WO2017150670A1 - Method for producing methane in formation using microorganisms - Google Patents

Method for producing methane in formation using microorganisms Download PDF

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WO2017150670A1
WO2017150670A1 PCT/JP2017/008286 JP2017008286W WO2017150670A1 WO 2017150670 A1 WO2017150670 A1 WO 2017150670A1 JP 2017008286 W JP2017008286 W JP 2017008286W WO 2017150670 A1 WO2017150670 A1 WO 2017150670A1
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methane
activator
microorganism
microorganism group
formation
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PCT/JP2017/008286
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French (fr)
Japanese (ja)
Inventor
鎌形　洋一
将 坂田
秀幸 玉木
大介 眞弓
聡 玉澤
英治 米林
治男 前田
樹 若山
雅之 五十嵐
典子 大坂
洋 押部
良和 白井
剛史 飯田
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国立研究開発法人産業技術総合研究所
国際石油開発帝石株式会社
東京瓦斯株式会社
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Publication of WO2017150670A1 publication Critical patent/WO2017150670A1/en

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P5/00—Preparation of hydrocarbons or halogenated hydrocarbons
    • C12P5/02—Preparation of hydrocarbons or halogenated hydrocarbons acyclic
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02E—REDUCTION OF GREENHOUSE GAS [GHG] EMISSIONS, RELATED TO ENERGY GENERATION, TRANSMISSION OR DISTRIBUTION
    • Y02E50/00—Technologies for the production of fuel of non-fossil origin
    • Y02E50/30—Fuel from waste, e.g. synthetic alcohol or diesel

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  • the present invention relates to a method for producing methane in a formation using microorganisms.
  • microbial attack is an attack that can increase efficiency and reduce costs because microorganisms grow and diffuse themselves within the formation and metabolize chemical substances and solvents necessary for enhanced recovery. Expected as a law.
  • microorganisms that have a methane-producing function are collected from within the geological formation and then re-injected into the basement, and then generated from petroleum-based underground resources by the dominant and dominant methane-producing microorganisms.
  • a method for recovering methane has been proposed (see, for example, Patent Document 1).
  • the present invention has been made in view of the above, and an object thereof is to provide a method for producing methane using a microorganism capable of increasing the amount of methane produced in a short period of time.
  • a methane production method using a microorganism capable of increasing the methane production amount in a short period of time is provided.
  • FIG. 4 is a diagram showing the amount of each crude oil component before and after the experiment in Example 2.
  • FIG. 4 is a diagram showing the amount of each crude oil component before and after the experiment in Example 2.
  • FIG. 4 is a diagram showing the amount of each crude oil component before and after the experiment in Example 2.
  • FIG. 5 is a diagram showing the amount of each crude oil component before and after the experiment in Example 3.
  • the method for producing methane using microorganisms includes an activator (microbe group activity) that activates a microorganism group in a formation in which a hydrocarbon-based underground resource and a microorganism group that generates methane from the underground resource exist.
  • the amount of methane produced can be increased in a short period of time by supplying an agent (also referred to simply as an activator).
  • the stratum refers to mud, earth, sand, gravel, ash, or the like, or a combination of these, or a laminate in which a plurality of these layers are combined.
  • artificially formed ones are also included.
  • FIG. 1 is a diagram for explaining a methane production method using microorganisms in the first embodiment.
  • the microorganism group 12 is activated in the underground layer 11 and the formation in which the microorganism group 12 that generates methane from the underground resource 11 exists.
  • Activating agent 100 is supplied.
  • the underground resources 11 are, for example, petroleum-based hydrocarbon resources such as crude oil, shale oil, and sand oil, and coal-based hydrocarbon resources such as lignite, peat, and subbituminous coal, and are buried in the formation.
  • the underground resource 11 may be in any state of solid, liquid, and gas.
  • the underground resource 11 is not particularly limited as long as it contains hydrocarbons that can be used as a substrate by the microorganism group, and is a chain or cyclic aliphatic hydrocarbon, monocyclic or polycyclic aromatic hydrocarbon It is sufficient that one or more of these are included.
  • the aliphatic hydrocarbon may be linear or branched, and may be a saturated hydrocarbon or an unsaturated hydrocarbon.
  • This embodiment is suitable for producing methane using one or more hydrocarbons having 6 to 70 carbon atoms, preferably one or more hydrocarbons having 9 to 34 carbon atoms as a substrate. Further, one or more of alkanes having 6 to 70 carbon atoms, preferably one or more of alkanes having 9 to 34 carbon atoms, more preferably one or more of alkanes having 6 to 40 carbon atoms and alkanes having 9 to 34 carbon atoms. More preferably, it is suitable for producing methane using at least one linear alkane having 9 to 34 carbon atoms as a substrate. Moreover, it is suitable for producing methane using one or more aromatic hydrocarbons having 6 or 7 carbon atoms, preferably toluene as a substrate.
  • Microorganism group 12 inhabits the formation and produces methane from hydrocarbon-based underground resources 11 as described above.
  • the microorganism group 12 includes, for example, a multitude of symbiotic microorganisms, and anaerobically decomposes hydrocarbons such as chain hydrocarbons or cyclic hydrocarbons to reduce organic acids generated in the process. Produces methane.
  • any microorganisms involved in methane production can be used.
  • the microorganism group preferably contains one or more methanogens.
  • the methanogen include archaebacteria such as the Methanobacteria class and the Methanomicrobia class.
  • the methanogen preferably contains at least one of the genus Methanothermobacter, Methanosaeta, and Methanoculleus, and the archaebacteria belonging to the genus Methanothermobacter And at least one archaebacteria belonging to the genus Methanosaeta.
  • the microorganism group preferably contains one or more bacteria belonging to any of the Firmicutes gate, Ca. Atribacteria gate, Ca.
  • Cloacimonetes gate and one species of bacteria belonging to the Firmicutes gate As described above, it is more preferable that one or more bacteria belonging to the Ca. Atribacteria gate and one or more bacteria belonging to the Ca. Cloacimonetes gate are included.
  • the microorganism group may be an uncultured bacterium.
  • the activator (also called nutrient source) 100 may contain vitamins (including vitamin-like substances) or derivatives thereof, preferably water-soluble vitamins or derivatives thereof.
  • vitamins including vitamin-like substances
  • Examples of the activator 100 include Biotin, p-ABA (p-aminobenzoic acid), Pantothenate, Ca salt, Pyridoxine-HCl, Nicotinic acid, Thiamine-HCl ⁇ 2H 2 O, Lipoic (Thioctic) acid, Folic acid, B12 And at least one vitamin selected from the group of vitamins such as Riboflavin.
  • the activator 100 may further contain a metal salt (including a metalloid salt).
  • the activator 100 is, for example, a metal salt such as FeCl 2 , CoCl 2 , MnCl 2 .4H 2 O, ZnCl 2 , H 3 BO 3 , NiCl 2 , AlCl 3 , Na 2 MoO 4 .2H 2 O, CuCl 2, etc. It contains at least one metal salt selected from the group.
  • the activator 100 may contain any one of vitamins and metal salts, or may contain both vitamins and metal salts. In addition, the activator 100 is not restricted to the vitamin group and metal salt group which were illustrated above, If the microorganism group 12 can be activated, it may contain substances other than the above. Further, the activator 100 can include salts such as NH 4 Cl, KH 2 PO 4 , MgCl 2 .6H 2 O, CaCl 2 .2H 2 O, NaCl, and the like.
  • archaea or a medium in which bacteria can be cultured preferably a medium in which anaerobic microorganisms can be cultured, for example, a so-called WS medium can be used.
  • the activator may contain a yeast extract.
  • Yeast extract is extracted by subjecting one or more selected from sake yeast, wine yeast, alcohol yeast, brewer's yeast, baker's yeast, mineral yeast, etc. to self-digestion, enzyme treatment, hot water treatment, etc. Refers to the extracted extract.
  • Yeast extracts include Alanine, Arginine, Aspartic Acid, Cystine, Glutamic Acid, Glycine, Histidine, Isoleucine, Leucine (Isoleucine) Leucine, Lysine, Methionine, Phenylalanine, Proline, Serine, Threonine, Tryptophan, Tyrosine, Valine, Biotin ( Biotin), Choline, Folic acid, Inositol, Nicotinic acid, 4-Aminobenzoic acid, Pantothenic acid, Pyridoxine, Contains one or more of riboflavin and thiamine.
  • one or more of the yeast extracts can be used as the activator 100, or one or more of the yeast extracts and one of the salts other than the above-mentioned vitamins, metal salts, and metal salts. It can also be used by mixing with the above.
  • the production rate of methane using aliphatic hydrocarbons, particularly alkanes, as a substrate can be increased by the microorganism group.
  • the production rate of methane using a straight-chain alkane having 6 to 70 carbon atoms, particularly 9 to 34 carbon atoms as a substrate can be increased.
