gas composition, light, etc., but it is the medium that directly affects it, and it is largely divided into liquid medium and solid medium.

A method for producing an organic acid according to an embodiment of the present invention includes a preparation step of preparing methanogenic bacteria; Repeated culturing step of culturing 30 or more times by introducing methanogenic bacteria into the medium; and an organic acid production step of producing an organic acid by reacting the methanogenic bacteria that have undergone the repeated culture step in a reaction vessel containing methane.

Hereinafter, each step will be described in detail.

First, in the preparation stage, methanogenic bacteria are prepared.

Methanogens are strains that produce organic acids using methane as the main carbon source. Specifically, Methylomonas , Methylobacter , Methylococcus , Methylomicrobium , Methylosphaera , Methylocaldum , genus Methyloglobus, genus Methylosarcina , genus Methyloprofundus , genus Methylothermus ( Methylothermus ), genus Methylohalobius ( Methylohalobius ) , methyl genus logea ( Methylogaea ), genus Methylomarinum ( Methylomarinum ), genus Methylovulum , genus Methylomarinovum ( Methylomarinovum ), genus Methylorubrum , genus Methyloparacoccus ) , Methylosinus , Methylocystis , Methylocella , Methylocapsa , Methylofurula , Methylacidiphilum And it may include one or more of the methyl acid di microbium genus ( Methylacidi microbium ). More specifically, it may be a methylomonas genus, and more specifically, it may be a methylomonas genus (Methylomonas sp.) DH-1 deposited with accession number KCTC18400P. More specifically, it may be a strain having a cre gene inserted into the genome of a wild-type strain, a deletion of the succinate dehydrogenase gene, and an isocitrate lyase gene and a malate synthase gene. Since the aforementioned recombinant methanogenic bacteria is described in detail in Korean Patent Registration No. 10-1954530, a detailed description thereof will be omitted.

Next, in the repeated culture step, methanogenic bacteria are introduced into the medium and cultured 30 times or more.

The medium is not particularly limited as long as it is a medium capable of culturing methanogenic bacteria, and may be a liquid medium or a solid medium. Specifically, it may be a nitrate mineral salt solution (NMS) medium.

In one embodiment of the present invention, by repeating culturing for 30 or more times, it is possible to increase the gas consumption rate of the methanogen and at the same time increase the production of organic acids. This is because the metabolism of microorganisms was more adapted to the culture conditions during repeated culturing of methanogenic bacteria.

The number of incubations is based on the separation from the medium after culturing of the methanogenic bacteria and introduction into a new medium. When the number of incubations is too small, it may be difficult to obtain a sufficient effect. Even if the number of cultures is increased, there may be a limit to increasing the gas consumption rate and increasing the production of organic acids. More specifically, the culture may be repeated 30 to 70 times. More specifically, the culture may be repeated 40 to 60 times. More specifically, the culture may be repeated 45 to 55 times.

In the repeated culture step, it may be cultured in an atmosphere containing 20 to 50% by volume of methane and 20% by volume or less of oxygen.

Methane is a carbon source necessary for the growth of methanogenic bacteria. If there is too little methane, it may be difficult for methanogens to grow. More specifically, it may contain 25 to 35% by volume of methane.

Oxygen is used to convert methane to methanol or as a final electron acceptor in microbial metabolism. More specifically, it may contain 1 to 20% by volume of oxygen.

The remainder may be an inert gas, specifically nitrogen. More specifically, it may contain 30 to 80% by volume of nitrogen.

In the repeated culture step, when methanogenic bacteria are introduced, OD600 may be added to 0.05 to 0.5. If there are too few or too many methanogenic bacteria, it may adversely affect the growth of methanogenic bacteria. More specifically, it may be added so that OD600 becomes 0.07 to 0.2 when methane magnetizing bacteria is added. Since OD600 is widely known, a detailed description thereof will be omitted.

In the repeated culture step, it can be cultured so that the OD600 is 1 to 2.5 per culture. By culturing in the above range, it is possible to further enhance the gas consumption rate of the methanogenic bacteria and further increase the production of organic acids. More specifically, it can be cultured so that the OD600 is 1.3 to 2.3.

In order to culture up to the above-mentioned OD600, it can be cultured for 1 to 5 days per culture.

The culture temperature of the methanogenic bacteria may be 15 °C to 45 °C, specifically 20 °C to 40 °C, more specifically 25 °C to 35 °C, and for smooth contact between methane and methanogenic bacteria, 150rpm to 300rpm, specifically 180 rpm to 270 rpm, more specifically 200 rpm to 250 rpm may be stirred, but is not limited thereto.

Next, in the organic acid production step, the methanogenic bacteria are reacted in a reaction vessel containing methane to produce an organic acid.

