KR20230143829A - Method for Producing Bio-Alcohol Using Clostridium sp. and Medium Comprising Acetate - Google Patents

Abstract

The present invention relates to a method for producing bioalcohol using culture of Clostridium strains and a medium for the same.
The bio-alcohol production method of the present invention uses a medium composition to which acetic acid is added, enabling the production of high-concentration bio-ethanol without the side effects of inhibiting bacterial growth of Clostridium sp. strains and reducing CO consumption.
Therefore, the method for producing bioalcohol and the medium composition for the same of the present invention can be usefully used in the process of selectively producing bioethanol from synthesis gas.

Description

Method for producing bio-alcohol using Clostridium genus strain and acetic acid-containing medium {Method for Producing Bio-Alcohol Using Clostridium sp. and Medium Comprising Acetate}

The present invention relates to a method for producing bioalcohol using a Clostridium genus strain and acetic acid-containing medium, and more specifically, by culturing Clostridium genus strains in acetic acid-containing medium, thereby inhibiting the growth of AWRP cells by CO gas and CO It relates to a method for producing bioalcohol that can overcome the phenomenon of reduced consumption and a medium composition for the same.

Carbon monoxide is generated through incomplete combustion during combustion of fossil fuels and incineration of waste, and is present in significant amounts in by-product gas or synthetic gas used in the steel mill process. Carbon monoxide is a toxic gas to most microorganisms, but it is well known that some microorganisms can grow using carbon monoxide as a single energy/carbon source. Using these characteristics, research is actively underway to convert carbon monoxide generated in various industrial processes into acetic acid, ethanol, etc. through microbial culture. Biological carbon monoxide conversion has the advantage that it can be operated at low temperature and pressure compared to chemical conversion using a catalyst, and can be produced using gases of various compositions.

In particular, acetogenic microorganisms are known to be able to rapidly convert carbon monoxide or carbon dioxide into acetic acid, ethanol, and/or C ₂ or higher compounds using the Wood-Ljungdahl pathway, and are used in biological carbon monoxide conversion research. Acetogenic microorganisms are widely used.

AWRP, a new strain of Clostridium genus, an acetogenic microorganism newly identified by the present inventors, has excellent alcohol selective production ability and has no side effects due to acetic acid accumulation during the alcohol production process, making it useful in the process of selectively producing bioethanol from synthesis gas. It has been shown to be the most promising strain that can be used effectively (Patent Publication No. 10-2021-0028504, March 12, 2021).

Despite these advantages, the culture of acetogenic microorganisms using CO (hereinafter used in the same sense as 'carbon monoxide') as a substrate has a low conversion rate due to the low solubility of CO, compared to fermentation using conventional carbohydrates and sugars. The disadvantage was low productivity, making it difficult to apply it industrially. In theory, improvement is possible by increasing the partial pressure of CO using an internal pressure reactor, but it is well known that growth of acetogenic microorganisms using CO is also inhibited at high concentrations of CO.

In particular, it is known that inhibition of the activity of hydrogenase, which is used for electron transfer and energy acquisition in the intracellular metabolism of acetogen, is the biggest cause, and in relation to this, formic acid dehydrogenase (formate), an intermediate reaction of the Wood-Ljungdahl pathway, dehydrogenase) is known to be inhibited, thereby reducing overall carbon fixation ability and growth rate (Wang et al., J. Bacteriol.).

In addition, inhibition of the growth of acetogenic microorganisms and inhibition of carbon fixation ability under high CO concentration partial pressure mainly occurs in the early stages of growth when the conversion rate of CO gas is low due to the low bacterial mass in the liquid phase.

Currently, as a method of solving growth inhibition by CO, a method of subculturing and adaptive evolution while gradually increasing the CO partial pressure is known, but there is no known acetogen strain developed in this way yet.

