KR20180077972A - Method of producing formate using Acetobacterium woodii - Google Patents

Abstract

According to the present invention, a formate production method comprises: a step of converting carbon monoxide contained in a carbon monoxide-containing gas into carbon dioxide; a step of bringing the carbon dioxide into contact with formate dehydrogenase (FDR) derived from microorganisms, Acetobacterium woodii; and a step of suppressing synthesis of sodium-dependent ATPase of the microorganisms, Acetobacterium woodii. Accordingly, the microorganisms, Acetobacterium woodii can be used to produce formate from CO, which can solve environmental problems and bring economic benefits.

Description

Technical Field [0001] The present invention relates to a method for producing formate using microorganism Acetobacterium woodii (Acetobacterium woodii)

The present invention relates to a process for the production of formates, and more particularly to a process for producing formate using carbon monoxide (CO) as a starting material by using the microorganism Acetobacterium woodii .

Carbon monoxide has a higher affinity for blood hemoglobin than for oxygen. Therefore, when CO is inhaled with air containing a certain concentration or more, it may cause vascular damage due to increase of blood pressure due to an increase in heart rate due to lack of oxygen, worst case unconsciousness and death.

Incomplete combustion of carbon in homes or industrial sites generates a considerable amount of CO. Among them, the by-product gas generated from steel mills contains less than 8% and more than 60% CO.

Korean Patent No. 10-1524340 (Publication date 2015.06.04)

Such CO-containing waste gas adversely affects the environment and requires a separate process to be performed, so that a great cost is consumed.

DISCLOSURE Technical Problem The present invention has been made to solve various problems including the above-mentioned problems, and it is an object of the present invention to provide a microbial Acetobacterium It is an object of the present invention to provide a method for producing formate using carbon monoxide (CO) as a starting material by using woodii .

The method for producing a formate according to the present invention comprises the steps of: converting the carbon monoxide of a gas containing carbon monoxide into carbon dioxide; Contacting the carbon dioxide with formate dehydrogenase (FDR) derived from microorganism Acetobacterium woodii to formate; And inhibiting the synthesis of the sodium-dependent ATP synthase of the microorganism Acetobacterium woodii .

The step of converting the carbon monoxide into the carbon dioxide may include contacting carbon monoxide dehydrogenase (CODH) derived from the microorganism Acetobacterium woodii .

The step of inhibiting the synthesis of the sodium-dependent ATP synthase may include the step of removing sodium ions using sodium ionophore.

The sodium ionophor may include any one of the group including ETH 157, ETH 227, ETH 2120 and ETH 4120.

The method of producing a formate according to an embodiment may further include adding potassium bicarbonate (KHCO ₃ ) before the step of inhibiting the synthesis of the sodium-dependent ATP synthase of the microorganism Acetobacterium woodii .

The volume ratio of the carbon monoxide in the gas may be from 40% to less than 60%.

The gas may be generated in a steel manufacturing process.

Other aspects, features, and advantages will become apparent from the following drawings, claims, and detailed description of the invention.

According to the present invention, the formate which is the anion of formic acid can be produced from toxic CO, in particular, from waste gas including CO generated in a steel manufacturing process and the like. Since formate is an industrially valuable organic material, the microbial Acetobacterium woodii can be used to produce formate from CO, which can solve environmental problems and bring economic benefits. Of course, the scope of the present invention is not limited by these effects.

FIG. 1 is a view showing the Wood-Ljungdahl pathway of the microorganism Acetobacterium woodii producing acetate (CH ₃ COO ^- ) from carbon dioxide.
2 is a flowchart showing a method of producing a formate according to the present invention.
3 is a graph showing the concentration of formate according to the concentration of CO.
4 is a graph showing the addition of potassium bicarbonate (KHCO ₃ ) and the concentration of formate according to the concentration of CO.
Figure 5 shows the microbial Acetobacterium FIG. 2 is a graph showing the concentration of formate according to the amount of cells of woodii . FIG.

BRIEF DESCRIPTION OF THE DRAWINGS The present invention is capable of various modifications and various embodiments, and specific embodiments are illustrated in the drawings and described in detail in the detailed description. The effects and features of the present invention and methods of achieving them will be apparent with reference to the embodiments described in detail below with reference to the drawings. However, the present invention is not limited to the embodiments described below, but may be implemented in various forms.

Hereinafter, exemplary embodiments of the present invention will be described in detail with reference to the accompanying drawings, wherein like reference numerals refer to like or corresponding components throughout the drawings, and a duplicate description thereof will be omitted .

