KR20220055830A - Method for Improving Metabolite Production of Microorganism having Ability to Metabolize C1 Gas using Organic Material based-Nanofluids - Google Patents

Abstract

The present invention relates to a method for increasing metabolite productivity using C1 gas of microorganisms by organic material based-nanofluids and, more specifically, to a method for increasing metabolite productivity through increase of a C1 gas consumption rate of microorganisms having C1 gas metabolizing ability by using a nanofluid containing a chitosan-lipid compound and/or a nanofluid containing a tannin-iron complex. According to the present invention, the method for including metabolite productivity by increasing efficiency of using C1 gas in microorganisms having C1 gas metabolism ability can increase economic feasibility of a C1 gas-based microbial culture process and can be applied to the field of production of high value-added materials through application of microbial culture processes using various shale gas, industrial by-product gas, and biogas containing carbon monoxide or methane in a variety of ways, thereby being useful. The method comprises an adsorption and adaptation step and a cultivation step.

Description

Method for Improving Metabolite Production of Microorganism having Ability to Metabolize C1 Gas using Organic Material based-Nanofluids}

The present invention relates to a method of improving the productivity of metabolites of microorganisms utilizing C1 gas using nanofluids, and more specifically, chitosan-lipid compound-containing nanofluids and/or tannin-iron complex-containing nanofluids. It relates to a method for improving metabolite productivity by improving the C1 gas consumption rate of microorganisms.

In the domestic industrial production ranking, the petroleum-based energy/chemical industry occupies a position as a representative key industry in Korea. The demand for securing source technology for this purpose is increasing rapidly. At present, C1 gas composed of one carbon atom, such as methane (CH ₄ ) and carbon monoxide (CO), which has a huge reserves and is only used by simple combustion, is composed of carbon and oxygen like petroleum in its molecular structure, so it is used for various transportation purposes. Since it can be converted to fuel and chemical materials, the technology for converting fuel and chemical materials for transportation using C1 gas has a huge ripple effect when development is successful.

However, the conventional C1 gas conversion process lacks process economics compared to the conventional petroleum-based chemical process due to low yield, high operating cost, and the like. The conventional chemical conversion process of C1 gas based on indirect conversion is conducted under harsh conditions of high temperature and high pressure. Without adequate discharge of the generated heat, local overheating occurs, and serious problems such as catalyst degradation and reactor cracking can occur. it is known In addition, since C1 gas such as CO or CH ₄ has a strong reactivity to an oxidizing agent, precise control of the reaction at the molecular level is essential to prevent a decrease in the yield of the target product due to the generation of by-products, so continuous research is being conducted (DJ Miller et al. al., Surface and Interface Analysis, Vol. 33(4), pp. 299-305, 2002; L. Li et al., Physics Letters A Vol. 372(25), pp.4541-4544, 2008.).

Recently, as basic research achievements on molecular biology or C1 chemistry have been accumulated, clues are being prepared for the design of high-performance biocatalysts and chemical catalysts that can economically convert C1 gas. In particular, the bioconversion process of C1 gas is considered to be a high value-added technology because it can produce useful substances through the C1 gas direct conversion route, which is the simplest reaction route in theory. In addition, unlike conventional chemical processes, the reaction proceeds under mild conditions of low temperature and low pressure. It is reported that a very diverse microbial species such as positive bacteria ), Methanogenic-bacteria , and Methanotrophic-bacteria can be used (DTN Nguyen et al., Metab Eng, Vol. 54, pp. 170-179, 2019; CA Henard et al., Front Microbiol Vol. 9, p. 2610, 2018; M.-S. Kim et al., International Journal of Hydrogen Energy, Vol. 42(45), pp. 27593 -27599, 2017; K. Shabestary et al., Metab Eng Commun, Vol. 3, pp. 216-226, 2016; DH Hur et al., Journal of Chemical Technology & Biotechnology, Vol. 92(2), pp. 311-318, 2017). Unlike microorganisms such as S. cerevisiae or E. coli , which grow or ferment using sugar as a substrate, they are specifically C1 gas or by-product gas emitted during combustion. Industrially valuable bio-methanol (CH ₃ OH), bio-hydrogen (H ₂ ), propanol (C) using carbon monoxide (CO), carbon dioxide (CO ₂ ), methane (CH ₄ ), propane (C ₃ H ₈ ), etc. ₃ H ₆ OH), acetic acid, succinic acid, maleic acid, formic acid, etc. are produced.

However, in the case of C1 gas conversion microbial process, the solubility of C1 gas is very low (20-25 mg/L for carbon monoxide, 15-20 mg/L for CH ₄ ), even if there are excellent bioconversion strains or catalysts, In the absence of process technology that can improve the mass transfer of C1 gas, which is a problem, there is a fundamental limit to productivity improvement. And in this respect, various high-concentration cell culture methods such as cell recycling and membrane reactor have been proposed, and various studies are being conducted to overcome the problem of mass transfer (EM de Medeiros et al., Chemical Engineering Science: X 5, 2020). RM Handler et al., Industrial & Engineering Chemistry Research, Vol. 55(12), pp.3253-3261, 2015;H. Chaumat et al., Chemical Engineering Science, Vol. 62(24), pp.7378- 7390, 2007).

On the other hand, according to the research results of Professor Gaddy's team at the University of Arkansas in the US, the carbon consumption rate of C. ljungdahlii , a microorganism that uses carbon monoxide (0.22 gcarbon/gcell/h), and that of S. cerecisiae that produce bioethanol ( 0.27 gcarbon/gcell/h) is reported not to be significantly lowered compared to (US 5173429A), but in the case of the actual C1 gas conversion microbial process, the growth and fermented water conversion rate are very slow compared to E. coli, making commercial application difficult.

For commercial use of the biological conversion technology using C1 gas, it is necessary to efficiently supply C1 gas to cells by overcoming the problems of low solubility of C1 gas in the liquid phase and low mass transfer efficiency (K _L a) in the reactor. And in this respect, approaches to improve mass transfer in microbial conversion process systems include 1) stable generation of microbubbles, 2) increase in flow rates of gases and liquids, 3) increase in solubility of gases by using solvents or high pressure 4) A method for solving the mass transfer barrier at the liquid/solid interface formed on the cell surface can be given.

Recently, research to improve the mass transfer coefficient and solubility in gas/liquid systems through nanofluids incorporating nanoparticles is being conducted. Nanofluid refers to a fluid containing nanometer-sized particles, in which nanoparticles having a diameter smaller than or similar to 100 nm are stably dispersed in a fluid. Since the research on nanofluids was first reported in 1995, research reports on nanofluids have been increasing, and the scope of application has been mainly focused on theoretical analysis related to the improvement of heat transfer efficiency, and recently mass transfer diffusion It has been reported that the effect of increasing the coefficient can also be expected.

Specifically, research using nanofluids is mainly focused on improving the mass transfer efficiency of oxygen, ammonia, and carbon dioxide by using inorganic-oriented oxide, nitride and carbide ceramics, metals and metal oxides, and carbon nanotube-based nanoparticles. , Boiling-phenomena, Catalyst, Surface-Area, Convection, Mass-Transfer, Bio-Medical, etc. In progress, in particular, in terms of mass transfer, several studies have been reported on the effect of improving the solubility of gases (Gas absorption) in the vapor-liquid part (Y. Kang et al., International Journal). of Coal Geology, s154-155, pp. 123-135, 2016; J. S. Lee et al., Applied Energy, Vol. 143, pp. 119-129, 2015).

There are several hypotheses as to the reasons for the improvement of mass transfer by nanofluids, but among them, the most influential hypotheses are 1) reduction in bubble size by nanofluid, 2) reduction in thickness of gas/liquid interface by nanofluid, 3) nanofluid It is believed to be due to the transport effect of C1 gas adsorption/desorption on the surface of nanoparticles constituting the fluid, and 4) mass transfer in the liquid phase by additional convective diffusion of nanoparticles.

Although nanofluids can be used in such a wide variety of fields and can be effectively used to improve heat transfer and mass transfer in various industrial environments, there are few reports of nanofluids applied to biological processes. It is judged that this is because it is known that inorganic-centered nanoparticles of less than nm enter the cells of microorganisms and cause cytotoxicity. In addition, in the case of nanofluids exhibiting efficiency in the productivity of microbial fermentation products, the applied concentration is relatively high, and in conjunction with the disadvantage of an increase in process operating cost, reuse through recovery is essential.

On the other hand, Korean Patent No. 10-2105851 discloses that the organic nanoparticle nanofluid is produced using a chitosan-based polymer organic material with good biocompatibility and an antibacterial lipid that is expected to interact with the cell membrane, so that microorganisms can use the C1 gas efficiently. It is described that the improvement is improved, and in Korean Patent No. 10-2139352, it is described that the C1 gas utilization efficiency of microorganisms is improved by manufacturing an organic nanoparticle nanofluid using nanocellulose and cell-friendly surface-treated nanocellulose. there is.

However, the nanofluid of No. 10-2105851 has a disadvantage in that cytotoxicity occurs when a high concentration is used for mass culture, and the nanofluid used in No. 10-2139352 has a disadvantage that the degree of improvement in C1 gas utilization efficiency is low. there is.

Accordingly, the inventors of the present inventors have worked diligently to solve the above problems, and to develop a method that can dramatically increase the productivity of metabolites of microorganisms having C1 gas metabolism without cytotoxicity. When applied sequentially and simultaneously to the seed culture and main culture of It was confirmed that the present invention was completed.

It is an object of the present invention to provide a method for improving metabolite productivity by improving the C1 gas utilization efficiency of microorganisms having C1 gas metabolizing ability using chitosan-lipid compound-based nanofluids.

Another object of the present invention is to provide a method for improving metabolite productivity by improving C1 gas utilization efficiency of microorganisms having C1 gas metabolizing ability using chitosan-lipid compound-based nanofluid and tannin-transition metal complex-based nanofluid.

In order to achieve the above object, the present invention (a) chitosan introduced lipid compound nanoparticles in a medium containing a nanofluid dispersed in the medium by culturing a microorganism having C1 gas metabolizing ability (seed culture) to the medium Adsorbing and adapting the nanoparticles dispersed in the cell membrane; And (b) the method of improving metabolite productivity through improvement of C1 gas utilization efficiency of microorganisms having C1 gas metabolizing ability comprising the step of culturing the cultured microorganisms in a medium not containing nanofluids to provide.

The present invention also provides (a) chitosan-introduced lipid compound nanoparticles dispersed in the medium by culturing a microorganism having a C1 gas metabolizing ability in a medium containing a nanofluid dispersed in the medium (seed culture) nanoparticles dispersed in the medium adsorption and adaptation to the cell membrane; And (b) the tannin-transition metal complex introduced biopolymer nanoparticles of the cultured microorganism in a medium containing the nanofluid dispersed in the medium (main culture) comprising the step of C1 gas metabolism To provide a method for improving metabolite productivity by improving the C1 gas utilization efficiency of microorganisms with

The method for improving metabolite productivity by improving the C1 gas utilization efficiency of microorganisms with C1 gas metabolizing ability according to the present invention can not only increase the economic feasibility of the C1 gas-based microbial culture process, but also various shale gases containing carbon monoxide or methane It is useful because it can be used in various fields such as the production of high value-added substances through the application of the microbial culture process using industrial by-product gas, biogas, etc.

