liquid composition

Abstract

To provide novel means capable of generating methane gas with higher efficiency than before, without using raw materials with a large environmental load, in single-phase treatment using a mixture of lumen fluid and liquid containing a methanogen.SOLUTION: A liquid composition which is a blend of rumen fluid collected from a ruminant, a methanogen-containing fluid, and an alkaline porous material.SELECTED DRAWING: None

Description

Technical Field

[0001] The present invention relates to a liquid composition.

Background Art

[0002] Ruminants typified by cows have a variety of microbial communities inside the rumen, and these microbial communities are used to decompose ingested lignocellulose. The rumen fluid collected from the rumen contains microorganisms and enzymes capable of decomposing lignocellulose, and the decomposition of lignocellulose produces organic acids that are easily utilized by methanogens. Therefore, this decomposition process can be used to produce methane gas. Thus, various studies have been conducted on the method for producing methane gas using rumen fluid.

[0003] As a method for producing methane gas using rumen fluid, there has been known a method in which a methane gas raw material containing cellulose (cellulose-based methane gas raw material) is decomposed with rumen fluid and then further fermented with a methanogen-containing liquid containing methanogens to produce methane gas. Such a method is advantageous in that the production of methane gas is promoted as compared with the simplest method for producing methane gas until then, in which the cellulose-based methane gas raw material is treated with a methanogen-containing liquid. However, such a method for producing methane gas requires two steps: the decomposition treatment of the cellulose-based methane gas raw material with rumen fluid and the fermentation treatment of the treated product (the decomposed product of the cellulose-based methane gas raw material) with the methanogen-containing liquid. There are problems in that the process is complicated and the equipment used becomes large-sized. In this specification, such a two-step treatment format is referred to as a "two-phase system".

[0004] In contrast, a method for producing methane gas is known in which the rumen liquid and a methane-producing bacteria-containing liquid are mixed in advance, and the methane gas raw material is then added to this mixture, thereby performing the process in a single step (see Non-Patent Documents 1-2). In this method, the processing of the methane gas raw material is completed in a single step, thus simplifying the process and allowing for the use of small-scale equipment. In this specification, such a single-step processing method is referred to as the "single-phase method". [Prior art documents] [Non-patent literature]

[0005] [Non-Patent Document 1] Zheng et al, Improving the Anaerobic Digestion of Switchgrass via Cofermentation of Rumen Microorganisms (Rumen Bacteria, Protozoa, and Fungi) and a Biogas Slurry, Energy Fuels 2019, 33, 1185-1195. [Non-Patent Document 2] Fonoll, et al, Understanding the Anaerobic Digestibility of Lignocellulosic Substrates Using Rumen Content as a Cosubstrate and an Inoculum, ACS EST Engg. 2021, 1, 424-435. [Overview of the Initiative] [Problems that the invention aims to solve]

[0006] However, the one-phase methane gas production method disclosed in Non-Patent Documents 1 and 2 had the problem of low methane gas production efficiency and low methane gas production volume. When producing methane gas using a mixture of rumen liquid and methane-producing bacteria-containing liquid, it is possible to increase the amount of methane gas produced by adding a strong base such as sodium hydroxide to make the mixture neutral or weakly alkaline. However, such strong bases are highly toxic, and from the perspective of environmental impact, their use is impractical, and their use in methane gas production is also impractical.

[0007] The present invention aims to provide a novel method for generating methane gas with higher efficiency than conventional methods, without using raw materials that have a heavy environmental impact, in a one-phase treatment using a mixture of rumen liquid and methane-producing bacteria-containing liquid. [Means for solving the problem]

[0008] To solve the above problems, the present invention adopts the following configuration. [1] A liquid composition comprising a rumen liquid, a methane-producing bacteria-containing liquid, and an alkaline porous material. [2] The liquid composition according to [1], wherein the porous material is electrically conductive. [Effects of the Invention]

[0009] According to the present invention, a novel method is provided for producing methane gas with higher efficiency than conventional methods in a one-phase process using a mixture of rumen liquid and methane-producing bacteria-containing liquid, without using raw materials that have a heavy burden on the environment. [Brief explanation of the drawing]

[0010] [Figure 1] This graph shows the amount of methane gas generated during the production of methane gas in Example 1 and Comparative Examples 1-4. [Figure 2] This graph shows the types and proportions of microorganisms identified during methane gas generation in Example 1, Comparative Example 1, and Comparative Example 2. [Figure 3] This is SEM imaging data of the powdered oak charcoal used in Example 1. [Figure 4] This is the SEM imaging data of the graphite used in Comparative Example 4. [Modes for carrying out the invention]

[0011] <<Liquid composition>> A liquid composition according to one embodiment of the present invention is a mixture of rumen fluid (sometimes simply referred to as "rumen fluid" in this specification) collected from ruminant animals, a methanogenic bacteria-containing liquid, and an alkaline porous material (sometimes simply referred to as "porous material" in this specification). According to the liquid composition of this embodiment, methane gas can be produced with high efficiency by a single-phase process without using raw materials that have a high environmental burden, such as strong base agents such as sodium hydroxide, and the amount of methane gas produced can be dramatically increased compared to conventional methods. Moreover, the residue remaining after processing the methane gas raw materials does not contain highly environmentally harmful substances, and its processing has an extremely low environmental burden, making it easy to, for example, return it to farmland.