Accordingly, the present inventors made extensive research efforts to overcome the limitations of acetogen strains in which carbon fixation ability and growth rate decrease at high CO concentrations, and as a result, the AWRP strain of Clostridium was cultured in a basic medium containing acetic acid (Acetate). In this case, it is possible to overcome the inhibition of bacterial growth by CO gas and increase the CO consumption of the medium by the strain. The maximum specific growth rate (μmax) increased by 83% in batch culture conditions and about 2.6 in bioreactor conditions. The present invention was completed after confirming that the maximum specific growth rate (μmax) was increased twofold and that the final concentration of ethanol and 2,3-butanediol could be increased by two to three times.

Therefore, the main purpose of the present invention is to provide a method for producing bioethanol at high concentration from synthesis gas using Clostridium strains without the side effect of reduced carbon fixation ability and growth rate at high CO concentration.

Other objects and advantages of the present invention will become clearer from the following detailed description, claims, and drawings.

According to one aspect of the present invention, the present invention includes the steps of (a) cultivating a Clostridium genus AWRP (Accession number: KCTC 13908BP) strain by supplying a gas containing carbon monoxide to a medium composition to which acetic acid has been added; and (b) separating alcohol from the culture.

In the present invention, the Clostridium AWRP (accession number: KCTC 13908BP) strain is characterized by the selective production of alcohol compared to acetic acid. As a result, ATP production efficiency is not reduced due to side effects caused by acetic acid accumulation, and cell growth is not inhibited due to acetic acid accumulation. Therefore, the Clostridium AWRP strain of the present invention is characterized by having a high alcohol production yield without acetic acid accumulation. The alcohol produced by the Clostridium AWRP (Accession Number: KCTC 13908BP) strain of the present invention is characterized in that it is ethanol and 2,3-butanediol.

In addition, in the present invention, a method of improving the side effect of reduced carbon fixation ability and growth rate at high CO concentration was exemplified by using the AWRP strain of the Clostridium genus among acetogenic microorganisms, but other acetogen strains capable of producing ethanol were used. Therefore, it will also be obvious to those skilled in the art to use a similar culture method.

In the method for producing bioalcohol of the present invention, the acetic acid concentration of the medium composition in step (a) may be 10mM to 80mM, and the partial pressure of carbon monoxide in the medium may be 50kPa to 250kPa. Acetic acid added to the medium composition is used as an electron donor for carbon monoxide (CO) in the metabolic process of the Clostridium AWRP strain, thereby consuming carbon monoxide, and as a result, the Clostridium AWRP strain It plays a role in alleviating early growth inhibition. Therefore, it is important to maintain the acetic acid concentration and the partial pressure of carbon monoxide in the medium composition at a constant ratio. When the partial pressure of carbon monoxide in the medium is 50 kPa to 170 kPa, adding acetic acid concentration to 10mM to 80mM reduces the toxicity of carbon monoxide and helps clostry. It can facilitate the growth of AWRP strains of the Dium genus.

In the present invention, the term 'gas' refers to a gas containing carbon monoxide generated by incomplete combustion of solids, and is a concept that includes 'synthetic gas'.

In the bio-alcohol production method of the present invention, the gas input to the bio-alcohol production method in step (a) essentially contains carbon monoxide, and may preferably include gases such as carbon monoxide, hydrogen, carbon dioxide, and nitrogen. there is.

In the bioalcohol production method of the present invention, the gas in step (a) may contain 50% (v/v) to 100% (v/v) of carbon monoxide. Additionally, the synthesis gas containing carbon monoxide can be compressed to 50 kPa to 250 kPa, preferably 75 kPa to 200 kPa, and continuously supplied.

In the method for producing bioalcohol of the present invention, the medium in step (a) can be cultured using any medium selected from RM medium, PETC medium, AM medium, and AMv2 medium, preferably RM medium or PETC medium. It can be cultured using medium.