  • the activator 100 is in a mixed solution containing vitamins and metals as described above, and is pressed into the formation through a conduit, for example, by a pump or the like, and supplied together with the underground resources 11 to the microorganism group 12 existing in the formation.
  • the microorganism group 12 is activated and the production of methane from the underground resource 11 is promoted.
  • the methane 13 generated from the underground resource 11 by the microorganism group 12 is recovered on the ground through a conduit provided so as to communicate with the underground resource 11 from the ground, for example.
  • the methane production method using microorganisms in the first embodiment by supplying the activator 100 to the microorganism group 12 that produces methane from the underground resource 11 in the formation, It becomes possible to increase the amount of methane produced in a short period of time by activation.
  • the activator 100 is supplied in the formation existing in the ground, but the formation does not necessarily exist underground. If an environment capable of producing methane can be prepared by underground resources and microbial groups capable of producing methane from the underground resources, even if natural strata raised above the ground are used, artificially formed strata are used. Also good.
  • An environment in which methane can be generated by an underground resource and a group of microorganisms that can generate methane from the underground resource is, for example, in an anaerobic atmosphere at a temperature of 10 to 120 ° C. and a pressure of 0.1 to 70 MPa. be able to. The temperature is more preferably 35 to 75 ° C. The pressure is more preferably 3 to 15 MPa.
  • the microorganism group is activated to produce methane in a short period of time.
  • the amount can be increased.
  • a group of microorganisms that generate methane from underground resources is supplied into a formation where there are no microorganism groups that generate methane from underground resources.
  • microbial groups collected from other formations are supplied into the formations that are small in quantity or low in activity and do not generate enough methane.
  • methane can be generated in a short period of time by supplying microbial groups into the formation where underground resources exist.
  • the activator that activates the microorganism group is insufficient in the formation, the amount of methane produced can be increased in a short period of time by supplying the activator together with the microorganism group into the formation. .
  • FIG. 2 is a diagram for explaining a methane production method using microorganisms in the second embodiment.
  • an activator that activates the microorganism group 210 and, if necessary, the microorganism group 210 in the formation in which the underground resource 21 exists. 220 is supplied.
  • the underground resources 21 are, for example, petroleum-based hydrocarbon resources such as crude oil, shale oil, and sand oil, and coal-based hydrocarbon resources such as lignite, peat, and subbituminous coal, and are buried in the formation.
  • the underground resource 21 is the same as that exemplified in the first embodiment.
  • the microorganism group 210 generates methane from the hydrocarbon-based underground resource 21 as described above.
  • the microorganism group 210 includes, for example, a large number of symbiotic microorganisms, and generates methane from the underground resource 21 by anaerobically decomposing organic substances and reducing organic acids generated in the process.
  • any microorganisms involved in the production of methane can be used.
  • the microorganism group 210 is collected from, for example, a formation different from the formation in which the underground resource 21 exists, and after being cultured in a culture solution containing a substrate (crude oil), water, and an activator that activates the microorganism, The resource 21 is supplied into the formation where it exists.
  • This culture can be performed outside the formation.
  • the microorganism group 210 is cultured in a culture tank maintained at an temperature of 10 to 120 ° C. under an anaerobic condition as in the formation, and at a normal pressure or a pressurized environment, for example, 0.1 to 70 MPa. In this state, it is pressed into the formation through a conduit by a pump or the like and supplied to the underground resource 21.
  • the microorganism group 210 cultured in this way preferably contains, for example, one or more methanogens.
  • the methanogen include archaebacteria such as the Methanobacteria class and the Methanomicrobia class.
  • the methanogen preferably contains at least one of the genus Methanothermobacter, Methanosaeta, and Methanoculleus, and the archaebacteria belonging to the genus Methanothermobacter And at least one archaebacteria belonging to the genus Methanosaeta.
  • the microorganism group preferably contains one or more bacteria belonging to any of the Firmicutes gate, Ca. Atribacteria gate, Ca.
  • Cloacimonetes gate and one species of bacteria belonging to the Firmicutes gate As described above, it is more preferable that one or more bacteria belonging to the Ca. Atribacteria gate and one or more bacteria belonging to the Ca. Cloacimonetes gate are included.
  • the microorganism group may be an uncultured bacterium.
  • the first activator depends on the type of microorganism contained in the microorganism group. Components contained in the activator exemplified in the embodiment can be used.
  • the activator 220 may be pressed into the formation together with the microorganism group 210. By supplying the activator 220, it becomes possible to activate the microorganism group 210 supplied to the underground resource 21 and increase the amount of methane produced in a short period of time.
  • the activator 220 is the same as the activator 100 exemplified in the first embodiment, and includes vitamins, metals, and the like that activate the microorganism group 210.
  • the activator 220 is pressed into the formation with a culture solution containing the microorganism group 210 by a pump or the like, for example, in a mixed solution containing vitamins and metal salts, and supplied to the underground resource 21.
  • the microorganism group 210 is activated and the amount of methane produced increases in a short period of time.
  • the methane 22 produced by the microorganism group 210 is collected on the ground through a conduit provided so as to communicate with the underground resource 21 from the ground.
  • the microorganism group 210 that generates methane from the underground resource 21 is supplied into the formation, so that the microorganism group 210 can generate methane from the underground resource 21. Can be generated in a short period of time.
  • the microorganism group 210 can be activated and the amount of methane produced can be increased in a short period of time.
  • microbes collected and cultured from a predetermined formation may be supplied again into the same formation, it may be preferable to use a group of microorganisms collected from a different formation.
  • microbes collected from the same strata have already undergone geological periods and subterranean resources that match their metabolic pathways have already been degraded, and other metabolites taken from strata different from crude oil and oil / water reservoirs. This is because there is a case where currently remaining underground resources cannot be decomposed without using a microorganism group having a route.
  • the amount of methane produced can be increased in a short period of time by supplying the microorganism group or the microorganism group and the activator to the underground resource. It becomes possible to make it.
  • Example 1 100 ml of crude oil (collected from oil fields in Japan), 300 ml of oil reservoir (collected from oil fields in Japan), 250 ml of activator (described later), and 5 ⁇ l of toluene labeled with stable isotope 13C in 1 L stainless steel Placed in a container. In the container, 250 ml of a porous material was previously placed.
  • the atmosphere inside the container was an anaerobic atmosphere, and the pressure and temperature inside the container were kept at 5 MPa and 55 ° C., respectively.
  • the atmosphere, pressure and temperature were the same conditions as the environment in the formation from which the crude oil and oil reservoir water were collected.
  • acetic acid includes acetate ion and acetate in addition to acetic acid.
  • a gas chromatograph and a high performance liquid chromatograph were used.
  • the stable isotope ratio (13C / 12C) of carbon in the produced methane was measured using a mass spectrometry gas chromatograph (GCMS).
  • the stable isotope ratio (atm%) was determined by the following formula.
  • Example 1 The crude oil and oil reservoir water used in Example 1 are collected from the same oil field and contain a group of microorganisms that produce methane from crude oil.
  • the bacteria of the Firmicutes, Ca.Atribacteria, Ca.Cloacimonetes, Methanothermobacter, and Methanosaeta was included.
  • WS medium was used as an activator. Specifically, vitamins (Biotin, p-ABA (p-aminobenzoic acid), Pantothenate, Ca salt, Pyridoxine-HCl, Nicotinic acid, Thiamine-HCl 2H 2 O, Lipoic (Thioctic) acid, Folic acid, B12 , Riboflavin) and metal salt group (FeCl 2 , CoCl 2 , MnCl 2 ⁇ 4H 2 O, ZnCl 2 , H 3 BO 3 , NiCl 2 , AlCl 3 , Na 2 MoO 4 ⁇ 2H 2 O, CuCl 2 ) It is. In addition to the above-mentioned vitamin group and metal salt group, NH 4 Cl, KH 2 PO 4 , MgCl 2 .6H 2 O, CaCl 2 .2H 2 O, and NaCl are added.
  • vitamins Biotin, p-ABA (p-aminobenzoic acid), Pantoth
  • Example 1 The experiment was carried out in the same manner as in Example 1 except that the amount of oil layer water was changed and no toluene labeled with an activator and a stable isotope was added.
  • the crude oil and oil reservoir water used in Comparative Example 1 are the same as the crude oil and oil reservoir water used in Example 1, and contain a group of microorganisms that produce methane from crude oil.
  • FIG. 3 is a graph showing changes in the amount of methane produced and the amount of acetic acid in the container in Example 1 and Comparative Example 1.
  • the horizontal axis represents the number of days elapsed from the start of measurement
  • the vertical axis represents the concentration of methane and the concentration (mM) of acetic acid in the container.
  • FIG. 4 is a diagram showing the measurement results of the stable isotope ratio of carbon in the produced methane in Example 1.
  • the horizontal axis represents the number of days elapsed from the start of measurement
  • the vertical axis represents the stable isotope ratio of carbon in the produced methane.