1 is a diagram showing the amount of change in the mass transfer coefficient (K _L a) measured when chitosan / oleamide nanofluid is injected into a microbial culture medium at a concentration of 0.1 * 10 ^-3 -100 * 10 ^-3 % (w / v) am.
2 is a diagram showing the amount of change in the mass transfer coefficient (K _L a) measured when the tannin-iron complex-introduced nanocellulose nanofluid was injected into a microbial culture medium at a concentration of 0.025-0.1% (w/v).
3 is a diagram showing the amount of change in CO solubility measured when the chitosan / oleamide nanofluid is injected into the 0.01*10 ^-3 -1*10 ^-3 % (w/v) microbial culture medium.
4 is a diagram showing the change in CH ₄ solubility measured when the chitosan / oleamide nanofluid was injected into 0.1*10 ^-3 -50*10 ^-3 % (w/v) microbial culture medium.
5 is a view showing the amount of change in CO gas solubility measured when the tannin-iron complex introduced nano-cellulose nanofluid was injected into a microbial culture medium at a concentration of 0.025-0.1% (w/v).
6 is a view showing the amount of change in CH ₄ gas solubility measured when the tannin-iron complex introduced nanocellulose nanofluid was injected into a microbial culture medium at a concentration of 0.025-0.1% (w/v).
7 is a view showing the gas bubble stabilization effect and gas bubble rise rate measured when the tannin-iron nanofluid was injected into the NMS culture medium at a concentration of 0.05% (w/v).
Figure 8 is a diagram showing the gas bubble generation and bubble breaking effect measured when the chitosan / oleamide nanofluid was injected into the NMS culture medium at a concentration of 1 * 10-3% (w / v).
FIG. 9 is a diagram of observation of microbial cell and cell membrane deformation measured when chitosan/oleamide nanofluid was injected at a concentration of 0.1*10 ^-3 -10*10 ^-3 % (w/v) during seed culture.
10 is a view of observing whether the cell membrane is deformed, measured when the tannin-iron complex introduced nano-cellulose nanofluid was injected at a concentration of 0.05% (w/v) during microbial culture.
11 is a diagram showing the amount of nanoparticles adsorbed to the microbial cell membrane measured when chitosan/oleamide nanofluid was injected at a concentration of 0.1*10 ^-3 -50*10 ^-3 % (w/v) in seed culture .
12 is a chitosan / oleamide nanofluid CH ₄ using gram-negative bacteria, methanogen Methylomonas sp. It is a diagram showing the amount of CH ₄ consumption and changes in the microbial growth concentration when the microbial main culture was performed without additional nanofluid after application at a concentration of 0.005 % (w/v) during seed culture of DH-1 .
13 is a chitosan / oleamide nanofluid using CH ₄ Gram-negative bacteria Methylomonas sp. It is a diagram showing the amount of change in the microbial growth concentration when the microbial main culture is continuously subcultured without additional nanofluid after application at a 0.001% (w/v) concentration during seed culture of DH-1 .
14 is a tannin-iron nanofluid using CH ₄ Gram-negative bacteria Methylomonas sp. This is a diagram showing the amount of change in the microbial growth concentration when DH-1 was applied at a concentration of 0.025-0.1 % (w/v) during seed culture, and the microbial main culture was continuously subcultured without applying additional nanofluid.
15 is a chitosan / oleamide nanofluid at a concentration of 0.0001%, 0.0005% (w / v) in the seed culture of the hydrogen-producing archaea Thermooccus onnurinues NA1 , which is an archaea using CO, after applying the main culture without applying an additional nanofluid. It is a diagram showing the amount of CO consumption when proceeding.
16 is a chitosan / oleamide nanofluid using CH ₄ Gram-negative bacteria Methylomonas sp. It is a diagram showing the amount of change in the microbial growth concentration when DH-1 was applied at a concentration of 0.005% (w/v) in seed culture and then cultured by applying the same concentration of chitosan/oleamide nanofluid during main culture.
17 is a gram-negative bacterium using CH ₄ Methylomonas sp. After culturing microbial cells without applying nanofluid during seed culture of DH-1 , chitosan/oleamide nanofluid was applied at a concentration of 0.005% (w/v) during main culture and tannin-iron nano It is a diagram showing the amount of change in the relative microbial growth concentration when the fluid is applied at a concentration of 0.05% (w/v).
18 is a gram-negative bacterium using CH ₄ Methylomonas sp. When chitosan/oleamide nanofluid was applied at a concentration of 0.005% (w/v) during DH-1 seed culture, and then tannin-iron nanofluid was applied at a concentration of 0.05% (w/v) during main culture, microorganism growth It is a diagram comparing the amount of change in concentration.
Figure 19 is chitosan / oleamide nanoparticles / nanofluid CH ₄ using gram-negative bacteria, methanogen Methylomonas sp. It is a diagram showing the amount of change in CH ₄ consumption and microbial growth concentration when DH-1 was applied at a concentration of 0.005% during seed culture, and then the tannin-iron nanofluid was applied to a 5L fermenter at a concentration of 0.05% and the main culture was carried out. .
20 is chitosan/oleamide nanoparticles/nanofluids CH ₄ Using gram-negative bacteria, methanogen Methylomonas sp. It is a quantitative index for comparison of methane consumption according to fermentation time when DH-1 was applied at a concentration of 0.005% during seed culture, and then tannin-iron nanofluid was applied to a 5L fermenter at a concentration of 0.05% and main culture was performed.
Figure 21 is chitosan / oleamide nanoparticles / nanofluid using CH ₄ gram-negative bacterium methanocytosis Methylomonas sp. A diagram showing the methanol production when DH-1 was applied at a concentration of 0.005% during seed culture, and then the tannin-iron nanofluid was applied to a 5L fermenter at a concentration of 0.05% and the main culture was performed.
Figure 22 is chitosan / oleamide nanoparticles / nanofluid using CH ₄ gram-negative bacteria methanocytosis Methylomonas sp. This is a diagram showing acetic acid production when DH-1 was applied at a concentration of 0.005% during seed culture, and then the tannin-iron nanofluid was applied to a 5L fermenter at a concentration of 0.05% and the main culture was performed.
23 shows chitosan/oleamide nanoparticles/nanofluids using CH ₄ , which is a gram-negative bacterium, methanogen Methylomonas sp. This is a diagram showing succinic acid production when DH-1 was applied at a concentration of 0.005% during seed culture, and then the tannin-iron nanofluid was applied to a 5L fermenter at a concentration of 0.05% and the main culture was performed.
Figure 24 is chitosan / oleamide nanoparticles / nanofluid CH ₄ using gram-negative bacteria methanocytosis Methylomonas sp. Quantitative index comparing the final production of fermented products with the negative control when DH-1 was applied at a concentration of 0.005% during seed culture, and then the tannin-iron nanofluid was applied to a 5L fermenter at a concentration of 0.05% and the main culture was performed. am.
Figure 25 is a chitosan / oleamide nanoparticles / nanofluid using CH ₄ methane magnetizing bacteria Methylosinus trichosporium. It is a diagram showing the change in methane consumption and the change in the concentration of microorganisms when the main culture was carried out by applying the tannin-iron nanofluid to a 3L fermenter at a concentration of 0.05% after application at a concentration of 0.005% during the seed culture of OB3b .
Figure 26 is chitosan / oleamide nanoparticles / nanofluid using CH ₄ methane magnetizing bacteria Methylosinus trichosporium. When OB3b was applied at a concentration of 0.005% during seed culture, and then the tannin-iron nanofluid was applied to a 3L fermenter at a concentration of 0.05%, and the main culture was carried out, the amount of production of poly-hydroxybutyrate (PHB), a metabolite, according to fermentation time It is a diagram showing the amount of change.
Figure 27 is a chitosan / oleamide nanoparticles / nanofluid using CH ₄ methane magnetizing bacteria Methylosinus trichosporium. This is a quantitative index for comparison of methane consumption according to fermentation time when OB3b was applied at a concentration of 0.005% during seed culture, and then the tannin-iron nanofluid was applied to a 3L fermenter at a concentration of 0.05% to proceed with the main culture.
Figure 28 is chitosan / oleamide nanoparticles / nanofluid using CH ₄ methane magnetizing bacteria Methylosinus trichosporium. When OB3b was applied at a concentration of 0.005% in the seed culture, the tannin-iron nanofluid was applied to a 3L fermenter at a concentration of 0.05%, and the main culture was performed. This is a quantitative index comparing the production of the fermentation product with the negative control.

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 and the experimental methods described below are well known and commonly used in the art.

In the present invention, even when culturing a microorganism having C1 gas metabolizing ability in a medium containing a nanofluid containing chitosan-introduced lipid compound, and then culturing in a medium not containing a nanofluid, the C1 of the microorganism It was intended to confirm that the gas metabolism capacity and the production capacity of metabolites were improved.

That is, in one embodiment of the present invention, the effect of improving the gas mass transfer efficiency of the nanofluid in which the chitosan-oleamide nanoparticles are dispersed in an aqueous solution was confirmed, and in the medium containing the nanofluid, DH-1 in Methylomonas genus Alternatively, when culturing Thermococcus onnurinus NA1, it was confirmed that the nanoparticles were adsorbed to the microbial cell membrane and modified a part of the cell membrane, and then the C1 gas consumption even when the main culture was performed in a medium not containing the nanofluid. And it was confirmed that the microbial growth rate is improved (FIGS. 1, 3, 4, 8, 9, 11 to 13 and 15)

Accordingly, the present invention in one aspect,

(a) In a medium containing a nanofluid in which chitosan-introduced lipid compound nanoparticles are dispersed in a medium, a microorganism having C1 gas metabolizing ability is seed cultured to adsorb the nanoparticles dispersed in the medium to the cell membrane and adapting; And (b) the step of culturing the main culture (main culture) in a medium that does not contain the nano-fluid in the cultured microorganism;

It relates to a method of improving metabolite productivity through improvement of C1 gas utilization efficiency of microorganisms having C1 gas metabolizing ability, including.

On the other hand, in another embodiment of the present invention, after culturing Methylomonas DH-1 or Methyllocinus tricosporium OB3b in a medium containing the nanofluid, tannin-iron complex is introduced into the medium. When the main culture was performed in a medium containing the nanofluid dispersed in 7, 10 and 16 to 28).

Accordingly, the present invention from another point of view,

(a) In a medium containing a nanofluid in which chitosan-introduced lipid compound nanoparticles are dispersed in a medium, a microorganism having C1 gas metabolizing ability is seed cultured to adsorb the nanoparticles dispersed in the medium to the cell membrane and adapting; and

(b) culturing the cultured microorganism in a medium containing a nanofluid in which the biopolymer nanoparticles into which the tannin-transition metal complex is introduced are dispersed in the medium (main culture);

It relates to a method of improving metabolite productivity through improvement of C1 gas utilization efficiency of microorganisms having C1 gas metabolizing ability, including.

