KR101895683B1 - A high yield of methanol production method using covalent immobilized methylosinus sporium on the chitosan and a composition therefor - Google Patents

Abstract

The present invention relates to a high yield methanol production method and a composition thereof using methyl Rhosenerosporium spontaneously immobilized on chitosan. According to the present invention, by using M. sporium cells covalently immobilized on chitosan, stability during methanol production And the efficiency of methanol production is not significantly lowered even after 6 cycles of reuse, so it can be reused many times. In addition, pH, incubation temperature of the culture medium, stirring rate, was through optimization and MDH inhibition of the process parameters of density, such as phosphate methanol production increase of about 6.7 times that of methanol, the production method using the conventional micro-organisms, pure CH ₄ as well as synthetic It is possible to efficiently produce methanol from the gas mixture, thereby effectively reducing the greenhouse gas.

Description

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a high yield methanol production method using methylosynthosphospheres which are covalently immobilized on chitosan and a composition therefor,

The present invention relates to a method for producing high yield methanol using methylosinus sporium covalently immobilized on chitosan and a composition thereof.

Increasing concentrations of greenhouse gases (GHG) such as methane (CH ₄ ) and carbon dioxide (CO ₂ ) are global environmental issues. CH ₄ is one of the most abundant natural gases and is an abundant energy source, but the CH 4 is a primary greenhouse gas and has recently received more attention than CO ₂ due to its severely harmful environmental impact. Therefore, to explore the feasibility of a process that in order to reduce the harmful effects of greenhouse gases CH _4, the CH ₄ conversion to value-added products such as methanol are necessary.

Methanol is the basic substrate for a wide range of chemical synthesis reactions and is used as fuel in gasoline mixtures. In addition to lower costs associated with the storage and movement of methanol, the methanol is I have a high energy density, which is higher than about 400 times the CH _4.

However, chemical conversion of CH ₄ to methanol has problems such as high cost of the process and low energy efficiency. Compared to these chemical methods, biological processes are more environmentally friendly and have the advantages of high conversion, selectivity, and low capital / energy costs. Thus, efforts to develop methods for the bioconversion of CH ₄ to methanol have been increasing recently.

Methanotrophic bacteria refers to a group of bacteria that grows by using methane as a carbon source and an energy source. Methylomonas, Methanomonas, Methylococcus, Methylosinus, Methylobacter, Methylomicrobium, and Methylocystis. The methanogenic bacteria are methane monooxygenase (hereinafter abbreviated as MMO), which oxidizes methane to methanol, and methanol dehydrogenase (hereinafter referred to as MDH), which oxidizes methanol to formaldehyde. ) To convert methane gas to carbon dioxide. Specifically, the methane oxidizing bacteria oxidize methane to methanol and oxidize the methanol again to formaldehyde. Then, formaldehyde is oxidized to formic acid, which ultimately converts to less toxic carbon dioxide. In addition, the methanogenic bacteria can not use an organic compound having a carbon-carbon bond as a growth material, but many alkanes and aromatics can be oxidized by the enzymatic action of MMO, an enzyme produced when methane is oxidized.

The MMOs are classified into three groups based on the type of MMO they produce: (i) Type I methanotrophic bacteria, which produce only particulate MMO (pMMO); (ii) Type II Methane oxidizing bacteria, the methane oxidizing bacteria produce both pMMO and soluble MMO (sMMO), and (iii) the type X methane oxidizing bacteria, the methane oxidizing bacteria are MMOs having specific properties common to the I and II organisms [Fei Q, et al., Biotechnol Adv 2014; 32: 596-614]. pMMO is associated with a membrane-bound microparticle enzyme system, while sMMO is located within the cytoplasmic complex. The expression of these enzymes is highly dependent on the copper (Cu) concentration in the growth medium. pMMO expression is dominant as Cu level is higher, and sMMO is predominant as Cu concentration is lower. pMMO has a higher affinity for CH ₄ oxidation than sMMO.

Et al., Biotechnol Bioeng 1991; 37: 551-6], and OB3b [Hwang IY, et al., Biochem Biotech 2004; , J Microbiol Biotechnol 2015; 25: 375-80] bioconversion of pure methanol from CH ₄ by Sinners tricot sports Solarium (Methylosinus trichosporium) in various species, including methyl have been widely studied. However, the initial methanol production was very low and the maximum yield ranged from 0.16 to 1.84 mM. Therefore, there is a need for an improved method of improving methanol production in a method of producing methanol by methane oxidizing bacteria.

