KR102165641B1 - Composition for the production of propanol containing methanotroph and propanol production method - Google Patents

Abstract

The present invention relates to a method of producing propanol at a high concentration by supplying methanol and culturing methanogens in the presence of a gas containing propane. Since it is possible to stably produce a high concentration of propanol according to the method, it is effectively high concentration. It can be used to produce propanol.

Description

Composition for the production of propanol containing methanotroph and propanol production method}

The present invention relates to a composition for producing propanol and a method for producing propanol containing methanogens, and more particularly, to a method for producing a high concentration of propanol by culturing the methanogens in the presence of methanol and propane-containing gas.

Methane monooxygenase (MMO), possessed by methane-sugar bacteria, has a weak substrate specificity and can produce propanol from propane. However, since the produced propanol is further converted to acetone or the like by methanol dehydrogenase (MDH), it is impossible to produce high-concentration propanol.

The propanol production process is a concept of obtaining a reducing power for oxidizing propane from cheap methane or methanol, and selectively recovering propanol. Methanol hydrogenase possessed by methanogens is helpful in supplying reducing power because it produces reducing power by decomposing the produced alcohol, but since it can also decompose the produced propanol, an appropriate operation strategy or strain development is required.

Accordingly, the present inventors confirmed that the production efficiency of propanol was excellent as a result of supplying methanol and culturing the methanogens in the presence of a gas containing propane.

Accordingly, the object of the present invention is the genus Methylomonas, the genus Methylomicrobium, the genus Methylobacter, the genus Methylococcus, the genus Methylosphaera, and methyllocal Methylocaldum, Methyloglobus, Methylosarcina, Methyloprofundus, Methylothermus, methylohalobius (Methylohalobius), Methylogaea, Methylomarinum, Methylovulum, Methylomarinovum, Methylorubrum, Methylorubrum, Methylopara Genus Methyloparacoccus, Genus Methylosinus, Genus Methylocystis, Genus Methylocella, Genus Methylocapsa, Genus Methylofurula, and MethylASidi It is to provide a composition for producing propanol comprising at least one strain selected from the group consisting of the genus Pilum (Methylacidiphilum) and the genus Methylacidimicrobium or a culture solution of the strain.

Another object of the present invention is the genus Methylomonas, the genus Methylomicrobium, the genus Methylobacter, the genus Methylococcus, the genus Methylospera, the genus Methylocaldom, the genus Methylglobus, the genus Methylosarcina, Genus Methylpropundus, Genus Methyloselmus, Genus Methylhalobius, Genus Methyllogea, Genus Methylomarinum, Genus Methylobulum, Genus Methylomarinovum, Genus Methylolovrum, Methyllo One selected from the group consisting of the genus Paracoccus, the genus Methylosinus, the genus Methylosis, the genus Methylocella, the genus Methylcapsa, the genus Methyloperula, the genus Methylacidiphilum, and the genus Methylacidimicrobium It is to provide a method for producing propanol comprising a conversion step of inducing a conversion reaction in the above strain or the culture medium of the strain.

The present invention relates to a composition for producing propanol and a method for producing propanol including methanogens, and the method according to the present invention can be utilized to produce a high concentration of propanol.

The present inventors cultivated DH-1 or Methylomicrobium alcaliphilum 20Z in Methylomonas genus using methanol and propane-containing gas, and methanol dehydrogenase preferentially uses methanol due to competitive substrate inhibition. It was confirmed that propanol was accumulated in the culture medium, and because the biocatalyst was continuously active through methanol metabolism, it was found that high value-added propanol can be stably produced.

Hereinafter, the present invention will be described in more detail.

One aspect of the present invention is the genus Methylomonas, the genus Methylomicrobium, the genus Methylobacter, the genus Methylococcus, the genus Methylosphaera, and methylosphaera Genus (Methylocaldum), Genus Methyloglobus, Genus Methylosarcina, Genus Methyloprofundus, Genus Methylothermus, Genus Methylhalobius ( Methylohalobius), Methylogaea, Methylomarinum, Methylovulum, Methylomarinovum, Methylorubrum, Methylorubrum, Methyloparacoccus Genus (Methyloparacoccus), Genus Methylosinus, Genus Methylocystis, Genus Methylocella, Genus Methylocapsa, Genus Methylofurula, Methylacidiphilum It is a composition for producing propanol containing at least one strain selected from the group consisting of the genus (Methylacidiphilum) and the genus methylacidimicrobium or a culture solution of the strain.