In the present invention, the chitosan is a very safe cationic polysaccharide that exists in nature having the structure of (1->4) 2-amino-2-deoxy-β. Chitosan is in the form of a polymer in which free amine groups are generated by deacetylating chitin obtained from natural crustaceans and the like. Here, the chitosan may have a molecular weight of 1 to 500 kDa and a free amine group of 50% or more, and preferably, the molecular weight of chitosan may be 150-400 kDa and 80% or more of the free amine group, but the present invention is not limited thereto. In terms of solubilization of chitosan, water-soluble chitosan, insoluble chitosan, and chitosan derivatives obtained by changing the molecules of chitosan may also be included in the scope of chitosan of the present invention, and water-soluble chitosan may be preferably used. The water-soluble chitosan may be at least one selected from chitosan HCl, chitosan acetate, chitosan glutamate, and chitosan lactate. Water-soluble chitosan is soluble in water because the amine group in the molecular structure forms a salt in an equivalent ratio with hydrochloric acid, acetic acid, glutamic acid, and acidic substances such as lactic acid. Water-soluble chitosan has advantages such as easy removal of foreign substances. In addition, chitosan having the same molecular structure may also be included without limitation.

In the present invention, the lipid compound refers to a lipid reported to have antibacterial properties and a lipid derivative functionally similar thereto. Preferably, fatty acids having an aliphatic residue having between 6-18 carbon atoms, such as oleic acid and lauric acid, oleamide and erucamide, and fatty alcohols Refers to fatty acid derivatives such as fatty acid derivatives, monoglycerides, lipid compounds such as sphingosine, and derivatives thereof, more preferably oleamide and erucamide, and most preferably having structural identity with oleamide, but It is not limited.

In the present invention, the microorganism having C1 gas metabolizing ability is a microorganism using C1 gas, more preferably, Gram-negative bacteria, Gram-positive bacteria, Archaea, Thermophilic-bacteria, Halobacteria, Methanogenic-bacteria, Methanotrophic-bacteria, Acetogenic-bacteria and Hydrogenic bacteria and more preferably, Methanotrophic-bacteria, Acetogenic-bacteria and Hydrogenic bacteria.

Methanotrophic-bacteria of the present invention are Methylomonas sp. DH-1 ( Methylomonas sp. DH-1 ), Methylosinus sporium type 5 ( Methylosinus sporium type 5 ), Methylosinus tricosporium OB3b ( Methylosinus tricosporium OB3b ), Methylomicrobium alkalipium 20z ( Methylomicrobium alcaliphilum 20z ), Methylococcus capsulatus ( Methylococcus capsulatus ), Methylosphaera hansonii ), Methylomicrobium agile) ( Methylomicrobium agile ) , Methylomicrobium album , Methylobacter whittenburyi , Methylobacter luteus , Methylobacter bovis, Methylobacter bovis , Methylobacter vinelandii Methylomicrobium pelagicum ( Methylomicrobium pelagicum ) , Methylomicrobium sporium ), Methylomonas strain ( Methylomonas Strain ), Methylomonas rubra ( Methylomonas rubra ), Methylomonas aurantiaca ( Methylomonas aurantiaca ) aurantiaca ), Methylomonas fodinarum ( Methylomonas fodinarum ), Methylomonas methanica ( Methylomonas methanica ), Methylococcus thermophilus ( Methylococcus thermophilus ), Methylococcus sporium , Methylococcus sporium , Methyllococcus sporium ) ( Methylococcus capsulatas ), Methylomonas trisporium ( Methylomonas trisporium ) , Methylosinus trichosporium ( Methylosinus trichosporium ), Methylocystis parvus ), Methylocella palustris ( Methylocella palustris ), Methylocapsa acidiphila ( Methylocapsa acidiphila ), methyllomonas ( methylomonas buryatense ), methyl cystis sporium ( Methylocystis sporium ), methyl locinus trichosporium ( Methylosinus trichosporium ) , Candidatus methanoperededens ( Candidatus Methanoperededens ), nitro reducense ( nitroreducens ), methyl Selected from the group consisting of fibrate ( Methylosarcina fibrate ), methylocaldum szegediense , Candidatus Methylomirabilis limnetica , and Methylovulum psychrotolerans . It may be any one or more, but is not limited thereto.

Acetogenic-bacteria using CO of the present invention are Acetobacterium woodii ( Acetobacterium woodii ), Clostridium autoethanogenum ( Clostridium autoethanogenum ), Clostridium carboxidivorans P7 ( Clostridium carboxidivorans P7 ), Clostridium Clostridium ljungdahlii, Moorella thermoacetica , Eubacterium limosum , carboxidivorans, Oxobacter pfennigii, Peptostreptococcus Tooth ( Peptostreptococcus productus ), Butyribacterium methylotrophicum ( Butyribacterium methylotrophicum ), Methanosarcina acetivorans ), Murella thermoautotropica ( Moorella thermoautotrophica ), disulfotomaculum kuznetsovii ), disulfotomaculum thermobenzoicum ( Desulfotomaculum thermobenzoicum ), thermosyntropicum ( Thermosyntrophicum ), Archaeoglobus fulgidus ). not.

Hydrogen-producing bacteria using CO of the present invention are Rubrivivax gelatinosus , Rhodopseudomonas palustris , Rhodospirillum rubrum , Citrobacter genus Y19 ( Citrobacter sp Y19 ), Murella AMP ( Moorella AMP ), Carboxydothermus hydrogenoformans ), Carboxydibrachium pacificus ( Carboxydibrachium pacificus ), Carboxydocella sporoproducensense ( Carboxydocella ) , Carboxydocella thermoautotrophica ( Carboxydocella thermoautotrophica ), Thermomincola carboxydiphila ( Thermincola carboxydiphila ), Thermolithobacter carboxydivorans ( Thermolithobacter carboxydivorans ), Thermosinus carboxydivorans ), disulfotoma Cullum ( Thermosinus ), carboxydivorans Carboxy diborans ( Desulfotomaculum carboxydivorans ), Thermococcus genus AM4 ( Thermococcus strain AM4 ), Thermococcus onnurineus ( Thermococcus onnurineus ) It may be any one or more selected from the group consisting of, but is not limited thereto.

In the present invention, the microorganism having the C1 gas metabolizing ability is Methylomonas genus DH-1 ( Methylomonas sp. DH-1 ), Methylosinus trichosporium OB3b ( Methylosinus trichosporium OB3b ), Methylomonas strain ( Methylomonas Strain ) ), Methylomonas rubra , Methylomonas aurantiaca , Methylomonas fodinarum , Methylomonas methanica , Methylomonas methanica type -5 ( Methylocystis sporium type 5 ), Methylosinus trichosporium ), Acetobacterium woodii ), Thermococcus genus AM4 ( ( Thermococcus strain AM4 ) and Thermococcus onnurineus ( Thermococcus onnur ) It may be characterized in that any one or more selected from the group consisting of.

In the present invention, the nanofluid in which the chitosan-introduced lipid compound nanoparticles are dispersed in a medium is (a) a solution prepared by mixing chitosan and a lipid compound in an acidic solvent into nanofibers by electrospinning ; And (b) it may be characterized in that it is prepared by a method comprising the step of crushing the prepared nanofiber in an aqueous solution of pH 9 or less.

In the present invention, the electrospinning method is a technology for forming fibers using electrical attraction and repulsion generated when a polymer solution or molten polymer is charged with a predetermined voltage. The electrospinning process has the advantages of being able to manufacture fibers having various diameters ranging from several nm to several thousand nm, simple equipment structure, applicable to a wide range of materials, and easy mass production. Specifically, in order to perform the electrospinning process, first, chitosan and a lipid compound may be dissolved in an organic solvent alone or in a mixed solvent of an organic solvent and an acidic solvent.

The radiation process may be performed using an appropriate radiation device while applying at an appropriate voltage. When the voltage applied during the spinning process is set within the range of 10 to 40 kV, more preferably, 20 to 30 kV, a stable spinning process can be performed. In addition, the ejection rate is preferably 0.2 ml/hr to 0.9 ml/hr, more preferably 0.4 ml/hr to 0.8 ml/hr.

In the present invention, the acidic solvent may include any one or more acids selected from the group consisting of trifluoroacetic acid, hexafluoroisopropanol, hexafluoropropanol, acetic acid, and hydrochloric acid.

In the present invention, the average diameter of the lipid compound nanoparticles into which the chitosan is introduced may be characterized in that it is 200 nm or less.

In the present invention, the medium may be characterized as a polar solvent.

In the present invention, the C1 gas may be used without limitation as long as it is a gas containing one carbon atom, and may preferably be carbon monoxide or methane, but is not limited thereto.

In the present invention, the biopolymer may be at least one selected from the group consisting of cellulose, chitosan, polygamma-glutamic acid, polylactic acid, polyhydroxyalkanoate, polyhydroxybutyric acid, and polyolefin.

In the present invention, the nanofluid in which the biopolymer nanoparticles into which the tannin-transition metal complex is introduced are dispersed in a medium is prepared by (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.

In the present invention, the transition metal may be any one or more selected from the group consisting of iron, nickel, cobalt, molybdenum and chromium.

In the present invention, the acid may be any one or more 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. 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). can

Example

Hereinafter, the present invention will be described in more detail through examples. These examples are only for illustrating the present invention, and it will be apparent to those of ordinary skill in the art that the scope of the present invention is not to be construed as being limited by these examples.

Preparation Example 1: Preparation of chitosan-lipid complex introduced nanofluid

After adding 0.5 g of chitosan and 0.5 g of oleamide as a lipid complex to a 10 ml glass vial containing 6.5 ml of 1:1 ratio of TFA:HFIP co-solvent, at 50° C. for 20 minutes. By stirring, an electrospinning solution was prepared. Then, 115uL of tween20 solution, a non-polar surfactant, was added to the prepared electrospinning solution, stirred at room temperature for 20 minutes, and electrospinning was performed for 10 hours to prepare nanofibers/nanoparticles into which the chitosan olemide complex was introduced. did

Thereafter, the prepared nanoparticles/nanofibers were added to sterilized distilled water to disperse the nanoparticles through an ultrasonic mill to prepare a nanofluid.

Preparation Example 2: Preparation of nanofluids introduced with tannin-iron complex

20 g of cellulose was added to 350 mL of H ₂ SO ₄ aqueous solution (H ₂ SO ₄ content: 64% by weight based on the aqueous solution), and the slurry containing cellulose nanoparticles was obtained by continuously stirring at a temperature of 50° C. for 45 minutes. prepared, 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, the phosphate buffer solution was added dropwise until the pH of the mixture became 7 or more to prepare a nanofluid containing cellulose nanoparticles into which the tannin-iron complex was introduced.