In the method for producing bioalcohol of the present invention, the RM medium contains 0.2 g of MgSO ₄ ·7H ₂ O per liter; NaCl 1g; KCl 0.2g; Yeast extract 0.5 g; 10 mL of phosphoric acid solution; 1 mL of trace element I solution; 1 mL of trace element II solution; and 10 mL of Wolfe's vitamin solution. Additionally, the phosphoric acid solution contains NaH ₂ PO ₄ ·2H ₂ O 0.8 g/L; and K ₂ HPO ₄ 0.4 g/L, and the trace element I solution is 35% HCl 10 mL/L; FeSO ₄ ·7H ₂ O 27.8 g/L; MnCl ₂ ·4H ₂ O 9.9g/L; ZnCl ₂ 4.09 g/L; CoCl ₂ ·6H ₂ O 11.9 g/L; and NiCl ₂ ·6H ₂ O 2.38 g/L; and the trace element II solution includes Na ₂ MoO ₄ ·2H ₂ O 0.24 g/L; Na ₂ SeO ₃ ·5H ₂ O 1.32 g/L; and Na ₂ WO ₄ ·2H ₂ O 3.3 g/L; and the Wolfe's vitamin solution contains 2 g/L biotin; Folic acid 2g/L; Pyridoxine·HCl 10g/L; Thiamine·HCl 5g/L; Riboflavin 5g/L; Nicotinic acid 5g/L; Calcium D-pantothenate 5g/L; Vitamin B12 0.1g/L; p-aminobenzoic acid 5g/L; and lipoic acid 5g/L, but is not limited thereto.

Additionally, the PETC medium contains 0.1 g of KH ₂ PO ₄ per liter; MgSO ₄ ·7H ₂ O 0.2 g; CaCl ₂ ·2H ₂ O 0.02 g; NaCl 0.8g; KCl 0.1g; Yeast extract 0.5 g; 10 mL of trace element solution (ATCC 1754); 10 mL of Wolfe's vitamin solution; and 10 mL of reducing agent, wherein the trace element solution (ATCC 1754) contains 25 g/L of nitrilotriacetic acid; 15 g of MnSO ₄ ·H ₂ O; Fe(SO ₄ ) ₂ (NH ₄ ) ₂ ·6H ₂ O 0.85 g; CoCl ₂ ·6H ₂ O 0.25 g; ZnSO ₄ ·7H ₂ O 0.00025 g; CuCl ₂ ·2H ₂ O 0.000025 g; NiCl ₂ ·6H ₂ O 0.000025 g; Na ₂ MoO ₄ ·2H ₂ O 0.000025 g; Na ₂ SeO ₄ 0.000025 g; Na ₂ WO ₄ 0.000025 g; and the Wolfe's vitamin solution contains 2 g/L biotin; Folic acid 2g/L; Pyridoxine·HCl 10g/L; Thiamine·HCl 5g/L; Riboflavin 5g/L; Nicotinic acid 5g/L; Calcium D-pantothenate 5g/L; Vitamin B12 0.1g/L; p-aminobenzoic acid 5g/L; and 5 g/L lipoic acid.

In the bioalcohol production method of the present invention, the RM medium can use a reducing agent that maintains the pH of the medium at a certain level, and the reducing agent is MES [2-(N-morpholino)ethanesulfonic acid] at a concentration of 30 to 50mM. It is preferable to use, and in this case, pH can be maintained at 4.5 to 6.0, most preferably pH 5.5.

According to another aspect of the present invention, the present invention uses any one medium selected from RM medium, PETC medium, AM medium and AMv2 medium as a base medium, and further contains acetic acid at a concentration of 10 to 80mM. Provides a medium composition for cultivating Clostridium genus AWRP (Accession number: KCTC 13908BP) strain for bio-alcohol production under carbon monoxide partial pressure conditions.