The strain may be methylomicrobium alcaliphilum 20Z deposited with Methylomonas sp. DH-1 deposited with accession number KCTC 13004BP or Methylomicrobium alcaliphilum 20Z deposited under accession number DSM 19304, but is not limited thereto.

The composition is 0.5 to 4.0 g/L, 0.5 to 3.0 g/L, 0.5 to 2.0 g/L, 1.0 to 4.0 g/L or 1.0 to 3.0 g/L, for example, 1.0 to 2.0 g/L of methanol It may be added to include, but is not limited thereto.

The composition is 5 to 30% (v/v), 5 to 25% (v/v), 5 to 20% (v/v), 10 to 30% (v/v), 10 to 25% (v/ v), 10 to 20% (v/v), 15 to 30% (v/v) or 15 to 25% (v/v), for example 15 to 20% (v/v) of propane The gas may be additionally included, but is not limited thereto.

The gas contains 5 to 30% (v/v), 5 to 25% (v/v), 5 to 20% (v/v), 10 to 30% (v/v), 10 to 25% ( v/v), 10 to 20% (v/v), 15 to 30% (v/v) or 15 to 25% (v/v), for example 15 to 20% (v/v). It may be, but is not limited thereto.

Another aspect of the present invention is the genus Methylomonas, the genus Methylomicrobium, the genus Methylobacter, the genus Methylococcus, the genus Methyllospera, the genus Methylocaldom, the genus Methylglobus, the genus Methylosarcina, Genus Methylpropundus, Genus Methyloselmus, Genus Methylhalobius, Genus Methyllogea, Genus Methylomarinum, Genus Methylobulum, Genus Methylomarinovum, Genus Methylolovrum, Methyllo One selected from the group consisting of the genus Paracoccus, the genus Methylosinus, the genus Methylosis, the genus Methylocella, the genus Methylcapsa, the genus Methyloperula, the genus Methylacidiphilum, and the genus Methylacidimicrobium It is a method for producing propanol comprising a conversion step of inducing a conversion reaction in the above strain or the culture medium of the strain.

The term "conversion" as used herein refers to a process in which a strain produces a specific substance by using a substance in a culture medium.

The strain may be DH-1 of the genus Methylomonas deposited under the accession number KCTC 13004BP, or the alkaline columnar methylomicrobium 20Z deposited under the accession number DSM 19304, but is not limited thereto.

The conversion step is 0.5 to 4.0 g / L, 0.5 to 3.0 g / L, 0.5 to 2.0 g / L, 1.0 to 4.0 g / L or 1.0 to 3.0 g / L, for example, 1.0 to 2.0 g / It may be performed by adding L of methanol, but is not limited thereto.

The conversion step may be performed by additionally adding 0.5 to 4.0 g/L of methanol to the culture solution when methanol among the components of the culture solution is depleted.

The conversion step is 5 to 30% (v/v), 5 to 25% (v/v), 5 to 20% (v/v), 10 to 30% (v/v), 10 to 25% (v /v), 10 to 20% (v/v), 15 to 30% (v/v) or 15 to 25% (v/v), for example 15 to 20% (v/v) of propane It may be performed in the presence of the containing gas, but is not limited thereto.

The gas contains 5 to 30% (v/v), 5 to 25% (v/v), 5 to 20% (v/v), 10 to 30% (v/v), 10 to 25% ( v/v), 10 to 20% (v/v), 15 to 30% (v/v) or 15 to 25% (v/v), for example 15 to 20% (v/v). It may be, but is not limited thereto.

The conversion step may be performed by exhausting the gas for 10 to 900 minutes, but is not limited thereto. The optimum time required for exhausting the gas may be appropriately adjusted according to fluctuations in conditions including the volume of the total culture medium, the process execution time, the type of strain to be used, and the composition of the medium.