  • Example 1 As shown in FIG. 3, in Example 1, the concentration of methane increased to about 4 mM between about 60 days after the start of measurement, and further increased by about 2 mM after about 60 days. The concentration of acetic acid increased to more than 2 mM between the start of measurement and about 20 days, but thereafter decreased below the lower limit of quantification.
  • Comparative Example 1 In contrast, in Comparative Example 1, a small amount of methane was produced after about 150 days. However, the amount of methane produced in Comparative Example 1 was about 0.4 mM or less, which was a very small amount. In Comparative Example 1, the amount of acetic acid in the container was almost zero.
  • Example 1 it can be seen that by adding the activator, the microbial group was remarkably activated with respect to the comparative example in which no activator was added, and the amount of methane produced increased in a short period of time. .
  • the amount of acetic acid in the container increased from the start of measurement to about 20 days. This is because the microorganism group in which components having low carbon chains such as easily degradable volatile fatty acids are activated. This is considered to be caused by decomposition into acetic acid or a derivative thereof.
  • methane corresponding to a concentration increase of about 4 mM was generated in the period from the start of measurement to about 60 days, and this period was almost the same as the period in which the amount of acetic acid in the container decreased. It matches. Therefore, it is considered that methane produced in a period of up to about 60 days is mainly derived from decomposition of volatile fatty acids and the like.
  • Example 1 methane corresponding to an increase in concentration of about 2 mM was further generated after about 60 days. As shown in FIG. 4, the stable isotope ratio of carbon in the produced methane gradually increases after about 60 days and reaches about 2 atm% after about 280 days. The generated methane is thought to originate mainly from the decomposition of toluene in crude oil and other crude oil components.
  • Example 2 In Example 2, the experiment was performed in the same manner as in Example 1 except that the amount of the activator was changed and a culture solution containing a microorganism group was additionally added.
  • 1L stainless steel in which 250 ml of sea sand was placed in 100 ml of crude oil, 300 ml of oil reservoir water, 260 ml of activator, 40 ml of culture solution containing microorganisms, and 5 ⁇ l of toluene in which all the constituent carbons were labeled with stable isotopes 13C. Placed in a container. In an anaerobic atmosphere, the pressure and temperature in the container were maintained at 5 MPa and 55 ° C., respectively, and the amount of methane produced, the amount of acetic acid in the container, and the stable isotope ratio of carbon in the produced methane were measured.
  • Example 2 The crude oil and oil reservoir water used in Example 2 are the same as those used in Example 1, both of which were collected from the same oil field.
  • the activator is the same as the activator used in Example 1, including vitamins (Biotin, p-ABA (p-aminobenzoic acid), Pantothenate, Ca salt, Pyridoxine-HCl, Nicotinic acid, Thiamine).
  • the microorganism group in the culture solution containing the microorganism group used in this example was collected from an oil field different from the oil field from which the crude oil and the oil reservoir water were collected, and the crude oil (substrate), water, and microorganism activation substance.
  • the liquid containing the lysate one cultured in an anaerobic environment at a pressure of 5 MPa and a temperature of 55 ° C was used.
  • the bacteria include the bacteria of the Firmicutes, Ca.Atribacteria, Ca.Cloacimonetes, Methanothermobacter, and Methanosaeta It was.
  • Example 2 Experiments were conducted in the same manner as in Example 2 except that the amounts of the oil reservoir water and the activator were changed and the culture solution containing the microorganism group and toluene labeled with a stable isotope were not added.
  • 100 ml of crude oil, 400 ml of oil reservoir water, and 300 ml of activator were placed in a 1 L stainless steel container, and the pressure and temperature in the container were maintained at 5 MPa and 55 ° C., respectively, and the amount of methane produced and the amount of acetic acid in the container were measured.
  • the crude oil, oil reservoir water, and activator used in Comparative Example 2 are the same as the crude oil, oil reservoir water, and activator used in Example 2.
  • FIG. 5 is a graph showing changes in the amount of methane produced and the amount of acetic acid in Example 2 and Comparative Example 2.
  • the horizontal axis represents the number of days elapsed from the start of measurement
  • the vertical axis represents the methane concentration and the acetic acid concentration (mM).
  • FIG. 6 is a graph showing the measurement results of the stable isotope ratio of carbon in the produced methane in Example 2.
  • the horizontal axis represents the number of days elapsed from the start of measurement, and the vertical axis represents the stable isotope ratio of carbon in the produced methane.
  • Example 2 the amounts of aliphatic and aromatic hydrocarbons were measured before and after the experiment, that is, at the time when 0 day had elapsed (before the start of the reaction) and at the time when 750 days had elapsed, using a gas chromatograph.
  • FIG. 7 shows the results for straight chain alkanes having 9 to 25 carbon atoms
  • FIG. 8 shows the results for monocyclic aromatic hydrocarbons
  • FIG. 9 shows the results for polycyclic aromatic hydrocarbons. Show.
  • Example 2 the methane concentration increased to about 10 mM from the start of measurement to about 40 days, and remained almost unchanged from about 40 days to about 250 days. .
  • the methane concentration further increased by about 40 mM between about 250 days and about 750 days.
  • the concentration of acetic acid decreased by about 5 mM between the start of measurement and about 20 days, and thereafter became lower than the lower limit of quantification.
  • Comparative Example 2 On the other hand, in Comparative Example 2, almost no methane was produced between about 50 days from the start of measurement and an increase in concentration of about 10 mM was observed from about 50 days to about 200 days. Thereafter, there was no change in the concentration of methane between about 200 days and about 750 days. In Comparative Example 2, the acetic acid concentration increased to about 10 mM from the start of measurement to about 20 days and decreased from about 20 days to about 200 days. The acetic acid concentration was below the lower limit of quantification after about 200 days.
  • toluene decreased from about 45 mM to about 7 mM after a predetermined period, but there was almost no change in other components.
  • Example 2 by supplying the microorganism group and the activator, methane generation from crude oil by the microorganism group activated by the activator is performed, and the amount of methane generated increases in a short period of time. I understand that.
  • methane corresponding to an increase in concentration of about 10 mM was generated in the period from the start of measurement to about 40 days, and this period was almost the same as the period in which the amount of acetic acid in the container decreased. It matches. Therefore, it is considered that methane produced in a period of up to about 40 days is mainly derived from decomposition of volatile fatty acids and the like.
  • Example 2 methane corresponding to a concentration increase of about 40 mM was further generated between about 250 days and about 750 days.
  • the stable isotope ratio of carbon in the generated methane has increased to about 1.3 atm% after about 290 days (Fig. 6), and a decrease in toluene in crude oil components has been observed before and after the experiment.
  • Fig. 8 methane produced after about 250 days is considered to originate mainly from the decomposition of toluene in crude oil because it almost matches the methane production calculated stoichiometrically from the reduction in toluene. It is done.
  • the amount of acetic acid in the container is increased from the start of measurement to about 20 days. This is considered to be caused by the fact that a component having a low carbon chain such as an easily decomposable volatile fatty acid was decomposed into acetic acid and / or a derivative thereof by an activated microorganism group. Further, in Comparative Example 2, methane corresponding to an increase in concentration of about 10 mM was generated in the period from about 50 days to about 200 days, and this period substantially coincides with the period in which the amount of acetic acid in the container decreased. I'm doing it. Therefore, it is considered that methane produced in a period from about 50 days to about 200 days is mainly derived from decomposition of volatile fatty acids and the like.
  • Example 2 the activator was supplied to the crude oil together with the microorganism group. However, if there is a substance that activates the microorganism in the ground, the activator is prepared by subtracting the existing amount and supplied. May be. Or you may supply only a microorganism group, without giving an activator. Also in this case, the supplied microorganism group generates methane from crude oil, so that the amount of methane generated increases in a short period of time.
  • Example 3 The experiment was performed in the same manner as in Example 1 except that the amount of oil layer water was changed, the composition and amount of the activator were changed, and toluene labeled with a stable isotope was not added.
  • Activators include vitamins (Biotin, p-ABA (p-aminobenzoic acid), Pantothenate, Ca salt, Pyridoxine-HCl, Nicotinic acid, Thiamine-HCl ⁇ 2H 2 O, Lipoic (Thioctic) acid, Folic acid, B12, Riboflavin) and metal salts (FeCl 2 , CoCl 2 , MnCl 2 ⁇ 4H 2 O, ZnCl 2 , H 3 BO 3 , NiCl 2 , AlCl 3 , Na 2 MoO 4 ⁇ 2H 2 O, CuCl 2 ) NH 4 Cl, KH 2 PO 4 , MgCl 2 .6H 2 O, CaCl 2 .2H 2 O, and NaCl are added to the aqueous solution, and 5 ml of yeast extract is added.
