KR20220040409A - Method of Relieving Toxicity of Carbon Monoxide and Enhancing Stability of Biological Conversion of Carbon Monoxide Using Nanofluid - Google Patents

Abstract

Related is a method for decreasing the toxicity of carbon monoxide using a nanofluid and enhancing the stability of carbon monoxide metabolism of microorganisms. More specifically, related is a method which inhibits the toxic effect of carbon monoxide by using a biopolymer-based nanofluid capable of capturing carbon monoxide, to reduce a substrate inhibition effect of the carbon monoxide on microorganisms and enhancing the substrate metabolic stability of carbon monoxide metabolizing microorganisms.

Description

Method of Relieving Toxicity of Carbon Monoxide and Enhancing Stability of Biological Conversion of Carbon Monoxide Using Nanofluid

The present invention relates to a method for reducing the toxicity of carbon monoxide and improving the stability of carbon monoxide metabolism of microorganisms using a nanofluid. It relates to a method for reducing the substrate inhibitory effect of the carbon monoxide and improving the substrate metabolic stability of carbon monoxide metabolizing microorganisms.

The production of chemicals using the microbial fermentation process is regarded as a technology that can overcome the limitations of the existing chemical production methods, and studies to utilize cheap and abundant carbon sources to increase the price competitiveness of the production of these microorganism-based chemicals are actively conducted. is in progress In particular, it is known that carbon monoxide is contained in a large amount in the by-product gas generated from various industrial processes including the steel smelting process, and is attracting attention as a next-generation carbon source because it is easy to obtain from the by-product gas (Office of Air and Radiation. Technical support document for the iron and steel sector: proposed rule for mandatory reporting of greenhouse gases. (US Environmental Protection Agency, 2009). Recently, as several microorganisms that can naturally metabolize carbon monoxide have been discovered and studied, the potential of carbon monoxide as a next-generation carbon source for microbial fermentation processes is being evaluated more highly (Im, HS et al., Biotechnol. Bioprocess Eng . 24, 232- 239 (2019); Liew, F. et al., Front. Microbiol . 7, 694 (2016); Molitor, B. et al., Bioresour. Technol . 215, 386-396 (2016)).

Nevertheless, carbon monoxide-based microbial culture has a limitation in that it is difficult to operate a continuous and stable process, and toxicity to microorganisms of carbon monoxide is considered to be a representative cause of instability of the carbon monoxide-based microbial culture. Carbon monoxide is strongly attached to the metal enzymes present in microorganisms and has a characteristic of reducing the activity of the enzymes, and by this mechanism, respiration of microorganisms, DNA replication, energy production, etc. may be inhibited (Davidge, KS et al. , J. Biol. Chem . 284, 4516-4524 (2009); Nobre, LS et al., Antimicrob. Agents Chemother . 51, 4303-4307 (2007)). The toxicity of carbon monoxide can be a major cause of a sharp decrease in the overall metabolic activity of microorganisms, which in turn causes instability of the carbon monoxide-based microbial fermentation process (Ainala, SK et al., Biotechnol. Biofuels 10, 80 (2017)). ). In order to reduce the toxicity of carbon monoxide to the microorganism, research has been conducted in the direction of optimizing the microbial culture conditions or improving the physiological characteristics of the microorganism. In order to reduce the toxicity of carbon monoxide through optimization of culture conditions, mass transfer limitation conditions can be used. The mass transfer limiting condition refers to a condition in which the rate at which the gaseous carbon monoxide is dissolved into the medium (mass transfer rate) is faster than the rate at which the microorganisms consume dissolved carbon monoxide in the medium (Chan, M. et al., Chem. Eng . Sci. 47, 3541-3548 (1992)). Under these conditions, the concentration of toxic dissolved carbon monoxide can be reduced to a very low level because the dissolved carbon monoxide dissolved in the medium is consumed at a fast rate by microorganisms, thereby minimizing the toxicity of carbon monoxide. However, the metabolic activity of carbon monoxide metabolizing microorganisms may gradually decrease over time due to various causes, and the decrease in the rate of carbon monoxide consumption by microorganisms makes it difficult to maintain continuous and stable mass transfer limiting conditions. On the other hand, the effects of carbon monoxide toxicity can be reduced by improving the properties of microorganisms themselves, and these studies have been conducted in the direction of improving the resistance of microorganisms to carbon monoxide through an evolutionary method ⁹ . However, there are not many studies that improve resistance to carbon monoxide through evolutionary improvement of microorganisms, and there is a limitation that specific improvement is required for each strain according to the characteristics of the strain. In addition, in the case of microbial strain improvement through the evolutionary method, there is a limitation in that the effect due to carbon monoxide toxicity was still shown at a high carbon monoxide concentration even in the evolved strain (Kang, S. et al., Front. Microbiol. 11, 402 (2020)).

Meanwhile, a nanofluid is a fluid containing nanometer-sized particles, and typically refers to a dispersion in which nanoparticles having a diameter smaller than or similar to 100 nm are stably dispersed. Recently, research is underway to introduce nanofluids with surface-functionalized nanoparticles and use them for various biological purposes (George, J. et al., Nanotechnol. Sci. Appl . 8, 45-). 54 (2015)). However, when conventional inorganic-based oxide, nitride and carbide ceramics, metal and metal oxide, and carbon nanotube-based nanoparticles were used, various applications for biological purposes were difficult due to toxicity to cells. . On the other hand, when cellulose nanoparticles (CNC) manufactured based on cellulose are used, there is an advantage that they can be applied for various biological purposes due to the cell-friendly properties of the nanoparticles (George, J. et al . ., Nanotechnol. Sci. Appl . 8, 45-54 (2015)). In the case of these cellulose nanoparticles, they have been used for various biological purposes, such as the immobilization of enzymes, the production of antimicrobial substances, and the development of environmentally friendly catalysts and drug delivery systems. Only the bar used for the purpose of increasing the transfer efficiency of intracellular C1 gas in the gas bioconversion process is described.

Accordingly, the present inventors have made diligent efforts to solve the above problems and improve the carbon monoxide metabolic stability of microorganisms by reducing the toxicity of carbon monoxide to microorganisms. It was confirmed that not only to reduce the toxicity of carbon monoxide, but also to improve the carbon monoxide metabolic stability of microorganisms, the present invention was completed.