The conversion step may be performed at a temperature of 25 to 35°C, 27 to 35°C, 29 to 35°C, 25 to 32°C, or 27 to 32°C, for example, 29 to 32°C, but is limited thereto. It is not.

The present invention relates to a method for producing propanol at a high concentration by culturing methanogens in the presence of methanol and propane-containing gas, and since it is possible to stably produce a high concentration of propanol according to the above method, it is possible to effectively produce high concentration propanol. Can be used to do.

1 is a schematic diagram showing the production route of propanol using methane magnetizing bacteria.
FIG. 2 shows Methylomonas sp. in the condition that methane and air were supplied at a volume ratio of 3:7 and 1 g of propanol was additionally supplied per liter. It is a graph showing the growth inhibition effect of propanol from the DH-1 strain.
3 is Methylomonas sp under the condition of supplying methane and air at a volume ratio of 3:7 and additionally supplying 0.5 g, 1 g, 2 g (0.5 g/L, 1 g/L, 2 g/L) of methanol per liter. . It is a graph showing the effect of inhibiting the growth of methanol from the DH-1 strain.
4 is a graph showing the growth curve of the DH-1 strain when using methanol 2 g/L and propane 30% as substrates.
5 is a graph showing the amount of metabolites when using methanol 2 g/L and propane 30% as substrates.
6 is a graph showing the growth curve of the DH-1 strain when an experiment in which methane and air are supplied in a volume ratio of 3:7 and methanol of various concentrations is used as the sole carbon source.
7 is a graph in which 2 g/L of methanol was added to the culture solution of the DH-1 strain having an initial OD of 10, and 30% of propane was supplied, and the concentrations of propanol, acetone, and methanol were measured every 6 hours.
8 is a graph in which 2 g/L of methanol was added to the culture medium of the DH-1 strain having an initial OD of 4, 30% of propane was supplied, and the concentrations of propanol, acetone, and methanol were measured every 3 hours.
9 is a graph in which 1 g/L of methanol was added to the culture medium of the DH-1 strain, 30% of propane was supplied, and the concentrations of propanol, acetone, and methanol were measured every 2 hours.
FIG. 10 is a graph showing the concentration of propanol, acetone, and methanol in an accumulation pattern every 2 hours, in which 1 g/L of methanol was added to the culture medium of the DH-1 strain and 30% of propane was supplied.
11 is a graph showing the production of propanol by additionally supplying 1 g/L of methanol after methanol is depleted in the culture medium of the DH-1 strain.
12 is a graph showing propanol production as an accumulation pattern by supplying 1 g/L of methanol after methanol is depleted in the culture medium of the DH-1 strain.
13 is a graph showing the production of propanol by supplying 1.5 g/L of methanol at 0.5 g/L increments after methanol is depleted in the culture medium of the DH-1 strain.
FIG. 14 is a graph showing propanol production as an accumulation pattern by supplying 1.5 g/L of methanol in 0.5 g/L increments after methanol is depleted in the culture medium of the DH-1 strain.
FIG. 15 is a graph showing the production of propanol by supplying 1.5 g/L of methanol at 0.3 g/L increments after methanol is depleted in the culture medium of the DH-1 strain.
FIG. 16 is a graph showing propanol production as an accumulation pattern by supplying 1.5 g/L of methanol at 0.3 g/L increments after methanol is depleted in the culture broth of the DH-1 strain.
17 is a graph showing the production amount of propanol by simultaneously supplying methane and propane to the culture medium of the DH-1 strain.
18 is Methylomicrobium alcaliphilum 20Z is a graph showing the production of propanol by simultaneously supplying methane and propane to the culture medium.

Hereinafter, the present invention will be described in more detail by the following examples. However, these examples are for illustrative purposes only, and the scope of the present invention is not limited by these examples.

