Method for suppressing methane production

Abstract

The present invention has: a culture step for culturing methane-suppressing microorganisms that decompose metabolites required for methane-producing microorganisms to produce methane or methane-suppressing microorganisms that decompose methane produced by methane-producing microorganisms; and an addition step for adding the cultured methane-suppressing microorganisms to an environment in which methane-producing microorganisms are present. In the culture step, methane-suppressing microorganisms that decompose organic matter as metabolites may be cultured. In the addition step, the cultured methane-suppressing microorganisms may be added to an environment in which methane-producing microorganisms are present.

Description

Methods for suppressing methane production

[0001] The present invention relates to a method for inhibiting methane production.

[0002] Various methods for reducing greenhouse gases have been proposed. For example, Patent Literature 1 describes a method in which a formulation containing methionine is sprayed on soil to promote arsenic methylation by microorganisms in the soil and inhibit methane production by these microorganisms.

[0003] Patent No. 7225466

[0004] The method described in Patent Document 1 has the problem that it is necessary to continuously spray the formulation into the soil in order to suppress the production of methane by microorganisms.

[0005] The present invention has been made in view of these points, and an object of the present invention is to provide a method capable of suppressing methane production.

[0006] The method for inhibiting methane production of the present invention includes a culturing step of culturing a methane-inhibiting microorganism that decomposes metabolic substances required for methanogenic microorganisms to produce methane, or a methane-inhibiting microorganism that decomposes methane produced by methanogenic microorganisms, and an addition step of adding the cultured methane-inhibiting microorganism to an environment where the methanogenic microorganisms exist.

[0007] In the culturing step, the methane-inhibiting microorganism may be cultured, which decomposes organic matter as the metabolic substance. In the culturing step, the methane-inhibiting microorganism may be cultured, which decomposes hydrogen, acetic acid, formic acid, or methyl compounds as the metabolic substance. In the culturing step, the methane-inhibiting microorganism may be cultured, which belongs to the genus Anaeromyxobacter, Geomonas, Geobacter, Rhodopseudomonas, Methylobacillus, Methylopila, Methylobacter, Methylobacterium, Methylocaldum, Methylocapsa, Methyloceanibacter, Methylocella, Methylocystis, Methyloferula, Methylogaea, Methylomagnum, Methylomicrobium, Methylomonas, Methylosinus, or Methylotuvimicrobium.

[0008] In the adding step, the cultured methane-inhibiting microorganism may be added to an anaerobic environment in which the methanogenic microorganism exists, such as paddy field soil, an animal intestinal environment, sludge, landfill soil, forest soil, swamp sediment, river sediment, marine sediment, an anaerobic wastewater treatment reactor, the digestive tract of an insect, or animal excrement.

[0009] According to the present invention, the effect of suppressing the production of methane is achieved.

[0010] 1 is a diagram for explaining an overview of a method for suppressing methane production using methane-inhibiting microorganisms. It shows the measurement of the partial pressure of methane gas by gas chromatography. It is a flowchart showing the steps of a screening method for identifying a target microorganism capable of reducing the amount of methane produced by methanogenic microorganisms. It shows the co-culture of methanogenic microorganisms, archaea, and candidate microorganisms. It shows the relationship between the number of days of culture and the amount of methane produced when methanogenic microorganisms and archaea are cultured without adding candidate microorganisms. It shows the methane production suppression effect of Methylobacillus arboreus.

[0011] [Principle of suppression of methane production] Through extensive research, the present inventors have discovered a method for suppressing methane production by methanogenic microorganisms capable of producing methane. Figure 1 is a diagram for explaining the principle by which methane-suppressing microorganisms suppress methane production. First, the metabolic pathway of methane production by methanogenic microorganisms will be described.