Korean Patent Publication No. 1020110006964 Korean Patent Publication No. 1020160026087

Xin J-Y, et al., Biocatal Biotrans 2004; 22: 225-9, Mehta PK, et al., Biotechnol Bioeng 1991; 37: 551-6, Hwang IY, et al., J Microbiol Biotechnol 2015; 25: 375-80

It is an object of the present invention to provide a method for biosynthesizing methanol from a methane or syngas mixture with methane oxidizing bacteria at a high yield.

It is another object of the present invention to provide a composition for the production of methanol from a methane or syngas mixture in high yield.

In order to achieve the above object, the present invention provides a method for producing high-yield methanol from a methane or syngas mixture using methylosinus sporium covalently immobilized on chitosan.

In addition, preferably, the methanol production method may include the step of culturing the methylosynthosporium cells covalently immobilized on chitosan and a methane or syngas mixture as a feedstock in a culture solution while stirring.

In addition, preferably, the culture liquid may include a phosphate buffer solution and a nitrate mineral salt (NMS).

Further, preferably, the culture liquid may further include at least one member selected from the group consisting of Cu ion, MDH inhibitor and formate salt.

Preferably, the concentration of the phosphate buffer solution may be 100 mM.

Preferably, the Cu ion is 5 [mu] M and the concentration of the formic acid salt is 40 mM.

Preferably, the MDH inhibitor is selected from the group consisting of EDTA, MgCl ₂ , NaCl and NH ₄ Cl, wherein the optimum concentration is 1.0 mM EDTA, 20 mM MgCl ₂ , 80 mM NaCl, and NH ₄ Cl Can be 15 mM.

Preferably, the culture is carried out at a pH of 6.8-7.0 and at a stirring rate of 150-180 rpm at 25-30 ° C for 24 hours.

Also, preferably the optimum reaction volume to top space ratio is 1: 5 and the feedstock injection concentration may be 30-50%.

Preferably, the syngas mixture may be prepared by mixing CH ₄ , CO ₂ , and H ₂ in a ratio of 6: 3: 1.

In addition, the present invention relates to a method for producing methanol from a methane or a syngas mixture, comprising the steps of: (a) cultivating a culture medium containing methanol as an active ingredient in a culture medium containing methyl rhinosporphosphate cells, phosphate buffer solution and nitrate mineral salt (NMS) By weight of methanol.

Preferably, the composition further comprises at least one member selected from the group consisting of Cu ion, MDH inhibitor and formate salt.

According to the present invention, by using the M. sporium cells covalently immobilized on chitosan, the stability is improved during methanol production, and the methanol production efficiency is not lowered even after 6 cycles of reuse, so that it can be reused many times. In addition, pH, incubation temperature of the culture medium, stirring rate, was through optimization and MDH inhibition of the process parameters of density, such as phosphate methanol production increase of about 6.7 times that of methanol, the production method using the conventional micro-organisms, pure CH ₄ as well as synthetic It is possible to efficiently produce methanol from the gas mixture, thereby effectively reducing the greenhouse gas.

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 is a graph showing the effect of phosphate buffer in the production of methanol by M. sporium in one embodiment of the present invention.
Figure 2 is a graph showing the effect of MDH inhibitors in phosphate buffer in the production of methanol by M. sporium in one embodiment of the present invention.
Figure 3 is a graph showing the relative inhibition of MDH and (b) the concentration of formate salt in methanol production by methyl sporinum ( M. sporium ) of one embodiment of the present invention (a) at the optimal inhibitor concentration .
FIG. 4 is a graph showing the methanol production profile according to different methane concentration and incubation time in the production of methanol by M. sporium in one embodiment of the present invention. FIG.
FIG. 5 is a scanning electron microscope (SEM) photograph ((a) single particle and (b) high resolution photograph) of methylosynthosporium ( M. sporium ) covalently immobilized on chitosan.
6 is a graph showing the reusability of free or immobilized methyl Rhosine spores in the production of methanol by M. sporium in one embodiment of the present invention.
Figure 7 is a graph showing the methanol production profile from the syngas mixture in the methanol production by M. sporium in one embodiment of the present invention.

Hereinafter, the present invention will be described in detail.

The method for producing high yield methanol according to the present invention is characterized in that methylosinus ( Methylosinus < RTI ID = 0.0 > sporium ; M. sporium ).

In the present invention, in order to confirm methanol production potential in Korean Patent Laid-Open Publication No. 1020160026087, methylene synthosphosphorium was identified as one of methanotrophic bacteria, and methanol production from various methanotrophic bacteria was examined. As a result, Leum was selected as the optimal strain by producing the highest methanol.

However, in this case methyl maximum methanol yield by Sinners sports Solarium is a 473 μM, so was lower, Sinners tricot sports Solarium (Methylosinus trichosporium) species production of methanol from pure CH ₄ by a different methane variety methyl-oxidizing bacteria The conversion also ranged from 0.16-1.84 mM in maximum yield.