Republic of Korea Patent Registration No. 10-2139352

1. Office of Air and Radiation. Technical support document for the iron and steel sector: proposed rule for mandatory reporting of greenhouse gases. (U.S. Environmental Protection Agency, 2009) 2. Im, H. S. et al. Isolation of novel CO converting microorganism using zero valent iron for a bioelectrochemical system(BES). Biotechnol. Bioprocess Eng. 24, 232-239 (2019) 3. Liew, F. et al. Gas fermentation-a flexible platform for commercial scale production of low-carbon-fuels and chemicals from waste and renewable feedstocks. Front. Microbiol. 7, 694 (2016) 4. Molitor, B. et al. Carbon recovery by fermentation of CO-rich off gases - turning steel mills into biorefineries. Bioresour. Technol. 215, 386-396 (2016) 5. Davidge, K. S. et al. Carbon monoxide-releasing antibacterial molecules target respiration and global transcriptional regulators. J. Biol. Chem. 284, 4516-4524 (2009) 6. Nobre, L. S., Seixas, J. D., Romγo, C. C. & Saraiva, L. M. Antimicrobial action of carbon monoxide-releasing compounds. Antimicrob. Agents Chemother. 51, 4303-4307 (2007) 7. Ainala, S. K., Seol, E., Kim, J. R. & Park, S. Citrobacter amalonaticus Y19 for constitutive expression of carbon monoxide-dependent hydrogen-production machinery. Biotechnol. Biofuels 10, 80 (2017) 8. Chan, M., Morsi, B. I. Solubilities and mass transfer coefficient of carbon monoxide in a gas-inducing reactor operating with organic liquids under high pressures and temperatures. Chem. Eng. Sci. 47, 3541-3548 (1992) 9. Kang, S., Song, Y et al. Adaptive laboratory evolution of Eubacterium limosum ATCC 8486 on carbon monoxide. Front. Microbiol. 11, 402 (2020) 10. George, J., Sabapathi, S. N. Cellulose nanocrystals: synthesis, functional properties, and applications. Nanotechnol. Sci. Appl. 8, 45-54 (2015)

An object of the present invention is to provide a method for improving the carbon monoxide metabolic stability of microorganisms by reducing the toxicity of carbon monoxide to microorganisms that can utilize CO as a carbon source using a biopolymer-based nanofluid.

In order to achieve the above object, the present invention includes the step of culturing a microorganism capable of utilizing CO as a carbon source in a medium containing a nanofluid in which biopolymer nanoparticles into which the tannin-transition metal complex is introduced are dispersed. To provide a method for reducing the toxicity of carbon monoxide and improving the carbon monoxide metabolic stability of microorganisms.

Reduction of carbon monoxide toxicity of microorganisms according to the present invention and improvement of carbon monoxide metabolic stability through this can increase the economic feasibility of culturing carbon monoxide-based microorganisms. can be used in a variety of ways.

1 is a schematic diagram showing the structure of tannin-iron nanoparticles prepared in Example 1 of the present invention.
2(a) is a graph showing the effect of reducing the toxicity of carbon monoxide on Eubacterium limosum according to the concentration of tannin-iron nanoparticles added according to an embodiment of the present invention.
2 (b) is a graph comparing the toxicity reduction effect of carbon monoxide on Eubacterium limosum under the condition that each component of tannin-iron nanoparticles is added according to an embodiment of the present invention.
3 is a graph showing the effect of reducing the toxicity of carbon monoxide to Clostridium autoethanogenum according to the concentration of tannin-iron nanoparticles according to an embodiment of the present invention.
4 is a graph showing a search result of a carbon monoxide mass transfer restriction condition according to an embodiment of the present invention.
FIG. 5 is a graph showing the effect of improving the carbon monoxide metabolic stability of Eubacterium limosum in a medium to which tannin-iron nanoparticles are introduced according to an embodiment of the present invention.
6 is a graph showing the effect of improving the mass transfer coefficient (K _L a) in a medium to which tannin-iron nanoparticles are introduced according to an embodiment of the present invention.
7 is a graph showing the effect of improving carbon monoxide solubility in a medium to which tannin-iron nanoparticles are introduced according to an embodiment of the present invention.
8 is a graph showing changes in viscosity and surface tension of a medium to which tannin-iron nanoparticles are introduced according to an embodiment of the present invention.
Figure 9 (a) is a graph showing the effect of stabilizing CO gas bubbles in the medium to which the tannin-iron nanoparticles and the tannin-transition metal complex and cellulose nanoparticles are introduced according to an embodiment of the present invention.
9 (b) is a graph showing the change in the rate of rise of CO gas bubbles in the medium to which the tannin-iron nanoparticles and the tannin-transition metal complex and cellulose nanoparticles constituting the tannin-iron nanoparticles are introduced according to an embodiment of the present invention.
10 (a) is a view showing carbon monoxide adsorption of tannin-iron nanoparticles according to the introduction of tannin-iron nanoparticles nanofluid into a culture medium using surface-enhanced Raman spectroscopy (SERS) according to an embodiment of the present invention; FIG. .
10 (b) is a diagram showing carbon monoxide desorption of tannin-iron nanoparticles according to the introduction of tannin-iron nanoparticles nanofluid into a culture medium using surface-enhanced Raman spectroscopy (SERS) according to an embodiment of the present invention; FIG. .

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In general, the nomenclature used herein is those well known and commonly used in the art.

The present invention is based on the fact that in the case of a cellulose-based nanofluid used in the C1 gas bioconversion process, the tannin-iron complex functionalized on the nanoparticle surface can adsorb/desorb C1 gas, and the tannin-transition metal complex introduced cellulose When the nanoparticle-based nanofluid is used for the purpose of reducing the toxicity of carbon monoxide to microorganisms, it was confirmed that carbon monoxide metabolic stability of microorganisms can be improved by reducing the toxicity of carbon monoxide to microorganisms.

Therefore, the present invention in one aspect tannin-transition metal complex introduced biopolymer nanoparticles in a medium containing a nanofluid dispersed in the medium of carbon monoxide comprising the step of culturing a microorganism that can utilize CO as a carbon source It relates to a method for reducing toxicity and improving the carbon monoxide metabolic stability of microorganisms.

Hereinafter, the present invention will be described in detail.

In the present invention, a nanofluid is a fluid containing nanometer-sized particles, and typically refers to a dispersion in which nanoparticles having a diameter smaller than or similar to 100 nm are stably dispersed, and nanoparticles ) means nanometer-sized particles.

In addition, in the present invention, a biopolymer is a polymer material produced by living organisms, and carbohydrates, fats, proteins, nucleic acids and complexes thereof constituting the living organism are synthesized in the organism and secreted outside the body of various types of polymers. refers to the substance.

The present invention uses a nanofluid containing biopolymer nanoparticles such as cellulose to which a tannin-transition metal complex is introduced, such as a tannin-iron complex, to reduce the toxicity of carbon monoxide to microorganisms and thereby improve the carbon monoxide metabolic stability of microorganisms characterized in that

In the present invention, in the culture, the rate at which gaseous carbon monoxide is transferred to the liquid medium is faster than the rate of carbon monoxide metabolism by microorganisms under enzyme activity limiting conditions or the rate of carbon monoxide metabolism by microorganisms is the rate at which gaseous carbon monoxide is transferred to the liquid medium It is characterized in that it is performed under faster mass transfer constraints.

