WO2017039106A1 - Novel methylomonas sp. strain and use thereof - Google Patents

Abstract

The present invention relates to a novel strain Methylomonas sp. DH-1 and a use thereof and, more specifically, to a strain Methylomonas sp. DH-1, deposited as Accession Number KCTC13004BP, synthesizing methanol from methane, to a composition containing a culture product of the strain for synthesizing methanol, a kit comprising the composition for synthesizing methanol, and to a method for producing methanol by using the strain. The use of the strain Methylomonas sp. DH-1 of the present invention can achieve a mass production of methanol more promptly, and the produced methanol can be widely utilized in the production of a biodegradable plastic and the application to a biofuel, such as a methanol fuel cell or a gasoline engine.

Description

Novel methyllomonas strains and uses thereof

The present invention is a novel strain of genus Methylomonas sp.) DH-1 strain and its use, and more specifically, the present invention relates to the genus Methylomonas deposited with the deposit number KCTC13004BP to synthesize methanol sp.) DH-1 strain, the composition for synthesizing methanol comprising the strain or culture of the strain, the kit for methanol synthesis comprising the strain or the composition, and a method for producing methanol using the strain.

Methane gas is a major component of natural gas and shale gas and is known to cause a greenhouse effect. Unlike liquid hydrocarbons represented by petroleum, methane is a gas on its own, making it difficult to convert to high value products and use as a fuel. Accordingly, efforts have been made to convert methane gas into a liquid compound. Representative compounds that can be produced using methane include methanol. Methanol is a reaction intermediate used in the production of dimethyl ether and biodiesel, and is used as a basic compound in many other fields. The methanol production process using methane, which is currently commercialized, consists of converting methane to syngas, a mixture of hydrogen and carbon monoxide, and then synthesizing methanol through high temperature and high pressure conditions. However, such a chemical process requires high temperature and high pressure conditions, and thus, a problem of stability has been proposed, and a high process cost and a low yield also pose a big problem. Accordingly, research has been conducted to synthesize methanol through environmentally and economically bioprocessing. Bio process has the advantages of less energy consumption, more selective reaction, and stable than the existing chemical process, and efforts to synthesize methanol from methane by replacing many chemical processes with bio process are ongoing.

Methanotrophic bacteria are microorganisms capable of growing methane as the only energy source and were first isolated by Sohngen in 1906. Then, the taxonomic study morphology of the cells to, methyl Pseudomonas genus (Methylomonas), bakteo in (Methylobacter), in (Methylococcus) Rhodococcus methyl methyl depending on the cell or cell type of inner structure of the stationary phase, micro-methyl Methylomicrobium , Methylosphaera , Methylocaldum , Methyloglobus , Methylosarcina , Methyloprofundus , in Plymouth tide methyl (Methylothermus), to Flavian in (Methylohalobius), as geah in (Methylogaea), horses crossed in (Methylomarinum), in Marino commits methyl in (Methylovulum), beolreom as methyl methyl to methyl Methylomarinovum , Methylorubrum , Methyloparacoccus , Methylosinus , Methylocystis , Methylocella , Methylocella Genus ( Methylo 25 species of capsa), methyl hydroperoxide in Lula (Methylofurula), methyl O CD pilreom in (Methylacidiphilum), methyl O CD micro byum in (Methylacidimicrobium) Classified. In addition, methane-oxidizing bacteria have a developed inner membrane structure in all species, and type I, in which the inner membrane of the superimposed vesicles is distributed throughout the cell, and the inner membrane are arranged along the periphery of the cell, according to the difference between the intracellular membranes. It is divided into two forms. In addition, it is reported that there are differences in the fatty acid composition constituting lipids in the intracellular membrane, and type I methane oxidizing bacteria contains 16-carbon chain fatty acids and type II methane oxidizing bacteria contains 18-carbon chain fatty acids. Many, both types have fatty acids containing one unsaturated bond. These methane oxidizing bacteria contain an enzyme called methane monooxygenases (MMO), so that methane can be easily converted to methanol even at normal temperature and pressure. Methane is oxidized to methanol by methane monooxygenase, and then methanol is oxidized to formaldehyde or formic acid by methanol dehydrogenase (MDH) to carbon dioxide. Biosynthesis of carbon compounds from this methane is carried out by the ribulose monophosphate cycle (RuMP cycle) and the serine cycle (serine cycle). , Type II methane-oxidizing bacteria are known to synthesize biomass using the serine circuit (Biocatalytic Conversion of Methane to Methanol as a Key Step for Development of Methane-Based Biorefineries J. Microbiol. Biotechnol.In Yeub Hwang et al (2014) ), 24 (12), 1597-605). Due to the physiological characteristics of methane oxidizing bacteria, efforts have been made to convert methane to methanol using methane oxidizing bacteria to reduce harmful methane gas and to utilize them as energy sources.