[0012] Intermediate-producing microorganisms and methanogens decompose organic matter in anaerobic environments to produce methane. Examples of intermediate-producing microorganisms include archaea. As shown in Figure 1, intermediate-producing microorganisms decompose organic matter to produce hydrogen, acetic acid, formic acid, or methyl compounds (methanol, methylamines, dimethyl sulfide), etc. Methylamines include monomethylamine, dimethylamine, and trimethylamine. Methanogens decompose the hydrogen, acetic acid, formic acid, or methyl compounds produced by intermediate-producing microorganisms to produce methane. Figure 1 shows an example of methane production by intermediate-producing microorganisms and methanogens in the soil of a rice paddy where rice is grown. As indicated by the dashed arrow in Figure 1, methane produced by methanogens is absorbed by rice roots and then released into the atmosphere from the rice leaves. Alternatively, as indicated by the solid curved arrow in Figure 1, methane produced by methanogens is released from the soil into the atmosphere.

[0013] The mechanism by which methane-inhibiting microorganisms inhibit methane production by methanogenic microorganisms is thought to be Type A mechanism, in which methane-inhibiting microorganisms decompose metabolic substances required for methane production by methanogenic microorganisms, and Type B mechanism, in which methane-inhibiting microorganisms decompose methane produced by methanogenic microorganisms. Type A mechanism is a new mechanism discovered by the present inventors.

[0014] [Method for inhibiting methane production] The method for inhibiting methane production of this embodiment includes at least the following steps (A1) and (A2): (A1) a culturing step of culturing a methane-inhibiting microorganism that inhibits methane production by a methanogenic microorganism by a type A mechanism or a type B mechanism; and (A2) an adding step of adding the cultured methane-inhibiting microorganism to an environment where the methanogenic microorganism is present.

[0015] In the culturing step (A1), methane-inhibiting microorganisms are cultured. Examples of methane-inhibiting microorganisms include anaerobic and aerobic microorganisms collected from soil. Examples of methane-inhibiting microorganisms that inhibit methane production by methanogenic microorganisms using the A-type mechanism include microorganisms belonging to the genera Anaeromyxobacter, Geomonas, Geobacter, Rhodopseudomonas, Methylobacillus, and Methylopila. The A-type mechanism methane-inhibiting microorganisms may also belong to genera other than those listed above. Examples of methane-inhibiting microorganisms that suppress methane production by methanogenic microorganisms using the type B mechanism include microorganisms belonging to the genera Methylobacter, Methylobacterium, Methylocaldum, Methylocapsa, Methyloceanibacter, Methylocella, Methylocystis, Methyloferula, Methylogaea, Methylomagnum, Methylomicrobium, Methylomonas, Methylosinus, and Methylotuvimicrobium. Methane-inhibiting microorganisms using the type B mechanism may also belong to genera other than those listed. An example of a methane-inhibiting microorganism that suppresses methane production using the type A mechanism is Methylobacillus arboreus. For example, methane-inhibiting microorganisms decompose organic matter into metabolites required for methanogenic microorganisms to produce methane. This organic matter is used by intermediate-product-producing microorganisms to produce hydrogen, acetic acid, formic acid, methyl compounds, etc.

[0016] Methanogenic microorganisms are acetogenic methanogenic microorganisms that decompose acetic acid to produce methane, or hydrogenogenic methanogenic microorganisms that decompose hydrogen to produce methane. Acetogenic methanogenic microorganisms belong, for example, to the genus Methanosarcina or Methanosaeta. Hydrogenogenic methanogenic microorganisms belong, for example, to the genus Methanobacterium.

[0017] In the culturing step (A1), a methane-reducing microorganism is cultured in a liquid medium. The composition of the liquid medium is determined depending on the nutritional requirements of the methane-reducing microorganism. For example, the liquid medium is a calcium-free mineral base medium. This mineral base medium contains 5 mM NaNO, 2 mM KHPO, 1 mM MgCl, 0.1 mM NaSO, 20 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), and 10 mL L of each trace element solution (without CaCl) and vitamin solution. The pH of the liquid medium is adjusted to 7.0 using 6N KOH.

