Screening method
The screening method identifies microorganisms that reduce methane production by depleting metabolic substances or decomposing methane, addressing the limitations of existing methods and enhancing methane mitigation efficacy.
Patent Information
- Application Number
- JP2024068400
- Authority / Receiving Office
- JP · JP
- Patent Type
- Applications
- Current Assignee / Owner
- Filing Date
- 2024-04-19
- Publication Date
- 2025-10-30
AI Technical Summary
Existing methods fail to identify microorganisms that can reduce methane production by depleting organic matter or intermediate products used by methanogenic microorganisms, limiting the ability to effectively mitigate methane emissions.
A screening method involving culturing candidate microorganisms with methanogenic microorganisms, co-culturing to measure methane production, and identifying those that reduce methane below a reference value, utilizing both Type A mechanisms that decompose metabolic substances and Type B mechanisms that decompose produced methane.
Effectively identifies microorganisms capable of reducing methane production, either by depleting metabolic substances or decomposing produced methane, thereby efficiently inhibiting methane generation in various environments.
Smart Images
Figure 2025164424000001_ABST
Abstract
Description
[Technical Field]
[0001] The present invention relates to a screening method for identifying microorganisms associated with greenhouse gases. [Background technology]
[0002] A technique for reducing the amount of methane produced using microorganisms has been proposed (for example, Non-Patent Document 1). Non-Patent Document 1 describes the identification of microorganisms that consumed 90% or more of the methane in a culture from multiple microorganisms collected from pond sediments. [Prior art documents] [Non-patent literature]
[0003] [Non-Patent Document 1] Isolation and genomic characterization of a proteobacterial methanotroph requiring lanthanides S Kato, M Takashino, K Igarashi, W KitagawaMicrobes and environments, 2020(URL: https: / / www.jstage.jst.go.jp / article / jsme2 / 35 / 1 / 35_ME19128 / _pdf / ) Summary of the Invention [Problem to be solved by the invention]
[0004] The microorganisms described in Non-Patent Document 1 can reduce the amount of methane produced by consuming the methane produced by methanogenic microorganisms. On the other hand, another possible mechanism is to reduce the amount of methane produced by depleting the organic matter or intermediate products that methanogenic microorganisms use to produce methane. The method described in Non-Patent Document 1 has the problem of being unable to identify microorganisms that can reduce the amount of methane produced by such a mechanism.
[0005] Therefore, the present invention has been made in consideration of these points, and an object of the present invention is to provide a screening method that can identify microorganisms that can reduce the amount of methane produced. [Means for solving the problem]
[0006] The screening method of the first aspect of the present invention is a screening method for identifying a target microorganism capable of reducing the amount of methane produced by a methanogenic microorganism, and comprises a first culture step of culturing a candidate microorganism that is a candidate for the target microorganism, a second culture step of co-culturing the methanogenic microorganism with the cultured candidate microorganism, a measurement step of measuring the amount of methane produced by the methanogenic microorganism, and an identification step of identifying as the target microorganism the candidate microorganism whose amount of methane produced by the methanogenic microorganism during co-culture with the candidate microorganism is less than a reference value.
[0007] In the identification step, the candidate microorganism may be identified as the target microorganism when the amount of methane produced by the methanogenic microorganism during co-culture with the candidate microorganism is less than the reference value, which is the amount of methane produced by the methanogenic microorganism when the candidate microorganism is not added.
[0008] In the second culture step, an intermediate-product-producing microorganism that produces an intermediate product including hydrogen, acetic acid, formic acid, or a methyl compound from organic matter, the methanogenic microorganism that produces the methane from the intermediate product, and the candidate microorganism may be co-cultured.
[0009] In the measuring step, the partial pressure of the methane gas produced by the methanogenic microorganism may be measured as the amount of the methane by gas chromatography. [Effects of the Invention]
[0010] According to the present invention, an effect of identifying a microorganism capable of reducing the amount of methane produced can be achieved. [Brief explanation of the drawings]
[0011] [Figure 1] FIG. 1 is a diagram for explaining an outline of a method by which methane-inhibiting microorganisms inhibit methane production. [Figure 2] This shows how the partial pressure of methane gas is measured using gas chromatography. [Figure 3] 1 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. [Figure 4] This shows the co-culture of methanogens, archaea, and candidate microorganisms. [Figure 5] This shows the relationship between the number of days of culture when methanogenic microorganisms and archaea were cultured without adding candidate microorganisms and the amount of methane produced. [Figure 6] This shows the methane production inhibitory effect of Methylobacillus arboreus. DETAILED DESCRIPTION OF THE INVENTION
[0012] [Principles of methane production inhibition] Through extensive research, the present inventors have discovered a method for inhibiting methane production by methanogenic microorganisms capable of producing methane. Figure 1 is a diagram illustrating the principle by which methane-inhibiting microorganisms inhibit methane production. First, the metabolic pathway of methane production by methanogenic microorganisms will be described.