Cultivation in an embodiment of the present invention was 1) under conditions of restriction of enzyme activity for toxicity evaluation, and 2) under conditions of restriction of mass transfer for evaluation of stability.

In the present invention, the transition metal may be one or more selected from the group consisting of iron (Fe), nickel (Ni), cobalt (Co), molybdenum (Mo) and chromium (Cr), preferably iron (Fe) ), but is not limited thereto.

In the present invention, the biological metabolism of carbon monoxide can be achieved through various microorganisms that naturally metabolize carbon monoxide, the microorganisms are carbon monoxide metabolizing microorganisms, and representative examples of the carbon monoxide metabolizing microorganisms include Eubacterium limosum , Moorella thermoacetica , Clostridium ljungdahlii , Citrobactor amalonaticus Y19, Clostridium autoethanogenum , Citrobactor amalonaticus Y19, Acetobacterium Woodii or Alkalibaculum bacchi . They have a carbon monoxide dehydrogenase that can convert carbon monoxide to carbon dioxide, and obtain reducing power by oxidizing carbon monoxide to carbon dioxide through the enzyme. It is known that using the reducing power obtained at this time, it is known to produce energy necessary for the growth and survival of microorganisms.

In a specific example of the present invention, the carbon monoxide metabolizing strain may be selected from the group consisting of bacteria and archaea, preferably Eubacterium genus microorganisms, Clostridium genus microorganisms, Citrobacter ( Citrobactor ) genus microorganisms, Murella ( Moorella ) genus microorganisms, alkaline baculum genus microorganisms and Acetobacterium ( Acetobacterium ) One or more species can be selected from the group consisting of microorganisms, preferably Eubacterium It may be Eubacterium limosum or Clostridium autoethanogenum . However, the carbon monoxide metabolizing strain used in the present invention is not particularly limited as long as it is a microorganism capable of biologically metabolizing carbon monoxide.

In the present invention, the biopolymer is cellulose, chitosan, poly-gamma-glutamic acid (PGA), polylactic acid (PLA), polyhydroxyalkanoate (polyhydroxyalkanoate) , PHA), polyhydroxybutyric acid (PHB) and polyolefin (polyolefin, PL) may be selected from the group consisting of at least one, preferably using cellulose, but is not limited thereto.

In the present invention, the nanofluid comprises the steps of (a) adding an acid to the biopolymer to prepare biopolymer nanoparticles; and (b) b) adding and dispersing the biopolymer nanoparticles to a tannin solution and a transition metal solution.

In the present invention, the acid may be selected from the group consisting of sulfuric acid, hydrochloric acid, nitric acid, phosphoric acid, hydrofluoric acid and formic acid.

In the present invention, the biopolymer nanoparticles may have a cylindrical shape having a diameter of 0.1 to 50 nm and a length of 10 to 1 μm. Preferably, the diameter of the cylinder may be 1 to 25 nm, and the length may be 40 to 800 nm. By depolymerizing cellulose by adding acid, cylindrical nanoparticles can be mass-produced, and the cylindrical shape has a large surface area along the longitudinal direction, which is advantageous for introducing tannin-transition metal complexes.

In the present invention, tannin is a kind of polyphenol synthesized by plants, and its molecular weight may be 500 to 20000, but is not limited thereto. It is known to have the function of preventing arteriosclerosis by removing free radicals that cause aging in the body and removing blood clots in the arteries.

In the nanofluid containing cellulose nanoparticles according to a preferred embodiment of the present invention, the tannin-iron complex may be one in which tannins form a coordination bond with iron ions. In FIG. 1 , a tannin-iron complex in which tannins form a coordination bond with iron ions is combined with cellulose nanoparticles. Tannic acid contains a plurality of galloyl groups in the molecule, and the group forms a coordination bond with iron ions generated from iron salts during the manufacturing process to form a stable complex (tannin-iron complex). At this time, the galloyl group of tannic acid may form a cross link by coordinating with different iron ions. The tannin-iron complex may form tannin-iron nanocellulose by bonding with cellulose through hydrogen bonding.

where iron is FeCl ₃ , FeCl ₂ , Fe(NO ₃ ) ₃ , Fe(NO ₃ ) ₂ , Fe ₂ (SO ₄ ) ₃ , FeSO ₄ , nano zerovalent iron, Fe ₂ O ₃ and Fe ₃ It may be derived from at least one selected from the group consisting of O ₄ , and may be iron particles having magnetism.

When a complex containing iron is introduced into cellulose nanoparticles in the nanofluid containing cellulose nanoparticles according to a preferred embodiment of the present invention, nanoparticles can be recovered from the nanofluid containing nanoparticles using a magnet. The complex may be formed by mixing tannin and iron or iron salt as described above in a weight ratio of 300:1 to 1:1.

In the present invention, the weight ratio of the biopolymer nanoparticles and the tannin-transition metal complex in the nanofluid may be 1:10 to 1:1000. In the nanofluid containing cellulose nanoparticles according to a preferred embodiment of the present invention, the cellulose nanoparticles and the composite may be present in a weight ratio of 1:10 to 1:1000, preferably 1:100 to 1:150 by weight, The most efficient coating of the composite on the surface of the cellulose nanoparticles is possible when the weight ratio in the above range is used.

In the nanofluid containing cellulose nanoparticles according to the present invention, the medium may be selected from the group consisting of a polar solvent such as water, ethanol, propanol and acetic acid, but is not limited thereto.

In the present invention, the weight ratio of the biopolymer nanoparticles to which the tannin-transition metal complex is introduced and the medium may be 1:5000 to 1:50. In the nanofluid containing cellulose nanoparticles according to a preferred embodiment of the present invention, the cellulose nanoparticles and the medium are present in a ratio of 1:5000 to 1:50 (weight), preferably 1:1000 to 1:200 ratio (weight). When the ratio of the biopolymer nanoparticles to which the tannin-transition metal complex is introduced and the medium is within the above range, the effect of reducing the toxicity of carbon monoxide to microorganisms is maximized, thereby improving the carbon monoxide metabolic stability of microorganisms has the effect of being When a ratio lower than the above range is used, the effect of reducing the toxicity of the nanofluid may be relatively reduced. Because the metabolic activity of microorganisms may be inhibited by

Hereinafter, preferred examples are presented to help the understanding of the present invention, but the following examples are merely illustrative of the present invention, and it will be apparent to those skilled in the art that various changes and modifications are possible within the scope and spirit of the present invention, It goes without saying that such variations and modifications fall within the scope of the appended claims.

[Example]

Preparation Example 1: Preparation of tannin-iron complex-introduced nanofluid

20 g of cellulose was added to 350 mL of H ₂ SO ₄ aqueous solution (H ₂ SO ₄ content: 64% by weight relative to the aqueous solution), and stirred continuously for 45 minutes at a temperature of 50° C. to prepare a slurry containing cellulose nanoparticles and cellulose nanoparticles were obtained therefrom. Cellulose nanoparticles and 5 mL of an aqueous tannin solution having a concentration of 1 mg/mL and 5 mL of an aqueous solution of iron chloride (FeCl ₃ ) having a concentration of 0.3 mg/mL were prepared and mixed, respectively. Then, a phosphate buffer solution was added dropwise until the pH of the mixture reached 7 or more to prepare a nanofluid containing cellulose nanoparticles into which the tannin-iron complex was introduced (FIG. 1).