[0018] In the addition step (A2), the methane-inhibiting microorganism is added to an environment in which methanogenic microorganisms exist. In the example of FIG. 1, the environment is soil in a rice paddy where rice is cultivated. The environment is not particularly limited as long as it is an environment in which methanogenic microorganisms exist, and may be, for example, the intestinal environment of an animal such as a cow. The environment may include sludge generated in a sewage treatment plant or the like. For example, the environment may be landfill soil. The environment may be forest soil, sediments such as those in a swamp or river, marine sediments, an anaerobic wastewater treatment reactor, or the digestive tract of an insect such as a termite. The environment may be animal excrement from a cow, pig, chicken, sheep, or the like. The environment may be wastewater such as palm oil. In the addition step (A2), the cultured methane-inhibiting microorganism may be added to an anaerobic environment in which methanogenic microorganisms exist.

[0019] In this way, the method for inhibiting methane production of this embodiment depletes metabolic substances used by methanogenic microorganisms to produce methane through mechanism A, thereby efficiently reducing the amount of methane generated even in environments where the methane produced by methanogenic microorganisms does not remain in the vicinity for long periods of time.The method for inhibiting methane production of this embodiment decomposes methane produced by methanogenic microorganisms through mechanism B, thereby enabling the methane produced by methanogenic microorganisms to be decomposed before it is released into the atmosphere.

[0020] [Screening method for identifying methane-inhibiting microorganisms] A screening method for identifying new methane-inhibiting microorganisms that can be used in the above-mentioned methane production inhibition method will be described. The screening method of this embodiment is intended to identify target microorganisms that can reduce the amount of methane produced by methanogens. The screening method of this embodiment includes at least the following steps (B1) to (B4): (B1) a first culture step of culturing a candidate microorganism that is a target microorganism candidate; (B2) a second culture step of co-culturing a methanogen with the cultured candidate microorganism; (B3) a measurement step of measuring the amount of methane produced by the methanogen; and (B4) an identification step of identifying as the target microorganism a candidate microorganism that produces an amount of methane that is smaller than a predetermined reference value during co-culture with the candidate microorganism.

[0021] In the first culturing step (B1), methane-reducing microorganisms are cultured in a liquid medium. The composition of the liquid medium is determined depending on the nutritional requirements of the methane-reducing microorganisms. For example, the liquid medium contains 2.0 g / L of KH2PO4, 2.0 g / L of (NH4)2SO4, 0.025 g / L of MgSO4.7H2O, 0.5 g / L of NaCl, 0.002 g / L of FeSO4.7H2O, and 0.5% (v / v) CH3OH. The pH of the liquid medium is adjusted to 7.2. In the culturing step, the methane-reducing microorganisms are cultured in an aerobic environment at 30°C. The liquid medium may be a calcium-free inorganic base medium. This mineral basal medium contains 5 mM NaNO3, 2 mM KH2PO4, 1 mM MgCl2, 0.1 mM Na2SO4, 20 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), and 10 mL L-1 of each trace element solution (without CaCl2) and vitamin solution. The pH of the liquid medium is adjusted to 7.0 using 6N KOH.

[0022] The liquid medium for co-cultivating the methanogen and the candidate microorganism in the second culturing step (B2) is, for example, the medium No. 1067 for culturing Methanobacterium bryantii (NBRC 104951).4 0.136g, NH4Cl 0.54g, MgCl2·6H2O 0.2g, CaCl2·2H2O 0.147g, NaHCO3 2.5g, Bacto yeast extract (Difco) 0.2g, sodium acetate 0.8g, vitamin solution 10ml, trace element solution 10ml, resazurin 1mg, cysteine ​​HCl 0.5g, Na2S·9H2O 0.5g, and distilled water 1L.