Example 1. Mass transfer coefficient (K) by introducing chitosan/oleamide nanofluid and tannin-iron complex-introduced nanocellulose nanofluid into a microbial culture medium _L a) Comparative analysis of the amount of change

In order to confirm the effect of improving the gas transfer rate in the culture medium to which the nanofluid is applied, the mass transfer coefficient (K _L a) according to the presence or absence of the application of the nanofluid was measured and analyzed. For mass transfer coefficient (K _L a) analysis according to nanofluid application, it was measured using a 1L culture system.

)로 유지하였다. 실험 장치는 세부적으로 공급기로부터 가스가 주입되어 용액과 반응이 일어나는 발효조(Reactor), 발효조 내부로 공급되는 가스의 압력 및 외부로 빠져나가는 가스의 유량을 측정, 조절해주는 유량 측정기 (Mass Flow meter) 및 실제 용액 내 용존 산소를 측정하는 용존산소 측정 장치 (Disolved oxygen probe), 공급 및 외부로 빠져나가는 가스의 유량을 측정 조절하는 유량 측정기로 이루어져 있고, 물질전달계수 측정 분석에 이용되는 에어 가스통과 메탄 가스통, 레귤레이터 등이 있으며, 발효조 내부에는 공급기로부터 공급되는 가스가 실제 용액과 접촉하도록 구경 1/5 inch 크기의 스테인리스 가스 공급관 (Sparger)이 설치 되어있다. In the case of the 1L culture system, it is largely divided into five types: gas supply, fermenter, flow meter, and oxygen measuring device in solution.

) was maintained. The experimental device is a fermenter (Reactor) where gas is injected from the feeder and reacts with the solution, a flow meter that measures and controls the pressure of the gas supplied into the fermenter and the flow rate of the gas exiting the fermenter (Mass Flow meter) and It consists of a dissolved oxygen probe that measures dissolved oxygen in the actual solution, and a flow meter that measures and controls the flow rate of supply and outflow gas, and an air gas cylinder and methane gas cylinder used for mass transfer coefficient measurement and analysis. , 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 comes into contact with the actual solution.

In the case of gas supply pipe, Alicat scientific's MFC (mass flow controller) MC series is connected to control the flow rate, and the microbial culture medium used for measuring the mass transfer coefficient is NMS medium (MgSO ₄ 7H ₂ O 1g per 1 L of sterile water, KNO ₃ 1 g, CaCl ₂ 2H ₂ O 0.2 g, iron-chelate 0.0038 g, Na ₂ MoO ₄ 2H ₂ O 0.0006 g, KH ₂ PO ₄ 0.26 g, Na ₂ HPO ₄ 7H ₂ O 0.62 g, CuSO ₄ 5H ₂ O 2.5 mg, biotin 0.02 mg, folic acid 0.02 mg, thiamine hydrochloride 0.05 mg Ca-pantothenate 0.05 mg, vitamin-B12 0.001 mg, riboflavin 0.05 mg, nicotiamide 0.05 mg, FeSO ₄ 7H ₂ O 0.5 mg, ZnSO4 7H ₂ O 0.4 mg, MnCl ₂ 7H ₂ O 0.02 mg, CoCl ₂ 6H ₂ O 0.05 mg, NiCl ₂ 6H ₂ O 0.01 mg, boric acid 0.015 mg, EDTA 0.25 mg) was used, and the actual substance The transfer coefficient was measured according to the presence or absence of nanofluid application in 0.3 L of NMS.

At this time, the flow rate range of the gas supplied through the gas supply pipe into the incubator was set to 0.2-0.4 L/min, and the effect of improving the mass transfer coefficient according to the introduction of the nanofluid system according to the gas flow rate was measured. Specifically, for the measurement method, an NMS medium was prepared, and a degassing reaction was performed to remove residual gas in the solution using an ultrasonic grinder (Ultrasonicator, Ulso Hitech, Korea).

After degassing, 0.3L of NMS medium was transferred to the inside of the fermenter, and the agitation-speed in the incubator was set to 400 rpm. Then, the mixed gas in the methane:air (4:6) ratio was fed at a flow rate of 0.4 L/ The maximum dissolved oxygen concentration is regarded as the maximum dissolved oxygen concentration when there is no difference in the flow rate of gas that is supplied to the inside of the fermenter at min speed and the amount of change in dissolved oxygen in the culture medium through the DO probe (Dissolved-Oxygen) probe. The mass transfer coefficient (K _L a) was measured by calculating the rate of change in dissolved oxygen.

First, in the case of microbial culture medium (NMS) to which nanofluid is not applied, which is a negative control, in the case of a mass transfer coefficient of 100% oxygen based on 0.3 L, 29.97 hr at 0.2 L/min, respectively, depending on the gas flow range At ^-1 , 0.3 L/min, it is 40.95 hr ^-1 , and at 0.4 L/min, it is 56.96 hr ^-1 At a feed rate of 0.2 L/min, 19.008 hr ^-1 , 23.67 hr ^-1 at 0.3 L/min, and 31.2 hr ^-1 at 0.4 L/min were measured, and based on this measurement, the nanofluid application concentration was Through the measurement of the mass transfer coefficient, the degree of improvement in the relative mass transfer coefficient was shown in the figure by comparison with the negative control group.

As a result, as shown in FIG. 1, in the case of the relative mass transfer coefficient according to the introduction of the chitosan/oleamide nanofluid into the NMS medium, as the applied concentration of the chitosan/oleamide nanofluid increased, the relative mass transfer coefficient was negative. It was confirmed that 50%, 65%, 85%, and 100% improvement compared to the control NMS.

In addition, as shown in FIG. 2, in the case of the nanocellulose nanofluid to which the tannin-iron complex was introduced, when the tannin-iron nanofluid was applied in the flow rate section of 0.4 L/min, the relative mass transfer coefficient was improved by 15% compared to NMS confirmed that.

Example 2. Comparative analysis of solubility change by introducing chitosan/oleamide nanofluid and tannin-iron complex-introduced nanocellulose nanofluid into a microbial culture medium

By measuring the solubility, which is the actual supply amount of C1 gas (CO, CH ₄ ), which is a nutrient source in the C1 gas conversion microorganism culture, it was confirmed whether the solubility was improved through the application of the nanofluid in the culture medium. In order to measure the change in solubility in the culture medium depending on whether or not the nanofluid was applied, the analysis was performed using a solubility analyzer.

)를 유지하여 시켜주었다. 용해도 분석 장치는 세부적으로 47 L 메탄(99.999%, vol), 에어(99.9%, vol), 일산화탄소 (99.999%, vol) 가스 용기로부터 가스를 개별적으로 공급받아 포집해 두는 공급기, 공급기로부터 가스가 주입되어 용액과 반응이 일어나는 반응기, 공급기와 반응기의 초기 및 반응 평형 후 압력을 나타내는 압력계측기로 이루어져 있으며, 별도로 반응 후, 반응기 및 공급기 내의 용액내 용존 가스를 제거해줄 헬륨 가스통과 레귤레이터 등으로 구성되어있다. The experimental equipment used for solubility analysis is largely divided into three types: gas supply, reactor, and pressure measuring instrument. In order to maintain a constant temperature and ambient pressure, it is installed in a fume hood,

) was maintained. In detail, the solubility analyzer is a 47 L methane (99.999%, vol), air (99.9%, vol), carbon monoxide (99.999%, vol) gas supply that is individually supplied and collected from a gas container, and the gas is injected from the supply. It consists of a reactor where the reaction takes place with a solution, a supply and a pressure gauge indicating the initial pressure of the reactor and after reaction equilibrium, and a helium gas cylinder and a regulator to remove dissolved gas in the solution after the reaction separately. .

The internal volume of the feeder and reactor is 101.11 cm ³ on average, and is made of stainless steel so that gas does not leak to the outside. An inch Teflon-tubing (1/8 inch interface tubing, Agilent, USA) was run from the outside of the feeder to the bottom of the reactor. In addition, the constant flow rate of gas supplied into the gas supply is 100 cc/min for methane, air, and CO gas using Alicat scientific's MFC (mass flow controller) MC series, and the flow rate for helium gas is 200 cc/min. was used for measurement.

PDK's PDR model pressure sensor having a precision of 0.0001 bar was used to measure the pressure inside the feeder and reactor, and in the case of actual solubility analysis measurement, the NMS medium of Example 1, MM1 medium (NaCl per 1 L of sterile water) into the reactor 35 g, KCl 0.7 g, CaCl ₂ ·2H ₂ O 0.4 g, NH ₄ Cl 0.3 g, Cystein-HCl 0.5 g, YeastExtract 10 g, CuSO ₄ ·5H ₂ O 0.04 g, ZnSO ₄ ·7H ₂ O 0.4 g, CoCl ₂ 6H ₂ O 0.020 g, MnCl ₂ 4H ₂ O 0.8 g, Na ₂ MoO ₄ 2H ₂ O 0.4 g, KBr 0.2 g, KI 0.2 g, H ₃ BO ₃ 0.4 g, NaF 0.2 g, LiCl 0.2 g, Al ₂ (SO ₄ ) ₃ 0.2 g, NiCl ₂ ·6H ₂ O 0.04 g, VOSO ₄ ·2H ₂ O 0.02 g, H ₂ WO ₄ 0.02 g, Na ₂ SeO ₄ 0.02 g, SrCl 6H ₂ O 0.02 g, BaCl ₂ 0.02g, P-aminobenzoicAcid 0.005g, Biotin 0.002g, CalciumPentothenate 0.005g, Cyanobalamin (B12) 0.0001g, Folicacid 0.002g, Nicotinicacid 0.005g, Pyridoxine 0.01g, Riboflavin 0.005g, Thiamine-poic Acid 0.005g 0.005 g), PBBM medium (NaCl 0.9 g per 1 L of sterile water, MgSO ₄ ·2H ₂ O 0.2 g, CaCl ₂ ·2H ₂ O 0.2 g, NH ₄ Cl 1.0 g, Na ₂ S 9H ₂ O 0.000625 g, Resasurin 0.0002g, Cystein-HCl 0.5g, YeastExtract 2g, CuSO ₄ 5H ₂ O 0.04 g, ZnSO ₄ 7H ₂ O 0.4 g, CoCl ₂ 6H ₂ O 0.020 g, MnCl ₂ 4H ₂ O 0.8 g, Na ₂ MoO ₄ 2H ₂ O 0.4g, KBr 0.2g, KI 0.2g, H ₃ BO ₃ 0.4g, NaF 0.2g, LiCl 0.2g, Al ₂ (SO ₄ ) ₃ 0.2g, NiCl ₂ 6H ₂ O 0.04 g, VOSO ₄ 2H ₂ O 0.02 g, H ₂ WO ₄ 0.02 g, Na ₂ SeO ₄ 0.02 g, SrCl 6H ₂ O 0.02 g, BaCl ₂ 0.02 g, P-aminobenzoicAcid 0.005 g, Biotin 0.002 g, CalciumPentothenate 0.005 g, Cyanobalamin (B12) ) 0.0001g, Folicacid 0.002g, Nicotinicacid 0.005g, Pyridoxine 0.01g, Riboflavin 0.005g, Thiamine-HCl 0.005g, and Lipoic Acid 0.005g) After 50 ml) is supplied, helium gas is supplied into the reactor for the reaction to proceed. And the residual gas in the feeder was removed as much as possible.