  • vitamins Biotin, p-ABA (p-aminobenzoic acid), Pantothenate, Ca salt, Pyridox
  • Yeast extracts include Alanine, Arginine, Aspartic Acid, Cystine, Glutamic Acid, Glycine, Histidine, Isoleucine, Leucine (Isoleucine) Leucine, Lysine, Methionine, Phenylalanine, Proline, Serine, Threonine, Tryptophan, Tyrosine, Valine, Biotin ( Biotin), Choline, Folic acid, Inositol, Nicotinic acid, 4-Aminobenzoic acid, Pantothenic acid, Pyridoxine, It contained riboflavin and Thiamine.
  • the concentration of methane and the concentration of acetic acid in the container were measured at arbitrary time intervals of several days to several tens of days over about 750 days.
  • the methane concentration was 0 mM, but after 124 days of culture, it was recorded that the methane concentration was 190 mM.
  • the concentration in the container was measured in the same manner as in Example 2 for a plurality of aliphatic hydrocarbons before and after the experiment, that is, at the time when 0 day had elapsed (before the start of reaction) and at the time when 750 days had elapsed.
  • the measurement results are shown in FIG.
  • the graph in FIG. 10 shows the results for a linear alkane (n-alkane) having 9 to 34 carbon atoms. After the experiment (after 750 days had elapsed), it was found that the concentration of all linear alkanes to be measured was lower than the concentration before the experiment (0 days).
  • Example 3 it was found that by using an activator containing a yeast extract, the microorganism group can produce methane in a short period of time using an aliphatic hydrocarbon as a substrate.

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Abstract

Provided is a method for producing methane using microorganisms, with which it is possible to increase the amount of methane produced in a short time. Provided is a method for producing methane using microorganisms, the method being characterized by having an activator supply step for supplying an activator to activate the microbial community in a formation in which hydrocarbon-based underground resources and a microbial community that produces methane from the underground resources are present.

Description

微生物を用いた地層内メタン生成方法Formation method of methane in the formation using microorganisms
　本発明は、微生物を用いた地層内メタン生成方法に関する。 The present invention relates to a method for producing methane in a formation using microorganisms.
　地層内の炭化水素系資源を回収するために、熱攻法、ガス攻法、ケミカル攻法、微生物攻法等といった様々な増進回収法の開発が進められている。この中でも微生物攻法は、地層内で微生物が自ら増殖して拡散していくこと、及び増進回収に必要な化学物質や溶媒を自ら代謝することから、高効率化、低コスト化が可能な攻法として期待されている。 Development of various enhanced recovery methods such as thermal attack, gas attack, chemical attack, microbial attack, etc. is underway in order to recover hydrocarbon resources in the formation. Among these, the microbial attack is an attack that can increase efficiency and reduce costs because microorganisms grow and diffuse themselves within the formation and metabolize chemical substances and solvents necessary for enhanced recovery. Expected as a law.
　微生物攻法としては、例えば、地層内からメタン生成機能を持つ微生物を採取して地上で増殖させた後に地下に再圧入し、優勢、優占化したメタン生成微生物により石油系地下資源から生成されたメタンを回収する方法が提案されている（例えば、特許文献１参照）。 For example, microorganisms that have a methane-producing function are collected from within the geological formation and then re-injected into the basement, and then generated from petroleum-based underground resources by the dominant and dominant methane-producing microorganisms. A method for recovering methane has been proposed (see, for example, Patent Document 1).
国際公開第２００６／１１８５７０号International Publication No. 2006/118570
　しかしながら、上記した特許文献１に係る方法では、地層内から微生物群を採取して増殖させた後に地層内に再圧入する必要があり、微生物群によって地下資源からメタンが生成されるまでに長い期間を要する。 However, in the method according to Patent Document 1 described above, it is necessary to reinject into the formation after collecting and growing the microorganism group from within the formation, and a long period of time until methane is generated from the underground resource by the microorganism group. Cost.
　本発明は上記に鑑みてなされたものであって、短期間でメタン生成量を増大させることが可能な微生物を用いたメタン生成方法を提供することを目的とする。 The present invention has been made in view of the above, and an object thereof is to provide a method for producing methane using a microorganism capable of increasing the amount of methane produced in a short period of time.
　本発明の一態様の微生物を用いたメタン生成方法によれば、炭化水素系の地下資源及び前記地下資源からメタンを生成する微生物群が存在する地層内に、前記微生物群を活性化させる活性化剤を供給する活性化剤供給工程を有する。 According to the method for producing methane using the microorganism of one embodiment of the present invention, activation for activating the microorganism group in a formation in which a hydrocarbon-based underground resource and a microorganism group that generates methane from the underground resource exist. An activator supplying step for supplying the agent.
　本発明の実施形態によれば、短期間でメタン生成量を増大させることが可能な微生物を用いたメタン生成方法が提供される。 According to the embodiment of the present invention, a methane production method using a microorganism capable of increasing the methane production amount in a short period of time is provided.
第１の実施形態における微生物を用いたメタン生成方法を説明するための図である。It is a figure for demonstrating the methane production | generation method using the microorganisms in 1st Embodiment. 第２の実施形態における微生物を用いたメタン生成方法を説明するための図である。It is a figure for demonstrating the methane production | generation method using the microorganisms in 2nd Embodiment. 実施例１及び比較例１におけるメタン生成量及び容器中の酢酸量を示す図である。It is a figure which shows the amount of methane production | generation in Example 1 and Comparative Example 1, and the amount of acetic acid in a container. 実施例１における生成メタン中の炭素の安定同位体比の測定結果を示す図である。It is a figure which shows the measurement result of the stable isotope ratio of carbon in the production | generation methane in Example 1. FIG. 実施例２及び比較例２におけるメタン生成量及び容器中の酢酸量を示す図である。It is a figure which shows the amount of methane production | generation in Example 2 and Comparative Example 2, and the amount of acetic acid in a container. 実施例２における生成メタン中の炭素の安定同位体比の測定結果を示す図である。It is a figure which shows the measurement result of the stable isotope ratio of carbon in the production | generation methane in Example 2. FIG. 実施例２における実験前後の各原油成分量を示す図である。FIG. 4 is a diagram showing the amount of each crude oil component before and after the experiment in Example 2. 実施例２における実験前後の各原油成分量を示す図である。FIG. 4 is a diagram showing the amount of each crude oil component before and after the experiment in Example 2. 実施例２における実験前後の各原油成分量を示す図である。FIG. 4 is a diagram showing the amount of each crude oil component before and after the experiment in Example 2. 実施例３における実験前後の各原油成分量を示す図である。FIG. 5 is a diagram showing the amount of each crude oil component before and after the experiment in Example 3.
　以下、図面を参照して発明を実施するための形態について説明する。各図面において、同一構成部分には同一符号を付し、重複した説明を省略する場合がある。 Hereinafter, embodiments for carrying out the invention will be described with reference to the drawings. In the drawings, the same components are denoted by the same reference numerals, and redundant description may be omitted.
　[第１の実施形態]
　第１の実施形態における微生物を用いたメタン生成方法は、炭化水素系の地下資源及び地下資源からメタンを生成する微生物群が存在する地層内に微生物群を活性化させる活性化剤（微生物群活性化剤、又は単に、活性化剤ともいう）を供給することで、メタン生成量を短期間で増大させることができる。 [First embodiment]
The method for producing methane using microorganisms according to the first embodiment includes an activator (microbe group activity) that activates a microorganism group in a formation in which a hydrocarbon-based underground resource and a microorganism group that generates methane from the underground resource exist. The amount of methane produced can be increased in a short period of time by supplying an agent (also referred to simply as an activator).
　本明細書において、地層とは、泥、土、砂、礫、灰、若しくはこれらに類するもの、又はこれらを組合せた層、或いはこれらの層が複数組み合わされた積層体を指す。地層には、天然に存在するものの他、人工的に形成されたものも含まれる。 In this specification, the stratum refers to mud, earth, sand, gravel, ash, or the like, or a combination of these, or a laminate in which a plurality of these layers are combined. In addition to the naturally occurring ones, artificially formed ones are also included.
　図１は、第１の実施形態における微生物を用いたメタン生成方法を説明するための図である。 FIG. 1 is a diagram for explaining a methane production method using microorganisms in the first embodiment.
　第１の実施形態における微生物を用いたメタン生成方法では、図１に示すように、地下資源１１及び地下資源１１からメタンを生成する微生物群１２が存在する地層内に、微生物群１２を活性化させる活性化剤１００を供給する。 In the methane generation method using microorganisms in the first embodiment, as shown in FIG. 1, the microorganism group 12 is activated in the underground layer 11 and the formation in which the microorganism group 12 that generates methane from the underground resource 11 exists. Activating agent 100 is supplied.