[0023] The vitamin solution contains biotin 2mg, folic acid 2mg, pyridoxine-HCl 10mg, thiamine-HCl 5mg, riboflavin 5mg, nicotinic acid 5mg, Ca-pantothenic acid 5mg, p-aminobenzoic acid 1mg, vitamin B 12 The trace element solution contains 12.8 g of nitrilotriacetic acid (NTA), 1.35 g of FeCl3·6H2O, 0.1 g of MnCl2·4H2O, 0.024 g of CoCl2·6H2O, 0.1 g of CaCl2·2H2O, 0.1 g of ZnCl2, 0.025 g of CuCl2·2H2O, and 1 L of distilled water. 3 The liquid medium used to co-culture the methanogenic microorganism and the candidate microorganism in the second culturing step (B2) may be medium number 1028 for culturing Methanolobus profundi (NBRC 104158).

[0024] In the second culturing step (B2), an intermediate-product-producing microorganism that produces intermediate products including hydrogen, acetic acid, formic acid, or methyl compounds from organic matter may be co-cultured with the methanogenic microorganism and the candidate microorganism. In this case, the methanogenic microorganism produces methane from the intermediate products produced by the intermediate-product-producing microorganism.

[0025] The methanogenic microorganisms and intermediate product-producing microorganisms used may be those present in soil, for example. In the second culturing step (B2), this soil may be added to the sealed container instead of the methanogenic microorganisms and intermediate product-producing microorganisms and cultured. In the second culturing step (B2), a liquid medium containing the candidate microorganisms is further added to the sealed container containing the methanogenic microorganisms and intermediate product-producing microorganisms, and cultured. The anaerobic environment is created, for example, by replacing oxygen in the sealed container with nitrogen, carbon dioxide, or the like. The anaerobic environment is created according to the oxygen requirement of the candidate microorganisms.

[0026] In the measurement step (B3), the amount of methane produced by the methanogenic microorganisms during co-cultivation of the methanogenic microorganisms and the candidate microorganisms is measured by gas chromatography. This gas chromatography utilizes the fact that different types of gas molecules move through a column at different speeds, and measures the amount of methane as the partial pressure of methane gas. Figures 2(a) and 2(b) show how the partial pressure of methane gas is measured by gas chromatography.

[0027] FIG. 2(a) shows an example of a gas chromatography measurement device. FIG. 2(b) shows the analysis results of the gas chromatography. The vertical axis of FIG. 2(b) shows the time from when the sample is introduced into the measurement device until a peak appears. The horizontal axis of FIG. 2(b) shows the signal intensity. In the example of FIG. 2, nitrogen (N ２ ), methane (CH ４ ) and carbon dioxide (CO ２ The partial pressure of each gas is calculated from the signal intensity of these peaks.

[0028] In the identification step (B4), it is determined whether the amount of methane measured in the measurement step (B3) is smaller than a reference value. The reference value is, for example, the amount of methane produced by a methanogen when the methanogen is cultured in the liquid medium (A1) in which the candidate microorganism has not been cultured or the liquid medium (A1) in which the candidate microorganism has been cultured and then autoclaved. In the identification step (B4), if the amount of methane measured is significantly smaller than the reference value, the candidate microorganism is identified as a target microorganism capable of reducing the amount of methane produced by the methanogen. In the identification step (B4), if the amount of methane measured in the measurement step (B3) is not significantly smaller than the reference value, the candidate microorganism is not identified as a target microorganism.

[0029] In this way, in the screening methods (B1) to (B4), by co-culturing methanogens and candidate microorganisms in the culturing step, it is possible to identify candidate microorganisms that can reduce the amount of methane produced by methanogens. This screening method makes it possible to identify both methane-suppressing microorganisms with a type A mechanism that decomposes metabolic substances required for methanogens to produce methane, and methane-suppressing microorganisms with a type B mechanism that decomposes methane produced by methanogens.