Therefore, an improved method for improving methanol production in a method for producing methanol by methane-oxidizing bacterium has been desired, and the present inventors have found that the growth conditions, production conditions, process parameters, and MDH inhibitors through optimization of the concentration of was confirmed that this can improve the methanol yield from CH ₄ (see Fig. 1 to 4), especially, using chitosan as the support material, with methyl the cells in the chitosan Sinners sports Solarium cells (See FIGS. 6 and 7). In addition, the present invention has been made in view of the above problems.

In the present invention, the method of immobilizing methylotrophosphate cells on chitosan is preferably a covalent immobilization. In the covalent immobilization, chitosan is first functionalized with glutaraldehyde to attach an aldehyde group to chitosan particles, and then functionalized Adding chitosan and methylosynthosphosphate cells and stirring. The shared immobilization can be confirmed by a scanning electron microscope or the like (see FIG. 5).

Methylosynergosporium cells covalently immobilized on the chitosan according to the present invention were prepared by the conventional method (1.37-2.34 mM) in which pure CH ₄ was used as a feedstock and the methanol yield was 4.61 mM and encapsulated using a polymer matrix as a support The methanol yield was improved by 2-3.5 times as compared with the methanol productivity. In addition, when the synthesis gas mixture was used as the feedstock, the methanol yield was 6.12 mM, which was higher than the yield by free cell (5.80 mM) (see Table 3).

Thus, the methanol production process using M. sporium covalently immobilized to the chitosan according to the present invention can lead to significantly higher methanol production from both pure CH ₄ and the synthesis gas mixture.

In the present invention, the methanol production method includes a step of culturing the methylolated synthosphosphate cells covalently immobilized on chitosan and a methane or syngas mixture as a feedstock in a culture broth while stirring.

At this time, the culture solution contains phosphate buffer solution and nitrate mineral salt (NMS), and may further include Cu ion, MDH inhibitor, formate salt and the like.

In the present invention, the concentration of the phosphate buffer solution is preferably 100 mM, the Cu ion is preferably 5 μM, and the concentration of the formic acid salt is preferably 40 mM.

In the present invention, EDTA, MgCl ₂ , NaCl or NH ₄ Cl may be used as the MDH inhibitor, wherein the optimum concentrations are 1.0 mM of EDTA, 20 mM of MgCl ₂ , 80 mM of NaCl, and NH ₄ Cl 15 mM.

In the present invention, the pH of the culture liquid is preferably 6.8-7.0.

In the present invention, it is preferable that the culturing is carried out at 25-30 DEG C for 24 hours at a stirring speed of 150-180 rpm.

In the present invention, the optimal reaction volume to space ratio is preferably 1: 5, and the feedstock injection concentration is preferably 30-50%.

In the present invention, the synthesis gas mixture is preferably mixed with CH ₄ , CO ₂ , and H ₂ in a ratio of 6: 3: 1.

In addition, the present invention relates to a method for producing a chitosan-containing microorganism which comprises, as an active ingredient, a methylhydrocinosphosphate cell, The present invention provides a high-yield composition for methanol production.

The composition may further comprise Cu ions, MDH inhibitors, formates, and the like.

The composition according to the present invention is a composition used in the above-mentioned high yield methanol production method, and the common description of both inventions is omitted in order to avoid excessive complexity of the specification according to the repeating material.

Hereinafter, the present invention will be described in more detail with reference to Examples. It will be apparent to those skilled in the art that these embodiments are merely illustrative of the present invention and that the scope of the present invention is not limited to these embodiments.

< Example  1> &lt; / RTI &gt; Methyl rosiners Sporeum  Production of Methanol from Used Methane

(1) On chitosan Methyl rosiners Sporium  Sharing Immobilization

M. sporium DSMZ 17706 was obtained from the German Collection of Microorganisms and Cell Cultures, and (gL ^-1 ) units of KH ₂ PO ₄ (0.26), Na _{2 HPO 4 · 12H 2 O (0.716} ), KNO 3 (1.0), CaCl 2 (0.20), MgSO 4 · 7H 2 O (1.0), Fe-EDTA (0.38), and _{Na 2 MO 4 · 2H 2 O} (0.026 ) On a nitrate mineral salt (NMS). Next, ZnSO ₄ .7H ₂ O (0.40) (gL ^-1 ), H ₃ BO ₃ (0.015), CoCl ₂ .6H ₂ O (0.050), Na ₂ -EDTA (0.250), MnCl ₂ .4H ₂ O (0.020), and NiCl ₂ .6H ₂ O (0.010) was added to the culture medium. The pH of the culture was adjusted to 7.0 using 1M H ₂ SO ₄ and 1M NaOH. Millipore water (18 MX) was used for all reagent production and for all measurements. All chemicals were analytical grade and were purchased from Sigma-Aldrich or other commercial sources. The M. sporium strains were maintained by secondary culture every 2 weeks and stored at 4 DEG C on NMS agar plates. The presence of contaminants was checked on R2 agar (Fluka, USA).