　地下資源１１は、例えば、原油、シェールオイル、サンドオイル等の石油系の炭化水素資源や、褐炭、泥炭、亜瀝青炭等の石炭系の炭化水素資源であり、地層内に埋蔵されている。地下資源１１は、固体、液体、及び気体のいずれの状態であってもよい。 The underground resources 11 are, for example, petroleum-based hydrocarbon resources such as crude oil, shale oil, and sand oil, and coal-based hydrocarbon resources such as lignite, peat, and subbituminous coal, and are buried in the formation. The underground resource 11 may be in any state of solid, liquid, and gas.
　地下資源１１は、微生物群が基質として利用し得る炭化水素が含まれていれば、特に限定されず、鎖式又は環式の脂肪族炭化水素、単環式又は多環式の芳香族炭化水素の１種以上が含まれていればよい。脂肪族炭化水素としては、直鎖であっても分枝していてもよく、飽和炭化水素であっても不飽和炭化水素であってもよい。 The underground resource 11 is not particularly limited as long as it contains hydrocarbons that can be used as a substrate by the microorganism group, and is a chain or cyclic aliphatic hydrocarbon, monocyclic or polycyclic aromatic hydrocarbon It is sufficient that one or more of these are included. The aliphatic hydrocarbon may be linear or branched, and may be a saturated hydrocarbon or an unsaturated hydrocarbon.
　本実施形態は、炭素数６～７０の炭化水素の１種以上、好ましくは炭素数９～３４の炭化水素の１種以上を基質としてメタンを生成するのに好適である。また、炭素数６～７０のアルカンの１種以上、好ましくは炭素数９～３４のアルカンの１種以上、より好ましくは炭素数６～４０のアルカン、炭素数９～３４のアルカンの１種以上、より好ましくは炭素数９～３４の直鎖のアルカンの１種以上を基質としてメタンを生成するのに好適である。また、炭素数６又は７の芳香族炭化水素の１種以上、好ましくはトルエンを基質としてメタンを生成するのに好適である。 This embodiment is suitable for producing methane using one or more hydrocarbons having 6 to 70 carbon atoms, preferably one or more hydrocarbons having 9 to 34 carbon atoms as a substrate. Further, one or more of alkanes having 6 to 70 carbon atoms, preferably one or more of alkanes having 9 to 34 carbon atoms, more preferably one or more of alkanes having 6 to 40 carbon atoms and alkanes having 9 to 34 carbon atoms. More preferably, it is suitable for producing methane using at least one linear alkane having 9 to 34 carbon atoms as a substrate. Moreover, it is suitable for producing methane using one or more aromatic hydrocarbons having 6 or 7 carbon atoms, preferably toluene as a substrate.
　微生物群１２は、地層内に生息し、上記したような炭化水素系の地下資源１１からメタンを生成する。微生物群１２は、例えば、共生する多数種の微生物を含み、炭化水素、例えば、鎖式炭化水素あるいは環式炭化水素等を嫌気的に分解してその過程で生成する有機酸等を還元することでメタンを生成する。微生物群１２としては、メタンの生成に関与するあらゆる微生物を利用することができる。 Microorganism group 12 inhabits the formation and produces methane from hydrocarbon-based underground resources 11 as described above. The microorganism group 12 includes, for example, a multitude of symbiotic microorganisms, and anaerobically decomposes hydrocarbons such as chain hydrocarbons or cyclic hydrocarbons to reduce organic acids generated in the process. Produces methane. As the microorganism group 12, any microorganisms involved in methane production can be used.
　微生物群は、１種以上のメタン生成菌を含むことが好ましい。メタン生成菌としては、メタノバクテリウム（Methanobacteria）綱、メタノミクロビウム（Methanomicrobia）綱等の古細菌が挙げられる。さらに、メタン生成菌が、メタノサーモバクター（Methanothermobacter）属、メタノサエタ（Methanosaeta）属、及びメタノキュレウス（Methanoculleus）属のうち１種以上を含んでいると好ましく、メタノサーモバクター（Methanothermobacter）属に属する古細菌の１種以上とメタノサエタ（Methanosaeta）属に属する古細菌の１種以上とを含んでいるとより好ましい。また、微生物群は、上記メタン生成菌以外に、Firmicutes門、Ca.Atribacteria門、Ca.Cloacimonetes門のいずれかに属する細菌のうち１種以上を含むことが好ましく、Firmicutes門に属する細菌の１種以上、Ca.Atribacteria門に属する細菌の１種以上、及びCa.Cloacimonetes門に属する細菌の１種以上を含んでいるとより好ましい。上記微生物群は、未培養菌であってよい。 The microorganism group preferably contains one or more methanogens. Examples of the methanogen include archaebacteria such as the Methanobacteria class and the Methanomicrobia class. Further, the methanogen preferably contains at least one of the genus Methanothermobacter, Methanosaeta, and Methanoculleus, and the archaebacteria belonging to the genus Methanothermobacter And at least one archaebacteria belonging to the genus Methanosaeta. In addition to the above methanogen, the microorganism group preferably contains one or more bacteria belonging to any of the Firmicutes gate, Ca. Atribacteria gate, Ca. Cloacimonetes gate, and one species of bacteria belonging to the Firmicutes gate As described above, it is more preferable that one or more bacteria belonging to the Ca. Atribacteria gate and one or more bacteria belonging to the Ca. Cloacimonetes gate are included. The microorganism group may be an uncultured bacterium.
　活性化剤（栄養源ともいう）１００は、ビタミン（ビタミン様物質を含む）又はその誘導体、好ましくは水溶性のビタミン又はその誘導体を含んでいてよい。活性化剤１００は、例えば、Biotin、p-ABA（p-aminobenzoic acid）、Pantothenate, Ca salt、Pyridoxine-HCl、Nicotinic acid、Thiamine-HCl・2H2O、Lipoic（Thioctic） acid、Folic acid、B12、Riboflavin等のビタミン群から選択される少なくとも１種以上のビタミンを含む。 The activator (also called nutrient source) 100 may contain vitamins (including vitamin-like substances) or derivatives thereof, preferably water-soluble vitamins or derivatives thereof. Examples of the activator 100 include Biotin, p-ABA (p-aminobenzoic acid), Pantothenate, Ca salt, Pyridoxine-HCl, Nicotinic acid, Thiamine-HCl · 2H 2 O, Lipoic (Thioctic) acid, Folic acid, B12 And at least one vitamin selected from the group of vitamins such as Riboflavin.
　また、活性化剤１００は、さらに金属塩（半金属塩を含む）を含んでいてよい。活性化剤１００は、例えば、FeCl2、CoCl2、MnCl2・4H2O、ZnCl2、H3BO3、NiCl2、AlCl3、Na2MoO4・2H2O、CuCl2等の金属塩群から選択される少なくとも１種以上の金属塩を含む。活性化剤１００は、ビタミン及び金属塩の何れか一方を含んでいてもよく、ビタミン及び金属塩の両方を含んでもよい。なお、活性化剤１００は、上記にて例示したビタミン群及び金属塩群に限られるものではなく、微生物群１２を活性化させることが可能であれば、上記以外の物質を含んでもよい。また、活性化剤１００は、NH4Cl、KH2PO4、MgCl2・6H2O、CaCl2・2H2O、NaCl等の塩を含むことができる。 The activator 100 may further contain a metal salt (including a metalloid salt). The activator 100 is, for example, a metal salt such as FeCl 2 , CoCl 2 , MnCl 2 .4H 2 O, ZnCl 2 , H 3 BO 3 , NiCl 2 , AlCl 3 , Na 2 MoO 4 .2H 2 O, CuCl 2, etc. It contains at least one metal salt selected from the group. The activator 100 may contain any one of vitamins and metal salts, or may contain both vitamins and metal salts. In addition, the activator 100 is not restricted to the vitamin group and metal salt group which were illustrated above, If the microorganism group 12 can be activated, it may contain substances other than the above. Further, the activator 100 can include salts such as NH 4 Cl, KH 2 PO 4 , MgCl 2 .6H 2 O, CaCl 2 .2H 2 O, NaCl, and the like.
　活性化剤１００として、古細菌又は細菌を培養することのできる培地、好ましくは嫌気性微生物を培養することのできる培地、例えば、いわゆるＷＳ培地等を用いることができる。 As the activator 100, archaea or a medium in which bacteria can be cultured, preferably a medium in which anaerobic microorganisms can be cultured, for example, a so-called WS medium can be used.