Then, after closing the valve connecting the feeder and the reactor, the gas (carbon monoxide, methane, air) for the reaction is collected from the 47L gas container to the feeder, and when the marked pressure of the pressure sensor connected to the feeder does not change for 1 minute The reaction preparation was completed by closing the valve between the feeder and the gas vessel. Thereafter, the valve connecting the feeder and the reactor was opened so that the capture gas of the feeder could be supplied to the inside of the solution, so that the reaction gas was absorbed into the solution.

At this time, the pressure sensor connected to the feeder decreases as the absorption reaction proceeds into the reactor, and then, when the change in pressure for 5 minutes is less than 0.01, it is considered that the absorption equilibrium has been reached, and the pressure at that time is measured and the feeder After calculating the pressure change of the and the reactor, the pressure of the reaction gas used for absorption equilibrium and the volume, pressure, and temperature of the culture medium are measured. Considering the range to be satisfactory, the solubility was measured by calculating using Henry's law and the ideal gas state equation.

For methane and air solubility in NMS culture medium as negative control, 0.501 (mmol/L) and 0.507 (mmol/L), respectively, for CO solubility in MM1 medium as another negative control, 0.49 (mmol/L), PBBM The CO solubility of the medium was determined to be 0.505 (mmol/L). Thereafter, the solubility of the chitosan/oleamide nanofluid and the tannin-iron cellulose nanofluid was measured, and the relative solubility compared to the solubility of the negative control was shown in the figure.

As a result, as described in FIGS. 3 and 4, chitosan/oleamide nanofluid 0.01*10 ^-3 -50*10 ^-3 % (w/v) in the concentration range for carbon monoxide, methane, and air gas, solubility It was confirmed that there was no influence on the improvement effect.

On the other hand, in the case of the nanocellulose nanofluid into which the tannin-iron complex is introduced, as the applied concentration increases in the range of 0.025-0.1% (w/v) of the applied concentration in the culture medium, as described in FIG. 5 , in the case of CO solubility , it was confirmed that the maximum solubility was improved by 20% compared to the negative control.

Specifically, in the case of the culture medium (MM1) of the archaea T.onnurinues NA1 using CO, when the tannin-iron nanocellulose nanofluid was applied, the solubility of CO was increased by 15% compared to the negative control at 0.05% (w/v) concentration. The effect was confirmed, and in the case of the CO-using Acetogen culture medium (PBBM), it was confirmed that the effect of increasing the solubility of CO by 20% compared to the negative control group was observed in the interval above the applied concentration of 0.05% (w/v).

In addition, as described in FIG. 6, in the case of air and methane solubility in the methanogen culture medium (NMS medium), the methane solubility was 17% compared to the negative control at the nanofluid application concentration of 0.035% (w/v). It was confirmed that, from 0.05% (w/v), it was confirmed that both air and methane were improved by 20%.

Through this, in the case of tannin-iron cellulose nanofluid, it was confirmed that the solubility of C1 gas, which is the substrate source of C1 gas (CO, CH ₄ ) in the culture medium, was improved by up to 20% depending on the dispersion state in the medium.

Example 3. Comparative analysis of the effect of stabilizing C1 gas bubbles in the culture medium after application of chitosan/oleamide and tannin-iron nanofluid in the microbial culture medium using C1 gas

In order to confirm the stable bubble supply of C1 gas in the actual culture medium according to the supply of the nanofluid, the change of the C1 gas bubble depending on whether chitosan/oleamide nanofluid and tannin-iron nanofluid were applied was recorded using a high-speed camera. Comparative analysis was performed.

Gas bubble stabilization comparison measurement is performed by filling a transparent 1L poly-styrene-based rectangular water tank with 0.5L upper NMS medium, and injecting CO gas and CH ₄ gas at a feed rate of 0.4 L/min, and the form of gas bubbles in the solution. was observed using a high-speed camera.

During the initial introduction of gas into the culture medium, a hole of 0.04cm ² -0.08cm ² in diameter was drilled in a 1/6 inch Teflon tube so that it could be introduced without aggregation between bubbles so that the solution could be supplied from the bottom. Then, depending on whether or not the nanofluid was applied, it was observed how the behavior of individually introduced gas bubbles in the microbial culture medium was changed.

As a result, in the NMS medium used as a negative control, there was no significant difference depending on the type of gas supply (CO, CH ₄ ), but gas bubbles that were individually dispersed when initially introduced into the NMS medium coagulate with each other within 0.03 seconds after introduction of the medium. It was confirmed that it was formed in the form of a large bubble and ascended to the upper part of the medium. In addition, although there is a difference depending on the height in the culture medium, it was confirmed that the bubble rising rate of C1 gas increased from the initial 0.8 m/s to 1.4 m/s in the flow rate range of 0.4 L/min.

On the other hand, as described in Figure 7, when the tannin-iron nanofluid in the NMS medium was applied at a concentration of 0.05% (w/v), unlike the negative control, the gas bubble did not aggregate in the medium, and a uniform dispersion state was obtained. Maintenance was confirmed, and as the gas bubble ascended to the upper part of the culture medium, it was confirmed that the rising speed of the gas bubble increased from 0.9 m/s to 1.6 m/s from the initial 0.9 m/s compared to the negative control NMS medium.

This means that in the case of tannin-iron nanofluids, the mass transfer coefficient (K _L A) and solubility of gases are increased, and the gas bubbles are stabilized so that they do not agglomerate, thereby increasing the mass transfer efficiency of gases in the solution.

On the other hand, as described in FIG. 8, when the chitosan/oleamide nanofluid in the NMS medium was applied at a concentration of 1*10 ^-3 % (w/v), the tannin-iron nanofluid was different from the gas bubble stabilization effect and The difference in the gas bubble rise rate could not be confirmed, but a relatively small bubble compared to the negative control was partially observed at the initial introduction of the NMS medium, and the bubble breaking effect of breaking the bubble surface and forming a small bubble could be partially confirmed. In the case of chitosan/oleamide nanofluid, the mass transfer coefficient (K _L A) was increased, but it was confirmed that there was not much gas solubility and bubble stabilization effect for increasing mass transfer efficiency.

Example 4. Comparative analysis of microbial cell membrane deformation by presence or absence of introduction of chitosan/oleamide nanofluid and tannin-iron complex-introduced nanocellulose nanofluid during microbial species culture

Tannin-iron complex nanocellulose nanofluid composition Nanoparticles and chitosan/oleamide nanofluid composition nanoparticles, whether or not nanofluid is applied through electron microscopy (SEM) According to Methylomonas sp. Differences in cell morphology and cell membrane morphology of DH-1 were analyzed.

4-1. seed culture

Nanofluids in which chitosan-lipid complex-containing nanoparticles are dispersed, and nanofluids in which tannin-iron complex nanocellulose is dispersed were put into a 50ml plastic Falcon tube, 10 ml each, and then ultraviolet (UV) was sterilized for 30 minutes to 2 hours using Then, the same NMS medium as in Example 2 was prepared.

Then, after dispensing 45 ㎖ of NMS medium in a 500 ㎖ flask, methane-consuming Gram-negative bacteria, Methanotroph belonging to Methlylomonas sp . After dispensing 4 ml of DH-1 (hereafter DH-1) Seed (Strain Patent No.: KCTC13004BP), in the case of sterilized chitosan/oleamide nanofluid, the final concentration is 0.1*10 ^-3 -10* in NMS medium. In the case of tannin-iron complex nanocellulose nanofluid, 500 ml so that the final concentration becomes 0.025-0.1% (w/v) in the solution, 1 ml each was injected into a 500 ml flask so as to be 10 ^-3 % (w/v). 1 ml was injected into the flask.

In the control group, 1 ㎖ of sterile water was injected to adjust the volume to 50 ㎖, and then, using a rubber stopper and flask cap, the inlet of the 500 ㎖ flask was closed, and then a 5 ㎖ syringe needle was inserted on one side of each 500 ㎖ flask. After inserting, a mixed gas in which methane (CH ₄ ) gas and air were mixed in a 4:6 ratio was supplied for 8 minutes and 30 seconds at a speed of 300 cc/min, and then cultured with shaking at 32 ° C. 200 rpm for 20 hours to terminate the seed culture. did

4-2. Observation of microbial cell membrane deformation

Recover the cell pellet using a centrifuge, resuspend the cell pellet in a vial containing 1 mL of PBS solution containing 25% GA (glutaraldehyde), and place it in a box with ice for 30 minutes. reacted while Then, after discarding the supernatant using a centrifuge, the process of resuspending only the microbial pellet in PBS solution was performed three times to remove the residual GA solution, and osmium crystals (OsO ₄ ) were 0.004 g After resuspending the cell pellet in 1 mL of the included 50% (v/v) PBS solution and reacting for 30 minutes in a box containing ice, the centrifuge and the resuspension process using PBS solution were repeated 3 times. was repeated to remove residual osmium crystals.

After that, 1 mL of PBS solution containing the microbial pellet was dispensed on a cover glass with a diameter of 8 mm in a 24 well plate of SPL, and the acetone content was increased by 10% in a stand incubator at 32 ° C, and the drying process was performed. It was repeated 9 times. Finally, the dried microbial pellets were analyzed for cell surface and cell shape according to the concentration of chitosan/oleamide nanofluid applied using a field-emission scanning electron microscope (Field-emission SEM) model of HITACHI.

As a result, as described in FIG. 9, Methylomonas sp. grown in a culture medium in which chitosan/oleamide nanofluid was applied at a concentration of 10*10 ^-3 % (w/v) during seed culture. It was confirmed that the overall shape of DH-1 was changed and the cell shape was partially damaged.

On the other hand, in the case of the tannin-iron complex nanocellulose nanofluid, as described in FIG. 10, when applying the seed culture, Methylomonas sp. It was confirmed that DH-1 had no significant effect on the cell membrane.

Example 5. Comparative analysis of microbial cell membrane adsorption by presence or absence of introduction of chitosan/oleamide nanofluid during microbial seed culture

5-1. Chitosan/oleamide nanofluid composition nanoparticles nile-red tag

In Example 4, the nanoparticles constituting the chitosan/oleamide nanofluid, which caused the transformation of the cell membrane, to confirm the adsorption to the microbial membrane during seed culture, a fluorescent tag to the nanoparticles constituting the chitosan/oleamide of the nanofluid To proceed, CH ₄ using strain Methylomonas sp. CFDA tag was applied to DH-1, and microbial membrane adsorption analysis of chitosan/oleamide nanoparticles was performed through nile-red intensity analysis around CFDA fluorescence.