[0030] In addition to steps (B1) to (B4), this screening method may include a step of periodically measuring the amount of methane while the identified target microorganism is cultured alone in a liquid medium containing only methane as a substrate. By periodically measuring the amount of methane in this screening method, it is possible to identify whether the target microorganism decomposes methane. In this way, it is possible to identify whether the target microorganism is a methane-inhibiting microorganism of the A-type mechanism, which decomposes metabolic substances required for methanogenic microorganisms to produce methane, or a methane-inhibiting microorganism of the B-type mechanism, which decomposes methane produced by methanogenic microorganisms.

[0031] 3 is a flowchart showing the procedure of a screening method for identifying a target microorganism capable of reducing the amount of methane produced by a methanogen. In this screening method, a candidate microorganism that is a target microorganism is first cultured (S101). Next, the cultured candidate microorganism and a methanogen are co-cultured in the same sealed container under an anaerobic environment (S102).

[0032] Next, the amount of methane produced by the methanogens during co-culture is measured by gas chromatography (S103). It is then determined whether the measured amount of methane is significantly less than the reference amount (S104). If the measured amount of methane is significantly less than the reference value (YES in S104), this candidate microorganism is identified as a target microorganism capable of reducing the amount of methane produced by the methanogens (S105).

[0033] If the amount of methane measured in the determination of S104 is not significantly smaller than the reference value (NO in S104), the process proceeds to the determination of S106, where it is determined whether there are other candidate microorganisms that have not been co-cultured with the methanogenic microorganisms (S106). If it is determined that there are no other candidate microorganisms (NO in S106), the process ends. If it is determined that there are other candidate microorganisms in the determination of S106 (YES in S106), the process returns to S101.

[0034] The present inventors screened for target microorganisms capable of reducing the amount of methane, a greenhouse gas. First, in order to reproduce the methane production process, the present inventors obtained soil containing methanogenic microorganisms capable of producing methane and archaea capable of producing hydrogen, acetic acid, formic acid, methyl compounds, or the like, which are used by the methanogenic microorganisms to produce methane.

[0035] Some of the candidate microorganisms were anaerobic, while others were aerobic. All of the candidate microorganisms were collected from soil. These candidate microorganisms were cultured in liquid media.

[0036] 22.5 mL of soil containing methanogens and archaea and 2.5 mL of liquid medium containing the cultured candidate microorganisms were added to a vial and sealed. Depending on the oxygen requirement of the candidate microorganism, in the case of anaerobic microorganisms, 44 mL of air in the vial was replaced with 44 mL of nitrogen to create an anaerobic environment. The vial was maintained at 30°C during culture. Figures 4(a) and 4(b) show the co-culture of methanogens, intermediate-product-producing microorganisms, and candidate microorganisms. Figure 4(a) shows a vial during co-culture of methanogens, intermediate-product-producing microorganisms, and candidate microorganisms. Figure 4(b) shows the culture apparatus used for co-culture.

[0037] During the co-cultivation, the gas in the vial was sampled periodically, and the partial pressure of methane gas was measured by gas chromatography (see Figure 2(b)). Figure 5 shows the relationship between the number of days of cultivation when methanogens and archaea were cultivated without adding candidate microorganisms and the amount of methane generated. The vertical axis in Figure 5 indicates the amount of methane generated. The unit of the vertical axis in Figure 5 is milligrams. The horizontal axis in Figure 5 indicates the number of days of cultivation of methanogens and archaea. As shown in Figure 5, it was confirmed that methane was generated stably for about one week from the fifth day onwards.

[0038] When each of multiple candidate microorganisms was co-cultured with a methanogen and an archaea, it was confirmed whether the amount of methane generated was significantly smaller than when the methanogen and archaea were cultured without the addition of the candidate microorganism. Candidate microorganisms that generated significantly less methane were identified as target microorganisms capable of reducing the amount of methane. In this example, the inventors identified Methylobacillus arboreus of the genus Methylobacillus as a target microorganism capable of reducing the amount of methane.