　また、活性化剤は、酵母抽出物を含んでいてよい。酵母抽出物とは、清酒酵母、ブドウ酒酵母、アルコール酵母、ビール酵母、パン酵母、ミネラル酵母等から選ばれる１種又は２種以上を、自己消化、酵素処理、熱水処理等することによって抽出された抽出物を指す。酵母抽出物は、アラニン（Alanine）、アルギニン（Arginine）、アスパラギン酸（Aspartic Acid）、シスチン（Cystine）、グルタミン酸（Glutamic Acid）、グリシン（Glycine）、ヒスチジン（Histidine）、イソロイシン（Isoleucine）、ロイシン（Leucine）、リジン（Lysine）、メチオニン（Methionine）、フェニルアラニン（Phenylalanine）、プロリン（Proline）、セリン（Serine）、トレオニン（Threonine）、トリプトファン（Tryptophan）、チロシン（Tyrosine）、バリン（Valine）、ビオチン（Biotin）、コリン（Choline）、葉酸（Folic acid）、イノシトール（Inositol）、ニコチン酸（Nicotinic Acid）、４－アミノ安息香酸（p-aminobenzoic acid）、パントテン酸（Pantothenic acid）、ピリドキシン（Pyridoxine）、リボフラビン（Riboflavin）、チアミン（Thiamine）の１種以上を含む。 Also, the activator may contain a yeast extract. Yeast extract is extracted by subjecting one or more selected from sake yeast, wine yeast, alcohol yeast, brewer's yeast, baker's yeast, mineral yeast, etc. to self-digestion, enzyme treatment, hot water treatment, etc. Refers to the extracted extract. Yeast extracts include Alanine, Arginine, Aspartic Acid, Cystine, Glutamic Acid, Glycine, Histidine, Isoleucine, Leucine (Isoleucine) Leucine, Lysine, Methionine, Phenylalanine, Proline, Serine, Threonine, Tryptophan, Tyrosine, Valine, Biotin ( Biotin), Choline, Folic acid, Inositol, Nicotinic acid, 4-Aminobenzoic acid, Pantothenic acid, Pyridoxine, Contains one or more of riboflavin and thiamine.
　また、上記酵母抽出物の１種以上を活性化剤１００として用いることもできるし、上記酵母抽出物の１種以上と、上述のビタミン群、金属塩群、金属塩群以外の塩の１つ以上と混合して用いることもできる。 In addition, one or more of the yeast extracts can be used as the activator 100, or one or more of the yeast extracts and one of the salts other than the above-mentioned vitamins, metal salts, and metal salts. It can also be used by mixing with the above.
　酵母抽出物を含む活性化剤を供給することにより、微生物群による脂肪族炭化水素、特にアルカンを基質とするメタンの生成速度を速めることができる。とりわけ、炭素数６～７０、特に炭素数９～３４の直鎖のアルカンを基質とするメタンの生成速度を速めることができる。 By supplying an activator containing a yeast extract, the production rate of methane using aliphatic hydrocarbons, particularly alkanes, as a substrate can be increased by the microorganism group. In particular, the production rate of methane using a straight-chain alkane having 6 to 70 carbon atoms, particularly 9 to 34 carbon atoms as a substrate can be increased.
　活性化剤１００は、上記したビタミンや金属を含む混合溶液の状態で、例えばポンプ等により導管を通じて地層内に圧入され、地下資源１１と共に地層内に存在する微生物群１２に供給される。活性化剤１００が供給されることで、微生物群１２が活性化して地下資源１１からのメタンの生成が促進される。 The activator 100 is in a mixed solution containing vitamins and metals as described above, and is pressed into the formation through a conduit, for example, by a pump or the like, and supplied together with the underground resources 11 to the microorganism group 12 existing in the formation. By supplying the activator 100, the microorganism group 12 is activated and the production of methane from the underground resource 11 is promoted.
　微生物群１２により地下資源１１から生成されたメタン１３は、例えば、地上から地下資源１１に通じるように設けられている導管を通じて地上にて回収される。 The methane 13 generated from the underground resource 11 by the microorganism group 12 is recovered on the ground through a conduit provided so as to communicate with the underground resource 11 from the ground, for example.
　上記したように、第１の実施形態における微生物を用いたメタン生成方法よれば、地層内で地下資源１１からメタンを生成する微生物群１２に活性化剤１００を供給することで、微生物群１２を活性化させて短期間でメタン生成量を増大させることが可能になる。 As described above, according to the methane production method using microorganisms in the first embodiment, by supplying the activator 100 to the microorganism group 12 that produces methane from the underground resource 11 in the formation, It becomes possible to increase the amount of methane produced in a short period of time by activation.
　なお、図示の例では、活性化剤１００を、地中に存在する地層内に供給しているが、地層は必ずしも地下に存在するものでなくてもよい。地下資源とその地下資源からメタンを生成可能な微生物群とによってメタンを生成できる環境を整えることができれば、地上に***した天然の地層を利用しても、人工的に形成した地層を利用してもよい。地下資源とその地下資源からメタンを生成可能な微生物群とによってメタンを生成できる環境とは、例えば、嫌気性雰囲気中、１０～１２０℃の温度で、０．１～７０ＭＰａの圧力という条件とすることができる。上記温度は３５～７５℃であるとより好ましい。また、上記圧力は３～１５ＭＰａであるとより好ましい。 In the example shown in the figure, the activator 100 is supplied in the formation existing in the ground, but the formation does not necessarily exist underground. If an environment capable of producing methane can be prepared by underground resources and microbial groups capable of producing methane from the underground resources, even if natural strata raised above the ground are used, artificially formed strata are used. Also good. An environment in which methane can be generated by an underground resource and a group of microorganisms that can generate methane from the underground resource is, for example, in an anaerobic atmosphere at a temperature of 10 to 120 ° C. and a pressure of 0.1 to 70 MPa. be able to. The temperature is more preferably 35 to 75 ° C. The pressure is more preferably 3 to 15 MPa.
　以上で説明したように、第１の実施形態における微生物を用いたメタン生成方法によれば、微生物群に微生物群活性化剤を供給することで、微生物群を活性化させて短期間でメタン生成量を増大させることが可能になる。 As explained above, according to the method for producing methane using microorganisms in the first embodiment, by supplying a microorganism group activator to the microorganism group, the microorganism group is activated to produce methane in a short period of time. The amount can be increased.
　[第２の実施形態]
　次に、第２の実施形態について図面に基づいて説明する。 [Second Embodiment]
Next, a second embodiment will be described based on the drawings.
　第２の実施形態における微生物を用いたメタン生成方法では、地下資源からメタンを生成する微生物群が存在しない地層内に、地下資源からメタンを生成する微生物群を供給する。もしくは、地下資源からメタンを生成する微生物群が存在しても量が少ないか活性が低くメタンが十分に生成されない地層内に、他の地層内から採取した微生物群を供給する。このように地下資源が存在する地層内に微生物群を供給することで、短期間でのメタン生成を可能にする。また、地層内に微生物群を活性化させる活性化剤が不足している場合には、微生物群と共に活性化剤を地層内に供給することで、短期間でメタン生成量を増大させることができる。 In the method for producing methane using microorganisms in the second embodiment, a group of microorganisms that generate methane from underground resources is supplied into a formation where there are no microorganism groups that generate methane from underground resources. Alternatively, even if there are microbial groups that produce methane from underground resources, microbial groups collected from other formations are supplied into the formations that are small in quantity or low in activity and do not generate enough methane. In this way, methane can be generated in a short period of time by supplying microbial groups into the formation where underground resources exist. Moreover, when the activator that activates the microorganism group is insufficient in the formation, the amount of methane produced can be increased in a short period of time by supplying the activator together with the microorganism group into the formation. .
　図２は、第２の実施形態における微生物を用いたメタン生成方法を説明するための図である。 FIG. 2 is a diagram for explaining a methane production method using microorganisms in the second embodiment.
　第２の実施形態における微生物を用いたメタン生成方法では、図２に示すように、地下資源２１が存在する地層内に、微生物群２１０及び必要に応じて微生物群２１０を活性化させる活性化剤２２０を供給する。 In the methane production method using microorganisms in the second embodiment, as shown in FIG. 2, an activator that activates the microorganism group 210 and, if necessary, the microorganism group 210 in the formation in which the underground resource 21 exists. 220 is supplied.
　地下資源２１は、例えば、原油、シェールオイル、サンドオイル等の石油系の炭化水素資源や、褐炭、泥炭、亜瀝青炭等の石炭系の炭化水素資源であり、地層内に埋蔵されている。地下資源２１は、第１の実施形態において例示したものと同様である。 The underground resources 21 are, for example, petroleum-based hydrocarbon resources such as crude oil, shale oil, and sand oil, and coal-based hydrocarbon resources such as lignite, peat, and subbituminous coal, and are buried in the formation. The underground resource 21 is the same as that exemplified in the first embodiment.
　微生物群２１０は、上記したような炭化水素系の地下資源２１からメタンを生成する。微生物群２１０は、例えば、共生する多数種の微生物を含み、有機物を嫌気的に分解してその過程で生成する有機酸等を還元することで、地下資源２１からメタンを生成する。微生物群２１０としては、メタンの生成に関与するあらゆる微生物を利用することができる。 The microorganism group 210 generates methane from the hydrocarbon-based underground resource 21 as described above. The microorganism group 210 includes, for example, a large number of symbiotic microorganisms, and generates methane from the underground resource 21 by anaerobically decomposing organic substances and reducing organic acids generated in the process. As the microorganism group 210, any microorganisms involved in the production of methane can be used.