In the case of the chitosan/oleamide nanofluid-constituting nanoparticle fluorescent tag, nile-red (Sigma Aldrich, USA) reagent showing red fluorescence was applied to perform fluorescence staining of the nanoparticles constituting the chitosan/oleamide nanofluid. In detail, 10 μg of the purchased nile-red powder was suspended in 1 mL of co-solvent in a 1:1 ratio of glycerol: PBS (phosphate buffer saline) to prepare a nile-red stock solution, and then the prepared nile- After reacting 10 μl of red stock solution with 1 mL of 0.1% (w/v) concentration of chitosan/oleamide nanofluid solution, Nikon's NIS-element model fluorescence microscope showed that some aggregated nanoparticles were treated with nile as a control. By confirming a clear difference in fluorescence intensity compared to the -red reagent, it was determined whether the chitosan/oleamide nanofluid constituent nanoparticles were fluorescently stained.

5-2. CH ₄ Microbial CFDA tags utilized

Then, in order to check whether the nanoparticles are actually bound to the microbial cell membrane, to be distinguished from the red fluorescence color of the nile-red tagged nanoparticles, the model CH ₄ using strain Methylomonas sp. A green fluorescent staining tag was applied to DH-1 using CFDA (6-carboxyfluorescein diacetate) reagent (Dojindo, Maryland, USA). Methylomonas sp. As an experimental method for CFDA tagging on DH-1, first, 10 μL of the purchased CFDA reagent was suspended in 10 mL PBS to prepare a CFDA stock solution, and 1 mL of the prepared CFDA stock solution was added to Methylomonas sp. It was applied 1:1 to 1 mL PBS solution in which DH-1 was suspended.

Thereafter, in an incubator equipped with a dark room condition at 37 ° C., after performing the CFDA tag reaction for 30 minutes, in order to remove the residual CFDA stock solution, the supernatant was discarded using a centrifuge, and the microbial pellet was washed with PBS solution. The resuspension process was carried out three times.

5-3. nile-red tagged chitosan/oleamide nanoparticles were obtained from CFDA-tagged Methylomonas sp. Analysis of microbial cell membrane adsorption by application to DH-1

CFDA-tagged Methylomonas sp. in a 1 mL vial. To 0.5 mL of DH-1 sample, nile-red-tagged chitosan/oleamide nanoparticle-constituting nanofluid was applied in 0.5 mL to a concentration of 0.1*10 ^-3 % (w/v). At this time, as a negative control, 0.5 ml of a stock solution (0.1*10 ^-6 % (w/v)) of nile-red concentration used for chitosan/oleamide nanoparticles nile-red tag was tagged with CFDA contained in a 1 ml vial. Methylomonas sp. Adsorption fluorescence images of the chitosan/oleamide nanoparticles with the microbial membrane were taken by applying to 0.5 mL of a DH-1 sample.

Fluorescence imaging was taken using the Eclipse Ni-U model (Nikon, Japan), and when nile-red tagged chitosan/oleamide nanoparticles were applied, the intensity of nile-red fluorescence around CFDA-tagged microorganisms compared to negative control was confirmed to be clearly distinguished. Then, while increasing the concentration of nile-red tagged chitosan/oleamide nanoparticles to 0.1*10 ^-3 -50*10 ^-3 % (w/v), through nile-red fluorescence intensity relative analysis, nanoparticles were applied The degree of microbial membrane adsorption was evaluated according to

As a result, as shown in Figure 11, as the concentration of the nile-red tagged nanoparticles increased, it was observed that the fluorescence intensity of the negative control increased up to 11 times, the chitosan / oleamide nanofluid composition nanoparticles were the microorganisms Adsorption to the cell membrane was confirmed.

Example 6. Methylomonas sp. After application of chitosan-based nanofluid in DH-1 seed culture, CH in main culture without application of nanofluid ₄ Consumption / Microorganism Growth Concentration Comparative Analysis

6-1. seed culture

During seed culture, when the nanoparticles constituting the chitosan lipid complex-containing nanofluid are adsorbed to the microbial cell membrane, and then cultured without applying the nanofluid during the main culture, what effect does it have on the microbial growth concentration and methane consumption In order to confirm whether the chitosan-lipid complex-containing nanoparticles are dispersed, 10 ml of each of the nanofluids is put into a 50 ml plastic Falcon tube, and then 30 minutes to 2 hours using ultraviolet light at a biosafety test bench. sterilized during Then, the same NMS medium as in Example 2 was prepared.

Then, after dispensing 45 ㎖ of NMS medium in a 500 ㎖ flask, methane-consuming Gram-negative bacteria, Methanotroph belonging to Methlylomonas sp . After dispensing 4 ml of DH-1 Seed, 1 ml of the sterilized nanofluid was injected into a 500 ml flask so that the final concentration became 0.005% (w/v) in the solution, and the control group was sterilized to adjust the volume to 50 ml. 1 ml of water was injected.

Then, using a rubber stopper and flask cap to close the inlet of the 500 ml flask, insert a 5 ml syringe needle into one side of each 500 ml flask, methane (CH ₄ ) gas and air in a 4:6 ratio The mixed gas was supplied at a speed of 300 cc/min for 8 minutes and 30 seconds, and then incubated with shaking at 32°C and 200 rpm for 32 hours.

6-2. main culture

The main culture was performed by dispensing 45 ml of NMS medium in a 500 ml flask, and during seed culture, the nanofluid was applied at a concentration of 0% (w/v) (control) and 0.005% (w/v), respectively, Methylomonas sp. After dispensing 5 ml of DH-1 into different flasks, block the inlet of the flask using a rubber stopper and a flask cap in the same way, and supply a mixed gas of methane and air equal to the upper composition ratio to the flask, then 32 Cultivation was performed with shaking at 200 rpm.

The cell growth rate was compared by measuring the absorbance at 600 nm using a spectrophotometer according to the incubation time. Methane consumption was measured using a 7890B GC (Agilent, CA, USA) connected to a Molsieve 5A (Agilent, 6ft, 1.8 X 2 mm) or Propak Q (Agilent, 6ft, 1.8 X 2 mm) column for methane, carbon dioxide, and oxygen in the flask. , methane consumption was measured by measuring the change in the nitrogen composition ratio.

As a result, as described in FIG. 12 , in the case of 4-hour culture microbial growth concentration, 0.1367 (OD ₆₀₀ ), 0.2640 (OD ₆₀₀ ) were shown, respectively, and in the case of CH ₄ consumption, 0.45 (mmol/L), 1.46 (mmol/L), and in the case of microbial growth concentration of 0.22 (OD ₆₀₀ ), 0.509 (OD ₆₀₀ ), and methane consumption 0.509 (mmol/L), 3.44 (mmol/L) in the result of 8-hour culture was confirmed to be.

Through this, even if the chitosan-based nanofluid is applied only during the seed culture, without additional application of the chitosan-based nanofluid during the main culture, through the adsorption of the microbial cell membrane of the nanoparticles constituting the chitosan-based nanofluid applied during the seed culture, additional It was confirmed that even without the application of chitosan-based nanofluid, a DH-1 strain with increased growth concentration and CH ₄ consumption could be obtained.

And the effect was confirmed that the same effect appeared without additional chitosan-based nanofluid application during the main culture, when 0.001% (w/v) chitosan-lipid complex-containing nanofluid was applied at a different concentration during seed culture (FIG. 13) .

Example 7. Methylomonas sp. Comparative analysis of microbial growth concentration during main culture without application of nanofluid after application of tannin-iron nanocellulose nanofluid in DH-1 seed culture

7-1. seed culture

During seed culture, 10 ml of each of the nanofluids in which the tannin-iron complex nanocellulose is dispersed is put into a 50 ml plastic Falcon tube, and then sterilized for 30 minutes to 2 hours using ultraviolet (UV) light on a biosafety test bench. made it Then, an NMS medium was prepared in the same manner as in Example 1. After dispensing 45 ㎖ of NMS medium into a 500 ㎖ flask, methane-consuming Gram-negative bacteria, Methanotroph belonging to Methlylomonas sp . After dispensing 4 ml of DH-1 Seed, 1 ml of sterilized nanofluid was injected into a 500 ml flask so that the final concentration became 0.025-0.1% (w/v) in the solution, and the volume was set to 50 ml for the control group. 1 ㎖ of sterile water was injected to adjust.

Then, using a rubber stopper and flask cap to close the inlet of the 500 ml flask, insert a 5 ml syringe needle into one side of each 500 ml flask, methane (CH ₄ ) gas and air in a 4:6 ratio The mixed gas was supplied at a speed of 300 cc/min for 8 minutes and 30 seconds, and then incubated with shaking at 32°C and 200 rpm for 32 hours.

7-2. main culture

For main culture, 45 ml of NMS medium was dispensed into 500 ml flasks, and during seed culture, the nanofluid was applied at a concentration of 0% (w/v) (control) and 0.025-0.1% (w/v), respectively, Methylomonas sp. . After dispensing 5 ml of DH-1 into different flasks, block the inlet of the flask using a rubber stopper and a flask cap in the same way, and supply a mixed gas of methane and air equal to the upper composition ratio to the flask, then 32 Shaking culture was performed at ℃ and 200 rpm conditions, and the cell growth rate was compared by measuring the absorbance at 600 nm using a spectrophotometer according to the incubation time.

As a result, as described in FIG. 14 , in the case of the tannin-iron complex nanocellulose nanofluid, it was confirmed that there was no effect of improving the microbial growth concentration without additional supply of the nanofluid during the main culture after the application of the seed culture.

Example 8. Thermococcus onnurineus After applying chitosan-based nanofluid in NA1 seed culture, without additional nanofluid application, CO consumption and product comparative analysis during main culture

In the case of CO-using microorganisms, in order to check whether microorganisms with improved CO gas consumption rate can be obtained only by application of seed culture without additional chitosan-based nanofluid application during main culture, seed culture of T.onnurinues NA1 156T, an archaeal species using CO By applying a chitosan-based nanofluid to , the CO consumption of T. onnurinues NA1 in main culture was comparatively analyzed.

8-1. seed culture

During seed culture, the prepared nanofluid was placed in a 50 ㎖ plastic centrifuge tube (Falcon tube), and then sterilized for 16 hours using ultraviolet (UV) light in a biosafety laboratory. Then, NM1 medium was prepared.

In order to maintain the prepared medium in an anaerobic state, nitrogen (N ₂ ) gas was supplied at a rate of 100 cc/min for 20 minutes to replace the air inside the glass serum bottle with nitrogen (N ₂ ) gas, and then, an anaerobic chamber After dispensing 36 ml of the microbial medium prepared in 110 ml glass serum bottles, the previously prepared 0.02%, 0.01% (w/v) chitosan-based nanofluid was finally added to the medium in 0.0001%, 0.0005% ( w/v) concentration was injected by 2 ml each, and in the case of a control group, 2 ml of sterile tertiary distilled water was injected instead of nanofluid.

After that, the entrance of the 110 ml glass serum bottle was blocked using a rubber stopper and an aluminum seal, and then carbon monoxide (CO) gas was supplied at a rate of 100 cc/min for 30 minutes, and then T. onnurinues NA1 156T seed (strain patent) No.: KR 2011-0094092A) was dispensed by 2 ml each, and control and experimental samples were prepared and completed. Thereafter, seed culture was performed at 70-85 °C for 20 hours.