In the medium composition for bioalcohol production of the present invention, the RM medium contains 0.2 g of MgSO ₄ ·7H ₂ O per liter; NaCl 1g; KCl 0.2g; Yeast extract 0.5 g; 10 mL of phosphoric acid solution; 1 mL of trace element I solution; 1 mL of trace element II solution; and 10 mL of Wolfe's vitamin solution. Additionally, the phosphoric acid solution contains NaH ₂ PO ₄ ·2H ₂ O 0.8 g/L; and K ₂ HPO ₄ 0.4 g/L, and the trace element I solution is 35% HCl 10 mL/L; FeSO ₄ ·7H ₂ O 27.8 g/L; MnCl ₂ ·4H ₂ O 9.9g/L; ZnCl ₂ 4.09 g/L; CoCl ₂ ·6H ₂ O 11.9 g/L; and NiCl ₂ ·6H ₂ O 2.38 g/L; and the trace element II solution includes Na ₂ MoO ₄ ·2H ₂ O 0.24 g/L; Na ₂ SeO ₃ ·5H ₂ O 1.32 g/L; and Na ₂ WO ₄ ·2H ₂ O 3.3 g/L; and the Wolfe's vitamin solution contains 2 g/L biotin; Folic acid 2g/L; Pyridoxine·HCl 10g/L; Thiamine·HCl 5g/L; Riboflavin 5g/L; Nicotinic acid 5g/L; Calcium D-pantothenate 5g/L; Vitamin B12 0.1g/L; p-aminobenzoic acid 5g/L; and lipoic acid 5g/L, but is not limited thereto.

In the medium composition for bioalcohol production of the present invention, the medium composition may further include MES [2-(N-morpholino)ethanesulfonic acid] at a concentration of 30 to 50mM as a reducing agent.

One of the points that differentiates the present invention from previous patents is that in Clostridium strains, which are acetogens, cell growth is inhibited when the partial pressure of carbon monoxide in the medium is high, and CO consumption of the medium by the strain is reduced, converting carbon monoxide into alcohol. Although the alcohol production is reduced, the medium composition for bio-alcohol production of the present invention produces high concentrations of carbon monoxide in the culture of the Clostridium AWRP (Accession number: KCTC 13908BP) strain even under high-concentration carbon monoxide conditions where the carbon monoxide partial pressure of the medium is 50 kPa to 250 kPa. Even when supplied, cell growth is not inhibited, and the alcohol conversion process is carried out without difficulty, making it possible to produce the final ethanol and 2,3-butanediol at high concentrations.

In an embodiment of the present invention, as the CO partial pressure increases, the growth of the Clostridium sp. AWRP strain is inhibited and the cumulative CO consumption decreases (see Figure 1), but as acetic acid (Acetate) is added to the medium, , it is possible to overcome the inhibition of AWRP bacterial growth by CO gas and increase the CO consumption of AWRP strains (see Figure 2). In batch culture conditions, the maximum specific growth rate (μmax) increased by 83% compared to the condition without acetic acid. ) (see Figure 3), and under bioreactor conditions, the maximum specific growth rate increases by about 2.6 times with the addition of acetic acid, and the final concentration of ethanol and 2,3-butanediol produced increases by 2 to 3 times. There was (see Figure 4).

As described above, the bio-alcohol production method of the present invention uses a medium composition to which acetic acid is added to produce high-concentration bio-ethanol without the side effects of inhibiting bacterial growth of Clostridium sp. strains and reducing CO consumption. This is possible.

Therefore, the method for producing bioalcohol and the medium composition for the same of the present invention can be usefully used in the process of selectively producing bioethanol from synthesis gas.

Figure 1 is a graph showing the growth change and cumulative CO consumption of Clostridium AWRP strains according to the partial pressure of CO in the medium and the presence or absence of MES.
Figure 2 is a graph showing the cumulative CO consumption of Clostridium AWRP strains with or without the addition of acetic acid (Acetate), formic acid (Formate), and MES (●: RM medium, ▲: RM + Formate, ■: RM + Acetate, ○: RM + MES, □: RM + MES + Acetate).
Figure 3 is a graph showing the cumulative CO consumption, cell mass, and concentration of metabolites in the culture medium of Clostridium AWRP strains with or without the addition of acetic acid under batch culture conditions.
Figure 4 is a graph showing the cumulative CO consumption, cell mass, and concentration of metabolites in the culture medium of Clostridium AWRP strains with or without the addition of acetic acid under bioreactor culture conditions (●■▼▲: addition of acetic acid, ○□▽△: No acetic acid added).