However, the bioprocess through methane oxidizing bacteria is a microbial reaction, which means that the reaction rate is slow, the mass production of microorganisms is difficult, the long-term stable culture is difficult, and the product cannot be efficiently recovered. Due to the problem, mass production of methanol through bioprocessing is difficult. Reportedly, in the synthesis of methanol using methylosinous tricosporium OB3b, a kind of conventional methane-oxidizing bacteria, its production capacity is about 30 μmol / min / g dry bacterium, which is not sufficient for commercial use, and thus is substantially (Optimization of Methanol Biosynthesis by Methylosinus) trichosporium OB3b, M. Tak eguti et al , Applied Biochemistry and Biotechnology vol68, 143-152 1997, Korea Patent Publication No. 2002-0073376 call).

As a solution to this problem, if methanol can be maintained or improved by oxidizing methane to methanol or selectively inhibiting the action of methanol dehydrogenase, the synthesis of methanol in high yield at room temperature and atmospheric conditions It is thought to be able to induce.

The present inventors have made diligent efforts to develop a method for producing methanol more efficiently using methane gas, and as a result, Methylomonas sp. The DH-1 strain was isolated and identified, and the DH-1 strain rapidly produced methanol in high yield, thus completing the present invention.

It is an object of the present invention to provide a Methylomonas sp. DH-1 strain deposited with accession number KCTC13004BP.

Another object of the present invention is to provide a composition for synthesizing methanol, comprising the DH-1 strain or a culture of the strain.

Still another object of the present invention is to provide a kit for methanol synthesis, comprising the DH-1 strain or the composition.

Still another object of the present invention is to provide a method for producing methanol, comprising the step of reacting the DH-1 strain or its culture product in a reaction vessel containing methane.

Since the DH-1 strain of the genus Methylmonas deposited with the accession number KCTC13004BP of the present invention can synthesize methanol, it can be widely used in biodegradable plastic production, methanol fuel cell, gasoline engine, and other biofuels. .

Figure 1 is an image of the DH-1 strain of the present invention observed under a microscope, Figure 1a is a scanning electron microscope, Figure 1b shows a result of observation with a projection electron microscope.

Figure 2 is a molecular biological phylogenetic diagram of the DH-1 strain of the present invention, Figure 2a is a phylogenetic diagram using the 16S rDNA gene sequence, Figure 2b is a particulate methane monooxygenase alpha subunit (particulate methane monooxygenase alpha subunit) , pmoA ) A phylogenetic map using the nucleotide sequence of a gene.

Figure 3 is a graph showing the methanol synthesis ability under various conditions of the DH-1 strain of the present invention, Figure 3a is methane concentration, Figure 3b is pH, Figure 3c is the dry cell weight of the DH-1 strain, Figure 3d is sodium formate (Sodium formate) concentration, Figure 3e shows the methanol synthesis ability of the DH-1 strain according to the EDTA concentration.

Figure 4 is a graph showing the methanol synthesis capacity in the optimum conditions for methanol production of the DH-1 strain of the present invention.

Figure 5 compares the methanol synthesis capability between the DH-1 strain and Sinners tricot sports Solarium in OB3b strain to another methane oxidizing bacteria methyl invention (Methylosinus trichosporium OB3b), Cellar silbeseuteuri methyl switch in BL2 strain (Methylocella silvestirs BL2) As a graph, the reaction was carried out at methanol production conditions optimized in the present invention.

As an embodiment for achieving the above object of the present invention, the present invention provides a Methylomonas sp. DH-1 strain deposited with accession number KCTC13004BP.