　微生物群２１０は、例えば、地下資源２１が存在する地層とは異なる地層内から採取され、基質（原油）、水、及び微生物を活性化する活性化剤を含む培養液において培養された後に、地下資源２１が存在する地層内に供給される。この培養は、地層外で行うことができる。微生物群２１０は、例えば、地層内と同様に嫌気的条件で、温度が１０～１２０℃に保たれた培養タンクにおいて常圧又は加圧環境、例えば０．１～７０ＭＰａで培養され、培養液の状態でポンプ等により導管を通じて地層内に圧入され、地下資源２１に供給される。 The microorganism group 210 is collected from, for example, a formation different from the formation in which the underground resource 21 exists, and after being cultured in a culture solution containing a substrate (crude oil), water, and an activator that activates the microorganism, The resource 21 is supplied into the formation where it exists. This culture can be performed outside the formation. For example, the microorganism group 210 is cultured in a culture tank maintained at an temperature of 10 to 120 ° C. under an anaerobic condition as in the formation, and at a normal pressure or a pressurized environment, for example, 0.1 to 70 MPa. In this state, it is pressed into the formation through a conduit by a pump or the like and supplied to the underground resource 21.
　このようにして培養される微生物群２１０は、例えば、１種以上のメタン生成菌を含むことが好ましい。メタン生成菌としては、メタノバクテリウム（Methanobacteria）綱、メタノミクロビウム（Methanomicrobia）綱等の古細菌が挙げられる。さらに、メタン生成菌が、メタノサーモバクター（Methanothermobacter）属、メタノサエタ（Methanosaeta）属、及びメタノキュレウス（Methanoculleus）属のうち１種以上を含んでいると好ましく、メタノサーモバクター（Methanothermobacter）属に属する古細菌の１種以上とメタノサエタ（Methanosaeta）属に属する古細菌の１種以上とを含んでいるとより好ましい。また、微生物群は、上記メタン生成菌以外に、Firmicutes門、Ca.Atribacteria門、Ca.Cloacimonetes門のいずれかに属する細菌のうち１種以上を含むことが好ましく、Firmicutes門に属する細菌の１種以上、Ca.Atribacteria門に属する細菌の１種以上、及びCa.Cloacimonetes門に属する細菌の１種以上を含んでいるとより好ましい。上記微生物群は、未培養菌であってよい。 The microorganism group 210 cultured in this way preferably contains, for example, one or more methanogens. Examples of the methanogen include archaebacteria such as the Methanobacteria class and the Methanomicrobia class. Further, the methanogen preferably contains at least one of the genus Methanothermobacter, Methanosaeta, and Methanoculleus, and the archaebacteria belonging to the genus Methanothermobacter And at least one archaebacteria belonging to the genus Methanosaeta. In addition to the above methanogen, the microorganism group preferably contains one or more bacteria belonging to any of the Firmicutes gate, Ca. Atribacteria gate, Ca. Cloacimonetes gate, and one species of bacteria belonging to the Firmicutes gate As described above, it is more preferable that one or more bacteria belonging to the Ca. Atribacteria gate and one or more bacteria belonging to the Ca. Cloacimonetes gate are included. The microorganism group may be an uncultured bacterium.
　地下資源２１が存在する地層とは異なる地層内から採取された微生物群を培養液中で活性化するための活性化剤としては、微生物群に含まれる微生物の種類にもよるが、第１の実施形態において例示した活性化剤に含まれる成分を用いることができる。 As an activator for activating a microorganism group collected from within a formation different from the formation in which the underground resource 21 exists in the culture solution, the first activator depends on the type of microorganism contained in the microorganism group. Components contained in the activator exemplified in the embodiment can be used.
　また、微生物群２１０と共に、活性化剤２２０を地層内に圧入してもよい。活性化剤２２０を供給することで、地下資源２１に供給した微生物群２１０を活性化させて短期間でメタン生成量を増大させることが可能になる。 Also, the activator 220 may be pressed into the formation together with the microorganism group 210. By supplying the activator 220, it becomes possible to activate the microorganism group 210 supplied to the underground resource 21 and increase the amount of methane produced in a short period of time.
　活性化剤２２０は、第１の実施形態において例示した活性化剤１００と同様であり、微生物群２１０を活性化させるビタミンや金属等を含む。活性化剤２２０は、上記したビタミンや金属塩を含む混合溶液の状態で、例えばポンプ等により微生物群２１０を含む培養液と共に地層内に圧入され、地下資源２１に供給される。活性化剤２２０が供給されることで、微生物群２１０が活性化して短期間でメタン生成量が増大する。 The activator 220 is the same as the activator 100 exemplified in the first embodiment, and includes vitamins, metals, and the like that activate the microorganism group 210. The activator 220 is pressed into the formation with a culture solution containing the microorganism group 210 by a pump or the like, for example, in a mixed solution containing vitamins and metal salts, and supplied to the underground resource 21. By supplying the activator 220, the microorganism group 210 is activated and the amount of methane produced increases in a short period of time.
　微生物群２１０により生成されたメタン２２は、地上から地下資源２１に通じるように設けられている導管を通じて地上にて回収される。 The methane 22 produced by the microorganism group 210 is collected on the ground through a conduit provided so as to communicate with the underground resource 21 from the ground.
　上記したように、第２の実施形態における微生物を用いたメタン生成方法よれば、地下資源２１からメタンを生成する微生物群２１０を地層内に供給することで、微生物群２１０により地下資源２１からメタンを短期間で生成させることが可能になる。また、微生物群２１０と共に活性化剤２２０を地層内に供給することで、微生物群２１０を活性化させて短期間でメタン生成量を増大させることが可能になる。 As described above, according to the methane generation method using microorganisms in the second embodiment, the microorganism group 210 that generates methane from the underground resource 21 is supplied into the formation, so that the microorganism group 210 can generate methane from the underground resource 21. Can be generated in a short period of time. In addition, by supplying the activator 220 together with the microorganism group 210 into the formation, the microorganism group 210 can be activated and the amount of methane produced can be increased in a short period of time.
　また、所定の地層から採取し培養した微生物群を、その同じ地層内に再度供給してもよいが、異なる地層から採取した微生物群を用いることが好ましい場合がある。これは、一般に同じ地層から採取した微生物群は地質学的期間を経て自身の代謝経路に合致した地下資源は既に分解を完了しており、原油及び油水層とは異なる地層から採取した他の代謝経路を持つ微生物群を用いなければ、現在残存している地下資源を分解できない場合があるためである。 In addition, although a group of microorganisms collected and cultured from a predetermined formation may be supplied again into the same formation, it may be preferable to use a group of microorganisms collected from a different formation. In general, microbes collected from the same strata have already undergone geological periods and subterranean resources that match their metabolic pathways have already been degraded, and other metabolites taken from strata different from crude oil and oil / water reservoirs. This is because there is a case where currently remaining underground resources cannot be decomposed without using a microorganism group having a route.
　以上で説明したように、第２の実施形態における微生物を用いたメタン生成方法では、微生物群、または微生物群及び活性化剤を地下資源に供給することで、短期間でメタンの生成量を増大させることが可能になる。 As described above, in the method for producing methane using microorganisms in the second embodiment, the amount of methane produced can be increased in a short period of time by supplying the microorganism group or the microorganism group and the activator to the underground resource. It becomes possible to make it.
　次に実施例及び比較例について説明する、なお、本発明は以下で説明する実施例に限定されるものではない。 Next, examples and comparative examples will be described. It should be noted that the present invention is not limited to the examples described below.
　（実施例１）
　原油（日本国内の油田より採取）１００ｍｌ、油層水（日本国内の油田より採取）３００ｍｌ、活性化剤（後述）２５０ｍｌ、及び構成炭素の全てを安定同位体１３Ｃで標識化したトルエン５μｌを１Ｌステンレス容器に入れた。容器内には、予め、多孔質体２５０ｍｌを入れておいた。 Example 1
100 ml of crude oil (collected from oil fields in Japan), 300 ml of oil reservoir (collected from oil fields in Japan), 250 ml of activator (described later), and 5 μl of toluene labeled with stable isotope 13C in 1 L stainless steel Placed in a container. In the container, 250 ml of a porous material was previously placed.
　容器内の雰囲気を嫌気性雰囲気とし、容器内の圧力及び温度をそれぞれ５ＭＰａ、５５℃に保った。この雰囲気、圧力及び温度は、上記の原油及び油層水を採取した地層内の環境と同等の条件であった。 The atmosphere inside the container was an anaerobic atmosphere, and the pressure and temperature inside the container were kept at 5 MPa and 55 ° C., respectively. The atmosphere, pressure and temperature were the same conditions as the environment in the formation from which the crude oil and oil reservoir water were collected.