Next, chitosan (90% deacetylation, crab shell) as a support was purchased from Bio Basic Inc. (Canada). The chitosan (20 g) was washed twice with distilled water and then with phosphate buffer (50 mM, pH 7.0). The chitosan was functionalized with glutaraldehyde to attach aldehyde groups to the particles as described in Patel SKS, et al., J Microbiol Biotechnol 2014; 24: 639-47. The chitosan was then separated by centrifugation at 4000 rpm for 30 minutes and the remaining glutaraldehyde was removed by washing it with distilled water three times and then with phosphate buffer (20 mM). All cells were fixed on the chitosan by adding 100 mg of M. sporium cells per g of functionalized chitosan in phosphate buffer (50 mM, pH 7.0) and stirring overnight at 100 rpm at 4 ° C. Unbound cells were separated by centrifugation at 4000 rpm for 20 min. The fixed cells were washed twice with phosphate buffer (20 mM, pH 7.0) containing MgCl ₂ (5 mM) &Lt; / RTI &gt;

For primary immobilization, M. sporium cells covalently immobilized on chitosan were transferred to Karnovsky's fixative solution. Next, the samples were dehydrated in graded ethanol (Duksan, South Korea; 30%, 50%, 70%, 80%, 90%, and 100%; 10 min each at 4 ° C) Lt; / RTI &gt; and coated with platinum. The images were analyzed using a field emission scanning electron microscope (JEOL, Japan) and shown in FIG.

In Fig. 5, (a) represents a single particle and (b) represents a high-resolution image.

As shown in Fig. 5, on the chitosan particles, M. sporium Cells were stably immobilized.

(2) a chitosan- Methyl rosiners Sporeum  Production of Methanol from Used Methane

30% CH under ₄ atmosphere containing containing yeomreul 200 mL of nitric acid, an inorganic, a closed screw cap (screw cap) (Suba seal) were positive cells in the attached 1L flasks (Duran-Schott, Germany), 30 (Lab Champion IS-971R, USA) at 200 rpm for 7 days to produce methanol. During the incubation, it was added to 30% CH ₄ by two days interval.

The methanol concentration produced was analyzed by enzymatic oxidation with alcohol oxidase (Sigma-Aldrich, USA) according to the method of Wood PJ, et al., Anal Biochem 1971; 39: 418-28. Specifically, the methanol concentration was analyzed using gas chromatography (GC (Agilent 7890A, USA) equipped with an HP-5 column (Agilent 19091J-413, USA) and an FID detector. Flow rate 25 mL of H ₂ and air in min ^{-1 -} helium with (300 mL min ¹⁾ was used as a carrier gas. The oven temperature was initially maintained at 35 ° C for 5 minutes, then increased to 150 ° C at a rate of 5 ° C min ^-1 , and finally to 250 ° C at a rate of 20 ° C min ^-1 . The injector and detector temperatures were set at 220 ° C and 250 ° C, respectively.

As a result, the maximum yield of methanol production by M. sporium cells covalently immobilized on chitosan was 4.61 mM (see Table 3).

< Example  &Lt; 2 &gt; &gt; Methyl rosiners Sporeum  Production of methanol from synthetic gas mixture

(1) Production of synthesis gas mixture

NK Co. Syngas mixture was prepared by mixing pure CH ₄ , CO ₂ , and H ₂ , obtained in a ratio of 6: 3: 1, obtained from Aldrich (Busan, South Korea).

(2) a chitosan- Methyl rosiners Sporeum  Production of Methanol from Used Methane

The prepared syngas mixture was used as feedstock for methanol production by M. sporium cells covalently immobilized on chitosan for up to 72 hours. For methanol production, the upper space in the air: this is similar to CH ₄ amount (30%) was retained and diluted (1: 1).

As a result, the maximum yield of methanol production by M. sporium cells covalently immobilized on chitosan was 6.12 mM (see FIG. 7 and Table 3).

< Comparative Example  1> Production of Methanol by Other Methane-oxidizing Bacteria

Except that the immobilized on chitosan covalently M. sporium cells in place of the M. trichosporium NCIB 111311 cells immobilized covalently on DEAE cellulose, and a result of measurement of methanol output from CH ₄ under the same conditions, 20 mM and 80 mM phosphate buffer to produce 0.40 and 2.10 μmol of methanol h ^-1 mg ^-1 , respectively.