As used herein, the term " Methylomonas sp. DH-1 strain" refers to a strain belonging to the genus Methylomonas sp., Which is derived from sewage sludge and has not been reported in the past , 2016. It means a strain deposited with the Korea Institute of Bioscience and Biotechnology Center on April 8, 2010 and given accession number KCTC13004BP. The strain was deposited on August 27, 2015 to the Korea Research Institute of Bioscience and Biotechnology, and converted to a deposit under the Budapest Treaty, which was assigned the deposit number KCTC18400P, and the strain of accession number KCTC13004BP and the strain number KCTC18400P It is obvious that the same thing.

In the present invention, in order to find a strain suitable for the production of methanol using microorganisms, the present inventors have focused on the sewage sludge which methane is continuously generated, and succeeded in identifying and identifying a new methane-oxidizing bacterium, the genus Methyluromonas.

The strain is not limited thereto, but may be derived from at least one selected from the group consisting of sewage sludge as well as groundwater, surface water, industrial water, sewage, wastewater, and sewage.

As a result of analyzing the characteristics of the isolated strain, it was confirmed that the bacillus having a size of 1x1.5 μm through a scanning electron microscope, and of type I having an intracytoplasmic membrane (ICM) structure inside through a projection microscope. It was confirmed that the methane oxidizing bacteria (Fig. 1a, Fig. 1b).

The strain has a 16S rDNA gene as set out in SEQ ID NO: 3 and a pmoA gene as set out in SEQ ID NO: 6.

As used herein, the term '16S rDNA', also called 16S ribosomal DNA, refers to rDNA constituting the 30S subunit of prokaryotic ribosomes. While the sequence of the rDNA has almost no diversity among homologous species, it is used for bioidentification because of its diversity among other species, and identification and classification of organisms that are impossible or difficult to cultivate or organisms that have not been reported. This is especially useful for.

According to one embodiment of the present invention, the amplification of the 16S rDNA gene to determine the nucleotide sequence and molecular systematic analysis, showing a similarity of 99% with other strains belonging to the genus Methylonomonas, said strain belonging to the genus Methylonomonas It was found that the novel strain (Fig. 2a).

As used herein, the term "particulate methane monooxygenase (pMMO)" means an enzyme that catalyzes a chemical reaction for oxidizing methane contained in methane oxidized bacteria to methanol.

As used herein, the term "particulate methane monooxygenase alpha subunit ( pmoA )" means an alpha subunit that is one of three subunits of particulate methane monooxygenase. By gene means a gene expressing the methane monooxygenase alpha subunit.

According to an embodiment of the present invention, pmoA of the strain Molecular systematic analysis of the gene, Methylomonas koyamae The pmoA gene of the Fw12E-Y strain showed a similarity of 98% or more, indicating that the strain was a novel strain belonging to the genus Methylonomonas (FIG. 2B). Thus, the strain was named " Methylomonas sp. DH-1" and was deposited on April 8, 2016 to the Korea Institute of Bioscience and Biotechnology Biological Resource Center was given accession number KCTC13004BP.

As a result of analyzing the morphological and biochemical characteristics of the strain of the present invention, it is a gram-negative microorganism, which is resistant to the antibiotics chloramphenicol (Cam), tetracycline (Tet) and rifampicin (Rif), and in culture Appropriate temperature is 30 ℃, the appropriate concentration of copper ion is 10 μM, it was confirmed that the growth well under the condition of the air: methane ratio of 7: 3 (Table 1). In addition, the strain was found to grow well in the carbon source of methane, methanol, methylamine (methylamine), esculin (Table 2).

In addition, the strain is a condition in which 30 to 70% of methane, specifically 40% of methane is present; PH conditions of 6 to 8, specifically pH 7 conditions; 0.6 to 2.4 g dry cell weight / L, specifically, 2.4 g dry cell weight / L; Conditions in which 20 to 200 mM sodium formate, specifically 40 mM sodium formate, is present; In the presence of 0.3 to 5 mM EDTA, specifically, 0.5 mM EDTA, it was confirmed that methanol production capacity was maximized (FIGS. 3A to 3E), and methanol production capacity was maximized within 4 hours of reaction. It was confirmed that the synthesis capacity was maintained up to 12 hours (Fig. 4).

In addition, the DH-1 strain was compared with Sinners tricot sports Solarium in OB3b strain (Methylosinus trichosporium OB3b) or methyl Cellar silbeseuteuri's in BL2 strain (Methylocella silvestirs BL2) to a different methane oxidizing bacteria, methyl, respectively, 130%, or It was confirmed to exhibit a 230% high methanol production capacity (FIG. 5). This suggests that the DH-1 strain of the genus Methylonomonas of the present invention can produce methanol from methane in a high yield in a fast reaction time compared to other methanogenic bacteria, and thus biofuel or various chemical processes using methanol. It suggests that it can be widely used for application as a material.