[0013] 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 cultivated. As indicated by the dashed arrow in Figure 1, methane produced by methanogens is absorbed by the rice plants and then released into the atmosphere. Alternatively, as indicated by the solid curved arrow in Figure 1, methane produced by methanogens is released from the soil into the atmosphere.
[0014] The mechanisms by which methane-suppressing microorganisms suppress methane production by methanogenic microorganisms are thought to be Type A mechanism, in which they decompose metabolic substances required for methane production by methanogenic microorganisms, and Type B mechanism, in which they decompose methane produced by methanogenic microorganisms. Type A mechanism is a new mechanism discovered by the present inventors.
[0015] [Methods for suppressing 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; (A2) an addition step of adding the cultured methane-inhibiting microorganism to an environment where methanogenic microorganisms are present; Equipped with.
[0016] 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. Methane-inhibiting microorganisms using the A-type mechanism may also belong to genera other than those listed. 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 then used by intermediate-product-producing microorganisms to produce hydrogen, acetic acid, formic acid, methyl compounds, etc.
[0017] 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.
[0018] 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.
[0019] In the addition step (A2), the methane-inhibiting microorganism is added to an environment in which the methanogenic microorganism exists. In the example of FIG. 1, the environment is soil in a paddy field where rice is grown. The environment is not particularly limited as long as it is an environment in which the methanogenic microorganism exists, 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 soil in a landfill. 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. In the addition step (A2), the cultured methane-inhibiting microorganism may be added to an anaerobic environment in which the methanogenic microorganism exists.
[0020] 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.
[0021] [Screening method for identifying methane-reducing 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 for identifying target microorganisms that can reduce the amount of methane produced by methanogenic microorganisms. The screening method of this embodiment includes at least the following steps (B1) to (B4): (B1) a first culturing step of culturing a candidate microorganism that is a candidate for the target microorganism; (B2) a second culturing step of co-culturing methanogenic microorganisms and the cultured candidate microorganisms; (B3) a measuring step of measuring the amount of methane produced by the methanogenic microorganism; (B4) A step of identifying as a target microorganism a methanogenic microorganism whose amount of methane produced by the methanogenic microorganism during co-culture with the candidate microorganism is less than a predetermined reference value; It has.
[0022] 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 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 with 6 N KOH.
[0023] The liquid medium for co-cultivating the methanogenic microorganism and the candidate microorganism in the second culturing step (B2) is, for example, medium number 1067 for culturing Methanobacterium bryantii (NBRC 104951). This medium contains 0.136 g of KH2PO4, 0.54 g of NH4Cl, 0.2 g of MgCl2·6H2O, 0.147 g of CaCl2·2H2O, 2.5 g of NaHCO3, 0.2 g of Bacto yeast extract (Difco), 0.8 g of sodium acetate, 10 ml of vitamin solution, 10 ml of trace element solution, 1 mg of resazurin, 0.5 g of cysteine HCl, 0.5 g of Na2S·9H2O, and 1 L of distilled water.
[0024] 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, 0.01 g of H3BO3, 0.024 g of Na2MoO4·2H2O, 1 g of NaCl, 0.12 g of NiCl2·6H2O, 0.004 g of Na2SeO4, 0.004 g of Na2WO4, 0.02 g of KAl(SO4)2·12H2O, and 1 L of distilled water. The liquid medium in which the methanogenic microorganism and the candidate microorganism are co-cultured in the second culture step (B2) may be medium number 1028 for culturing Methanolobus profundi (NBRC 104158).
[0025] 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.
[0026] 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 the oxygen in the sealed container with nitrogen, carbon dioxide, etc. The anaerobic environment is created according to the oxygen requirement of the candidate microorganisms.
[0027] In the measurement step (B3), the amount of methane produced by the methanogenic microorganisms during co-cultivation with the candidate microorganisms is measured by gas chromatography. This gas chromatography utilizes the fact that different types of gas molecules move at different speeds through the column to measure 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.