Hereinafter, the present invention will be described in more detail through examples. Since these examples are merely for illustrating the present invention, the scope of the present invention is not to be construed as limited by these examples.

Example 1. Confirmation of inhibition due to increase in CO partial pressure in acetogenic microorganisms

As an acetogenic microorganism, the present inventors used Clostridium sp. AWRP strain (accession number: KCTC 13908BP) (hereinafter referred to as AWRP strain), a new acetogenic microorganism isolated from Ansan reed wetlands. AWRP strain is known to have excellent ethanol production ability from synthetic gas mixed with CO, CO ₂ and H ₂ gases (Lee et al., Biotechnol. Biofuels).

First, to confirm the degree of inhibition when the AWRP strain uses only carbon monoxide, medium and CO were added to a sealed 125 mL serum bottle at partial pressures of 75, 125, and 200 kPa, respectively, and cultured to determine the CO of the AWRP strain. The degree of inhibition of use was compared. As a culture medium, 20 mL of RM medium to which 1 g/L yeast extract was added (Lee et al., Biotechnol. Biofuels) was used, and a buffer to suppress the decrease in culture pH caused by microbial fermentation products. 40mM MES (2-(N-morpholino)ethanesulfonic acid, pH 5.5) was used. Seed culture was performed in the same manner as in the existing literature. During culture, the bacterial mass (OD ₆₀₀ ) and gaseous CO partial pressure were measured periodically and are shown in [Figure 1].

The measured CO partial pressure was converted to cumulative CO consumption per unit volume of culture medium using the ideal gas equation, and the gas chromatography analysis conditions for measuring CO partial pressure are as follows.

- Column: Molecular Sieve 13X + Porapack N

- Gas flow rate: Argon 15mL/min

- Oven temperature: 40℃, 9.5 min

- Injector temperature: 150℃

- Thermal conductivity detector temperature: 150℃

- Flame ionization detector temperature: 250℃

- Methanizer temperature: 350℃

Referring to [Figure 1], which summarizes the experimental results, it was confirmed that as the CO partial pressure increased, the amount of bacterial cells and cumulative CO consumption at the same time decreased, and it was confirmed that the CO inhibition effect was strong in the condition without MES. Since MES is well known as a buffer that is not metabolized within cells, it is assumed that this difference is caused by the difference in initial pH depending on whether or not the buffer is added.

Through the above experimental results, it can be confirmed that as the CO partial pressure increases, the growth of Clostridium sp. AWRP strains is inhibited, the cumulative CO consumption decreases, and the addition of MES, a buffer, alleviates this inhibition and reduction phenomenon. there was.

Example 2. CO inhibition improvement activity according to medium additives

Acetic acid (Acetate) or formic acid (Formate) and MES were selectively added to the culture medium of Clostridium sp. AWRP strain, and the CO inhibition improvement effect of these substances was confirmed.

In the case of formic acid, it is related to the inhibition of formic acid dehydrogenase, which is a representative cause of bacterial growth inhibition by CO. When formic acid, which is the reaction product of formic acid dehydrogenase, the target enzyme related to bacterial growth inhibition, is added It is well known that carbon fixation in the Wood-Ljungdahl pathway can proceed smoothly.

Acetic acid (Acetate) or formic acid (Formate) was prepared as a 1M pH 5.5 solution by adding NaOH, which was diluted to a final concentration of 40mM and added to the medium. Cultivation was performed in the same manner as in Example 1, CO was adjusted to a partial pressure of 125 kPa, and CO partial pressure was measured periodically during cultivation, and the cumulative CO consumption is shown in [Figure 2].

Referring to [Figure 2], which summarizes the experimental results, no CO consumption occurred under conditions without the addition of any organic acid or MES. In addition, under conditions in which only MES, a reducing agent, was added to the medium, it took about 13 days for CO gas to be completely consumed, which is consistent with the results observed in the experiment in Example 1. However, unlike the case of Acetobacterium woodii , another acetogenic microorganism, it was confirmed that the addition of formic acid had no effect on improving the inhibition of AWRP cell growth by CO gas.