In the following examples, the singular forms "a", "an" and "the" include plural referents unless the context clearly dictates otherwise.

In the following embodiments, terms such as inclusive or possessive are intended to mean that a feature, or element, described in the specification is present, and does not preclude the possibility that one or more other features or elements may be added.

Hereinafter, exemplary embodiments of the present invention will be described in detail with reference to the accompanying drawings, wherein like reference numerals refer to like or corresponding components throughout the drawings, and a duplicate description thereof will be omitted .

1 is a view showing a Wood-Ljungdahl pathway of a microorganism Acetobacterium woodii that produces acetate from carbon dioxide (CO ₂ ).

Acetobacterium woodii is an anaerobic microorganism that converts carbon dioxide to acetate or CO through the wood-firming pathway of FIG. 1 and then converts the carbon monoxide dehydrogenase / acetyl-CoA synthase (CODH / ACS) Lt; RTI ID = 0.0 > acetate. ≪ / RTI > Referring to Part A of FIG. 1, formate is formed as an intermediate product in this process.

Formate is an anion of formic acid (HCOOH) and has the formula HCOO ^- . Formic acid is a toxic organic acid used in animal feeds to kill bacteria or used as a coagulant in rubber manufacturing.

2 is a flowchart showing a method for producing a formate according to the present invention.

The method for producing a formate according to the present invention comprises the steps of: converting the carbon monoxide of a gas containing carbon monoxide into carbon dioxide; Contacting the carbon dioxide with formate dehydrogenase (FDR) derived from microorganism Acetobacterium woodii to formate; And inhibiting the synthesis of the sodium-dependent ATP synthase of the microorganism Acetobacterium woodii .

Referring to FIG. 2 (a), CO is converted to CO ₂ . Such CO oxidation can occur spontaneously in small amounts. When CO is oxidized to CO ₂ , two electrons and two protons are generated. On the other hand, when CO ₂ is reduced to formate as shown in FIG. 2 (b), two electrons and protons are consumed. 2 (a) and 2 (b), a continuous reaction from CO to CO ₂ and from CO ₂ to formate is possible.

Since CO exists in a stable state in the atmosphere, the rate of conversion to CO ₂ by spontaneous oxidation is low. Therefore, in order to convert CO to CO ₂ , it is preferable to employ a catalytic oxidation method.

According to one embodiment, the step of converting carbon monoxide to carbon dioxide comprises contacting carbon monoxide dehydrogenase (CODH) derived from the microorganism Acetobacterium woodii .

Microorganism Acetobacterium a carbon monoxide dehydrogenase derived from woodii (CODH, enzyme code: EC : 1.2.99.2) was Acetobacterium It is encoded by the acase gene of woodii. Referring to Figures 2 (a) and 2 (b), CODH and FDR are used in the step of converting CO to CO ₂ and the step of converting CO ₂ to formate, respectively. When such CODH is present, the oxidation reaction from CO to CO ₂ is actively carried out, so that the yield of the final product formate is increased. At this time, Acetobacterium Since woodii can synthesize both enzymes, all steps of Fig. 2 can be performed with only one microorganism.

On the other hand, referring to part B of FIG. 2, Acetobacterium Woodii has a sodium-dependent ATP synthase that synthesizes ATP in proportion to the amount of sodium ions. The synthesized ATP is used to synthesize formyl-THF (Formyl-THF), the next step of formate. That is, when ATP is present, Acetobacterium Since the wood-firming pathway of woodii progresses intact, formate is present only as an intermediate product and can not be mass produced. Therefore, if the sodium ion can be removed, the synthesis of ATP is inhibited as shown in Fig. 2 (c), and as a result, formyl-THF synthesis is inhibited as shown in Fig. 2 (d), and formate is accumulated. That is, the microorganism Acetobacterium It is only necessary to inhibit the synthesis of sodium-dependent ATP synthase in woodii to produce formate as the final product.

According to one embodiment, the step of inhibiting the synthesis of a sodium-dependent ATP synthase comprises the step of removing sodium ions using sodium ionophore. Ionophor is a chemical substance that can bind to ions, usually dissolved in lipids and passes ions through the cell membrane.

According to one embodiment, the sodium ionophor may comprise any one of the group comprising ETH 157, ETH 227, ETH 2120 and ETH 4120. ETH 157, ETH 227, ETH 2120 and ETH 4120 have the following Chemical Formulas 1 to 4, respectively.