Example 1: Preparation and experimental method of strain

1-1. Methylomonas sp. DH-1

After inoculating 1 mL of DH-1 stock (KCTC 13004BP) in a 500 mL baffled flask containing 50 mL NMS medium, periodically inject methane/air to the desired cell concentration at 30°C in a shaker incubator. , Incubated at 230 rpm. In order to prevent the inflow of outside air and the outflow of internal methane or propane during culture, a stopper with a septum made of butyl rubber was used on the flask. The composition of the NMS medium is shown in Table 1 below.

NMS composition Amount for 1L MgSO ₄ 7H ₂ O 1 g KNO ₃ 1 g CaCl ₂ 2H ₂ O 0.228 g Fe-EDTA 0.0038 g Na ₂ MoO ₄ 0.0006 g 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 H ₃ BO ₃ 0.015 mg EDTA 0.25 mg KH ₂ PO ₄ 0.26 g Na ₂ HPO ₄ 7 (H ₂ O) 0.62 g Biotin 0.02 mg Folic acid 0.02 mg Thiamine HCl 0.05 mg Ca pantothenate 0.05 mg Vitamin B12 0.001 mg Riboflavin 0.05 mg Nicotiamide 0.05 mg CuSO ₄ 5H ₂ O 2.5 mg

When the initial OD was 10 or more, the culture solution with OD 4 was centrifuged and concentrated, and then NMS medium was added to adjust the cell concentration.

In the propanol production experiment, a certain amount of methanol, which is a reducing power, was supplied to the above-mentioned DH-1 culture solution, and a gas having a ratio of propane:air=3:7 was exhausted for 30 minutes, and then stirred in a shaking incubator at 30°C at 230 rpm.

In the case of providing methane as a reducing power, the gas of methane:propane:air=1.5:1.5:7 was exhausted for 30 minutes without adding methanol, and the mixture was stirred at 30°C at 230 rpm in a shaker incubator.

1-2. Methylomicrobium alcaliphilum 20Z

Prepared under the same conditions as in 1-1, but the composition of the medium is shown in Table 2 below.

Medium composition Amount for 1L NaCl 30 g MgSO ₄ 7H ₂ O 0.2 KNO ₃ 1 g CaCl ₂ 2H ₂ O 0.02 g EDTA 5 mg CuCl ₂ 5H ₂ O 0.1 mg FeSO ₄ 7H ₂ O 2 mg Na ₂ MoO ₄ 0.03 mg ZnSO ₄ 7H ₂ O 0.1 mg MnCl ₂ 4H ₂ O 0.03 mg CoCl ₂ 6H ₂ O 0.2 mg NiCl ₂ 6H ₂ O 0.02 mg H ₃ BO ₃ 0.03 mg KH ₂ PO ₄ 0.28 g Na ₂ HPO ₄ 12 (H ₂ O) 0.6 g 1M NaHCO ₃ 50 mL 1M Na ₂ CO ₃ 5 mL

Example 2: Analysis of propanol metabolism from strain

As in Figure 1, Methylomonas sp. The DH-1 strain is a methane metabolizing strain and has a characteristic of converting methane to methanol using MMO. The strain was identified, and the production of propanol was analyzed when propane or a mixed gas of methane-propane or methanol-propane was supplied to the strain.

Prior to the study on the production of propanol, methane and air were supplied in a volume ratio of 3: 7 to confirm whether the strain can metabolize propane and that the growth of the strain is not inhibited by the produced propanol, and 1 g per liter (1 g/L) of propanol was additionally supplied, and the growth curve of the bacteria was confirmed.

As can be seen in FIG. 2, a slight inhibition of growth occurred, but it was determined that the degree was not large, and subsequent experiments were conducted.

To determine the optimal methanol feed concentration, supply methane and air at an initial OD of 2 at a volume ratio of 3:7 and 0.5 g, 1 g, 2 g (0.5 g/L, 1 g/L, 2 g/L) of methanol per liter. After supplying additionally, the growth curve of the bacteria was confirmed.

As can be seen in Figure 3, it was determined that there was almost no inhibitory effect by methanol even at a methanol concentration of 2 g/L as shown in the figure below.