[0012] <Lumen fluid> Examples of ruminant animals from which the rumen fluid is collected include cattle.

[0013] The rumen (first stomach) is a large sac in ruminants that utilizes plant fiber as an energy source. For example, in cattle, it occupies almost the entire left side of the abdominal cavity and the posterior half of the right side. The rumen accounts for 80% of the entire complex stomach, which consists of the first to fourth stomachs, and is about 200 liters in size in dairy cows (adult cows). The rumen stores a large amount of feed, which is broken down by microorganisms living within it. About 90% of the rumen contents are water, and fermentation by microorganisms takes place sufficiently.

[0014] The lumen is home to countless microorganisms, with over 60 species per gram of lumen contents, totaling approximately 1 x 10¹⁶ 9~1×10 11 individual bacteria (cellulose decomposing bacteria, starch decomposing bacteria, protein decomposing bacteria, etc.) and about 1×10 5 ~1×10 6 individual protozoa (protozoans) inhabit, and due to the action of these microorganisms (cellulose decomposing bacteria, starch decomposing bacteria, protein decomposing bacteria, etc.), the decomposition and synthesis of feed components are actively carried out in the rumen.

[0015] Rumen fluid is an aqueous solution (rumen fluid in the first stomach) containing various microorganisms inhabiting the rumen of such ruminants, and its amount is, for example, about 200 L per cow. Rumen fluid can be easily collected from the rumen of ruminants by any method known to those skilled in the art. In the present embodiment, for example, rumen fluid collected from live livestock, rumen fluid that was conventionally treated as waste after slaughter of livestock, etc. can be used.

[0016] The collected rumen fluid may be used after performing known pretreatment, or may be used as it is in the state of "raw rumen fluid" without performing pretreatment. Also, the collected rumen fluid may be used immediately after collection, or may be used after storage such as freezing preservation. Note that the rumen fluid may be appropriately diluted.

[0017] Rumen fluid contains microorganisms having methane-producing ability (which may be referred to as "methanogens" in this specification) and microorganisms having cellulose-decomposing ability (which may be referred to as "cellulose decomposing bacteria" in this specification), and further may contain microorganisms having hemicellulose-decomposing ability (which may be referred to as "hemicellulose decomposing bacteria" in this specification). Even if the methanogen-containing liquid is not formulated, methane can be produced. However, by formulating the methanogen-containing liquid, the amount of methane gas produced can be increased, and methane gas can be produced with high efficiency. Further, by also formulating the porous substance exhibiting the alkalinity, the amount of methane gas produced can be further increased, and methane gas can be produced with even higher efficiency.

[0018] Examples of the methanogens contained in the rumen fluid include microorganisms belonging to the genus Methanobrevibacter, microorganisms belonging to the genus Methanobacterium, microorganisms belonging to the genus Methanoculleus, microorganisms belonging to the genus Methanosarcina, and the like.

[0019] Examples of the cellulolytic bacteria contained in the rumen fluid include microorganisms belonging to the genus Prevotella, and the like. Examples of the hemicellulolytic bacteria contained in the rumen fluid include microorganisms belonging to the genus Butyrivibrio, microorganisms belonging to the genus Ruminococcus, and the like.

[0020] <Liquid containing methanogens> The liquid containing methanogens is a liquid material necessary for methane fermentation and contains microorganisms (methanogens) having the ability to produce methane. Examples of the liquid containing methanogens include extracts from cow dung; digestion liquids generated after methane fermentation of food waste; sludge and the like generated in various treatment facilities for domestic waste or industrial waste, such as digestion sludge from anaerobic wastewater treatment facilities.

[0021] The methanogens are anaerobic microorganisms having the ability to produce methane. Among the methanogens, there are bacteria that produce methane gas from acetic acid (decompose acetic acid to produce methane gas) and bacteria that produce methane gas from hydrogen and carbon dioxide (synthesize methane gas from hydrogen and carbon dioxide).

[0022] Examples of the methanogens contained in the liquid containing methanogens include the same ones as the methanogens contained in the above-mentioned rumen fluid.

[0023] In the liquid composition, the ratio of the amount of rumen liquid (L) to the total amount of the rumen liquid (L) and the amount of methane-producing bacteria-containing liquid (L) is preferably 4% by volume or more, and may be, for example, 6% by volume or more, or 8% by volume or more. By having this ratio above the lower limit, methane can be produced with higher efficiency. The upper limit of the aforementioned ratio is not particularly limited. For example, in order to avoid excessive use of lumen fluid, the ratio may be either 30% by volume or less, or 15% by volume or less. The aforementioned percentages may be, for example, 4-30% by volume, 6-30% by volume, and 8-30% by volume, or 4-15% by volume, 6-15% by volume, and 8-15% by volume. However, these are merely examples of the aforementioned percentages.