On the other hand, when only acetic acid was added to the medium, CO gas consumption was completed on the 6th day of culture, which shortened the culture time by about 50% compared to the condition in which only MES was added, showing the best effect in the single addition condition. . In addition, it was confirmed that when acetic acid and MES were added simultaneously, the culture time was shortened to 4 days.

The gas consumption and generation amount at the beginning of the culture were measured when only MES was added to the medium and when MES and acetic acid were added simultaneously, and are summarized in Table 1 below.

[Table 1]

The net reaction equations when acetic acid, ethanol, and 2,3-butanediol, which are metabolites of the AWRP strain, are produced from CO through the Wood-Ljungdahl pathway are respectively as follows.

4 CO + 2 H ₂ O → CH ₃ COOH + 2 CO ₂ (CO ₂ /CO = 50 mol%)

6 CO + 3 H ₂ O → C ₂ H ₅ OH + 4 CO ₂ (CO ₂ /CO = 67 mol%)

11 CO + 5 H ₂ O → C ₄ H ₁₀ O ₂ + 7 CO ₂ (CO ₂ /CO = 64 mol%)

Referring to [Table 1], the CO ₂ /CO ratio at the beginning of the culture (0 to 1 day) when acetic acid is added is more than 67%, which means that additional CO is produced along with fermentation through the Wood-Jjungdahl pathway at the beginning of the culture when acetic acid is added. This means that oxidation has occurred. In particular, the CO ₂ /CO ratio on days 0-1 is close to 100% considering the analysis error, which means that CO is not converted to compounds with less than 2 carbon atoms (> C ₂ compounds) through the Wood-Jjungdahl pathway, but rather acts as an electron donor. This means that it was oxidized to CO ₂ through the action of When acetic acid is not added, it appears close to the theoretical CO ₂ /CO range, which means that CO metabolism occurred only through the Wood-Ljungdahl pathway. In conclusion, the oxidation of CO is promoted by adding acetic acid to the culture medium, which can be interpreted as reducing the inhibition of cell metabolism by CO.

Through the above experimental results, it was confirmed that adding acetic acid (Acetate) as an organic acid can overcome the inhibition of AWRP cell growth by CO gas and increase CO consumption by the AWRP strain.

Example 3. Effect of improving CO inhibition phenomenon by adding acetic acid in batch culture

The improvement in inhibition of AWRP bacterial growth by CO gas following the addition of acetic acid was confirmed under batch culture conditions using a flask.

The experiment was conducted using a 1L capacity serum bottle to compare conditions with and without acetic acid. To buffer the pH during culture, 40mM MES was added to 100mL of RM medium for the experiment, and seed culture was performed under the same conditions as Examples 1 and 2. The initial CO partial pressure in the gas phase was set to 200 kPa, and samples were collected periodically to measure the CO partial pressure in the gas phase, the amount of bacteria, and the concentration of metabolites in the culture medium, and the measurement results are shown in [Figure 3]. Measurement of CO gas concentration was performed in the same manner as Example 1. The YL 9100 HPLC system was used to measure the concentration of the culture product, and the analysis conditions were as follows.

- Column: Rezex ROA (300 × 7.8 mm)

- Mobile phase: 0.005NH ₂ SO ₄

- Oven temperature: 60℃, 30min

- Injection volume: 20μL

Referring to [Figure 3], which summarizes the experimental results, it was confirmed that when acetic acid was added, CO conversion and bacterial growth actively occurred immediately after inoculation, and the incubation time was reduced to less than 50% compared to the unadded condition to about 6 days. It was confirmed that this was the case (see FIGS. 3A to 3D). In particular, the maximum specific growth rate (μmax) was confirmed to be 1.73d ^-1 when acetic acid was added, an 83% increase compared to 0.95d ^-1 in the condition without addition. Meanwhile, there was no significant difference in the final bacterial mass at the end of culture.

In addition, gas consumption and generation at the beginning of the culture were measured when only MES was added to the medium and when MES and acetic acid were added simultaneously, and these were compared with metabolite production and summarized in Table 2 below.