Example 3: Analysis of production of propanol from strains

Propane and methanol were supplied with an initial OD of 10, and 2 g/L of methanol and 30% of propane were given as substrates to confirm whether propanol was actually produced in the strain.

As can be seen in Figure 4, the cell concentration decreased over time.

As can be seen in FIG. 5, 116 mg/L of propanol was produced, but it was found that there was little change in metabolites after 6 hours of incubation, so further experiments were conducted on growth and metabolic inhibitory factors. As a result of the analysis, the production of acetone, a decomposition product of isopropanol, was confirmed.

Example 4: Confirmation of growth inhibition by metabolite or methanol

In order to check whether there is growth inhibition due to metabolites or methanol when methanol was used as the sole carbon source, cultivation was conducted by supplying only methanol and air of various concentrations (2 g/L, 3 g/L, 4 g/L). . For comparison, an experiment in which methane and air were supplied in a volume ratio of 3:7 was performed, and the result was compared with this result when methanol was used as the sole carbon source.

As can be seen in FIG. 6, at low cell concentrations of 0.1 initial OD and 1 OD below final OD 1, the overall metabolite concentration was not high, so it appeared that it was not inhibited even at 4 g/L of methanol, but the intermediate metabolites accumulated and DH It was confirmed that metabolism and growth of -1 strain were inhibited. When the experiment was started at an initial OD of 2, the effects of these two products were more evident.

The influence of the initial cell concentration on the production of propanol was observed. 2 g/L of methanol was added to cells with initial ODs of 10 and 4, respectively, and 30% of propane was supplied, and concentrations of propanol, acetone, and methanol were measured every 6 hours.

As can be seen in FIG. 7, when the initial OD is 10, the propanol concentration recorded 0.11 g/L at 6 hours of incubation, and there was no significant change. OD continued to decrease from the beginning of the culture and dropped to 6 at 24 hours.

As can be seen from FIG. 8, even when the initial OD was 4, propanol did not increase any more after reaching the maximum value of 0.08 g/L at 6 hours of culture. Judging from these results, it seems that the inhibitory effect on excess methanol had a greater effect than the initial cell concentration.

Since excess methanol affects cell activity as well as propanol production, the initial methanol concentration was reduced to 1 g/L and propanol production experiments were conducted.

As can be seen in FIG. 9, the propanol concentration recorded the highest concentration of 0.18 g/L at 4 hours of incubation and did not increase any more. When the initial methanol concentration was 2 g/L, it increased about twice as much, and it was confirmed once again that an excess of methanol affects the cell activity. Methanol was depleted after 4 hours of incubation, and as the methanol was depleted, as the MDH enzyme decomposed the produced propanol, no more propanol production was made, and acetone was accumulated.

Example 5: Characterization of DH-1 biocatalyst

The properties of the DH-1 biocatalyst related to methanol consumption and propane conversion were analyzed by expressing propanol and acetone in molar concentrations.

As can be seen in Figure 10, after methanol is depleted, the total amount of propanol and acetone is partially increased, but most of it is due to acetone, and even if methanol is depleted, reducing power can be provided by oxidation of formaldehyde or formic acid accumulated in the culture medium.

When methanol was depleted, it was additionally supplied to promote a continuous increase of propanol.

As can be seen in FIG. 11, 1 g/L of methanol was additionally supplied at 4 hours when methanol was depleted, and propanol production was observed. As a result of additionally supplying methanol at 4 hours of incubation, the final propanol concentration was 0.20 g/L by preventing a decrease in propanol, but the increase was not large.

As can be seen from FIG. 12, rather, the accumulation of acetone became more active, and the amount of cells decreased due to the accumulation of acetone or the accumulation of formaldehyde, which is a methanol by-product, so that the effect of the additional supply of methanol was not significant.

The total methanol input was 1.5 g/L, and the concentration of the one-time input was changed to 0.5 and 0.3 g/L to observe the production of propanol.

As can be seen in FIGS. 13 to 16, the influence of the methanol concentration was not significant, so that propanol of about 0.17 g/L was produced in both cases.