As known from Korean Patent Laid-Open Publication No. 2002-0073376, it is disclosed that the methanogenic bacterium, Methylus sinus tricosporium OB3b strain, produces 14 mM methanol after 40 hours of reaction at 0.6 mg dry cell weight / mL. In addition, according to the existing literature (Biotechnology and Bioprocess Engineering 15: 476-480, 2010. Hee Gon Kim), the OB3b strain produced 13.2 mM methanol after 12 hours of reaction at 0.6 mg dry cell weight / mL. Although disclosed, the DH-1 strain of the present invention can produce 41.5 mM of methanol within 4 hours, so that the DH-1 strain of the present invention can produce a large amount of methanol much faster than conventional methanogenic bacteria. It suggests that it can be used more effectively for methanol synthesis. In addition, the DH-1 strain of the present invention This suggests that it can be a solution to overcome the problems of slow reaction speed and difficulty of mass production, which have been suggested as problems of methanol production using existing microorganisms.

As another embodiment for achieving the above object of the present invention, the present invention provides a composition for synthesizing methanol comprising the methylomonas DH-1 strain or a culture of the strain.

Since the strain of methyl genus DH-1 provided by the present invention can synthesize methanol, the culture product of the strain of methylomonas DH-1 included in the composition is not particularly limited as long as it can synthesize methanol. Specific examples may be a culture of the strain, a culture supernatant, a lysate, fractions thereof, and the like. In this case, the culture supernatant may be obtained by centrifugation of the culture of DH-1 strain of Methylmonas, and the lysate may be obtained by physically or ultrasonically treating the DH-1 strain of Methylmonas, Fractions can be obtained by applying the culture, culture supernatant, lysate and the like to a method such as centrifugation, chromatography.

In addition, the composition for synthesizing methanol of the present invention may further include other strains that may increase the methanol production rate or increase the methanol production rate, in addition to the methylomonas DH-1 strain. Strains that can be included in the add is in particular to this the but are not limited and preferably methane, methane, methyl-oxidizing bacteria capable of oxidizing methanol Pseudomonas genus (Methylomonas), bakteo methyl in (Methylobacter), methyl Rhodococcus ( Methylococcus ), Methylomicrobium , Methylosphaera , Methylocaldum , Methyloglobus , Methylosarcina , Methylosarcina , Methylosarcina Methyloprofundus , Methylothermus , Methylohalobius , Methylogaea , Methylomarinum , Methylovulum , Marino penalized in (Methylomarinovum), methyl Love column in (Methylorubrum), methyl Paracoccus genus (Methyloparacoccus), Sinners in (Methylosinus), seutiseu in (Methylocystis) when a methyl methyl, methyl Cellar in (Methylocella), kaepsa in (Methylocapsa) methyl, methyl hydroperoxide in Lula (Methylofurula), methyl O CD pilreom in (Methylacidiphilum), methyl O CD micro byum in (Methylacidimicrobium) And the like may be combined.

As another embodiment for achieving the object of the present invention, the present invention provides a kit for methanol synthesis comprising the composition.

The kit of the present invention may be used to synthesize methanol, including the composition, but is not particularly limited thereto, and may include one or more other component compositions, solutions, or devices suitable for the reaction, and in particular, the composition. DH-1 strain culture medium of the genus Methylonomonas contained in, a buffer for methanol synthesis, a reaction vessel for performing the methanol synthesis, a temperature controller for performing the methanol synthesis, for performing the methanol synthesis reaction A timer may be included.

More specifically, the kit for synthesizing methanol of the present invention may include a DH-1 strain of the genus Methylonomonas, the culture medium for the strain, a buffer used for methanol synthesis, and a constant temperature reaction vessel used for methanol synthesis.

As another embodiment for achieving the object of the present invention, the present invention comprises the step of producing methanol, comprising the step of reacting the genus DH-1 strain or culture products thereof in a reaction vessel containing methane to provide.