< Experimental Example  1> Optimization of methanol production process parameters

(1) Influence of metals

The effect of supplementation of two metal ions, Cu ²⁺ (1-10 μM) and Fe ^{2 +} (2.5-20 μM) in the production of methanol by M. sporium according to the present invention was examined in 20 mM phosphate buffer (pH 7.0 ). As a result, the methanol production was significantly improved from 0.86 mM to 1.14 mM as the Cu concentration increased from 0 to 5 μM. At 10 μM Cu, yield slightly decreased (up to 1.03 mM), but at 10 μM Fe increased methanol production to 1.02 mM. Thus, both metal ions affect methanol production, and these effects appear to be due to improved MMO activity.

In addition, we evaluated the effect of Cu ^{2 +} concentration (0-10 μM) for M. sporium Cell Growth and sMMO activity under these same conditions.

First, cell growth was measured by determining the absorbance (OD) at 595 nm with a UV / Vis spectrophotometer (JENWAY Scientific, UK). Well-grown cells were harvested at 10,000 rpm for 15 minutes at 4 ° C via centrifugation (Gyrozen 1580 MGR, South Korea) and then washed twice with phosphate buffer (20 mM, pH 7.0). The collected cells were stored at 4 ° C until use. Dry cell mass (DCM) was calculated after incubation at 70 ° C for 48 hours.

Next, naphthalene oxidation was used to evaluate relative sMMO activity using a modified assay according to Brusseau GA, et al., Biodegradation 1990; 1: 19-29. First, naphthalene was oxidized by sMMO to produce naphthol. The presence of a complex of violet containing naphthol and tetraazoa o-dianisidine (Sigma-Aldrich, USA) was determined spectrophotometrically at 530 nm using a UV / Vis spectrophotometer. Briefly, the assay was performed using a suspension containing DCM's resting cells in phosphate buffer (50 mM, pH 7.0) containing sodium formate (2 mM). Next, a small amount of naphthalene crystals were added, and the reaction was incubated at 30 DEG C for 2 hours with stirring at 180 rpm. After incubation, 10 mg mL- ¹ tetrahedral o-dianisidine was added to observe the color change.

The results are shown in Table 1 below.

Cu
(μM) Dry cell mass
(mg mL ^-1 ) Non-growth rate
(h ^-1 ) Naphthalene oxide ^a Methanol production
(mM) 0 0.281 + 0.021 0.019 ± 0.002 ++++ 1.44 ± 0.12 One 0.300 0.023 0.020 0.002 +++ 1.65 + 0.13 2.5 0.320 + 0.025 0.020 0.002 ++ 2.16 ± 0.17 5 0.358 + 0.028 0.021 0.002 + 2.52 0.21 10 0.241 + 0.020 0.018 0.001 - 2.18 ± 0.19 Strength of ^a purple development: + (presence) and - (absence)

As shown in Table 1, at 5 μM Cu, a specific growth rate was observed to increase from 0.019 h ^-1 (at 0 μM Cu) to 0.021 h ^-1 , and at this Cu concentration, methanol production The yield increased to 2.52 mM. At 10 μM Cu, the rate of non-growth decreased to 0.018 h ^-1 . 5 μM Cu in the maximum specific growth rate of M. sporium was lower than 0.059 h ^-1 The value reported by the CH ₄ (30%) of the same concentration with respect to the M. trichosporium OB3b as feedstock [IY Hwang, et al., J Microbiol Biotechnol 2015; 25: 375-80]. A similar effect of Cu concentration on relative reduction in naphthalene oxidation was observed due to the dominance of pMMO [Takeguchi M, et al., Catal. Surv. Jpn 2000; 4: 51-63].

(2) Optimization of process parameters

The physiological characteristics of methanogenic bacteria are strongly influenced by various process parameters such as pH, temperature, and agitation during growth and methanol production. Thus, in order to optimize the process parameters in the methanol production by M. sporium according to the present invention, the methanol production amount was determined by measuring pH, temperature, and stirring conditions.

Specifically, after culturing for 24 hours, 30% CH ₄ was added as a feedstock in sodium acetate (pH 5.5) and phosphate (pH 6.0-8.0) buffer, pH 5.5-8.0 , and added to M. sporium Methanol production.

As a result, a maximum methanol production of 2.7 mM was observed at pH 6.8. When the pH was outside the range, for example, at pH 5.5 and 8.0, the methanol yields were reduced to about 12% and 28%, respectively. These results suggested that methanol production was optimized at a pH near neutral.

The cultures were also incubated at pH 6.8 and at different temperatures (25-40 ° C).

As a result, the optimum temperature was 30 캜, and when the temperature was outside the above range, specifically, a temperature change from 30 캜 to 25 캜 resulted in a slightly lower yield of 2.5 mM, while at 35 캜 and 40 캜, The maximum values were significantly reduced to 44% and 23%, respectively.