8-2. main culture

After incubation, the samples were cooled at room temperature for about 1 hour, and then added to each glass serum bottle to carry out the main culture with 0 % (w/v), 0.0001 % (w/v), 0.0005% (w/v) After dispensing 2 ㎖ of only T. onnurinues NA1 156T, which was applied during seed culture of chitosan-based nanofluid at a concentration, carbon monoxide (CO) gas was supplied in the same manner as above, and after 2 hours of incubation, Molsieve 5A ( Agilent, 6ft, 1.8 X 2 mm), Propak Q (Agilent, 6ft, 1.8 X 2 mm) column-connected 7890B GC (Agilent, CA, USA) was used to change the composition ratio of carbon monoxide, hydrogen, and carbon dioxide inside a glass serum bottle was measured to compare the consumption of CO.

As a result, as described in FIG. 15 , in the case of T.onnurineus NA1 156T grown in NM1 medium containing nanofluids at a concentration of 0.0001% (w/v) and 0.0005% (w/v) during seed culture, nano Compared to the negative control to which no fluid was applied, it was confirmed that the amount of CO consumption increased by about 2 times compared to the negative control in the initial 2 hour incubation period during the main culture.

Through this, it was confirmed that the chitosan-based nanofluid could be applied to the species culture of archaea using CO to obtain a strain with increased CO consumption in the main culture.

Example 9. Methylomonas sp. After application of chitosan/oleamide nanofluid during DH-1 seed culture, when the nanofluid was continuously applied during main culture, cells Growth rate comparison

9-1. seed culture

During seed culture, Methylomonas sp. In order to confirm the change in the microbial growth concentration when the chitosan-based nanofluid of the same concentration was additionally applied to DH-1 during main culture, in the case of seed culture, chitosan-based nanofluid was used as in the seed culture in Example 4 At an applied concentration of 0.005% (w/v), Methylomonas sp. Applied to DH-1 culture and proceeded.

9-2. main culture

Thereafter, the main culture was performed by dispensing 45 ml of NMS medium in a 500 ml flask, and during seed culture, the nanofluid was applied at a concentration of 0% (w/v) (control) and 0.005% (w/v), respectively, Methylomonas sp. . After dispensing 5 ml of DH-1 into different flasks, 1 mL of sterile distilled water was supplied for the control group, and Methylomonas sp. In the flask containing DH-1, apply the same chitosan-based nanofluid at 0.005 % (w/v), then block the inlet of the flask using a rubber stopper and flask cap, and then methane and air mixed gas equal to the upper composition ratio was supplied to the flask, and then cultured with shaking at 32° C. and 200 rpm. The cell growth rate was compared by measuring the absorbance at 600 nm using a spectrophotometer according to the incubation time.

As a result, as described in FIG. 16 , when the chitosan-based nanofluid was applied at the same concentration during the main culture after the seed culture was applied, it was confirmed that the microbial growth concentration decreased compared to the negative control.

Example 10. Methylomonas sp. Cells when tannin-iron nanofluid was individually applied only during DH-1 main culture and when tannin-iron nanofluid was applied during main culture after application of chitosan-based nanofluid in seed culture Comparison of growth rate improvement effect

In order to compare how the effect of improving cell growth rate is different when tannin-iron nanofluid is individually applied only during main culture and when tannin-iron nanofluid is applied during main culture after application of chitosan-based nanofluid in seed culture, The effect of improving the cell growth rate was compared by performing the following analysis.

10-1. Individual application of nanofluids only during main culture

In the case of individual application of the nanofluid only during the main culture, 44 ml of NMS medium was dispensed into a 500 ml flask, and then Methylomonas sp. 5 ml of DH-1 was dispensed into different flasks, 1 ml of sterile distilled water was supplied for the control group, and 1 ml of chitosan-based nanofluid was supplied at a concentration of 0.005 % (w/v) applied, and tannin-iron nanofluid was supplied. For fluids, 1 mL was supplied at an application concentration of 0.05 % (w/v). After blocking the inlet of the flask using a rubber stopper and a flask cap, methane and air mixed gas having the same composition ratio as in Example 6 were supplied to the flask, and then cultured with shaking at 32° C. and 200 rpm conditions, and the cell growth rate was Absorbance was measured and compared at 600 nm using a spectrophotometer based on 20 hours of incubation.

10-2. During seed culture, chitosan-based nanofluid is applied during seed culture, and then tannin-iron nanofluid is applied during main culture.

After applying the chitosan-based nanofluid during the seed culture, the tannin -iron nanofluid was applied during the main culture. It was applied to DH-1 culture, and distilled water was applied to the negative control group, and seed culture was performed at 32° C. and 200 rpm for 32 hours.

After that, the main culture was dispensed by 44 ml of NMS medium in a 500 ml flask, and chitosan-based nanofluid was applied at a concentration of 0% (w/v) (control) and 0.005% (w/v), respectively, during seed culture. Methylomonas sp. After dispensing 5 ml of DH-1 into different flasks, control Methylomonas sp. 1mL of sterile water was supplied to the flask containing DH-1, and Methylomonas sp. In the flask containing DH-1, 1 mL of tannin-iron nanofluid was supplied so that the applied concentration was 0.05% (w/v). After that, the inlet of the flask was blocked using a rubber stopper and a flask cap, and a mixed gas of methane and air equal to the upper composition ratio was supplied to the flask, followed by shaking culture at 32°C and 200rpm conditions, and the cell growth rate was cultured at 20 Absorbance was measured and compared at 600 nm using a time-based spectrophotometer.

10-3. Comparative analysis results of Examples 10-1 and 10-2

The effect of improving the microbial growth concentration in the case of individual application of nanofluid during main culture (10-1) and when tannin-iron nanofluid was applied as main culture after application of chitosan-based nanofluid during seed culture (10-2) For comparison, the relative microbial growth concentration by the application of the nanofluid compared to the negative control in (10-1) and the relative microbial growth concentration by the application of the nanofluid compared to the negative control in (10-2) were compared relative to each other, and only during the main culture We compared how the cell growth rate improvement effect differed when nanofluids were individually applied and when chitosan-based nanofluids were applied to seed culture and then tannin-iron nanofluids were applied to main culture.

As a result, as shown in FIG. 17, when comparing the effect of improving the relative microbial growth concentration when the nanofluid was individually applied during the main culture, in the case of the chitosan-oleamide nanofluid, it was rather reduced by 25% compared to the control, and the tannin- In the case of the iron nanofluid application, it was confirmed that the improvement was 26% compared to the control.

In addition, as described in FIG. 18, after applying the chitosan-based nanofluid during seed culture, when tannin-iron nanofluid was applied during the main culture, the effect of improving the relative microbial growth concentration was improved by 50% of the control. In contrast to the case of applying the nanofluid individually, it was confirmed that the microbial growth concentration was further increased when the chitosan-oleamide nanofluid was applied during the seed culture and then the tannin-iron nanofluid was applied during the main culture.

Example 11. Adapted to efficiently use C1 gas through chitosan-based nanofluids Methylomonas sp. CH when tannin-iron nanofluid was applied during main culture of DH-1 ₄ Consumption/microbial growth concentration and metabolite comparison

When performing the main culture fermentation of microorganisms adapted to efficiently use CH ₄ gas through chitosan-based organic nanoparticles/nanofluids during seed culture, when tannin-iron nanofluids are applied, conventional CH ₄ gas The purpose of this study was to compare microbial culture fermentation and CH ₄ consumption/microorganism growth concentration and fermentation product amount.

First, during the seed culture, the same NMS medium as the NMS medium of Example 1 was prepared, and each 40 ml was dispensed into a 500 ml flask with a screw cap, and the chitosan-based organic nanoparticles/nanofluid was injected to a final concentration of 0.005%. Then, after inoculating 10 ㎖ of DH-1 seed, as in Example 1, supplying methane air mixed gas, and performing seed culture for 2 days, adapted microorganisms that can efficiently use CH ₄ gas prepared. In the case of the control group, tertiary sterile distilled water was added as much as the volume containing the chitosan-based organic nanoparticles/nanofluid during seed culture, and culture was performed in the same manner.

During the main culture fermentation, 3L of NMS medium prepared in each 5L fermenter was dispensed by 100ml each, and 100ml of tannin-iron nanofluid was injected so that the final concentration in the medium was 0.05%, and 100ml of sterilized distilled water was used for the control medium After injection, 100 ml of DH-1 seeds according to each culture method were inoculated into each 5L reactor. At this time, in order to continuously supply methane and oxygen in the reactor, MFC (MC-500SCCM-D, MC-200SCCM-D, Alicat Scientific, US ) was set so that a mixed gas in which methane (CH ₄ ) gas and air were mixed in a 4:6 ratio at a rate of 300 ml/min could be supplied. Fermentation conditions were 30° C., pH 6.75-6.95, and batch culture was performed at 800 rpm.

The amount of consumed gas (CH ₄ , O ₂ ) was compared and analyzed at 6-minute intervals by connecting the gas outflow line exiting through the condenser connected to the fermenter to the QMS system, and the microbial growth concentration and fermentation over time Products (methanol, acetic acid, succinic acid) were recovered and analyzed using a sampling port connected to the reactor.

In the case of microbial growth concentration, it was observed in the absorption region of 600 nm using a spectrophotometer (Shimadzu, Kyoto, Japan). In the case of organic acid analysis generated during fermentation, 5 mM sulfuric acid (H ₂ SO ₄ ) was used as a mobile phase for analysis.

In the case of HPLC analysis, the analysis was performed in the absorption region 210 nm using 0.6 ml/min, 50° C. DAD without change in the composition of the mobile phase. At this time, pyruvic acid, succinic acid, formic acid, fumaric acid, It was confirmed that acetic acid was separated, and the concentration was calculated using each standard quantitative sample, and the concentration of the fermentation product according to the DH-1 fermentation time was analyzed.

As a result, as described in FIG. 19, in the case of microbial growth concentration and methane consumption during fermentation of DH-1 over time, when nanofluid was applied, it was confirmed that the methane consumption was increased and the microbial growth concentration was improved. there is. In particular, it was confirmed that the increase in initial methane consumption according to the presence or absence of application of nanoparticles/nanofluids was observed more clearly in the results up to 24 hours of initial culture.

A quantitative index for comparison of methane consumption according to fermentation time is described in FIG. 20 . As described in Figure 20, there is a difference in the CH _{4 consumption rate and the CH 4 consumption} rate according to the unit microbial growth concentration according to the time interval, but the DH-1 fermentation was performed by applying the nanofluid compared to the negative control that did not apply the nanofluid. case, it was confirmed that all of them showed high quantitative values.

Through this, the nanofluid in the fermentation process of CH ₄ using microorganisms It was confirmed that the effect of improving the microbial growth concentration was shown.

In addition, as a result of analyzing the amount of production change of the fermentation products, methanol, acetic acid, and succinic acid, as shown in FIG. 21 , methanol, one of the intermediate fermentation products of methanogenic bacteria, was produced according to the application of nanofluid compared to the negative control in all sections. It was confirmed that this was high, and as shown in FIG. 22, it was confirmed that the production amount of acetic acid, a fermentation intermediate product, was also high according to the application of the nanofluid, and as described in FIG. 23, succinic acid, a representative product of DH-1 fermentation, was also nano It can be seen that it appears high depending on the application of the fluid.