[0024] <Porous material exhibiting alkalinity> The aforementioned liquid composition contains an alkaline porous material, that is, a material that is not only porous but also alkaline, enabling highly efficient methane production. The methanogenic bacteria grow slowly under acidic conditions and grow more actively under weakly alkaline conditions. On the other hand, rumen fluid is acidic, and a mixture of rumen fluid and methane-producing bacteria-containing fluid is usually weakly acidic. Therefore, methane-producing bacteria do not grow actively in such a mixture. In contrast, the aforementioned liquid composition contains an alkaline porous material in addition to rumen fluid and methane-producing bacteria-containing fluid, so its pH is leaning towards weakly alkaline. In such a liquid composition, methane-producing bacteria grow actively, and as a result, methane can be produced with high efficiency. Furthermore, in this embodiment, by using an alkaline porous material, the use of raw materials that have a heavy burden on the environment, such as strong bases like sodium hydroxide, is eliminated.

[0025] In this specification, a porous material exhibiting alkalinity means a porous material in which, when the pH of a mixture obtained by thoroughly mixing 1 part by mass of the porous material with 10 parts by mass of water is measured at room temperature, the pH of the mixture is greater than 7.

[0026] In this specification, "room temperature" means a temperature that is neither cooled nor heated, i.e., a normal temperature, such as 15-25°C.

[0027] In this specification, "porous" means having a large number of pores, for example, whose maximum opening diameter is 30 μm or less. Here, "maximum diameter" means the diameter of the circle if the shape of the pore opening is circular, and the maximum length of the line segment obtained by connecting two different points on the opening with a straight line if the shape of the pore opening is non-circular.

[0028] Examples of porous materials exhibiting alkalinity include charcoal. Examples of the aforementioned charcoal include oak charcoal, Japanese evergreen charcoal, Japanese oak charcoal, sawtooth oak charcoal, Japanese cedar charcoal, willow charcoal, and bamboo charcoal.

[0029] The porous material blended in the liquid composition may be one type or two or more types, and if there are two or more types, their combination and ratio can be arbitrarily selected according to the purpose.

[0030] The charcoal is preferably obtained by burning woody raw materials at a high temperature, more preferably obtained by burning at a temperature of 800°C or higher (charcoal with a carbonization temperature of 800°C or higher), and even more preferably white charcoal. Examples of the white charcoal include oak white charcoal, evergreen oak white charcoal, Japanese evergreen oak white charcoal, and sawtooth oak white charcoal.

[0031] The aforementioned alkaline porous material is preferably in powder form. By using the powdered porous material, methane gas can be produced with higher efficiency.

[0032] The particle size of the alkaline porous material is preferably 10 to 500 μm, more preferably 20 to 350 μm, and may be, for example, 30 to 300 μm and 40 to 275 μm. Having the particle size of the porous material within this range allows for more efficient methane gas production. The particle size of the porous material can be confirmed, for example, by observing the porous material using known analytical instruments such as an optical microscope or an electron microscope. Alternatively, for example, a mixture of particles of various particle sizes can be passed through a sieve with a mesh size of X, and the particles that pass through are then passed through a sieve with a mesh size of Y (where X > Y) to select the particles that do not pass through, thereby obtaining particles with a particle size greater than Y and less than or equal to X. In this embodiment, porous material with a particle size within the desired range can be obtained and used by such a method.

[0033] The alkaline porous material is preferably conductive, more preferably white charcoal, and even more preferably one or more selected from the group consisting of oak white charcoal, Japanese oak white charcoal, Japanese evergreen oak white charcoal, and sawtooth oak white charcoal. By using the liquid composition containing such a porous material, methane gas can be produced with higher efficiency. As described above, the production of methane gas by the methanogenic bacteria involves two pathways: one that produces methane gas from acetic acid (CH3COOH → CH4 + CO2) and another that produces methane gas from hydrogen and carbon dioxide (4H2 + CO2 → CH4 + 2H2O). In both pathways, electron transfer between molecules occurs in the reaction that produces methane gas. When the porous material is conductive, methane gas can be produced with higher efficiency, but it is possible that the porous material facilitates electron transfer during methane gas production.

[0034] In this specification, "conductive" means that the conductivity of the object is 100 S / m or higher, unless otherwise specified. For example, the conductivity may be 250 S / m or higher, or 350 S / m or higher. Conductivity can be measured using a conductivity meter.

[0035] In the liquid composition, the amount of the porous material per 1 L of the total amount of rumen liquid and methane-producing bacteria-containing liquid is preferably 0.5 g or more, more preferably 1 g or more, and even more preferably 1.5 g or more. When the amount is above the lower limit, methane gas can be produced with higher efficiency. In particular, since the porous material is alkaline, the higher the amount within an appropriate range, the more easily the pH of the liquid composition and the pH of the mixture of the liquid composition and the cellulosic methane gas raw material, as described later, will exceed 7, and as a result, the efficiency of methane gas production will be higher. The upper limit of the aforementioned blending amount is not particularly limited. In terms of not using an excessive amount of the porous material, it is preferable that the blending amount be 10 g or less. The aforementioned blending amount may be, for example, 0.5 to 10 g, 1 to 10 g, or 1.5 to 10 g. However, these are just examples of the blending amount.