The effect of different agitation speeds (90-200 rpm) on methanol production at pH 6.8 and 30 ° C after 24 hours of culture was evaluated.

As a result, the optimum stirring condition was 150 rpm. Likewise, very similar yields of 2.70 mM and 2.68 mM methanol were observed at stirring ratios of 150 rpm and 180 rpm, respectively, but a decrease in methanol yield was observed at lower (90) and higher (200) rpm.

Thus, it can be seen that the optimal conditions for methanol production by M. sporium according to the present invention are pH 6.8-7.0, 25-30 ° C and 150-180 rpm.

(3) Methanol Phosphate buffer for production  Effect of Concentration

In methanol production by M. sporium according to the present invention, the effect of phosphate buffer concentration was evaluated.

Specifically, the effect on methanol production under optimum conditions was measured using phosphate buffer (10-120 mM, pH 6.8) at different concentrations and is shown in FIG.

As a result, as shown in Fig. 1, the methanol production increased from 2.70 mM to 3.66 mM as the concentration of the phosphate buffer increased from 20 mM to 100 mM. However, at a higher concentration (120 mM), a slight decrease in methanol production (3.48 mM) was observed.

Therefore, it can be seen that the optimum concentration of phosphoric acid buffer for methanol production by M. sporium according to the present invention is 100 mM.

(4) for methanol production MDH  Influence of inhibitor

In order to biosynthesize methanol efficiently from methane, it is necessary to increase the activity of MMO and to inhibit oxidation of the produced methanol to formaldehyde. In other words, the activity of MDH must be inhibited in order to prevent oxidation of methanol.

Thus, the effect of MDH inhibitors was evaluated in order to improve the production of methanol by M. sporium according to the present invention.

Specifically, different inhibitors to MDH After incubation for 24 hours using 30% CH ₄ under optimum conditions: EDTA (0.1-1.5 mM), MgCl 2 (5-50 mM), NaCl (10-120 mM), and NH ₄ Cl (5-25 mM) was added to phosphate buffer (100 mM, pH 6.8) and the amount of methanol produced was measured and shown in Fig.

As shown in FIG. 2, the optimum concentration (mM) of the MDH inhibitor was 1.0 mM for EDTA, 20 mM for MgCl ₂ , 80 mM for NaCl, and 15 mM for NH ₄ Cl.

To measure the inhibitory activity of the MDH inhibitors, MDH activity was assayed using 2,6-dichlorophenolindophenol (DCPIP) at 600 nm according to Kalyuzhnaya MG, et al., J Bacteriol 2008; 190: 3817-23. The results are shown in FIG. 3A, and the results are shown in FIG. 3A. FIG. 3A shows the results of spectrophotometric monitoring of the absorbance induced by phenazine methosulfate-mediated reduction.

As shown in Figure 3A, in phosphate buffer, these inhibitors inhibited MDH activity by 38.7%, 32.5%, 30.2%, and 26.4%, respectively. In contrast, phosphate buffer (100 mM) alone inhibited MDH activity by only 17.6%. These inhibitors had a mixed effect on methanol production at different concentrations and after 24 hours of incubation under optimal conditions, the methanol production increased from 3.66 mM to 4.20 mM. Of the inhibitors tested, the addition of MgCl ₂ (20 mM) led to maximum methanol production to 4.20 mM. Interestingly, the greater inhibition of MDH activity (38.7%) in M. sporium did not induce the presence of EDTA to improve methanol production, probably due to the chelation of metal ions in MMO.

(5) Formic acid  effect

In methanol production by M. sporium according to the present invention, the possible supportive role of formate salt for co-factor (NADH) regeneration during methanol production (at 10-120 mM) was evaluated.

Specifically, methanol production according to the concentration (mM) of sodium formate in the presence of MgCl ₂ (20 mM) and phosphate buffer (100 mM) was measured and shown in FIG.

As shown in Figure 3b, formate had a positive effect on methanol production and maximum methanol production (5.4 mM) was observed in 40 mM formate. However, increasing the formate salt concentration to 120 mM did not significantly affect methanol production.

Mehta et al. [Biotechnol Bioeng 1991; 37: 551-6] noted that the rate of methanol production decreases over time because of the consumption of endogenous NADH ₂ . Therefore, it is necessary to regenerate supplementary factors in order to maintain the high yield conversion. Therefore, in the methanol production by M. sporium according to the present invention, the addition of 40 mM formic acid salt can improve methanol production 1.3 times.

(6) Influence of reaction volume versus upper space ratio

In the production of methanol by M. sporium according to the present invention, the methanol content in methanol production was measured at five different ratios (1: 1, 1: 3, 1: 5, 1: 7, and 1: The effect of space ratio was evaluated. Experiments were conducted under optimal conditions using 30% CH ₄ as feedstock. These ratios were achieved by changing the reaction volume from 12 mL to 60 mL in a serum bottle.