[Chemical Formula 1]

(2)

(3)

[Chemical Formula 4]

Such sodium ionophores inhibit the synthesis of ATP synthase by binding sodium ions to remove sodium ions. This inhibits the metabolism of formate in the woody-firming pathway, so that the formate can be produced as a final product.

< Example >

1. Materials and Methods

1-1. Culture conditions

The strain Acetobacterium woodii (KCTC 1655) were used in the form of freeze-dry cells.

The culture medium for the culture of the strain has the following composition. KH ₂ PO ₄ 0.2 g / L; NH ₄ Cl 0.25 g / L; NaCl 1.16 g / L; MgSO ₄揃 7H ₂ O 1.45 g / L; CaCl ₂ 0.11 g / L; KCl 0.5 g / L; Yeast Extract 2.0 g / L; 0.5 ml / L 0.1% w / v resazurin solution; SL9 Trace element solution 1.0 ml / L; Selenite-Tungstate Solution 1.0 ml / L; 0.4 g / L Cysteine-hydrochloride monohydrate; 6.0 g / L KHCO ₃ ; And titrate with HCl to pH 7.0 - 7.1. Then add 20 ml / L DSMZ 141 vitamin solution by filter sterilization. Medium is prepared by placing 100 ml in a 500 ml serum bottle and head space purging with N ₂ / CO ₂ (80:20).

Fructose (20mM) was used as a carbon source of the strain and filter sterilization or autoclave was performed separately from the medium to be inoculated before cell inoculation.

Materials such as fructose, formate, and acetate, which are produced or consumed during cell growth, were measured by high performance liquid chromatography (Agilent) analysis.

1-2. Acetobacterium woodii Dormant cell ( resting cell ) From CO Formate  produce

Cells were harvested by centrifugation at 4 ° C and 11.300 g for 10 min at OD600 = 1.5. The harvested cells were washed once with 50 mM imidazole buffer (50 mM imidazole, 20 mM MgSO ₄ , 20 mM KCl, and 4 mM dithioerythritol), and then added to the same buffer solution as necessary using 50 mM potassium bicarbonate (KHCO ₃ ). Subsequently, head space purging was performed on CO gas (10%, 50%, 100%, N ₂ mixed) having different compositions and the reaction was carried out in a shaking water bath at 4 ° C. and 100 rpm .

The entire process was carried out under strictly anaerobic conditions using an anaerobic chamber (COY Laboratory). The resultant formate and acetate were measured by HPLC analysis as above. On the other hand, ETH 2120 was used to inhibit the synthesis of ATP synthase.

2. Results

2-1. Of CO  Depending on concentration Formate  output

3 is a graph showing the concentration of formate according to the concentration of CO. The changes in formate production with CO concentration were observed by head space purging 100%, 50%, 10% CO (N ₂ mixed) gas in 13 mg dry cell. As shown in FIG. 3, when 50% CO gas was used, the yield was about twice that of 100% CO gas. When 10% CO gas was used, 2 mM was produced after 25 hours, Did not increase. Acetate, on the other hand, did not increase after small amounts (not shown).

When the concentration of CO approaches 100%, metabolism of Acetobacterium woodii is inhibited by the toxicity of CO itself. On the other hand, in the case of 10% CO, the production amount does not increase at a certain level because the amount of CO as a starting material is small. There is therefore an optimal CO concentration in formate production.

According to one embodiment, the volume ratio of carbon monoxide may be between 40% and less than 60%. Referring to FIG. 3, since the yield of formate is maximized when the concentration of CO is about 50%, there is an optimum CO concentration of formate near the above value. In other words, when the concentration of CO is less than 60%, which is about 50%, the yield of formate can be maximized. The concentration of CO can be controlled by mixing N ₂ with CO gas.

2-2. KHCO ₃  Depending on availability Formate  output

4 is a graph showing the addition of potassium bicarbonate (KHCO ₃ ) and the concentration of formate according to the concentration of CO. In this case, the experiment was carried out by varying the addition of 50 mM KHCO ₃ to 14 mg dry cell and 5 ml imidazole buffer. At this time, the same CO gas composition (10%, 50%, 100%) was used as in the above experiment. The added KHCO ₃ is intended to supply CO ₂ . As a control, CO ₂ man in this case was not observed formate produced but a single experiment the CO ₂ purging the head space (headspace purging) a set (set) to see the effects of the presence of such a. At this time, nearly showed the highest yield and to the addition of 50mM KHCO ₃ in 50% CO gas generated in the formate 40.47mM the basis of 190 hours, when put gave the KHCO ₃ in every set of two or three times the Showed an increase in formate production. Acetate in all sets did not increase after small amounts were generated (not shown).