In this case, the organic acid may include succinic acid and acetic acid.

At this time, the atmosphere may contain 20 to 50% by volume of methane and 20% by volume or less of oxygen.

Methane is the carbon source needed to produce organic acids. Too little methane can make it difficult to adequately produce organic acids. If there is too much methane, there may be a problem in that the amount of methane gas that is not consumed by microorganisms during culture and is discarded increases. More specifically, it may contain 25 to 35% by volume of methane.

Oxygen is required to convert methane to methanol or is used as a final electron acceptor in microbial metabolism. If there is too much oxygen, there may be a problem in that the amount of oxygen that is not consumed by microorganisms during culture and is discarded increases. More specifically, it may contain 1 to 20% by volume of oxygen.

The remainder may be an inert gas, specifically nitrogen. More specifically, it may contain 30 to 80% by volume of nitrogen.

The above-described mixed gas may be supplied at a rate of 100 to 500 mL/min.

The temperature may be 15° C. to 45° C., specifically 20° C. to 40° C., more specifically 25° C. to 35° C., and for smooth contact between methane and methanobacteria, stirring at 500 rpm to 1000 rpm, specifically 750 rpm to 900 rpm can, but is not limited to.

At this time, the weight ratio of the produced succinic acid and acetic acid (succinic acid: acetic acid) may be 1:0.7 to 1:1.

Compared to the methanogenic bacteria that have not undergone the above-described repeated culture step, the methanogenic bacteria that have undergone the repeated culture step can produce succinic acid 1.5 to 3 times and acetic acid 1.5 to 3 times.

Hereinafter, the present invention will be described in more detail through experimental examples. However, these experimental examples are only for illustrating the present invention, and the present invention is not limited thereto.

Example 1

The following NMS medium, methanogenic bacteria was prepared.

NMS media (MgSO4·7H ₂ O: 1.0 g/L, KNO ₃ : 1.0 g/L, CaCl ₂ ·H ₂ O 0.2 g/L, Fe-EDTA (wrapping by aluminum foil): 0.0038 g/L, NaMoO ₄ 2H ₂ O: 0.0005 g/L, FeSO ₄ 7H ₂ O: 0.0005 g/L, ZnSO ₄ 7H ₂ O: 0.0004 g/L, MnCl ₂ : 0.00001 g/L, CoCl ₂ 6H ₂ O: 0.00005 g/L, NiCl ₂ 6H ₂ O: 0.00001 g/L, H ₃ BO ₃ (boric acid) 0.000015 g/L, EDTA 0.00025 g/L, KH ₂ PO ₄ 0.26 g/L, Na ₂ HPO ₄ 7 (H ₂ O): 0.62 g/L, Biotin: 0.00002 g/L, Folic acid: 0.00002 g/L, Thiamine-HCl: 0.00005 g/L, Ca-pantothenate: 0.00005 g/L, Vitamin B12: 0.00001 g/L L, Riboflavin: 0.00005 g/L, Nicotiamide: 0.00005 g/L)

Methylomonas sp . DH-1 (Accession No. KCTC18400P) Wild-type strain strain with cre gene inserted into genome, succinate dehydrogenase gene deleted, and isocitrate lyase gene and malate synthase gene. Hereinafter expressed as CS strains.

Repeat culture Adaptive laboratory evolution (ALE)

The CS strain is inoculated into NMS medium so that the initial OD600 becomes 0.1, and the head space of the serum bottle is replaced with 30v% CH ₄ , 3.5v% O ₂ , and 66.5v% N ₂ gas. After culturing the serum bottle in a shaking incubator at 30°C for 2-3 days, inoculate it in a new NMS medium, replace the head space with the gas of the same composition as above, and incubate for 2-3 days again. In this way, the culture was repeated 50 times. 1 shows a graph of OD600 according to incubation time.

microbial gas fermentation

2ml of the strain 50 passaged was inoculated into a baffled flask containing 200ml NMS medium, and the head space of the flask was replaced with 30v% CH ₄ , 3.5v% O ₂ , 66.5v% N ₂ gas. This was cultured for 2 to 3 days while shaking at 200 rpm in a shaking incubator at 30°C and raised to an OD600 of 1.5 or higher. The flask culture grown in this way was set to an initial OD600 of 0.1 and inoculated into a fermeter containing 3L NMS medium. 30v% CH ₄ , 3.5v% O ₂ , 66.5v% N ₂ A mixed gas having a composition was supplied through the sparger at a rate of 300 ml/min. Culture was continued while maintaining a stirring speed of 30°C and 800 rpm. The OD600 value was measured using a UV Spectrophotometer to determine the growth of microorganisms, and the rate at which the microorganisms consumed the supplied gas was measured using the Quadrupole Mass. HPLC analysis was performed using some samples during the culture to monitor the production of organic acids.