Example 1: Use of tannin-iron complex introduced nanofluid for reducing the toxicity of carbon monoxide to microorganisms

1-1. Selection of carbon monoxide metabolizing strains and cellulose nanoparticles

An attempt was made to reduce the toxicity of carbon monoxide to carbon monoxide metabolizing microorganisms and improve the carbon monoxide metabolic stability of microorganisms through the use of nanofluids introduced with cellulose nanoparticles.

As carbon monoxide metabolizing strains, Eubacterium rimosum and Clostridium autoethanogenum were used. These strains are representative carbon monoxide metabolizing strains that can use carbon monoxide as a single carbon source, and have a Wood-Ljungdahl pathway that produces acetic acid as a major fermentation product from carbon monoxide. In the case of cellulose nanoparticles, nanoparticles into which the tannin-iron complex was introduced were selected. The present inventors found that when cellulose nanoparticles in which the tannin-iron complex is functionalized are used, as dissolved carbon monoxide in the medium is adsorbed to the tannin-iron complex, it is possible to reduce the influence of dissolved carbon monoxide on microorganisms, thereby reducing the toxic effect of carbon monoxide. It was assumed that it could be reduced. In addition, it was hypothesized that carbon monoxide metabolic stability of microorganisms could be improved by alleviating the decrease in metabolic activity of microorganisms through the toxicity reduction effect of carbon monoxide.

1-2. Microbial culture

, 200 rpm 조건에서 수행되었으며, pH 값을 7.0 수준으로 유지하기 위해서 10 M NaOH를 사용하였다.Cultures of Eubacterium limosum and Clostridium autoethanogenum were performed under anaerobic conditions in the absence of molecular oxygen. As a medium for strain growth, RCM medium (10 g/L peptone, 10 g/L beef extract, 3 g/L yeast extract, 5 g/L dextrose, 5 g/L NaCl, 0.5 g/L L-cysteine· HCl, 3 g/L sodium acetate, and 0.02 g/L resazurin) were used. As a medium for carbon monoxide metabolism, in the case of Eubacterium limosum, modified PBBM medium (3 g/L yeast extract, 0.9 g/L NaCl, 0.3 g/L MgSO ₄ .7H ₂ O, 0.2 g/L CaCl ₂ 2H ₂ O, 1.0 g/L NH ₄ Cl, 50 mM potassium phosphate buffer (pH 7.0), 1.0 g/L L-cysteine HCl, 20 mL trace mineral solution, 20 mL vitamin solution, 0.02 g/L resazurin) , for Clostridium autoethanogenum, modified minimal medium (0.5 g/L yeast extract, 1.0 g/L NaCl, 0.52 g/L MgCl ₂ , 0.1 g/L CaCl ₂ .2H ₂ O; 1.0 g/L NH ₄ Cl, 0.33 g/L KCl, 1.0 g/L NaHCO ₃ , 50 mM potassium phosphate buffer (pH 7.0), 1 g/L L-cysteine HCl, 20 mL trace mineral solution, 20 mL vitamin solution, 0.02 g/L resazurin) was used. For anaerobic strain culture, the experiment was carried out in a sealed serum vial of 75 mL or 500 mL size. In order to provide carbon monoxide as a carbon source, the gas part of the syrup vial was replaced with high-purity nitrogen gas or carbon monoxide/nitrogen mixed gas, and a Model 1000 O ₂ Filter was used to remove all trace oxygen molecules contained in the provided gas. Strain culture 37

, was performed under 200 rpm conditions, and 10 M NaOH was used to maintain the pH value at a level of 7.0.

RCM medium was used to obtain the cell amounts of Eubacterium rimosum and Clostridium autoethanogenum required for the experiment. The culture was started from a glycerol stock, and when a desired amount of cells was obtained through RCM medium-based culture, the expression of an enzyme (carbon monoxide dehydrogenase) required for carbon monoxide metabolism was induced. In order to induce the expression of the enzyme, the strain obtained through culture was concentrated 30-fold and then a carbon monoxide/nitrogen mixed gas (10:90, v/v) was provided at 2 hour intervals for a total of 16 hours. This process is called carbon monoxide adaptation process. The strain adapted to carbon monoxide was obtained through centrifugation, and then resuspended in a modified PBBM medium or a modified minimal medium at a desired concentration and used for the main culture.

Cultures of Eubacterium limosum and Clostridium autoethanogenum strains were carried out under enzyme activity-restricting conditions or mass transfer-restricting conditions, respectively. In the present invention, the enzyme activity limiting condition refers to a state in which the rate of carbon monoxide metabolism by the microorganism is slower than the mass transfer rate of carbon monoxide from the gas to the liquid medium. will take effect In the present invention, the enzyme activity limiting condition means a condition in which the cell concentration of Eubacterium rimosum and Clostridium autoethanogenum is 0.5 or less based on OD ₆₀₀ , and the carbon monoxide metabolic activity of microorganisms and through this It was used to evaluate the toxicity of carbon monoxide. In the present invention, the mass transfer limiting condition refers to a state in which the rate of carbon monoxide metabolism by microorganisms is faster than the mass transfer rate of carbon monoxide from the gas to the liquid medium. Since the dissolved carbon monoxide concentration is kept very low, it is hardly affected by the toxic effects of carbon monoxide. In the present invention, the mass transfer restriction condition means a condition in which the cell concentration of Eubacterium limosum is 20 based on OD ₆₀₀ when the partial pressure of carbon monoxide is 20.3 kPa, which is for evaluation of carbon monoxide metabolic stability of Eubacterium limosum was used

Cell growth during culture was quantified by measuring absorbance at 600 nm using a UV-1700 spectrophotometer. The result was calculated as an OD ₆₀₀ value or 0.31 g DCW/L per OD ₆₀₀ unit. In addition, in order to evaluate the carbon monoxide metabolic activity and stability according to the conditions of culturing microorganisms, a gas sample present in the gas phase of the serum vial was analyzed. A Clarus 680 GC system was used for the analysis of gas samples, and a Carboxen 1010 PLOT column (30 m x 0.53 mm) and a thermal conductivity detector (TCD) were used for separation and quantification of each material.