Example 6: Confirmation of a method of inhibiting propane degradation through simultaneous supply of methane and propane

By supplying methane and propane at the same time, the reducing power required for propanol production was obtained from methane and at the same time, it was attempted to prevent propanol decomposition through the presence of methanol.

As can be seen in Figure 17, it can be seen that propanol is produced when methane and propane are supplied simultaneously. The final propanol concentration was 0.021 g/L, and the yield was very low compared to when methanol was supplied with reducing power. The acetone concentration was 0.08 g/L. When methane was used, sufficient reducing power was not supplied and the accumulated methanol concentration was low, making it difficult to prevent the activity of the MDH enzyme.

Example 7: Methylomicrobium alcaliphilum Propanol production using 20Z strain

The DH-1 strain has the advantage of fast growth and high methane consumption rate as the specific growth rate reaches 0.25 h ^-1 , whereas the consumption characteristics for methanol were not good, and the specificity of the MDH enzyme was not as good as the propane conversion results. There was a problem of decomposing propanol produced off into acetone.

Methylomonas sp. In order to investigate the possibility of propanol production in other strains besides DH-1, the same experiment was conducted using the same type 1 strain and methylomicrobium alcaliphilum 20Z (DSM 19304) that consumes methanol well.

First, in order to examine the effect of the initial methanol concentration on the production of propanol, propane conversion was performed by varying the methanol concentration to 1 to 4 g/L, and shown in Table 3 below.

The final OD was highest with 3 g/L methanol, but in terms of propanol production, 1 g/L recorded the highest propanol concentration of 1.10 mM (65 mg/L), making the initial methanol concentration 1 g/L and subsequent The experiment was carried out.

Methanol concentration 1 g/L 2 g/L 3 g/L 4 g/L Initial OD (g/L) 3.30 3.24 3.18 3.47 Final OD (g/L) 5.06 5.70 5.98 5.29 Initial propanol (mM) 0.13 0.18 0.15 0.16 Final propanol (mM) 1.10 0.55 0.42 0.38

When the initial methanol concentration was 1 g/L, an experiment for producing propanol by propane conversion was conducted.

As can be seen in FIG. 18, propanol was continuously increased until 16 hours of culture, resulting in a final 0.15 g/L. In the figure below, it can be seen that propanol continues to be produced even after methanol is depleted. As mentioned above, formate generated by decomposition of methanol seems to have provided the reducing power necessary for propane oxidation.

Compared to DH-1, a noteworthy point in the production of propanol using 20Z is that acetone accumulation is relatively less. This phenomenon is presumed to occur because the MDH specificity is different depending on the methanogen strains, and this is a result suggesting that propanol productivity can be maintained high if the methanol concentration is optimally maintained through appropriate methanol supply.

Claims (13)

delete A composition for producing propanol from propane containing a methylomicrobium alcaliphilum 20Z strain or a culture solution of the strain deposited with accession number DSM 19304. The composition for producing propanol according to claim 2, wherein the composition further comprises 0.5 to 4.0 g/L of methanol. The composition for producing propanol according to claim 2, wherein the composition additionally comprises a gas containing 5 to 30% (v/v) of propane. The composition for producing propanol according to claim 4, wherein the gas contains 5 to 30% (v/v) of methane. Preparing a composition for producing propanol comprising a methylomicrobium alcaliphilum 20Z strain or a culture solution of the strain deposited under the accession number DSM 19304; And
A conversion step of inducing a conversion reaction of propane into propanol by contacting methanol or methane, and propane with a composition for producing propanol;
Containing, propanol production method. delete The method of claim 6, wherein the concentration of methanol is 0.5% to 4.0% g/L. The method of claim 6, wherein the conversion step is performed by additionally adding 0.5 to 4.0 g/L of methanol to the culture solution when methanol among the components of the culture solution is depleted. The method of claim 6, wherein the conversion step is carried out in the presence of a gas containing 5 to 30% (v/v) propane. The method of claim 10, wherein the gas contains 5 to 30% (v/v) of methane. The method of claim 10, wherein the conversion step is performed by exhausting the gas for 10 to 900 minutes. The method of claim 6, wherein the conversion step is carried out at a temperature of 25 to 35°C.

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