[0036] Both methane-producing bacteria originating from the rumen fluid and methane-producing bacteria originating from the methane-producing bacteria-containing solution adhere to (survive) the alkaline porous material in the liquid composition. It is presumed that both of these methane-producing bacteria can stably survive in the voids within the porous material. In other words, it is presumed that by using the porous material, these methane-producing bacteria can grow while stably coexisting. Furthermore, cellulose-degrading bacteria originating from the rumen fluid also adhere to (survive) the alkaline porous material in the liquid composition. In other words, it is presumed that the coexistence of methane-producing bacteria originating from the rumen fluid, methane-producing bacteria originating from a methane-producing bacteria-containing solution, and cellulose-degrading bacteria originating from the rumen fluid in the porous material in the liquid composition increases the efficiency of methane gas production in the liquid composition.

[0037] <Other ingredients (1)> The liquid composition may contain other raw materials (which may be referred to as "other raw materials (1)" in this specification) that do not fall under the rumen liquid, the methane-producing bacteria-containing liquid, or the alkaline porous material, as long as the effects of the present invention are not impaired. The aforementioned other raw materials (1) can be arbitrarily selected depending on the purpose and are not particularly limited.

[0038] The other raw materials (1) blended in the liquid composition may consist of only one type or two or more types, and if there are two or more types, their combination and ratio can be arbitrarily selected according to the purpose.

[0039] Other preferred raw materials (1) include, for example, non-porous conductive components (sometimes referred to as "non-porous conductive components" in this specification), microbial culture materials, solvents, and the like.

[0040] [Non-porous conductive component] By using the liquid composition containing the non-porous conductive component, methane gas can be produced with higher efficiency than when using the liquid composition without the non-porous conductive component.

[0041] The liquid composition may, for example, contain the porous material that does not have conductivity and the non-porous conductive component, and may not contain the porous material that does have conductivity; or it may contain the porous material that does have conductivity and the non-porous conductive component, and may not contain the porous material that does not have conductivity; or it may contain the porous material that has conductivity, the porous material that does not have conductivity, and the non-porous conductive component.

[0042] Examples of the non-porous conductive components include carbon materials such as graphite, graphene, carbon nanotubes (CNTs), carbon nanohorns (CNHs), and fullerenes; elemental metals such as iron, copper, and zinc; and metal salts such as sodium chloride, potassium chloride, copper(II) chloride, and zinc chloride. The metal salts may be hydrated or unhydrated anhydrous salts. These non-porous conductive components may also include components equivalent to the microbial culture materials described later. Among these, graphite is preferred because it is inexpensive, safer, highly versatile, and highly effective in improving the efficiency of methane gas production.

[0043] The non-porous conductive component is preferably in powder form, similar to the case of the alkaline porous material described above. By using a powdered non-porous conductive component, methane gas can be produced with higher efficiency.

[0044] [Materials for culturing microorganisms] By using the liquid composition containing the culture material for the aforementioned microorganisms, the efficiency of methane gas production may be increased.

[0045] Examples of culture materials for microorganisms include culture medium materials such as yeast extract and inorganic salts, which are materials known in this field. Examples of the inorganic salts include sodium acetate, potassium acetate, ammonium chloride, potassium dihydrogen phosphate, sodium dihydrogen phosphate, calcium chloride, sodium chloride, magnesium chloride, ethylenediaminetetraacetate-iron complex (Fe-EDTA), ethylenediaminetetraacetate-calcium complex (Ca-EDTA), ethylenediaminetetraacetate-magnesium complex (Mg-EDTA), cobalt chloride, manganese chloride, and nickel chloride. The inorganic salts may be hydrated or unhydrated anhydrous salts.

[0046] [solvent] The liquid composition containing the aforementioned solvent may have improved handling properties. In this specification, unless otherwise specified, the term "solvent" refers not only to components capable of dissolving solutes in solution, but also to components that act as a dispersion medium in a dispersion.

[0047] Examples of the solvent include water and organic solvents. The organic solvent is preferably a hydrophilic solvent that can be uniformly mixed with the same volume of water at room temperature.

[0048] The amount of the other raw material (1) in the liquid composition can be arbitrarily selected depending on the type of the other raw material (1). For example, if the other raw material (1) is a raw material other than a solvent, the ratio of the amount (parts by mass) of the other raw material (1) to the total amount (parts by mass) of all raw materials in the liquid composition is preferably 20% by mass or less, and may be, for example, 10% by mass or less, 5% by mass or less, or 1% by mass or less. By keeping the ratio below the upper limit, methane gas can be produced with higher efficiency. On the other hand, the ratio is 0% by mass or more. When the other raw material (1) is a solvent, the ratio of the amount (parts by mass) of the other raw material (1) (i.e., the solvent) to the total amount (parts by mass) of all raw materials in the liquid composition is preferably 70% by mass or less, and may be, for example, 50% by mass or less, 30% by mass or less, or 10% by mass or less. A ratio below the upper limit allows for the production of methane gas with higher efficiency. On the other hand, the ratio is 0% by mass or more.