[Table 2]

When acetic acid was added, it was confirmed that acetic acid decreased and ethanol production increased immediately after culture until the second day. The CO ₂ /CO ratio during this period is 66 to 87%, which exceeds the maximum value of 67% that can be obtained through fermentation through the Oud-Ljungdahl route. In conclusion, it can be seen that acetic acid is used as an electron donor for CO and consumes CO, thereby alleviating the initial growth inhibition caused by CO.

Example 4. Effect of improving CO inhibition phenomenon under CO continuous supply conditions using a bioreactor

Similar to the batch culture conditions using a flask, it was confirmed whether the inhibition of AWRP bacterial growth and reduction of CO consumption due to CO gas can be overcome by adding acetic acid under continuous CO supply conditions using a bioreactor.

Cultivation was performed by adding 1L of RM medium containing 2g/L yeast extract to a 2.5L reactor. MES was not added because the automatic pH control function of the bioreactor was used. The detailed operating conditions of the reactor are as follows.

- Agitation: 500 RPM

- Gas: 99.999% CO

- Inlet CO flow rate: 10mL/min through 1 microsparger

(turned on from 3 h after inoculation)

- Temperature: 37℃

- Automatic pH adjustment: 5.5 (using 7.5N NH ₄ OH and 2N HCl)

- Inoculum: 10%

Referring to [Figure 4], which summarizes the experimental results, the maximum specific growth rate (μmax) of the AWRP strain was approximately 2.2 ^d-1 under acetic acid addition conditions, and approximately 0.83 ^d-1 under conditions without the addition of acetic acid. It was confirmed that the specific growth rate increased by about 2.6 times.

In addition, the final concentration of ethanol produced under acetic acid addition conditions was 232mM, which was approximately 3 times higher than the 80mM under acetic acid-free conditions. In addition, the final concentration of 2,3-butanediol produced under acetic acid addition conditions was confirmed to be 23mM, a 2.3-fold increase compared to 10mM under acetic acid-free conditions.

Summarizing the above experimental results, as the CO partial pressure increases, the growth of Clostridium sp. AWRP strains is inhibited and the cumulative CO consumption decreases. However, as acetic acid is added to the medium, CO gas increases. It can overcome the inhibition of AWRP bacterial cell growth by AWRP bacteria and increase the CO consumption of AWRP strains. In batch culture conditions, the maximum specific growth rate (μmax) is increased by 83% compared to the condition without acetic acid, and in bioreactor conditions, acetic acid Depending on the addition, the maximum specific growth rate increased by about 2.6 times, and the final concentration of ethanol and 2,3-butanediol produced increased by 2 to 3 times.

As the specific parts of the present invention have been described in detail above, it is clear to those skilled in the art that these specific techniques are merely preferred embodiments and do not limit the scope of the present invention. Accordingly, the substantial scope of the present invention will be defined by the appended claims and their equivalents.

Korea Research Institute of Bioscience and Biotechnology KCTC13908BP 20190806

Claims (10)