Experimental Example 1: Change in gas consumption rate

2 shows (A) methane consumption rate before ALE, (B) oxygen consumption rate before ALE, (C) methane consumption rate after ALE, and (D) oxygen consumption rate after ALE.

As shown in FIG. 2, the methane gas consumption rate before ALE increased to 4.47 mmol/L/hr at the time of incubation ~27 hr. Then the gas consumption rate starts to decrease rapidly. On the other hand, in the case of culturing cells that have undergone the ALE process, a high gas consumption rate is maintained between 13 and 65 hours of culture, and the maximum value is 6.30 mmol/L/h, which is increased by about 40%.

As shown in Experimental Example 1, a stable gas consumption pattern for a long period of time is a very useful feature in industrial application of methanogens.

Experimental Example 2: Growth change

For the growth curve of microorganisms, optical density (OD600) was measured at a wavelength of 600 nm using a UV spectrophotometer.

3 shows (A) microbial growth curve (OD600, linear scale) (B) microbial growth curve (log scale) before and after ALE.

As shown in FIG. 3 , when comparing the growth curves before and after ALE, there appears to be no significant difference in the early growth period up to 7 hours after inoculation, but in the mid-exponential phase thereafter, the microorganisms that passed ALE 50 showed faster growth and the maximum OD600 also increased 3.6 times from 3.3 to 11.9.

Experimental Example 3: Organic acid generation pattern change

4 shows the organic acid production pattern before (A) ALE, (B) after ALE 50.

As shown in FIG. 4 , the microorganisms before ALE had a maximum titer of 230 mg/L for succinic acid production and 143 mg/L for acetic acid production in fermenter culture. On the other hand, when the microorganisms subjected to ALE 50 times were used as seeds, the succinic acid titer increased 2.2 times to a maximum of 509 mg/L, and acetic acid showed a pattern of increasing 2.93 times to 419 mg/L.

As such, it can be confirmed that the production of succinic acid and acetic acid can be simultaneously increased through fermenter culture used in ALE and experimental methods.

The present invention is not limited to the above embodiments, but can be manufactured in various different forms, and those of ordinary skill in the art to which the present invention pertains can take other specific forms without changing the technical spirit or essential features of the present invention. It will be understood that it can be implemented as Therefore, it should be understood that the embodiments described above are illustrative in all respects and not restrictive.

Claims (9)

A preparatory step for preparing methanogens;
Repeated culturing step of introducing the methanogenic bacteria into the medium and culturing 30 or more times; and
A method for producing an organic acid using methanogenic bacteria, comprising a step of producing an organic acid by reacting the methanogenic bacteria that have undergone the repeated culturing step in a reaction vessel containing methane to produce an organic acid. According to claim 1,
The methanogenic bacteria are genus Methylomonas, genus Methylobacter , genus Methylococcus , genus Methylomicrobium , genus Methylosphaera , genus methylocaldum ( Methylocaldum ), methyloglobus ( Methyloglobus ), methylosarcina , methylopropundus genus ( Methyloprofundus ), methylothulmus genus ( Methylothermus ), methylohalobius genus ), Methylogaea , Methylomarinum , Methylovulum , Methylomarinovum, Methylorubrum , Methyloparacoccus ( Methyloparacoccus ), genus Methylosinus , genus Methylocystis , genus Methylocella , genus methylocapsa ( Methylocapsa ), genus Methylofurula , genus Methylacidiphyllum ( Methylacidiphilum ) and methylacidimicrobium genus ( Methylacidimicrobium ) Method for producing an organic acid using methanogenic bacteria including at least one of them. According to claim 1,
The method for producing an organic acid using the methanogenic bacteria, the methanogenic bacteria comprising the genus Methylomonas DH-1. According to claim 1,
A method for producing an organic acid using methanogenic bacteria culturing 40 to 60 times in the repeated culturing step. According to claim 1,
A method for producing an organic acid using methanogenic bacteria, wherein in the repeated culturing step, methane is cultured in an atmosphere containing 20 to 50% by volume of methane and 20% by volume or less of oxygen. According to claim 1,
A method for producing an organic acid using methanogenic bacteria in which OD600 becomes 0.05 to 0.5 when methanogenic bacteria are added in the repeated culture step. According to claim 1,
A method for producing an organic acid using methanogenic bacteria, which is cultured so that the OD600 becomes 1 to 2.5 during one culture in the repeated culture step. According to claim 1,
A method for producing an organic acid using methanogenic bacteria, which is cultured for 1 to 5 days per culture in the repeated culture step. According to claim 1,
A method for producing an organic acid using methanogens in which the reaction vessel contains 20 to 50% by volume of methane and 20% by volume or less of oxygen in the organic acid production step.

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