1-3. Confirmation of the effect of reducing the toxicity of carbon monoxide through tannin-iron nanofluid

It was confirmed whether the toxicity of carbon monoxide to microorganisms could be reduced by using the cellulose nanofluid into which the tannin-iron complex was introduced. In the case of Eubacterium limosum, although carbon monoxide can be used as a carbon source, when the partial pressure of carbon monoxide is increased, it was confirmed that the carbon monoxide metabolic activity of microorganisms is inhibited, as shown in FIG. 2(a). As shown in Fig. 2(a), when the partial pressure of carbon monoxide was higher than a certain level (about 20 kPa), as the partial pressure of carbon monoxide increased, the carbon monoxide metabolic activity of Eubacterium limosum showed a tendency to decrease. This shows the toxicity of carbon monoxide to Eubacterium limosum, which causes efficient and stable carbon monoxide metabolism by Eubacterium limosum when the microorganism is exposed to dissolved carbon monoxide.

In order to solve the decrease in metabolic activity of Eubacterium limosom due to the carbon monoxide toxicity, tannin-iron complex-introduced nanoparticles were used. To determine whether the toxicity of carbon monoxide to Eubacterium limosum was reduced when using tannin-iron nanoparticles, various concentrations (0, 0.25, 0.5 1 g/L) of tannin-iron nanoparticles were added. The carbon monoxide metabolic activity of Eubacterium was measured according to the partial pressure of carbon monoxide (0, 5.1, 10.1, 30.4, 60.8, 101.3 kPa) under the conditions. The experiment was conducted under enzyme activity limiting conditions. As shown in Fig. 2(a), when the partial pressure of carbon monoxide was increased to a certain level (about 20 kPa) or more in the condition in which tannin-iron nanoparticles were not added, the metabolic activity of Eubacterium limosum decreased, whereas tannin- It was confirmed that the carbon monoxide metabolic activity was restored at a relatively high partial pressure of carbon monoxide (about 20 kPa or more) under the condition in which iron nanoparticles were added. As shown in Fig. 2(a), it was confirmed that the effect of restoring the carbon monoxide metabolic activity of Eubacterium was more pronounced as the concentration of the added tannin-iron nanoparticles increased. The results of this study indicate the effect of reducing carbon monoxide toxicity through the use of tannin-iron nanoparticles.

In order to quantitatively evaluate the reduction effect of carbon monoxide toxicity through the addition of tannin-iron nanoparticles, the results were applied to the monod type substrate inhibition model. In the present invention, by applying the obtained results to the above model and calculating the Ks and Ki values, the binding affinity of carbon monoxide to Eubacterium limossum and the substrate inhibitory effect of carbon monoxide when nanoparticles introduced with tannin-iron complexes were used. were evaluated for each. To this end, the Lineweaver-Burke equation was plotted with the variables 1/m and 1/S as each axis from the substrate inhibition model of the Monod equation, and Ks and Ki values were calculated based on the plots (Table 1). When comparing the Ks values according to the concentration of tannin-iron nanoparticles added, it was confirmed that the Ks value did not change significantly (up to a 1.2-fold increase) until the concentration of the nanoparticles increased to about 0.5 g/L. did These results show that even when the corresponding nanoparticles are used, the binding affinity of carbon monoxide to Eubacterium limosum does not significantly decrease up to a certain level of concentration. On the other hand, the Ki value showed a tendency to increase as the concentration of tannin-iron nanoparticles increased, and when comparing the case where the nanoparticles were not added and the case where the nanoparticles were added at a level of 1 g/L, the Ki value This maximum increase of about 8.6 times was confirmed. Accordingly, in the present invention, it was quantitatively confirmed that the substrate-inhibiting effect caused by carbon monoxide could be reduced through the introduction of tannin-iron nanoparticles.

Tannin-iron nanoparticles added concentration (g/L) 0 0.25 0.5 One Ks value (mM) 22.18 26.94 25.27 39.94 Ki value (mM) 5.54 7.58 31.02 47.62

The carbon monoxide reduction effect of tannin-iron nanoparticles confirmed in the culture conditions of Eubacterium limosum is not a phenomenon that occurs independently by cellulose nanoparticles or tannin-iron complex, but appears only when tannin-iron and cellulose nanoparticles are added together. A study was conducted to confirm the phenomenon.

To this end, the carbon monoxide metabolic activity of Eubacterium limosum under the conditions in which cellulose nanoparticles, tannin-iron complex, and tannin-iron nanoparticles were added was confirmed as shown in FIG. 2(b). As shown in Fig. 2(b), compared to the condition in which only cellulose nanoparticles or tannin-iron complex were added, carbon monoxide metabolic activity of Eubacterium limosum in the condition in which cellulose nanoparticles and tannin-iron complex were added together It was confirmed that the improvement was significantly greater than this. This result means that the effect of reducing carbon monoxide toxicity of tannin-iron nanoparticles to microorganisms is not independently exhibited by the tannin-iron complex or cellulose nanoparticles, but by the synergistic effect of the two substances.

To confirm the versatility of the toxicity reduction effect, another carbon monoxide metabolizing strain, Clostridium autoethanogenum, was also tested with a tannin-iron nanoparticle-based nanofluid. Even though the Clostridium autoethanogenum strain is a strain that can utilize carbon monoxide as a carbon source as in the case of Eubacterium rimosum, when the partial pressure of carbon monoxide is increased to a certain level or more, the carbon monoxide metabolic activity of the microorganism is abruptly increased. It was confirmed that the inhibition was (FIG. 3).

In order to solve the problem of lowering metabolic activity of the Clostridium autoethanogenum strain due to carbon monoxide toxicity, tannin-iron complex-introduced nanoparticles were used. Carbon monoxide metabolic activity of Clostridium autoethanogenum according to carbon monoxide partial pressure (0, 10.1, 50.7, 101.3 kPa) when tannin-iron nanoparticles were added at various concentrations (0, 1, 1.5, 2 g/L) was measured. The experiment was conducted under enzyme activity limiting conditions. As shown in FIG. 3 , in the condition in which tannin-iron nanoparticles were not added, when the partial pressure of carbon monoxide was increased to a certain level (50.7 kPa) or more, the metabolic activity of microorganisms was greatly reduced, whereas in the condition in which tannin-iron nanoparticles were added In this study, it was confirmed that the metabolic activity at the corresponding partial pressure of carbon monoxide was significantly restored. The results of this study mean that tannin-iron nanoparticles have a universal carbon monoxide toxicity reduction effect on microorganisms.