As a result, at different ratios, 3.63-5.40 mM methanol was produced and the optimum reaction volume to top space ratio was 1: 5. Interestingly, higher ratios of 1: 1 and 1: 3 reduced methanol production to 33% and 23%, respectively. At lower ratios of 1: 7 and 1: 9, methanol production was constant. These results suggested that a suitable reaction volume to top space ratio is required to achieve high methanol production.

(7) Influence of feed concentration of feedstock

In the methanol production by M. sporium according to the present invention, the influence of the feed concentration of the feedstock was evaluated.

Methanol production by M. sporium was measured by injecting CH ₄ at various concentrations (10-50%) and culturing until 96 hours, and is shown in FIG.

As shown in Fig. 4, the methanol production was increased as the incubation time increased from 12 hours to 24 hours, and then decreased sharply in the 96 hour culture. Also. At 30% CH ₄ , the methanol production increased sharply from 2.78 mM (at 10% CH ₄ ) to 5.40 mM and at 40% and 50% higher concentrations, yields after incubation for 24 hours were 5.74 mM and 5.80 mM. From this, it can be seen that the feedstock injection concentration is preferably 30-50%, and the incubation time is preferably 24 hours.

< Experimental Example  2> Effects of Support and Immobilization Methods on Methanol Production

The effect of the support and immobilization method on the methanol production by M. sporium according to the present invention was evaluated.

Specifically, M. sporium was immobilized on various supports such as Amberlite XAD-2, XAD-4, and XAD-7HP as well as Duolite A7 and chitosan by different immobilization methods of adsorption or covalent bonds under batch culture conditions.

The immobilized cells were washed twice with phosphate buffer (20 mM, pH 7.0) containing MgCl ₂ (5 mM) and stored at 4 ° C until use. The immobilization yield (IY) was calculated according to the following equation:

[Equation 1]

IY (%) = amount of cells bound on the support / amount of total cells loaded 100

The results are shown in Table 2 below.

Support size
(μm) Surface area
(m ² g ^-1 ) Immobilization yield (IY)
(%) ^a Relative methanol
(%) ^b Amberlite XAD-2 682 336 19.5 ± 1.4 70.8 ± 5.0 Amberlite XAD-4 564 725 17.4 ± 1.2 64.9 ± 4.7 Amberlite XAD-7HP 580 450 18.1 ± 1.3 73.5 ± 5.2 Duolite A7 600-800 36 27.2 ± 2.2 80.0 ± 5.5 Chitosan 45-165 180 33.5 ± 3.4 85.4 ± 4.6 cell load per ^a support of 100 g was mg DCM Im
^b 5.40 ± 0.42 mM of free-cell methanol production is considered 100%.

As shown in Table 2, the maximum IY of M. sporium ranged from 17.4 to 33.5%. These cell loading yields were much higher than adsorption (IY: 7.15-13.4%) under the same conditions. Methanol production efficiency of covalently immobilized cells was 64.9-85.4%. Production of adsorbed cells was much lower (40.7-69.3%) than free cells (100%). Among these scaffolds, chitosan-immobilized cells through adsorption and covalent immobilization produced the highest methanol production, with a maximum cell loading of 13.4 and 33.5 mg DCM per gram of support, respectively. Immobilized cells were stable on supports during up to 96 hours of culture.

From this, it can be seen that when M. sporium immobilized on chitosan is used for methanol production by M. sporium according to the present invention, methanol production with improved yield can be obtained compared with the conventional method .

The prior art and according to the invention Methanol production by the M. sporium according to, are shown with the type of M. sporium, kind of immobilization method and the feedstock, to organize the methanol yield of the process parameters in Table 3.

Strain Feedstock
(%) Immobilization mode Temperature
(° C) pH Methanol
Yield (mM) Support Way M.sporium B2119 CH ₄ (22) Polymer matrix Encapsulation Batch 28 7.0 1.68 M.sporium B2120 CH ₄ (22) Polymer matrix Encapsulation Batch 28 7.0 1.43 M.sporium B2122 CH ₄ (22) Polymer matrix Encapsulation Batch 28 7.0 1.37 M.sporium B2123 CH ₄ (22) Polymer matrix Encapsulation Batch 28 7.0 1.84 M.sporium B2121 CH ₄ (22) Free cell - ^a Batch 28 7.0 1.56 M.sporium B2121 CH ₄ (22) Polymer matrix Encapsulation Continuous 28 7.0 2.34 M.sporium B2121 CH ₄ (22) Free cell - Batch 28 7.0 0.35 M.sporium B2121 CH ₄ (22) Polyvinyl alcohol Encapsulation Batch 28 7.0 1.94 M.sporium KCTC 22312 CH ₄ : CO ₂ (40) Free cell - Batch 35 7.0 0.71 M.sporium DSMZ 17706 CH ₄ (50) Free cell - Batch 30 6.8 5.80 M.sporium DSMZ 17706 CH ₄ : CO ₂ : H ₂ (50) Free cell - Batch 30 6.8 5.80 M.sporium DSMZ 17706 CH ₄ (30) Chitosan share Batch 30 6.8 4.61 M.sporium DSMZ 17706 CH ₄ : CO ₂ : H ₂ (50) Chitosan share Batch 30 6.8 6.12 ^{a Not} applicable