[0049] In the liquid composition, the ratio of the total amount (parts by mass) of the rumen liquid, the methane-producing bacteria-containing liquid, and the alkaline porous material to the total amount (parts by mass) of all raw materials is preferably 80% by mass or more, and may be, for example, 90% by mass or more, 95% by mass or more, or 99% by mass or more. When the ratio is above the lower limit, methane gas can be produced with higher efficiency. On the other hand, the ratio is 100% by mass or less.

[0050] <Method for producing a liquid composition> The liquid composition can be produced by blending rumen fluid, a methane-producing bacteria-containing solution, an alkaline porous material, and, if necessary, the other raw materials (1). After blending each component, the resulting mixture may be used as the liquid composition, or, if necessary, a known post-treatment or purification operation may be performed to obtain the resulting liquid composition. The post-treatment and purification operations are not particularly limited, as long as the liquid composition maintains a state in which it contains methane-producing bacteria derived from the rumen fluid, methane-producing bacteria derived from the methane-producing bacteria-containing solution, cellulose-degrading bacteria derived from the rumen fluid, and the porous material.

[0051] The order in which each raw material (rumen liquid, methane-producing bacteria-containing liquid, alkaline porous material, and the other raw materials (1)) are blended is not particularly limited.

[0052] The method of mixing each raw material and the mixture after all raw materials have been combined (for example, the liquid composition) is not particularly limited as long as it is thoroughly mixed, and any known method such as mixing by rotating a stirring bar or stirring blade can be appropriately selected. The temperature at the time of blending each raw material and the temperature of the blended product (e.g., the liquid composition) after all raw materials have been blended are not particularly limited as long as the individual raw materials or the blended product do not deteriorate, but are preferably between 15 and 40°C.

[0053] <<Method for producing methane gas (Method for using liquid composition)>> The aforementioned liquid composition can be used to produce methane gas. A method for producing methane gas using the liquid composition includes, for example, a step (A) in which a methane gas raw material containing cellulose (sometimes referred to herein as "cellulose-based methane gas raw material") is introduced into a treatment tank containing the liquid composition, thereby decomposing the cellulose-based methane gas raw material, and a step (B) in which the decomposed cellulose-based methane gas raw material is subjected to methane fermentation, wherein steps (A) and (B) are carried out in parallel.

[0054] <Process (A)> In step (A) above, the cellulosic methane gas raw material is decomposed by contacting the liquid composition with the cellulosic methane gas raw material. The cellulosic methane gas raw material (cellulose in the cellulosic methane gas raw material) is the target of decomposition by the cellulose-degrading microorganisms (cellulose-degrading bacteria, more specifically cellulose-degrading bacteria derived from rumen fluid) in the liquid composition.

[0055] [Liquid composition] The aforementioned liquid composition is as described above. The liquid composition may consist of only one type or two or more types. If there are two or more types, their combination and ratio can be arbitrarily selected according to the purpose.

[0056] [Cellulose-based methane gas raw material] The cellulosic methane gas raw material is not particularly limited as long as it contains cellulose and is a raw material that enables the production of methane gas by methane fermentation. The cellulosic methane gas raw material may be, for example, a plant containing lignocellulose (also referred to as "lignocellulosic biomass") itself, a processed product obtained by processing the plant (for example, processed food and its waste), or paper obtained by processing the plant in a known manner. The type of plant is not particularly limited. It is preferable that the plant be herbaceous, as it can be more easily used for methane gas production. The plant is preferably one or more species selected from the group consisting of its stem, leaves, fruits, and roots, or processed products thereof. The aforementioned paper materials may, for example, be recycled paper (recycled paper) produced for purposes other than those of the present invention, or they may be waste paper.

[0057] The aforementioned cellulosic methane gas raw material may be one type or two or more types, and if there are two or more types, their combination and ratio can be arbitrarily selected according to the purpose.

[0058] In step (A), the cellulosic methane gas raw material is preferably in the form of a pulverized material. By using the pulverized material, methane gas can be produced with higher efficiency. The maximum diameter of the pulverized material is not particularly limited, but is preferably 10 mm or less. By using such pulverized material, cellulose can be decomposed with higher efficiency, and methane gas can be generated with higher efficiency in step (B) described later. The lower limit of the maximum diameter of the pulverized material is not particularly limited. In terms of making the pulverized material easier, the maximum diameter of the pulverized material may be 0.1 mm or more. In this specification, "maximum diameter of the pulverized material" means the maximum length of a line segment obtained by drawing a straight line between two different points on the outer circumference of the image of the pulverized material when the pulverized material is observed using means such as an optical microscope.

[0059] The amount of the cellulosic methane gas raw material added per liter of the total amount of rumen liquid and methane-producing bacteria-containing liquid, calculated from the amount of the liquid composition, is preferably 1.25 g or more, and may be, for example, 2.5 g or more, or 3.75 g or more. By having the amount added be greater than or equal to the lower limit, methane gas can be produced with higher efficiency. The upper limit of the input amount is not particularly limited. In terms of not exceeding the amount of cellulosic methane gas raw material, it is preferable that the input amount be 25g or less. The amount to be added may be, for example, 1.25 to 25 g, 2.5 to 25 g, or 3.75 to 25 g. However, these are just examples of the amount to be added.