Example 2. Confirmation of the effect of improving the carbon monoxide metabolic stability of microorganisms by reducing the toxicity of carbon monoxide based on tannin-iron nanofluid

2-1. Establishment of mass transfer restrictions

As described above, various microorganisms including Eubacterium limosum are affected by metabolic activity inhibition by carbon monoxide. Therefore, the bioconversion process of carbon monoxide is performed under mass transfer limiting conditions to minimize the toxicity of carbon monoxide. In the present invention, when the effect of reducing the toxicity of carbon monoxide by nanoparticles into which the tannin-iron complex is introduced is utilized, it was attempted to check whether the metabolic stability of carbon monoxide is also improved under conditions of restriction of mass transfer similar to the actual process. In the present invention, before evaluating the carbon monoxide metabolic stability of Eubacterium limosum, it was attempted to explore the mass transfer restriction conditions. For this, various concentrations of Eubacterium limosum (0, 5, 10, 20, 50, and 100, respectively, based on OD ₆₀₀ ) were cultured under a carbon monoxide partial pressure of 20.3 kPa, and carbon monoxide metabolic activity at this time was measured. As shown in FIG. 4 , it was confirmed that even if the concentration of the strain was increased, the total carbon monoxide metabolic activity did not increase when the concentration of Eubacterium rimosum used was higher than about 20 levels based on OD ₆₀₀ . This means that even if the total carbon monoxide metabolism rate by Eubacterium limosum is increased, the amount of carbon monoxide reduced from the gas does not change, so it means the mass transfer restriction conditions specified above. Through this experiment, we succeeded in exploring the mass transfer restriction condition at a specific partial pressure of carbon monoxide (20.3 kPa), and thus, in the present invention, for the evaluation of carbon monoxide metabolic stability under the mass transfer restriction condition, Eubacterium at a level of 20 based on OD ₆₀₀ The cell concentration of limosum was used.

2-2. Improved carbon monoxide metabolic stability of tannin-iron nanofluid-based microorganisms

The purpose of this study was to evaluate the carbon monoxide metabolic stability of Eubacterium limosum according to the concentration of tannin-iron nanoparticles under the searched carbon monoxide mass transfer restriction conditions. To this end, Eubacterium limosum (20 based on cell concentration OD ₆₀₀ ) was cultured under conditions of 0 g/L or 5 g/L of tannin-iron nanoparticles added with carbon monoxide partial pressure of 20. kPa. During incubation, gas samples were analyzed every 12 or 24 hours, and carbon monoxide at a partial pressure of 20.3 kPa was provided in the gas phase of the syrup vial at every analysis time. At this time, the amount of carbon monoxide consumption according to time of the Eubacterium limosum is shown in FIG. 5 . As shown in FIG. 5 , it was confirmed that the carbon monoxide metabolic stability of Eubacterium limosum was improved as the tannin-iron nanoparticles were introduced. Under the condition in which the nanoparticles were not introduced, a very rapid decrease in carbon monoxide metabolism was observed from 24 hours after the start of culture, which is presumed to be because the microorganism was greatly affected by carbon monoxide toxicity due to the breakdown of the carbon monoxide mass transfer restriction condition. On the other hand, it was confirmed that, in the case of the condition in which the tannin-iron nanoparticles were introduced, the decrease in the carbon monoxide metabolism of Eubacterium limosum with time was significantly less. This is thought to be because, after the start of culture, when the mass transfer restriction condition was disrupted and the concentration of dissolved carbon monoxide in the medium increased, the tannin-iron nanoparticles adsorbed the dissolved carbon monoxide, which reduced the substrate inhibitory effect of the dissolved carbon monoxide. The effect of improving the carbon monoxide metabolic stability was more pronounced in the latter half of the culture. In particular, it was confirmed that the amount of carbon monoxide metabolism increased by about 6.6 times compared to the control group between 48 and 72 hours after the start of the culture. As a result, when the tannin-iron nanoparticles were added at a level of about 5 g/L, the total carbon monoxide metabolism increased by about 1.7 times compared to the control during a total of 96 hours of incubation. These study results show that the effect of reducing the toxicity of carbon monoxide through tannin-iron nanoparticles is a very efficient approach in the stable conversion process of carbon monoxide through microbial culture.

Example 3. Tannin-Iron nanoparticle-based nanofluid according to the introduction of the mass transfer coefficient into the microbial medium (K _L a) Comparative analysis of change amount and solubility

3-1. The mass transfer coefficient (K) according to the introduction of the tannin-iron nanoparticle-based nanofluid _L a) change

A study was conducted to understand the effect of reducing carbon monoxide toxicity and improving culture stability of microorganisms according to the addition of the tannin-iron nanoparticle-based nanofluid shown in Example 2. First, the relative mass transfer coefficient (K _L a) according to the concentration of nanoparticles applied was measured and analyzed in order to confirm the change in the gas transfer effect due to the introduction of tannin-iron nanoparticles in the culture medium. The mass transfer coefficient (K _L a) was measured using a 5 L culture system, and in the case of the 5 L culture system, it is largely divided into four types: gas supply, fermenter, flow meter, and oxygen measuring device in solution. A fermenter where gas is injected and reacts with a solution, a flow meter that measures and controls the gas flow into and out of the fermenter, and a dissolved oxygen probe that measures dissolved oxygen in the actual solution, oxygen storage container, gas There is a regulator, etc., and a stainless steel gas supply pipe (Sparger) with a diameter of 1/5 inch is installed inside the fermenter so that the gas supplied from the feeder can be supplied from the inside of the actual medium. Then, in order to constantly supply oxygen to the inside of the fermenter, Alicat scientific's flow controller was used, and the microbial culture medium for measuring the mass transfer coefficient was tannin- Iron nanoparticles were used by applying a concentration of 0-0.1% (w/v). In addition, in order to check the difference according to the gas flow rate, the flow rate range was 0.2-0.3 L/min in two sections. Specifically, the measurement was carried out after preparing a PBBM medium as in Example 1-2 above, suspending tannin-iron nanoparticles in the PBBM medium, and removing residual gas in the solution using an Ultrasonicator. . Then, after 1.5 L of PBBM medium was brought into the fermenter, the stirring speed (Agitation) in the incubator was set to 400 rpm, and then oxygen was supplied to the inside of the fermenter and the flow rate of the gas per unit time and the dissolved oxygen in the solution By measuring the amount of change, the mass transfer coefficient (K _L a) was measured. Then, based on the mass transfer coefficient of the conventional PBBM medium to which tannin-iron nanoparticles were not introduced, the relative mass transfer coefficient according to the concentration of tannin-iron nanoparticles introduced is shown in FIG. 6 . As shown in FIG. 6 , as the concentration of tannin-iron nanoparticles increased, it was confirmed that the mass transfer coefficient was relatively increased by 23 to 123% compared to the existing mass transfer coefficient. The increase in the mass transfer coefficient due to the introduction of the nanoparticles is expected to contribute to maintaining stable culture conditions without depleting carbon monoxide in the medium, even under conditions of restriction of mass transfer during culturing of carbon monoxide-using microorganisms.