The reason that the amount of formate produced changes with the addition of KHCO ₃ is due to an increase in the substrate of formate dehyrdogenase (FDR), which converts CO ₂ to formate. However, formate is not produced when only CO ₂ exists alone (the graph in Fig. 3). In the process of reducing CO ₂ to formate, protons and electrons are needed. When CO is used together, formate can be formed because protons and electrons generated when CO is oxidized can be used. However, since no proton or electron can be obtained with CO ₂ alone, the reaction does not proceed.

On the other hand, when CO ₂ , which is an intermediate substrate, is increased, the reaction rate is expected to increase, and the reaction proceeds while changing the amount of KHCO ₃ to 50, 100 and 200 mM. This is thought to be due to the somewhat lower oxidation rate of CO supplying the protons and electrons needed to reduce existing CO ₂ . Therefore, in order to increase the CO oxidation rate, Acetobacterium woodii When the derived CODH is added, the conversion of CO to CO ₂ becomes active, resulting in an increase in the amount of formate produced.

2-3. Cell ( cell Depending on the amount Formate  output

Figure 5 shows the microbial Acetobacterium FIG. 2 is a graph showing the concentration of formate according to the amount of cells of woodii . FIG.

Finally, the experiment was carried out by varying the amount of cells (26 mg, 77 mg). In both cases, imidazole buffer was used, 50 mM KHCO ₃ was added, and 50% CO gas was purged. After 24 hours, the reaction proceeded steadily with the formation of 15.80 mM and 42.64 mM formate, respectively. After that, the formation rate was decreased to produce 32.62 mM and 84.34 mM formate after 91 hours, respectively. Acetate did not increase any more after 1.90 mM and 5.80 mM, respectively (not shown).

Naturally, increasing the amount of cells increases the amount of formate produced. However, the rate of formation of formate per cell was about 0.0253 mM / hr mg-DCW (dry cell weight) when using 26 mg and 0.0231 mg / hr mg-DCW at 77 mg . There are two reasons why the generation rate decreases after 24 hours. The first is that as the reaction progresses, the mass transfer decreases and the rate of production decreases. Second, the cell viability decreases and gradually fails to function. First, even if the amount of cells is increased, the rate of formation of formate per unit cell does not show a large difference. Therefore, it is considered that the effect of the first mass transfer is small. Thus, the viability of the cell can be increased to increase the rate of formate formation.

According to the present invention, the formate which is the anion of formic acid can be produced from toxic CO, in particular, from waste gas including CO generated in a steel manufacturing process and the like. Since formate is an industrially valuable organic material, the microbial Acetobacterium woodii can be used to produce formate from CO, which can solve environmental problems and bring economic benefits.

While the present invention has been described with reference to exemplary embodiments thereof, it is to be understood that the invention is not limited to the disclosed exemplary embodiments, but, on the contrary, is intended to cover various modifications and equivalent arrangements included within the spirit and scope of the appended claims. Accordingly, the true scope of the present invention should be determined by the technical idea of the appended claims.

Claims (7)

Converting the carbon monoxide in the gas containing carbon monoxide (CO) to carbon dioxide;
The above-mentioned microorganisms Acetobacterium contacting a formate dehydrogenase (FDR) derived from woodii to produce a formate; And
The microorganism Acetobacterium inhibiting the synthesis of a sodium-dependent ATP synthase of woodii . The method according to claim 1,
Wherein the step of converting the carbon monoxide into carbon dioxide comprises:
The microorganism Acetobacterium a carbon monoxide dehydrogenase derived from woodii; comprising the step of contacting the (carbon monoxide dehydrogenase CODH), the production method of the formate. The method according to claim 1,
The step of inhibiting the synthesis of the sodium-dependent ATP synthase comprises:
A process for the production of formates, comprising the step of removing sodium ions using a sodium ionophore. The method of claim 3,
Wherein the sodium ionophores comprise any one of the group consisting of ETH 157, ETH 227, ETH 2120 and ETH 4120. The method according to claim 1,
The microorganism Acetobacterium Before the step of inhibiting the synthesis of sodium-dependent ATP synthase in woodii ,
Further comprising the step of adding potassium bicarbonate (KHCO ₃ ). The method according to claim 1,
Wherein a volume ratio of the carbon monoxide in the gas is 40% or more and less than 60%. The method according to claim 1,
Wherein the gas is generated in a steel manufacturing process.

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