[0060] [pH of a mixture of liquid composition and cellulosic methane gas raw material] The pH of the mixture obtained by contacting the liquid composition with the cellulosic methane gas raw material is not particularly limited, but is preferably greater than 7, and more preferably 7.1 or higher, at room temperature. Setting the pH in this way increases the efficiency of methane gas production. On the other hand, the upper limit of the pH at room temperature is not particularly limited. For example, the pH can be easily adjusted to 9 or less. The pH at room temperature may be, for example, greater than 7 and less than or equal to 9, or between 7.1 and 9. However, these are just examples of the pH range. The pH shown herein may be the pH of the mixture at room temperature immediately after contacting the cellulosic methane gas raw material with the liquid composition (this may be referred to as the "initial pH" in this specification). Here, "immediately after contact" means within 12 hours from the start of contact between the cellulosic methane gas raw material and the liquid composition.

[0061] The method of contacting the cellulosic methane gas raw material and the liquid composition in step (A) is not particularly limited as long as they can come into sufficient contact, and any known method such as mixing by rotating a stirring bar or stirring blade can be appropriately selected. The temperature of the mixture in step (A) is not particularly limited as long as the mixture does not deteriorate, but it is preferably 15 to 40°C.

[0062] <Process (B)> In step (B) above, methane gas is produced by fermenting the decomposed cellulosic methane gas raw material. For example, methane gas can be produced by growing methane-producing bacteria derived from rumen fluid, methane-producing bacteria derived from a methane-producing bacteria-containing solution, and cellulose-degrading bacteria derived from rumen fluid in the decomposed cellulosic methane gas raw material (in other words, in the decomposition products of the cellulosic methane gas raw material). To do this, the decomposed cellulosic methane gas raw material (or the mixture thereof) should be prepared under conditions suitable for the cultivation of these microorganisms.

[0063] More specifically, in step (B), the decomposed cellulosic methane gas raw material (or the mixture) can be placed under static culture or osmotic culture conditions. That is, in step (B), the mixture of the cellulosic methane gas raw material and the liquid composition, on which the above-mentioned microorganisms (methane-producing bacteria, cellulose-degrading bacteria) have grown, also serves as the culture medium. For example, in step (B), the mixture can be placed under a temperature condition of preferably 30 to 45°C, more preferably 32 to 42°C.

[0064] In the porous material exhibiting alkalinity in the culture medium where methane gas production has begun, both methane-producing bacteria originating from the rumen liquid and methane-producing bacteria originating from the methane-producing bacteria-containing liquid adhere to (survive) the porous material exhibiting alkalinity in the same way as in the porous material exhibiting alkalinity in the liquid composition. It is presumed that both of these methane-producing bacteria stably survive in the voids within the porous material. It is presumed that by using the porous material, these methane-producing bacteria can grow while stably coexisting. In the porous material exhibiting alkalinity in the culture medium where methane gas generation has begun, cellulose-degrading bacteria originating from the rumen fluid also adhere to (survive) in the same way as in the porous material exhibiting alkalinity in the liquid composition. In other words, it is presumed that the coexistence of methane-producing bacteria originating from the rumen fluid, methane-producing bacteria originating from the methane-producing bacteria-containing solution, and cellulose-degrading bacteria originating from the rumen fluid on the porous material increases the efficiency of methane gas generation in step (B).

[0065] Step (B) is preferably carried out under anaerobic conditions. More specifically, the concentration of oxygen gas in the gas phase region in contact with the culture medium is preferably 0.01% by volume or less, more preferably 0.007% by volume or less, and may even be 0% by volume. To achieve this, the gas phase region can be replaced with a non-oxygen gas such as carbon dioxide, nitrogen, helium, or argon.

[0066] In step (B), for example, by reducing the concentration of hydrogen gas in the gas phase region in contact with the culture medium, the amount of methane gas produced in the pathway for generating methane gas from hydrogen and carbon dioxide can be increased. To achieve this, for example, step (B) can be carried out while circulating the non-oxygen gas other than hydrogen gas in the gas phase region in contact with the culture medium so that the concentration of hydrogen gas in the gas phase region in contact with the culture medium can be maintained at a low level.

[0067] The method for producing methane gas described above may include other steps that do not fall under either step (A) or step (B), as long as they do not impair the effects of the present invention. The aforementioned other steps can be arbitrarily selected depending on the purpose and are not particularly limited. The number of other steps in the methane gas production method may be only one or two or more. If there are two or more steps, the types of these other steps may be only one or two or more. If there are two or more steps, their combinations and ratios can be arbitrarily selected according to the purpose.