3-2. Changes in solubility in the medium according to the introduction of tannin-iron nanoparticle-based nanofluids

In order to confirm whether carbon monoxide solubility is increased by the introduction of tannin-iron nanoparticles into the culture medium, solubility was measured and analyzed in a gas-liquid absorption equilibrium. The solubility analyzer used for solubility measurement is largely divided into three types: gas supply, reactor, and pressure gauge. In order to maintain a constant temperature and ambient pressure, it is installed in a fume-hood and maintained at a constant temperature (25℃). made it In detail, it consists of a gas supply that stores carbon monoxide gas (99.999%), a reactor where the actual gas-liquid absorption equilibrium reaction takes place, a supply before and after gas-liquid equilibrium reaction, and a pressure gauge indicating the internal pressure of the reactor. It was manufactured in a cylindrical shape with an internal volume of 101.11 cm ³ made of stainless steel to prevent gas leakage to the outside during the gas-liquid absorption equilibrium reaction. In addition, a 1/8 inch Teflon-tube (1/8 inch interface tubing) was connected from the outside of the feeder to the lower part of the reactor so that carbon monoxide could sufficiently contact the actual reaction solution. In addition, a certain amount of carbon monoxide and helium gas was supplied into the gas supply through a flow regulator of Alicat scientific. Specifically, in the case of solubility measurement in the gas-liquid absorption equilibrium state, after preparing a PBBM medium as in Example 1-2 above, tannin-iron nanoparticles are dispensed into the PBBM, and 50 mL is supplied into the reactor, and then helium After gas was supplied at a rate of 200 mL/min for 30 minutes to remove all residual gas inside the reactor, all valves connected to the feeder and the outside of the reactor were closed to minimize exposure to outside air. After that, carbon monoxide is quantitatively injected from the carbon monoxide gas container to the feeder at a partial pressure between 0 and 1 bar. A liquid absorption equilibrium reaction was prepared. Then, by opening the valve between the reactor and the feeder, the carbon monoxide collected in the feeder is supplied to the inside of the reactor, and the gas-liquid absorption reaction proceeds. At this time, it is considered that the gas-liquid absorption equilibrium has been reached, and the pressure difference before and after the gas-liquid absorption equilibrium reaction, the volume of the culture medium, and the temperature in the reaction state are measured to introduce tannin-iron nanoparticles through Henry's law. Solubility in gas-liquid absorption equilibrium in PBBM medium was calculated according to Thereafter, the solubility value of the PBBM medium in which tannin-iron nanoparticles were not introduced was regarded as 100%, and the relative solubility according to the concentration of tannin-iron nanoparticles introduced was shown in FIG. 7 . As shown in FIG. 7 , at the tannin-iron nanoparticle introduction concentration of 0.025% (w/v), the solubility did not increase compared to the conventional culture medium PBBM, but the tannin-iron nanoparticle introduction concentration of 0.05% (w/v) and 0.1% (w/v) showed an increase of about 16% compared to the previous one.

Example 4. Observation and comparative analysis of fluid properties and CO gas bubbles according to the introduction of tannin-iron nanoparticle-based nanofluid into a microbial medium

4-1. Observation of changes in surface tension and viscosity of microbial culture medium according to the introduction of tannin-iron nanoparticle-based nanofluids

In order to confirm whether the tannin-iron nanoparticles of Example 3 increase the mass transfer coefficient (K _L a) effect and the solubility improvement effect in the culture medium by the introduction of the tannin-iron nanoparticles are due to the change in the fluid properties of the culture medium by the introduction of the tannin-iron nanoparticles , the surface tension and viscosity were measured. Surface tension was measured using a surface tension meter, and viscosity was measured using a rheometer. Specifically, tannin-iron nanoparticles were dispensed at a concentration of 0-0.1% (w/v) in the PBBM culture medium prepared in the same manner as in Example 1-2, and then placed in a 100 mL beaker inside the SEO's surface tension meter. 50 mL of PBBM culture medium was supplied, and the surface tension was calculated and measured based on the buoyancy method. Subsequently, for viscosity measurement, 10 mL of PBBM culture medium injected at a concentration of 0-0.1% (w/v) of tannin-iron nanoparticles was supplied to an anton paar rheometer, followed by a shear rate of 100-700 s ^-1 By measuring and calculating the amount of change in shear stress in the range, the change in surface tension and viscosity of the PBBM culture medium according to the concentration of tannin-iron nanoparticles introduced is shown in FIG. 8 . As shown in FIG. 8 , it was confirmed that there was no difference in the surface tension and viscosity of the PBBM culture medium according to the presence or absence of introduction of tannin-iron nanoparticles. It was confirmed that the solubility synergistic effect and the mass transfer coefficient (K _L a) improvement by the introduction of tannin-iron nanoparticles of Example 3 were not due to the change in fluid properties.

4-2. Comparative observation of carbon monoxide gas bubble form and movement speed in microbial medium following introduction of tannin-iron nanoparticle-based nanofluid

To determine whether the tannin-iron nanoparticles of Example 3 increase the mass transfer coefficient (KLa) and solubility improvement effect in the culture medium by the introduction of the tannin-iron nanoparticles are a phenomenon due to the interaction between the tannin-iron nanoparticles and carbon monoxide gas in the culture medium , The shape of carbon monoxide gas bubbles (gas-bubble) and the rate of rise of gas bubbles in the culture medium according to the introduction of components constituting the tannin-iron nanoparticles and tannin-iron nanoparticles in the culture medium were recorded using a high-speed camera (high-speed camera). camera) was used for filming and analysis.

Specifically, in the PBBM culture medium prepared in the same manner as in Example 1-2, tannin-iron nanoparticles and tannin-iron nanoparticles constituting tannin-iron (Ta-Fe ³⁺ ) complexes, cellulose nanoparticles (CNC ) at 0.05 % (w/v), respectively, to prepare the sample, and then 1 L of 1/6-inch Teflon tube with a 0.04-0.08 cm ² diameter gas supply hole so that carbon monoxide gas can be supplied into the solution. After installation in a volumetric polystyrene material tank, 0.5 L of the sample was supplied. Then, using a gas flow controller connected from the carbon monoxide gas storage container, supplying gas at a constant flow rate of 400 mL/min, using a high-speed camera, observe the behavior of gas bubbles in the culture medium at 24000 frames per second, every 0.03 seconds As shown in FIG. 9, the shape of the gas bubble and the rate of rise were shown. As shown in FIG. 9(a), in the conventional culture medium, carbon monoxide gas bubbles aggregate at the same time as supply, whereas when tannin-iron nanoparticles (TA-Fe ³⁺ CNC) 0.05% (w/v) are applied, gas bubbles are Uniformly supplied in a relatively small size without agglomeration, which is a result based on gas bubble stabilization of surface tannin-iron (TA-Fe ³⁺ ) constituting tannin-iron nanoparticles, could check Subsequently, tannin-iron nanoparticles (TA-Fe ³⁺ CNC) 0.05% (w/v) and tannin-iron complex (TA-Fe ³⁺ ), carbon monoxide gas bubble rise rate according to the introduction of cellulose nanoparticles into the medium As shown in FIG. 9(b), tannin-iron nanoparticles (TA-Fe ³⁺ CNC) 0.05% (w/v) and tannin-iron complex (TA-Fe ³⁺ ) 0.05% (w/v) When applied to the medium, it was confirmed that the bubble rising rate was slower than that of the conventional medium (PBBM) due to interaction with gas bubbles. The result of uniform gas bubble supply and reduction of the gas bubble rising rate according to the introduction of tannin-iron nanoparticles of FIG. 9 shows the tannin-surface ^tannin -iron complex (TA-Fe ³⁺ ) is assumed to be a phenomenon that occurs while interacting with carbon monoxide gas bubbles.