In the present invention, the gas in the reaction vessel may contain methane 30 to 70% (v / v), specifically 40% (v / v); PH in the reaction vessel may be 6.0 to 8.0, specifically pH 7.0; The strain may comprise 0.6 to 2.4 g dry cell weight / L, specifically 2.4 g dry cell weight / L; The reaction vessel may further comprise 20 to 200 mM sodium formate, specifically 40 mM sodium formate; The reaction vessel may further include 0.3 to 5 mM EDTA, specifically, 0.5 mM EDTA, but is not limited thereto.

According to one embodiment of the present invention, when the reaction is carried out by including 40% methane, sodium phosphate pH 7, 2.4 g dry cell weight / L, 40 mM sodium formate, 0.5 mM EDTA in the reaction vessel, It was confirmed that the methanol production capacity is maximized compared to the conditions (FIGS. 3A to 3E).

Hereinafter, the present invention will be described in more detail by examples. However, the following examples are merely to illustrate the invention, but the content of the present invention is not limited to the following examples.

Example 1 Isolation of Methane Oxidized Bacteria from Sewage Sludge

The method for separating methane oxidizing bacteria from sewage sludge is as follows. The collected sewage sludge is nitrate-mineral-salt (NMS) medium (per liter; 1 g MgSO ₄ · 7H ₂ O, 1 g KNO ₃ , 0.2 g CaCl ₂ · H ₂ O, 0.0038 g Fe-EDTA, 0.0005 g NaMo · 4H ₂ O) in the trace elements solution (1x) 1 mL ( per _{liter; 500 mg FeSO 4 · 7H} 2 O, 400 mg ZnSO 4 · 7H 2 O, 20 mg MnCl 2 · 7H 2 O, 50 mg CoCl 2 · 6H ₂ O, 10 mg NiCl ₂ .6H ₂ O, 15 mg H ₃ BO ₃ , 250 mg EDTA), phosphate stock solution (1x) 10 mL (per liter; 26 g KH ₂ PO ₄ , 62 g Na ₂ HPO ₄ 7H ₂ O), vitamin stock (1x) 2 mL (per liter; 2 mg Biotin, 2 mg Folic acid, 5 mg Thimine HCl, 5 mg Ca pantothenate, 0.1 mg Vitamin B12, 5 mg Riboflavin, 5 mg Nicotinamide) After addition to the contained test tube, the test tube was sealed using a rubber stopper and insulating tape. Then, 30% of air was removed using a syringe, and methane gas was injected to adjust the final concentration of methane gas in the test tube to 30%. Then, it was incubated at 30 ℃, 230 rpm. Once the microorganisms were sufficiently cultured in the medium, the microorganisms were plated in a solid NMS agar. The smeared solid NMS agar was placed in a vacuum desiccator, sealed to maintain a ratio of air and methane 7: 3, and then incubated at 30 ° C. for several days. After several days, the resulting strains were harvested and subcultured two to three times to isolate single strains.

In order to observe the appearance of the isolated methane-oxidized bacteria, the results were observed using a scanning electron microscope and a projection electron microscope, and the results are shown in FIGS. 1A and B. As shown in Figure 1a, b it was confirmed that the bacterium having a size of 1x1.5 ㎛ through a scanning microscope, type I methane oxidizing bacteria having an intracytoplasmic membrane (ICM) structure inside through a projection microscope. .

Example 2: Identification and Naming of Novel Methylonomonas Strains

In order to identify the methane-oxidized bacteria isolated in Example 1, sequencing of the 16S rDNA gene was performed as follows. After amplification by PCR using primers described in SEQ ID NO: 1 and SEQ ID NO: 2 for 16S rDNA gene amplification, the amplified PCR product was analyzed by sequencing reaction.

Forward primer 27F (5'-AGAGTTTGATCCTGGCTCAG-3 '): (SEQ ID NO: 1)

Reverse primer 1492R (5'-GGTTACCTTGTTACGACTT-3 '): (SEQ ID NO: 2)

As a result of examining the homology of the microorganisms using the BLAST of the NCBI (National Center for Biotechnology Information (NCBI)), the nucleotide sequence of the strain isolated from the present invention was Methylomonas. koyamae At least 99% similarity with the Fw12E-Y strain. The result of analysis of the 16S rDNA base sequence of the strain was described as SEQ ID NO: 3, and the systematic result using the analyzed base sequence was inferred by the evolutionary distance and the phylogeny between the base sequences by the Kimura 2-parameter model and the neighbor-joining method. (FIG. 2A).