(a) cultivating Clostridium AWRP (accession number: KCTC 13908BP) strain by supplying gas containing carbon monoxide to a medium composition to which acetic acid was added; and
(b) separating alcohol from the culture. A method for producing bioalcohol, comprising: According to paragraph 1,
A method for producing bioalcohol, characterized in that the acetic acid concentration of the medium composition in step (a) is 10 to 80mM, and the carbon monoxide partial pressure is 50kPa to 250kPa. According to paragraph 1,
A method for producing bioalcohol, wherein the gas containing carbon monoxide in step (a) further contains hydrogen, carbon dioxide, and nitrogen. According to paragraph 1,
A method for producing bioalcohol, wherein the gas in step (a) contains 50% (v/v) to 100% (v/v) of carbon monoxide. According to paragraph 1,
A method for producing bioalcohol, characterized in that the medium in step (a) is any one medium selected from RM medium, PETC medium, AM medium, and AMv2 medium. According to clause 5,
The RM medium contained 0.2 g of MgSO ₄ ·7H ₂ O per liter; NaCl 1g; KCl 0.2g; Yeast extract 0.5 g; 10 mL of phosphoric acid solution; 1 mL of trace element I solution; 1 mL of trace element II solution; and 10 mL of Wolfe's vitamin solution;
The phosphoric acid solution contains NaH ₂ PO ₄ ·2H ₂ O 0.8 g/L; and K ₂ HPO ₄ 0.4 g/L,
The trace element I solution is 35% HCl 10mL/L; FeSO ₄ ·7H ₂ O 27.8 g/L; MnCl ₂ ·4H ₂ O 9.9g/L; ZnCl ₂ 4.09 g/L; CoCl ₂ ·6H ₂ O 11.9 g/L; and NiCl ₂ ·6H ₂ O 2.38 g/L;
The trace element II solution contains Na ₂ MoO ₄ ·2H ₂ O 0.24 g/L; Na ₂ SeO ₃ ·5H ₂ O 1.32 g/L; and Na ₂ WO ₄ ·2H ₂ O 3.3 g/L;
The Wolfe's vitamin solution contains 2 g/L of biotin; Folic acid 2g/L; Pyridoxine·HCl 10g/L; Thiamine·HCl 5g/L; Riboflavin 5g/L; Nicotinic acid 5g/L; Calcium D-pantothenate 5g/L; Vitamin B12 0.1g/L; p-aminobenzoic acid 5g/L; and a method for producing bioalcohol, characterized in that it consists of 5g/L lipoic acid. According to clause 6,
The RM medium is a method for producing bioalcohol, characterized in that it further contains MES [2-(N-morpholino)ethanesulfonic acid] at a concentration of 30 to 50mM as a reducing agent. Cloth for bio-alcohol production under carbon monoxide partial pressure conditions of 50 to 250 kPa, characterized in that it further contains acetic acid at a concentration of 10 to 80mM using any one medium selected from RM medium, PETC medium, AM medium and AMv2 medium as a base medium. Medium composition for cultivating strains of Tridium genus AWRP (Accession number: KCTC 13908BP). According to clause 8,
The RM medium contained 0.2 g of MgSO ₄ ·7H ₂ O per liter; NaCl 1g; KCl 0.2g; Yeast extract 0.5 g; 10 mL of phosphoric acid solution; 1 mL of trace element I solution; 1 mL of trace element II solution; and 10 mL of Wolfe's vitamin solution;
The phosphoric acid solution contains NaH ₂ PO ₄ ·2H ₂ O 0.8 g/L; and K ₂ HPO ₄ 0.4 g/L,
The trace element I solution is 35% HCl 10mL/L; FeSO ₄ ·7H ₂ O 27.8 g/L; MnCl ₂ ·4H ₂ O 9.9g/L; ZnCl ₂ 4.09 g/L; CoCl ₂ ·6H ₂ O 11.9 g/L; and NiCl ₂ ·6H ₂ O 2.38 g/L;
The trace element II solution contains Na ₂ MoO ₄ ·2H ₂ O 0.24 g/L; Na ₂ SeO ₃ ·5H ₂ O 1.32 g/L; and Na ₂ WO ₄ ·2H ₂ O 3.3 g/L;
The Wolfe's vitamin solution contains 2 g/L of biotin; Folic acid 2g/L; Pyridoxine·HCl 10g/L; Thiamine·HCl 5g/L; Riboflavin 5g/L; Nicotinic acid 5g/L; Calcium D-pantothenate 5g/L; Vitamin B12 0.1g/L; p-aminobenzoic acid 5g/L; and 5 g/L of lipoic acid. A medium composition for cultivating Clostridium genus AWRP (Accession number: KCTC 13908BP) strain for bio-alcohol production. According to clause 8,
A medium composition for cultivating Clostridium genus AWRP (Accession number: KCTC 13908BP) strain for bio-alcohol production, further comprising MES [2-(N-morpholino)ethanesulfonic acid] at a concentration of 30 to 50mM as a reducing agent.