[0028] Figure 2(a) shows an example of a gas chromatography measurement device. Figure 2(b) shows the results of the gas chromatography analysis. The vertical axis of Figure 2(b) represents the time from when the sample is introduced into the measurement device until a peak appears. The horizontal axis of Figure 2(b) represents the signal intensity. In the example of Figure 2, the peaks of nitrogen (N2), methane (CH4), and carbon dioxide (CO2) contained in a sealed container are shown. The partial pressure of each gas is calculated from the signal intensity values of these peaks.
[0029] 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 a liquid medium (A1) in which the candidate microorganism has not been cultured or in which the candidate microorganism has been cultured and then autoclaved, and the methanogen is then cultured. 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.
[0030] 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.
[0031] 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-reducing microorganism of the A-type mechanism, which decomposes metabolic substances required for methanogenic microorganisms to produce methane, or a methane-reducing microorganism of the B-type mechanism, which decomposes methane produced by methanogenic microorganisms.
[0032] [Procedure for screening target microorganisms] 3 is a flowchart showing the steps of a screening method for identifying a target microorganism capable of reducing the amount of methane produced by a methanogenic microorganism. In this screening method, a candidate microorganism that is a target microorganism is first cultured (S101). Next, the cultured candidate microorganism and a methanogenic microorganism are co-cultured in the same sealed container under an anaerobic environment (S102).
[0033] Next, the amount of methane produced by the methanogenic microorganism during co-cultivation is measured by gas chromatography (S103). Next, it is 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 methanogenic microorganism (S105).
[0034] If the amount of methane measured in the determination of S104 is not significantly smaller than the reference value (NO in S104), proceed to the determination of S106 to determine whether there are other candidate microorganisms that are not 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), return to S101.
[0035] <Example> 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 the methanogenic microorganisms use to produce methane.
[0036] The candidate microorganisms were anaerobic and aerobic microorganisms collected from soil and cultured in liquid media.
[0037] 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 the culture. Figures 4(a) and 4(b) show the co-culture of methanogens, intermediate-product-producing microorganisms, and candidate microorganisms. Figure 4(a) shows the vial during co-culture of methanogens, intermediate-product-producing microorganisms, and candidate microorganisms. Figure 4(b) shows the culture apparatus used for the co-culture.
[0038] 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 methanogenic microorganisms and archaea were cultivated without adding candidate microorganisms and the amount of methane produced. The vertical axis in Figure 5 shows the amount of methane produced. The unit of the vertical axis in Figure 5 is milligrams. The horizontal axis in Figure 5 shows the number of days of cultivation of methanogenic microorganisms and archaea. As shown in Figure 5, it was confirmed that methane was produced stably for about one week from the fifth day onwards.
[0039] 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.
[0040] 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 in Figure 6 is mg / m²·h. The inventors confirmed that the amount of methane produced was 8% less when methanogenic microorganisms and archaea were co-cultured with Methylobacillus arboreus (right side of Figure 6) than when methanogenic microorganisms and archaea were cultured without the addition of Methylobacillus arboreus (left side of Figure 6).
[0041] 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.
[0042] [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-suppressing microorganisms with a type A mechanism that suppresses metabolites required for methane production by methanogenic microorganisms, and methane-suppressing microorganisms with a type B mechanism that decomposes methane produced by methanogenic microorganisms.
[0043] 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.
[0044] 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 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. 1. A screening method for identifying a target microorganism capable of reducing the amount of methane produced by a methanogenic microorganism, comprising: a first culturing step of culturing a candidate microorganism that is a candidate for the target microorganism; a second culturing step of co-culturing the methanogenic microorganism and the cultured candidate microorganism; a measuring step of measuring the amount of methane produced by the methanogenic microorganisms; an identifying step of identifying, as the target microorganism, a methanogenic microorganism whose amount of methane produced during co-culture with the candidate microorganism is less than a reference value; A screening method comprising:
2. In the identifying step, the candidate microorganism is identified as the target microorganism when the amount of methane produced by the methanogen during co-culture with the candidate microorganism is smaller than the reference value, which is the amount of methane produced by the methanogen in the absence of the candidate microorganism. The screening method according to claim 1.
3. In the second culturing step, an intermediate-product-producing microorganism that produces an intermediate product containing hydrogen, acetic acid, formic acid, or a methyl compound from organic matter, the methanogenic microorganism that produces the methane from the intermediate product, and the candidate microorganism are co-cultured. The screening method according to claim 1 or 2.
4. In the measuring step, the partial pressure of the methane gas produced by the methanogenic microorganism is measured as the amount of the methane by gas chromatography. The screening method according to claim 1 or 2.