In addition, after the amplification by PCR using the primers described in SEQ ID NO: 4 and SEQ ID NO: 5 to analyze the nucleotide sequence of the particulate methane monooxygenase alpha subunit ( pmoA ) gene of the strain The sequence was analyzed by sequencing the amplified PCR product.

Forward primer A189F (5'-GGNGACTGGGACTTCTGG-3 '): (SEQ ID NO: 4)

Reverse primer mb661R (5'-CCGGMGCAACGTCYTTACC-3 '): (SEQ ID NO: 5)

As a result of examining the homology of the microorganisms using the BLAST of the NCBI (National Center for Biotechnology Information), the pmoA gene of the strain was Methylomonas. koyamae At least 98% similarity with the pmoA gene of Fw12E-Y strain. The nucleotide sequence of the pmoA gene of the strain was described as SEQ ID NO: 6, and the phylogenetic results using the analyzed nucleotide sequence were inferred by the evolutionary distance between the nucleotide sequences and the phylogenetic tree by the Kimura 2-parameter model and the neighbor-joining method ( 2b).

Based on the above results, it was confirmed that the microorganism isolated in the present invention is Methylomonas sp. Which is widely known as a microorganism for oxidizing methane to methanol.

Accordingly, the present inventors named the strain " Metthlyomonas sp. DH-1", and deposited it on the biological resource setter of the Korea Research Institute of Bioscience and Biotechnology on April 8, 2016 and received the accession number KCTC13004BP. .

Example 3: Morphological and Biochemical Properties of DH-1 Strains

Morphological characteristics of DH-1 strain of genus Meromonas were observed by scanning electron microscope (Scanning Electron Microscope, SEM) and transmission electron microscope (Transmission Electron Microscope, TEM) (Fig. 1). As shown in FIG. 1, the DH-1 strain showed a rod form, which is a typical form of methane oxidizing bacteria, and showed a colony color of Light Yellow-> Yellow-> Orange-> Brown in a solid medium.

In addition, as shown in Table 1, as a biochemical property, the DH-1 strain is a Gram-negative microorganism, and is resistant to antibiotics chloramphenicol (Cam), tetracycline (Tet), and rifampicin (Rif). In addition, it was confirmed that the proper temperature in the culture was 30 ° C, the appropriate concentration of copper ions was 10 µM, and well-cultivated under the condition that the air: methane ratio was 7: 3.

Example 4: Substrate Availability of DH-1 Strains

Substrate utilization of the DH-1 strain of the genus Methyluromonas isolated in Example 1 and Example 2 was investigated, and the results are shown in Table 2 below.

As shown in Table 2, the DH-1 strain was found to grow well in the carbon source of methane, methanol, methylamine (Methylamine) or esculin (Esculin).

Example 5 Comparison of Fatty Acid Compositions of Various Methanogenic Bacteria and DH-1 Strains

The fatty acid composition of the methyl of the type Ⅰ methane oxidizing bacteria Pseudomonas genus (Methylosinus), bakteo in (Methylobacter), micro-away in (Methylomicarobium) and Rhodococcus in a methyl (Methylococcu s) and, DH-1 strain of methyl methyl Were compared, and the results are shown in Table 3.

In Table 3, 14: 0 means 14 carbon atoms and saturated fatty acids. In the table, 16: 1ω8c means an unsaturated fatty acid having 16 carbon atoms and having a double bond of cis structure on carbon 8.

In addition, as shown in Table 3, the saturated fatty acid 14: 0 of the DH-1 strain showed 23.59%, showing a similar result in the genus methylomonas and fatty acid composition. However, the unsaturated fatty acids 16: 1ω8c, 16: 1ω7c, 16: 1ω6c, 16: 1ω5c and 16: 1ω8t showed different fatty acid compositions from the genus Methylonomonas, and 16: 1ω7c (31.82%) and 16: 1ω6c (3.52% ) Showed similar results for the genus Methyloccus and fatty acid composition.

Example 6 Establishment of Optimal Conditions for Methanol Synthesis of DH-1 Strains

In order to analyze the methanol synthesis ability of the isolated and identified methyl monascus DH-1 strains in Examples 1 and 2, in the reaction solution of different growth conditions through the following Examples 6-1 to 6-5 The optimum conditions for methanol production were confirmed by measuring the methanol production amount of.