As a result of analyzing the quantitative index of the fermentation product, as described in FIG. 24, in the case of the microbial dry weight, the negative control was 1.6 g/L, whereas when the nanofluid was applied, the microbial concentration was improved to 2.38 g/L. In the case of methanol, it was confirmed that the methanol production amount was also significantly increased to 2.29 g/L and 5.54 g/L, respectively, and in the case of acetic acid, it was confirmed that it increased to 0.045 g/L and 0.065 g/L, respectively, In the case of succinic acid, the main fermentation product of DH-1, it was confirmed that the negative control produced 0.1 g/L, but increased 1.5 times to 0.15 g/L when the nanofluid was applied.

Through this, it was confirmed that when the nanofluid was applied to the fermentation process of methane magnetizing bacteria, the effect of improving the methane consumption and microbial growth concentration and the production of the fermentation product were also remarkably increased.

Example 12. Adapted to Efficient Use of C1 Gas Through Chitosan-Based Nanofluids Methylosinus trichosporium CH when tannin-iron nanofluid was applied during main culture of OB3b ₄ Comparison of consumption/microorganism growth concentration and metabolite production amount

In the fermentation of Methylosinus trichosporium OB3b (hereinafter OB3b), which is a methanogenic bacterium having a metabolic process different from that of DH-1, the fermentation was carried out through the application of seed culture and main culture application of nanofluids. The medium used for the OB3b seed culture and main culture fermentation was NMS medium 2 (per 1 L of sterile water, MgSO ₄ 7H ₂ O 1 g, KNO ₃ 1 g, CaCl ₂ 2H ₂ O 0.2 g, iron-chelate 0.0038 g, Na ₂ MoO ₄ 2H ₂ O 0.0006 g, KH ₂ PO ₄ 0.26 g, Na ₂ HPO ₄ 7H ₂ O 0.62 g, biotin 0.02 mg, folic acid 0.02 mg, thiamine hydrochloride 0.05 mg Ca-pantothenate 0.05 mg, vitamin-B12 0.001 mg , riboflavin 0.05mg, nicotiamide 0.05mg, FeSO ₄ 7H ₂ O 0.5 mg, ZnSO 4 7H ₂ O 0.4 mg, MnCl ₂ 7H ₂ O 0.02 mg, CoCl ₂ 6H ₂ O 0.05 mg, NiCl ₂ 6H ₂ O 0.01 mg, boric acid 0.015 mg, EDTA 0.25 mg) was used.

In the case of seed culture, prepare upper NMS medium 2, dispense 38 ml of NMS medium 2 into a 500 ml flask with a screw cap, and then inject 2 ml so that the chitosan-oleamide nanofluid has a final concentration of 0.005%, or control After injecting 2ml of tertiary distilled water, OB3b seeds were inoculated at 10ml each.

After that, after inserting a syringe needle of 5 ml to one side of each flask, a mixed gas in which methane (CH ₄ ) gas and air were mixed in a 3:7 ratio was supplied at a rate of 300 cc/min for 8 minutes and 30 seconds. Then, the seed culture was performed at 30 ℃ 200rpm for 2 days.

In the case of OB3b fermentation, in which 1.4L of NMS medium 2 prepared in a 3L fermenter (Kobiotech, Incheon, Korea) was dispensed each, and nanofluid was applied during seed culture, tannin-iron nanofluid was added to the final concentration of 0.05% in the medium. Fermentation was carried out by applying 100 ml to become At this time, in order to continuously supply methane and oxygen in the reactor, MFC (MC-500SCCM-D, MC-200SCCM-D, Alicat Scientific, US ) was set so that a mixed gas in which methane (CH ₄ ) gas and air were mixed in a 3:7 ratio at a rate of 300 ㎖/min can be supplied. Thereafter, batch culture was performed at 30° C., pH 6.8-7.00, and 400 rpm as fermentation conditions.

At this time, the amount of consumed gas (CH ₄ , O ₂ ) is compared to the gas consumption at least 60 minutes intervals by connecting the gas outflow line exiting through the condenser connected to the fermenter to the 7890A GC system (Agilent, CA, USA). analyzed. The microbial growth concentration was analyzed using a spectrophotometer (Biochrom, Cambridge, UK) using the absorbance of the absorption wavelength in the 600nm wavelength region.

In addition, in the case of poly-hydroxybutyrate (PHB), which is the main metabolite of OB3b, the concentration was calculated using a standard quantitative sample, and the concentration was analyzed according to fermentation time. Concentration analysis was performed using the FID of the 7890A model connected to Agilent's DB-Wax column (30m x 0.32 mm, Agilent).

As a result, as described in FIG. 25 , it was confirmed that the methane consumption rate and microbial growth rate of OB3b to which the nanofluid was applied were excellent, and as shown in FIG. 26 , the production amount of PHB, the main metabolite, also, when the nanofluid was applied, all sections of the culture , and based on the results, methane consumption according to unit microbial growth concentration of OB3b and oxygen consumption quantitative index according to unit microbial growth concentration were calculated based on the results, as described in FIG. 27 Likewise, the methane consumption was 2.53 (g/g) in the case of the negative control, but 3.27 (g/g) when the nanofluid was applied, and in the case of the oxygen consumption rate, 3.67 (g/g) and 3.68, respectively. (g/g), and as a result of calculating the quantitative index of production of PHB, the main metabolite of OB3b, according to the dry weight of the unit microorganism, as described in FIG. 28, in the case of the negative control, it was 0.442 (g/g) whereas , when the nanofluid was applied, it was 0.478 (g/g), and it was confirmed that the amount of methane consumption and PHB production per unit microorganism in the case of applying the nanofluid was higher than in the negative control group without the application of the nanofluid.

As a specific part of the present invention has been described in detail above, for those of ordinary skill in the art, it is clear that this specific description is only a preferred embodiment, 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 appended claims and their equivalents.

Claims (16)

(a) In a medium containing a nanofluid in which chitosan-introduced lipid compound nanoparticles are dispersed in a medium, a microorganism having C1 gas metabolizing ability is seed cultured to adsorb the nanoparticles dispersed in the medium to the cell membrane and adapting; and
(b) the step of culturing the main culture (main culture) in a medium that does not contain the nano-fluid in the cultured microorganism;
A method of improving metabolite productivity through improvement of C1 gas utilization efficiency of microorganisms having C1 gas metabolizing ability, comprising:
(a) In a medium containing a nanofluid in which chitosan-introduced lipid compound nanoparticles are dispersed in a medium, a microorganism having C1 gas metabolizing ability is seed cultured to adsorb the nanoparticles dispersed in the medium to the cell membrane and adapting; and
(b) culturing the cultured microorganism in a medium containing a nanofluid in which the biopolymer nanoparticles into which the tannin-transition metal complex is introduced are dispersed in the medium (main culture);
A method of improving metabolite productivity through improvement of C1 gas utilization efficiency of microorganisms having C1 gas metabolizing ability, comprising:
The method according to claim 1 or 2, wherein the chitosan is at least one selected from the group consisting of water-soluble chitosan, insoluble chitosan, and chitosan derivatives. How to improve product productivity.
The method according to claim 3, wherein the water-soluble chitosan is at least one selected from the group consisting of chitosan HCL, chitosan acetate, chitosan glutamate and chitosan lactate. Methods for improving metabolite productivity.
According to claim 1 or 2, wherein the lipid compound is oleic acid, lauric acid, oleamide and erucamide, characterized in that any one or more selected from the group consisting of C1 gas utilization efficiency of microorganisms with C1 gas metabolizing ability Methods of improving metabolite productivity through enhancement.
According to claim 1 or 2, wherein the microorganism having the C1 gas metabolizing ability is Methylomonas genus DH-1 ( Methylomonas sp. DH-1 ), Methylosinus trichosporium OB3b ( Methylosinus trichosporium OB3b ), methyl Monas strain ( Methylomonas Strain ), Methylomonas rubra ( Methylomonas rubra ), Methylomonas aurantiaca ( Methylomonas aurantiaca ), Methylomonas fodinarum ), Methylomonas methanica ( Methylomonas methanica ) Rosinus sporium type-5 ( Methylosinus sporium type 5 ), Methylosinus trichosporium ), Acetobacterium woodii ), Thermococcus genus AM4 ( ( Thermococcus strain AM4 ) and Thermococcus onnuri Nus ( Thermococcus onnurineus ) Method of improving metabolite productivity through improvement of C1 gas utilization efficiency of microorganisms with C1 gas metabolic ability, characterized in that at least one selected from the group consisting of.
The nanofluid according to claim 1 or 2, wherein the chitosan-introduced lipid compound nanoparticles are dispersed in a medium (a) a solution prepared by mixing chitosan and a lipid compound in an acidic solvent by electrospinning. manufacturing into fibers; And (b) method for improving metabolite productivity through improving the efficiency of using C1 gas of microorganisms having C1 gas metabolizing ability, characterized in that it is prepared by a method comprising the step of crushing the prepared nanofibers in an aqueous solution of pH 9 or less .
According to claim 7, wherein the acidic solvent is trifluoroacetic acid, hexafluoroisopropanol, hexafluoropropanol, acetic acid, and hydrochloric acid, characterized in that it contains any one or more acids selected from the group consisting of C1 gas metabolizing capacity A method of improving metabolite productivity by improving the C1 gas utilization efficiency of microorganisms with
The method of claim 1 or 2, wherein the average diameter of the lipid compound nanoparticles into which the chitosan is introduced is 200 nm or less.
The method of claim 1 or 2, wherein the medium is a polar solvent.
The method of claim 1 or 2, wherein the C1 gas is carbon monoxide or methane.
According to claim 2, wherein the transition metal is iron, nickel, cobalt, molybdenum and chromium, characterized in that any one or more selected from the group consisting of C1 gas metabolizing ability of microorganisms with C1 gas utilization efficiency improvement through metabolite productivity How to improve.
The C1 gas of claim 2, wherein the biopolymer is at least one selected from the group consisting of cellulose, chitosan, polygamma-glutamic acid, polylactic acid, polyhydroxyalkanoate, polyhydroxybutyric acid, and polyolefin. A method of improving metabolite productivity by improving the C1 gas utilization efficiency of microorganisms with metabolic ability.
The method according to claim 2, wherein the tannin-transition metal complex is introduced into the nanofluid in which the biopolymer nanoparticles are dispersed in a medium, comprising the steps of: (a) adding an acid to the biopolymer to prepare the biopolymer nanoparticles; and (b) adding and dispersing the biopolymer nanoparticles to a tannin solution and a transition metal solution. How to improve product productivity.
15. The method according to claim 14, wherein the acid is at least one selected from the group consisting of sulfuric acid, hydrochloric acid, nitric acid, phosphoric acid, hydrofluoric acid and formic acid. How to improve.
According to claim 2, wherein the biopolymer nanoparticles are in the form of cylinders having a diameter of 0.1 to 50 nm and a length of 10 to 1 μm. How to improve.

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