[0039] Figure 6 shows the methane production inhibitory effect of Methylobacillus arboreus. The vertical axis of Figure 6 shows the amount of methane produced per hour. The unit of the vertical axis of Figure 6 is mg / m ２The present inventors confirmed that the amount of methane generated when methanogens and archaea were co-cultured with Methylobacillus arboreus (right side of FIG. 6 ) was 8% less than when methanogens and archaea were cultured without the addition of Methylobacillus arboreus (left side of FIG. 6 ).

[0040] The inventors confirmed that the amount of methane did not decrease when Methylobacillus arboreus was cultured alone in the presence of methane. This suggests that Methylobacillus arboreus suppresses methane production not through a type B mechanism, which decomposes methane produced by methanogenic microorganisms, but through a type A mechanism, which decomposes and depletes the metabolites required for methane production by methanogenic microorganisms.

[0041] [Effects of the Present Invention] The screening method of this embodiment can identify target microorganisms that reduce the amount of methane produced by methanogenic microorganisms. This screening method can identify both methane-inhibiting microorganisms with a type A mechanism that inhibits metabolites required for methane production by methanogenic microorganisms, and methane-inhibiting microorganisms with a type B mechanism that decomposes methane produced by methanogenic microorganisms.

[0042] According to the method for inhibiting methane production of this embodiment, methane production by methanogenic microorganisms is inhibited by a Type A mechanism that inhibits the metabolites required for methane production by methanogenic microorganisms, so the amount of methane generated can be efficiently reduced even in environments where the methane produced by methanogenic microorganisms does not remain in the surrounding area for long periods of time.

[0043] The present invention has been described above using embodiments, but the technical scope of the present invention is not limited to the scope described in the above embodiments, and various modifications and changes are possible within the scope of the gist of the present invention. For example, all or part of the device can be configured by functionally or physically distributing or integrating in any unit. Furthermore, new embodiments resulting from any combination of multiple embodiments are also included in the embodiments of the present invention. The effects of the new embodiments resulting from the combination also have the effects of the original embodiments.

Claims

1. A method for inhibiting methane production, comprising: a culturing step of culturing a methane-inhibiting microorganism that decomposes a metabolic substance required for methane-producing microorganisms, or a methane-inhibiting microorganism that decomposes methane produced by a methanogenic microorganism; and an adding step of adding the cultured methane-inhibiting microorganism to an environment where the methanogenic microorganism exists.

2. The method for inhibiting methane production according to claim 1, wherein the culturing step cultures the methane-inhibiting microorganism that decomposes organic matter as the metabolic substance.

3. The method for inhibiting methane production according to claim 1 or 2, wherein the culturing step cultures the methane-inhibiting microorganism that decomposes hydrogen, acetic acid, formic acid, or a methyl compound as the metabolic substance.

4. The method for inhibiting methane production according to claim 1 or 2, wherein the culturing step cultures the methane-inhibiting microorganism belonging to the genus Anaeromyxobacter, Geomonas, Geobacter, Rhodopseudomonas, Methylobacillus, Methylopila, Methylobacter, Methylobacterium, Methylocaldum, Methylocapsa, Methyloceanibacter, Methylocella, Methylocystis, Methyloferula, Methylogaea, Methylomagnum, Methylomicrobium, Methylomonas, Methylosinus, or Methylotuvimicrobium.

5. The method for inhibiting methane production according to claim 1 or 2, wherein in the adding step, the cultured methane-inhibiting microorganism is added to an anaerobic environment in which the methanogenic microorganism exists.

6. The method for inhibiting methane production according to claim 5, wherein the anaerobic environment is paddy field soil, the intestinal environment of an animal, sludge, landfill soil, forest soil, swamp sediments, river sediments, marine sediments, anaerobic wastewater treatment reactors, the digestive tract of an insect, or animal excrement.

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