Example 6-1 Analysis of Methanol Production According to Methane Concentration

1 ml of a reaction solution containing 20 mM sodium phosphate buffer (pH 6.3), 0.6 g dry cell weight / L of DH-1 strain, 0.5 mM EDTA, and 40 mM sodium formate was placed in a reaction vessel having a volume of 24 ml. After varying the methane concentration to 10 to 90%, methanol production was carried out for 12 hours at 30 rpm at 230 rpm. After the reaction was completed, the amount of methanol produced was measured. At this time, the amount of methanol produced was left to react at 90 ℃ heating block (heating block) 30 minutes to terminate the reaction, centrifuged at 13000 rpm to obtain a supernatant, and then the methanol production of the reaction solution through GC analysis Measured.

As a result, as shown in Figure 3a, it was confirmed that the methanol synthesis ability is excellent when providing 30 to 70% of methane, in particular, 0.706 g / L methanol is produced when providing 40% of methane, It was confirmed that the methanol synthesis capacity was maximized compared to the concentration.

Example 6-2 Analysis of Methanol Production According to pH

1 ml of a reaction solution containing 20 mM sodium phosphate buffer solution having a pH of 4 to 10, 0.6 g dry cell weight / L of DH-1 strain, 0.5 mM EDTA, and 40 mM sodium formate is placed in a reaction vessel having a volume of 24 ml. Methane concentration was set to 40% at the top, and methanol production was performed for 4 hours at 230 ° C. at 230 rpm. After the reaction was completed, the amount of methanol produced by the method according to Example 6-1 was measured.

As a result, as shown in Figure 3b, it was confirmed that the methanol synthesis ability is excellent in the pH conditions of 6 to 8, in particular, when using a sodium phosphate buffer solution of pH 7.0 as the reaction solution to produce 0.302 g / L methanol As a result, it was confirmed that methanol synthesis ability was maximized compared to other pH conditions.

Example 6-3 Analysis of Methanol Production According to Dry Cell Weight

1 ml of a reaction solution containing 20 mM sodium phosphate buffer (pH 7.0), 0.15 to 4.8 g dry cell weight / L of DH-1 strain, 0.5 mM EDTA, and 40 mM sodium formate was placed in a reaction vessel having a volume of 24 ml. After the upper methane concentration was set to 40%, methanol production was performed at 30 ° C. at 230 rpm for 1 hour. After the reaction was completed, the amount of methanol produced by the method according to Example 6-1 was measured.

As a result, as shown in Figure 3c, it was confirmed that methanol synthesis ability is excellent under the conditions of 0.6 to 2.4g dry cell weight / L, in particular, 0.256 g / L when 2.4 g dry cell weight / L DH-1 strain is present By producing methanol, it was confirmed that the methanol synthesis capacity was maximized compared to other dry cell weight conditions.

Example  6-4: Sodium formate  Methanol production by concentration

1 ml of a reaction solution containing 20 mM sodium phosphate buffer (pH 7.0), 2.4 g dry cell weight / L of DH-1 strain, 0.5 mM EDTA, and 0 to 200 mM sodium formate was placed in a reaction vessel having a volume of 24 ml. After setting the methane concentration of the upper portion to 40%, methanol production reaction was carried out for 12 hours at 230 rpm at 30 ℃. After the reaction was completed, the amount of methanol produced by the method according to Example 6-1 was measured.

As a result, as shown in Figure 3d, it was confirmed that the methanol synthesis ability is excellent under the conditions of 20 to 200 mM sodium formate, in particular 0.785 g / L methanol is produced in the presence of 40 mM sodium formate, different sodium formate concentration Compared to the methanol synthesis capacity was confirmed to the maximum.

Example 6-5 Analysis of Methanol Production According to EDTA Concentration

1 ml of a reaction solution containing 20 mM sodium phosphate buffer (pH 7.0), 2.4 g dry cell weight / L of DH-1 strain, 0 to 15 mM EDTA, and 40 mM sodium formate was placed in a reaction vessel having a volume of 24 ml. After setting the methane concentration of the upper portion to 40%, methanol production reaction was carried out for 12 hours at 230 rpm at 30 ℃. After the reaction was completed, the amount of methanol produced by the method according to Example 6-1 was measured.