[0068] In the culture medium, if either or both of the above-mentioned alkaline and conductive porous material and the non-porous conductive component are included (i.e., if any conductive component is included), then in the culture medium where methane gas production has started, the growth of methane-producing bacteria that produce methane gas from hydrogen and carbon dioxide is typically dominant over that of methane-producing bacteria that produce methane gas from acetic acid. In other words, in step (B), it is possible to make the pathway for producing methane gas from hydrogen and carbon dioxide dominant in the culture medium containing either or both of the conductive porous material and the non-porous conductive component.

[0069] According to the above-described methane gas production method, by contacting the cellulosic methane gas raw material with the liquid composition, the cellulosic methane gas raw material comes into contact with the mixture of rumen liquid and methane-producing bacteria-containing liquid. This allows the cellulosic methane gas raw material to be processed in one step in the above-described treatment tank, enabling methane gas production in a single phase. Therefore, unlike the two-phase method which requires two steps—treatment of the cellulosic methane gas raw material in the rumen liquid and treatment of the resulting product with methane-producing bacteria-containing liquid—the above-described methane gas production method allows methane gas to be produced using a simplified process and compact equipment. Moreover, unlike conventional single-phase methane gas production methods, by using an alkaline porous material, the amount of methane gas produced can be increased without using raw materials that have a heavy environmental burden, such as strong bases like sodium hydroxide, despite being a single-phase method, and methane gas can be produced with high efficiency. [Examples]

[0070] The present invention will be described in more detail below with reference to specific examples. However, the present invention is not limited in any way to the examples shown below.

[0071] <<Formation of liquid composition, generation of methane gas>> [Example 1] By filtering raw rumen liquid derived from cattle collected from a meat processing plant using a sieve with holes having a diameter of 0.5 mm, solid components were removed from the raw rumen liquid, and a rumen solution was obtained. We prepared powdered oak charcoal with a particle size of 100-250 μm (manufactured by "Sumiya no Kurashi" in Shichikashuku-cho, Miyagi Prefecture). Using a blender, the strawberry stems and leaves were crushed into pieces about 1-2 mm square.

[0072] At room temperature, a liquid composition was obtained by adding a starter culture solution for methane fermentation derived from cow manure (45 mL), the above-mentioned rumen liquid (5 mL), and the above-mentioned powdered oak charcoal (0.1 g) to a 50 mL vial and stirring thoroughly. At room temperature, the entire volume of the liquid composition was further mixed with the pulverized strawberry stems and leaves (0.25 g) and thoroughly stirred. Immediately after stirring, the pH (initial pH) of the mixture of the liquid composition and the pulverized strawberry stems and leaves was measured and found to be 7.2.

[0073] Next, the vial containing the mixture was sealed with a rubber stopper, and the air in the gas phase inside the vial was replaced with carbon dioxide gas at a concentration of 99.995% by volume. Then, the vial was placed inside an incubator adjusted to a temperature of 35°C, and static culture was performed. During static culture, the contents of the vial were agitated once a day by shaking the vial by hand. Also during static culture, a syringe was inserted into the rubber stopper attached to the vial, and the amount of gas generated inside the vial was read from the change in the syringe scale. A portion of the generated gas was then collected as a sample, and the amount of methane gas was measured by gas chromatography. More specifically, the methane concentration of the collected sample was measured by gas chromatography, and the increase in methane gas was calculated by multiplying the increase in gas by this measured methane concentration. Furthermore, since methane gas is generated from both the methane fermentation starter culture itself and the rumen liquid itself, the amount of methane gas generated from only the methane fermentation starter culture and the amount of methane gas generated from only the rumen liquid at this point were determined, and the sum of these amounts of methane gas was subtracted from the increase in methane gas calculated above. This calculated value was then adopted as the amount of methane gas generated at this point. This measurement of methane gas generation was performed regularly every few days until 21 days after the start of static culture. The results are shown in Figure 1.

[0074] Furthermore, 21 days after the start of static culture, the seal on the vial was removed and the pH of the culture medium was measured. The results are shown in Table 1.

[0075] Furthermore, a sample was taken from the culture medium 21 days after the start of static culture, and oak charcoal was isolated by centrifugation. A portion of this isolated oak charcoal was washed three times with sterile water to obtain washed oak charcoal. Using a DNA extraction kit (Fast DNA SPIN Kit FOR Soil), microbial DNA was extracted from these oak charcoal (unwashed) (approximately 0.5g) and washed oak charcoal (approximately 0.5g). The extracted DNA was subjected to amplicon sequencing analysis of the 16S rRNA V4 region to identify the archaeal and bacterial community structure in the culture medium. The data obtained using the microbiome analysis software "Qiime2" was analyzed, and for those that were divided into several genera, a Blast search was performed to determine the most closely related species name for the target microorganism. The identified microorganisms are shown in Figure 2. In Figure 2, "Example 1-1" shows the microorganisms from the washed oak charcoal, and "Example 1-2" shows the microorganisms from the unwashed oak charcoal.

[0076] [Comparative Example 1] Except for not using the rumen solution and powdered oak charcoal described above, the comparative liquid composition and crushed strawberry stems and leaves were mixed in the same manner as in Example 1, and static culture was performed to measure the amount of methane gas produced. The results are shown in Figure 1. Furthermore, the microorganisms in the obtained culture solution were analyzed in the same manner as in Example 1. The results are shown in Figure 2.