As shown in Table 3, the methanol production method using M. sporium covalently immobilized on the chitosan according to the present invention is characterized in that when the feed is pure CH ₄ , the methanol yield is 4.61 mM, encapsulated using a polymer matrix as a support The methanol yield was improved by 2-3.5 times as compared with the conventional method (1.37-2.34 mM). In addition, when the synthesis gas mixture was used as the feedstock, the methanol yield was 6.12 mM, which was higher than the yield by free cell (5.80 mM).

Thus, the methanol production process using M. sporium covalently immobilized to the chitosan according to the present invention can lead to significantly higher methanol production from both pure CH ₄ and the synthesis gas mixture.

< Experimental Example  3> Chitosan-bound &lt; RTI ID = 0.0 &gt; Methyl rosiners Sporium  Reusability

In the production of methanol M. sporium according to the invention, it was evaluated in the re-usability of the M. sporium immobilized on the chitosan.

Specifically, the free M. sporium cells washed after each cycle of the methanol produced by the cells of M. sporium immobilized through M. sporium cells, and covalently bonded immobilized through adsorption, by separating them by centrifugation with phosphate buffer And used as inoculums in the next cycle. The methanol production efficiency was considered to be 100% for the initial (0) cycle.

The results are shown in Fig.

As shown in Figure 6, the M. sporium cells immobilized through the adsorption or sharing method after 6 cycles of reuse maintained the production efficiencies of 26.6% and 63.4%, respectively. However, under the same conditions, the free cells were 17.3% Methanol yield of methanol was significantly lower than that of methanol. From these results, it can be seen that the shared immobilization of M. sporium is highly effective for improved stability and reusability.

The present invention has been described with reference to the preferred embodiments. It will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention as defined by the appended claims. Therefore, the disclosed embodiments should be considered in an illustrative rather than a restrictive sense. The scope of the present invention is defined by the appended claims rather than by the foregoing description, and all differences within the scope of equivalents thereof should be construed as being included in the present invention.

Claims (12)

Process for the production of high yield methanol from a methane or syngas mixture using cetyl -immobilized methylosinus sporium in chitosan.
The method according to claim 1, wherein the methanol production method comprises culturing the methylosynthosphospheric cells covalently immobilized on chitosan and a methane or syngas mixture as a feedstock in a culture medium while stirring.
3. The method of claim 2, wherein the culture comprises a phosphate buffer and a nitrate mineral salt (NMS).
4. The method of claim 3, wherein the culture further comprises at least one member selected from the group consisting of Cu ions, MDH inhibitors and formates.
4. The method according to claim 3, wherein the concentration of the phosphate buffer is 100 mM.
5. The method of claim 4, wherein the Cu ion is 5 [mu] M and the concentration of the formic acid salt is 40 mM.
The method of claim 4, wherein the MDH inhibitors EDTA, MgCl _2, is selected from the group consisting of NaCl and NH ₄ Cl, The optimal concentration of EDTA is 1.0 mM, MgCl ₂ is 20 mM, NaCl is 80 mM, and NH ₄ Cl &lt; / RTI &gt; is 15 mM.
3. The method according to claim 2, wherein the culture is carried out at a pH of 6.8-7.0 and a stirring speed of 150-180 rpm at 25-30 DEG C for 24 hours.
3. The process of claim 2 wherein the optimum reaction volume to top space ratio is 1: 5 and the feedstock injection concentration is 30-50%.
The method of claim 2, wherein the syngas mixture is prepared by mixing CH ₄ , CO ₂ , and H ₂ in a ratio of 6: 3: 1.
A method for producing high yield methanol containing an active ingredient such as methylcellulose membrane, phosphate buffer, and nitrate mineral salt (NMS), which is covalently immobilized on chitosan, is added to a culture medium for producing methanol from a methane or syngas mixture. Composition.
12. The composition of claim 11, wherein the composition further comprises at least one member selected from the group consisting of Cu ions, MDH inhibitors, and formates.

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