As a result, as shown in Figure 3e, it was confirmed that methanol synthesis ability is excellent under the conditions of 0.3 to 5 mM EDTA, in particular, 0.809 g / L methanol is produced in the presence of 0.5 mM EDTA, methanol compared to other EDTA concentrations It was confirmed that the synthetic capacity was maximum.

Example 7: Confirmation of maximum methanol synthesis capacity of DH-1 strain

In order to analyze the methanol synthesis ability of the DH-1 strain of the genus M. monomonas isolated and identified in Examples 1 and 2, the DH-1 strain was reacted under the optimum growth conditions established in Example 6 above. .

Specifically, 2.4 g dry cell weight / L DH-1 strain, 0.5 mM EDTA, in 1 ml of a reaction solvent containing 40 mM sodium formate, 0.5 mM EDTA, and 20 mM sodium phosphate (pH 7.0) relative to the total reaction solution. And 40 mM sodium formate was added to a reaction container having a volume of 24 ml, and the upper methane concentration was set to 40%. Then, methanol production reaction was performed at 230 ° C. at 230 rpm for 0 to 24 hours. After the reaction was completed, the amount of methanol produced by the method according to Example 6-1 was measured.

As a result, as shown in Fig. 4, the methanol production capacity was maximized within 4 hours of the reaction when incubated in the above conditions containing sodium formate and EDTA at an appropriate concentration, as compared with the case where nothing was added or only sodium formate was added. It was confirmed that the methanol synthesis ability of the DH-1 strain was confirmed to be maintained up to 12 hours.

Example 8 Comparison of Methanol Synthesis Ability between Methane Oxidized Bacteria

In order to analyze the methanol synthesis ability of the isolates identified in Example 1 and Example 2 of the genus Methylone Monas DH-1, other methanogenic bacteria and methanol synthesis ability was analyzed.

To be specific, the embodiment optimal growth conditions established through 6 with DH-1 strain, Sinners tricot sports Solarium in OB3b strain as methyl (Methylosinus trichosporium OB3b), Cellar silbeseuteuri's in BL2 strain as methyl (Methylocella silvestirs BL2) Each was reacted for 4 hours.

As a result, as shown in Figure 5, the DH-1 strain was able to confirm that the methanol production capacity of 130%, 230% higher than the other methane oxidizing bacteria OB3b strain and BL2 strain, respectively. Through the above results, it can be seen that the genus MH of the present invention DH-1 strain can produce methanol from methane in a high yield in a fast reaction time.

From the above description, those skilled in the art will appreciate that the present invention can be implemented in other specific forms without changing the technical spirit or essential features. In this regard, the embodiments described above are to be understood in all respects as illustrative and not restrictive. The scope of the present invention should be construed that all changes or modifications derived from the meaning and scope of the following claims and equivalent concepts rather than the detailed description are included in the scope of the present invention.

Claims (14)

1. Methylomonas sp. DH-1 strain deposited with accession number KCTC13004BP.
2. According to claim 1, The strain is characterized in that it has a 16S rDNA gene represented by SEQ ID NO: 3.
3. The strain according to claim 1, wherein the strain has a particulate methane monooxygenase alpha subunit ( pmoA ) gene represented by SEQ ID NO: 6.
4. The strain of claim 1, wherein the strain is isolated from sewage sludge.
5. The strain of claim 1, wherein the strain synthesizes methanol from methane.
6. Claim 1 of the methylomonas genus DH-1 strain comprising a culture product of the strain, the composition for methanol synthesis.
7. The composition of claim 6, wherein the culture product is selected from the group consisting of cultures, culture supernatants, lysates, and fractions thereof of the DH-1 strain of Methylonomonas.
8. A kit for synthesizing methanol comprising the DH-1 strain of the genus Methylone of claim 1 or the composition of claim 6.
9. The method of claim 1, comprising the step of reacting the strain or the culture product thereof in a reaction vessel containing methane.
10. The method of claim 9, wherein the air in the reaction vessel contains 30 to 70% (v / v) of methane.
11. The method of claim 9, wherein the pH in the reaction vessel is 6.0 to 8.0, the production method of methanol.
12. The method of claim 9, wherein the strain is comprised of 0.6 to 2.4 g dry cell weight / L.
13. The method of claim 9, wherein the reaction vessel further comprises 20 to 200 mM sodium formate.
14. 10. The method of claim 9, wherein the reaction vessel further comprises 0.3 to 5 mM EDTA.

Images