[0077] [Comparative Example 2] Except for not using the powdered oak charcoal mentioned above, the comparative liquid composition and the crushed strawberry stems and leaves were mixed in the same manner as in Example 1, and static culture was performed to measure the amount of methane gas produced. The results are shown in Figure 1. Furthermore, the microorganisms in the obtained culture solution were analyzed in the same manner as in Example 1. The results are shown in Figure 2.

[0078] [Comparative Example 3] Except for using a 12N sodium hydroxide aqueous solution (30 μL) instead of the powdered oak charcoal (0.1 g) mentioned above, the comparative liquid composition and the crushed strawberry stems and leaves were mixed in the same manner as in Example 1, and static culture was performed to measure the amount of methane gas generated. The results are shown in Figure 1.

[0079] [Comparative Example 4] Instead of the above powdered oak charcoal (0.1g), use graphite (Wako Pure Chemical Industries, Ltd. special grade reagent, conductivity 10). 6 Except for using S / m) (0.1g), the comparative liquid composition and the crushed strawberry stems and leaves were mixed in the same manner as in Example 1, and static culture was performed to measure the amount of methane gas generated. The graphite was in powder form, and of that, powder with a particle size of less than 45 μm accounted for more than 95% by mass. Note that graphite, unlike oak charcoal, is a neutral, non-porous conductive component. The results are shown in Figure 1.

[0080] [Comparative Example 5] Except for using finely shredded melamine sponge (manufactured by Lec Co., Ltd.) (0.5g) instead of the powdered oak charcoal (0.1g) mentioned above, the comparative liquid composition and the crushed strawberry stems and leaves were mixed in the same manner as in Example 1, and static culture was performed to measure the amount of methane gas generated.

[0081] [Table 1]

[0082] As is clear from Figure 1, in Comparative Example 1, which used a comparative liquid composition containing only methane fermentation starter culture, the amount of methane gas produced was the lowest 21 days after the start of static culture. In Comparative Example 2, which used a comparative liquid composition containing rumen liquid and methane fermentation starter culture, the amount of methane gas produced was higher than in Comparative Example 1, but still insufficient. In contrast, in Example 1, which used a liquid composition containing rumen liquid, methane fermentation starter culture, and oak charcoal, the amount of methane gas produced was significantly higher than in Comparative Example 1. In Comparative Example 4, which used a comparative liquid composition containing graphite instead of oak charcoal, the amount of methane gas produced was higher than in Comparative Example 2, confirming the effect of using conductive components, but the amount of methane gas produced was lower than in Example 1. In Comparative Example 3, which used a comparative liquid composition containing sodium hydroxide instead of oak charcoal, the amount of methane gas produced was the highest. However, this liquid composition was not suitable for practical use because it contained a raw material (sodium hydroxide) that had a high environmental impact. Although not shown in Figure 1, Comparative Example 5, which used a comparative liquid composition in which melamine sponge was used instead of oak charcoal, showed almost the same amount of methane gas production as Comparative Example 2 at the same measurement timing.

[0083] As shown in Figure 2, it was confirmed that the washed oak charcoal in Example 1 (Example 1-1), like the unwashed oak charcoal (Example 1-2), contained both methane-producing bacteria derived from the rumen fluid and methane-producing bacteria derived from the methane fermentation starter culture. Here, the main methane-producing bacteria derived from the rumen fluid are microorganisms belonging to the genus Methanobrevibacter, and the main methane-producing bacteria derived from the methane fermentation starter culture are microorganisms belonging to the genus Methanbacterium, microorganisms belonging to the genus Methanoculleus, and Methanosarcina mazei. In other words, in Example 1, it was confirmed that methane gas was produced under the activity of these microorganisms in the culture medium.

[0084] Furthermore, it was confirmed that the washed oak charcoal in Example 1 also contained Butyrivibrio hungatei and Ruminococcus albus, microorganisms involved in the decomposition of hemicellulose (hemicellulose-degrading bacteria), as well as Prevotella ruminocola, a microorganism involved in the decomposition of cellulose (cellulose-degrading bacteria). In the oak charcoal (unwashed) in Example 1, the survival of Prevotella ruminocola was confirmed, and in the culture solutions of Comparative Examples 1 and 2, the survival of Ruminococcus albus was confirmed.

[0085] Figure 3 shows scanning electron microscope (SEM) imaging data of the powdered oak charcoal used in Example 1. Figure 4 shows SEM imaging data of the graphite used in Comparative Example 4. As is clear from these imaging data, oak charcoal is a porous material with numerous voids with an aperture diameter of approximately 5 to 10 μm, while graphite is a non-porous, plate-like material that does not have such voids. [Industrial applicability]

[0086] This invention can be used for single-phase methane production using rumen liquid and methane-producing bacteria-containing liquid, without using raw materials that have a high environmental impact.

Claims

[Claim 1] A liquid composition comprising a rumen fluid, a methane-producing bacteria-containing solution, and an alkaline porous material, The porous material is electrically conductive, A liquid composition in which the particle size of the porous material is 500 μm or less.

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