Example 5: Comparative results of carbon monoxide adsorption/desorption of tannin-iron nanoparticle-based nanofluid in microbial culture medium

A study was conducted to confirm that the result of Example 4 was a phenomenon caused by the adsorption/desorption of tannin-iron nanofluids with carbon monoxide gas in the actual culture medium. Surface-enhanced Raman spectroscopy (SERS) was used to analyze the carbon monoxide adsorption/desorption phenomenon following the introduction of tannin-iron nanoparticle-based nanofluids into the culture medium. Specifically, a single layer of gold-palladium core-shell nanoparticles was transferred to a glass substrate having a size of 0.5 cm x 0.5 cm in order to use surface-enhanced Raman spectroscopy. The prepared nanostructure was introduced into the liquid phase of the gas-liquid reactor, and the dissolved carbon monoxide concentration was measured through the Raman signal of the carbon monoxide molecule generated when the nanostructure was irradiated with a 785 nm laser.

First, 1.2 ml of PBBM buffer was injected into the liquid phase of the gas-liquid reactor to confirm the carbon monoxide adsorption phenomenon following the introduction of tannin-iron nanoparticle-based nanofluid, and then 10% carbon monoxide gas was added to the gas phase at 30 ml/min. was supplied at a constant flow rate of After reaching the gas-liquid equilibrium, the case of adding 0.8 ml of PBBM buffer and the case of adding 0.8 ml of tannin-iron nanoparticle-based nanofluid were compared. As shown in FIG. 10( a ), when the PBBM buffer was added, the intensity of the Raman signal of carbon monoxide did not change. On the other hand, when the tannin-iron nanoparticle-based nanofluid was added, it was confirmed that the intensity of the Raman signal of carbon monoxide was reduced. The result of the reduction of the Raman signal intensity of carbon monoxide according to the introduction of the tannin-iron nanoparticle-based nanofluid in FIG.

To confirm the reversible desorption of carbon monoxide adsorbed to the tannin-iron nanoparticle-based nanofluid, one of the two gas-liquid reactors injected 1.2 ml of PBBM buffer into the liquid phase, and the other was the liquid phase with 1.2 ml of tannin. -Iron nanoparticle-based nanofluid was injected. 99.9% carbon monoxide gas was injected into the gas phase of each gas-liquid reactor and then sealed. After reaching the gas-liquid equilibrium, two gas-liquid reactors were exposed to a carbon monoxide-free atmosphere and the concentration of dissolved carbon monoxide over time was measured. As shown in FIG. 10(b) , when the PBBM buffer was injected, it was observed that the Raman signal of the dissolved carbon monoxide decreased as the dissolved carbon monoxide diffused through mass transfer into the gas phase. On the other hand, when the tannin-iron nanoparticle-based nanofluid was injected, it was observed that the Raman signal of dissolved carbon monoxide increased. This is presumed to be a phenomenon that occurs when carbon monoxide adsorbed to the tannin-iron complex on the surface of the tannin-iron nanoparticles is desorbed and the dissolved carbon monoxide concentration increases.

Accordingly, in this example, it was confirmed that the tannin-iron nanoparticle-based nanofluid had adsorption and desorption properties for carbon monoxide.

In conclusion, the mass transfer coefficient of carbon monoxide and the stabilization of carbon monoxide gas bubbles in the medium and the mass transfer coefficient of carbon monoxide according to the introduction of the tannin-iron nanoparticle-based nanofluid confirmed in Examples 3 and 4 were tannin-iron nanoparticle-based nanofluids. It is believed to be due to the reversible carbon monoxide adsorption/desorption of

As the specific parts of the present invention have been described in detail above, it will be clear to those of ordinary skill in the art that these specific descriptions are only preferred embodiments, and the scope of the present invention is not limited thereby. will be. Accordingly, it is intended that the substantial scope of the present invention be defined by the claims and their equivalents.

Claims (11)

Reduction of carbon monoxide toxicity and microbial stability of carbon monoxide metabolism, including the step of culturing microorganisms that can utilize CO as a carbon source in a medium containing a nanofluid in which biopolymer nanoparticles into which the tannin-transition metal complex is introduced are dispersed How to improve.
The method according to claim 1, wherein in the culture, the rate of gaseous carbon monoxide transferred to the liquid medium is faster than the rate of carbon monoxide metabolism by microorganisms under enzyme activity limiting conditions or the rate of carbon monoxide metabolism by microorganisms is gaseous carbon monoxide transferred to the liquid medium A method for reducing the toxicity of carbon monoxide and improving the stability of carbon monoxide metabolism of microorganisms, characterized in that it is carried out under the limiting conditions for mass transfer faster than the rate.
The method of claim 1, wherein the transition metal is selected from the group consisting of iron, nickel, cobalt, molybdenum and chromium.
According to claim 1, wherein the microorganism is Eubacterium limosum , Moorella thermoacetica , Clostridium ljungdahlii , Clostridium autoethanogenum , Citrobactor amalonaticus Y19, Acetobacterium Woodii and Alkalibaculum bacchi Reduction of toxicity and carbon monoxide of microorganisms characterized in that at least one selected from the group consisting of bacchi A method for improving carbon monoxide metabolic stability.
According to claim 1, wherein the biopolymer is cellulose, chitosan, polygamma glutamic acid, polylactic acid, polyhydroxy alkanoate, polyhydroxy butyric acid and polyolefin, characterized in that at least one selected from the group consisting of carbon monoxide A method for reducing toxicity and improving the stability of microbial carbon monoxide metabolism.
The method of claim 1 , wherein the nanofluid comprises: (a) adding an acid to the biopolymer to prepare biopolymer nanoparticles; and (b) adding and dispersing the biopolymer nanoparticles to a tannin solution and a transition metal solution.
The method of claim 6, wherein the acid is selected from the group consisting of sulfuric acid, hydrochloric acid, nitric acid, phosphoric acid, hydrofluoric acid and formic acid.
The method of claim 1, wherein the biopolymer nanoparticles have a cylindrical shape having a diameter of 0.1 to 50 nm and a length of 10 to 1 μm.
The method of claim 1, wherein the weight ratio of the biopolymer nanoparticles and the tannin-transition metal complex in the nanofluid is 1:10 to 1:1000.
The method of claim 1, wherein the medium is a polar solvent.
The method of claim 1, wherein the weight ratio of the biopolymer nanoparticles into which the tannin-transition metal complex is introduced and the medium is 1:5000 to 1:50.

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