　メタン生成量、容器中の酢酸の量の経時的な変化を調べるために、容器内のメタンの濃度及び酢酸の濃度を、約５５０日にわたり、数日から数十日の任意の時間間隔で測定した。本明細書において、酢酸には、酢酸以外に、酢酸イオン及び酢酸塩が含まれる。また、濃度の測定においては、ガスクロマトグラフ及び高速液体クロマトグラフを用いた。 In order to investigate changes over time in the amount of methane produced and the amount of acetic acid in the container, the concentration of methane and the concentration of acetic acid in the container were measured at an arbitrary time interval of several days to several tens of days over about 550 days. did. In this specification, acetic acid includes acetate ion and acetate in addition to acetic acid. In the measurement of concentration, a gas chromatograph and a high performance liquid chromatograph were used.
　また、生成メタン中の炭素の安定同位体比（１３Ｃ／１２Ｃ）を、質量分析ガスクロマトグラフ（ＧＣＭＳ）を用いて測定した。なお、安定同位体比（ａｔｍ％）は、以下の式で求めた。 Moreover, the stable isotope ratio (13C / 12C) of carbon in the produced methane was measured using a mass spectrometry gas chromatograph (GCMS). The stable isotope ratio (atm%) was determined by the following formula.
　　安定同位体比（ａｔｍ％）＝（１＋δ／１０００）×１．１２４
　　δ＝｛（試料中の１３Ｃの原子数）／（試料中の１２Ｃの原子数）｝／｛（標準物質の１３Ｃの原子数）／（標準物質の１２Ｃの原子数）－１｝×１０００ Stable isotope ratio (atm%) = (1 + δ / 1000) × 1.124
δ = {(number of 13 C atoms in sample) / (number of 12 C atoms in sample)} / {(number of 13 C atoms in standard material) / (number of 12 C atoms in standard material) −1 } × 1000
　実施例１において使用した原油及び油層水は、同じ油田から採取したものであり、原油からメタンを生成する微生物群を含んでいる。本実施例で用いた原油及び油層水中の微生物群を構造解析した結果、Firmicutes門の細菌、Ca.Atribacteria門の細菌、Ca.Cloacimonetes門の細菌、Methanothermobacter属の古細菌、及びMethanosaeta属の古細菌が含まれていた。 The crude oil and oil reservoir water used in Example 1 are collected from the same oil field and contain a group of microorganisms that produce methane from crude oil. As a result of structural analysis of the microbial community in the crude oil and oil reservoir used in this example, the bacteria of the Firmicutes, Ca.Atribacteria, Ca.Cloacimonetes, Methanothermobacter, and Methanosaeta Was included.
　活性化剤としては、ＷＳ培地を用いた。具体的には、ビタミン群（Biotin、p-ABA（p-aminobenzoic acid）、Pantothenate, Ca salt、Pyridoxine-HCl、Nicotinic acid、Thiamine-HCl・2H2O、Lipoic（Thioctic） acid、Folic acid、B12、Riboflavin）及び金属塩群（FeCl2、CoCl2、MnCl2・4H2O、ZnCl2、H3BO3、NiCl2、AlCl3、Na2MoO4・2H2O、CuCl2）を含む溶液である。上記したビタミン群及び金属塩群に加えて、NH4Cl、KH2PO4、MgCl2・6H2O、CaCl2・2H2O、NaClを添加したものである。 WS medium was used as an activator. Specifically, vitamins (Biotin, p-ABA (p-aminobenzoic acid), Pantothenate, Ca salt, Pyridoxine-HCl, Nicotinic acid, Thiamine-HCl 2H 2 O, Lipoic (Thioctic) acid, Folic acid, B12 , Riboflavin) and metal salt group (FeCl 2 , CoCl 2 , MnCl 2 · 4H 2 O, ZnCl 2 , H 3 BO 3 , NiCl 2 , AlCl 3 , Na 2 MoO 4 · 2H 2 O, CuCl 2 ) It is. In addition to the above-mentioned vitamin group and metal salt group, NH 4 Cl, KH 2 PO 4 , MgCl 2 .6H 2 O, CaCl 2 .2H 2 O, and NaCl are added.
　（比較例１）
　油層水の量を変更し、活性化剤及び安定同位体で標識化したトルエンを加えなかったこと以外は、実施例１と同様に実験を行った。 (Comparative Example 1)
The experiment was carried out in the same manner as in Example 1 except that the amount of oil layer water was changed and no toluene labeled with an activator and a stable isotope was added.
　すなわち、原油１００ｍｌ、油層水７００ｍｌを、多孔質体２５０ｍｌを入れておいた１Ｌステンレス容器に入れ、嫌気性雰囲気中で、容器内の圧力及び温度をそれぞれ５ＭＰａ、５５℃に保った。容器内のメタンの濃度及び酢酸の濃度を測定することにより、メタン生成量及び容器中の酢酸量を測定した。 That is, 100 ml of crude oil and 700 ml of oil layer water were placed in a 1 L stainless steel container in which 250 ml of a porous body had been placed, and the pressure and temperature in the container were maintained at 5 MPa and 55 ° C., respectively, in an anaerobic atmosphere. The amount of methane produced and the amount of acetic acid in the container were measured by measuring the concentration of methane and the concentration of acetic acid in the container.
　比較例１において使用した原油及び油層水は、実施例１において使用した原油及び油層水と同じものであり、原油からメタンを生成する微生物群を含んでいる。 The crude oil and oil reservoir water used in Comparative Example 1 are the same as the crude oil and oil reservoir water used in Example 1, and contain a group of microorganisms that produce methane from crude oil.
　図３は、実施例１及び比較例１におけるメタン生成量及び容器中の酢酸量の変化を示す図である。図３に示されるグラフでは、横軸が測定開始からの経過日数であり、縦軸が、容器中のメタンの濃度及び酢酸の濃度（ｍＭ）である。 FIG. 3 is a graph showing changes in the amount of methane produced and the amount of acetic acid in the container in Example 1 and Comparative Example 1. In the graph shown in FIG. 3, the horizontal axis represents the number of days elapsed from the start of measurement, and the vertical axis represents the concentration of methane and the concentration (mM) of acetic acid in the container.
　また、図４は、実施例１における生成メタン中の炭素の安定同位体比の測定結果を示す図である。図４に示されるグラフでは、横軸が測定開始からの経過日数、縦軸が生成メタン中の炭素の安定同位体比である。 FIG. 4 is a diagram showing the measurement results of the stable isotope ratio of carbon in the produced methane in Example 1. In the graph shown in FIG. 4, the horizontal axis represents the number of days elapsed from the start of measurement, and the vertical axis represents the stable isotope ratio of carbon in the produced methane.
　図３に示されるように、実施例１では、メタンの濃度が、測定開始から約６０日までの間に約４ｍＭまで上昇し、約６０日以降にさらに約２ｍＭ上昇した。酢酸の濃度は、測定開始から約２０日までの間に２ｍＭ超まで上昇したが、その後は定量下限値以下に減少した。 As shown in FIG. 3, in Example 1, the concentration of methane increased to about 4 mM between about 60 days after the start of measurement, and further increased by about 2 mM after about 60 days. The concentration of acetic acid increased to more than 2 mM between the start of measurement and about 20 days, but thereafter decreased below the lower limit of quantification.
　これに対して、比較例１では、約１５０日以降において微量のメタンが生成された。ただし、比較例１におけるメタンの生成量は約０．４ｍＭ以下であり極微量であった。また、比較例１では、容器中の酢酸量はほぼゼロであった。 In contrast, in Comparative Example 1, a small amount of methane was produced after about 150 days. However, the amount of methane produced in Comparative Example 1 was about 0.4 mM or less, which was a very small amount. In Comparative Example 1, the amount of acetic acid in the container was almost zero.
　このように、実施例１では、活性化剤を添加したことにより、活性化剤を添加していない比較例に対して著しく微生物群が活性化し、短期間でメタン生成量が増大したことが分かる。なお、実施例１において測定開始から約２０日までの間に容器中の酢酸量が増加したが、これは易分解性の揮発性脂肪酸等の低炭素鎖を持つ成分が、活性化した微生物群によって酢酸又はその誘導体に分解されたために生じたものと考えられる。 Thus, in Example 1, it can be seen that by adding the activator, the microbial group was remarkably activated with respect to the comparative example in which no activator was added, and the amount of methane produced increased in a short period of time. . In Example 1, the amount of acetic acid in the container increased from the start of measurement to about 20 days. This is because the microorganism group in which components having low carbon chains such as easily degradable volatile fatty acids are activated. This is considered to be caused by decomposition into acetic acid or a derivative thereof.
　上述のように、実施例１において、測定開始から約６０日までの期間に約４ｍＭの濃度増加に相当するメタンが生成されており、この期間は、容器中の酢酸量が減少した期間とほぼ合致している。そのため、約６０日までの期間に生成されたメタンは、主に揮発性脂肪酸等の分解由来であると考えられる。 As described above, in Example 1, methane corresponding to a concentration increase of about 4 mM was generated in the period from the start of measurement to about 60 days, and this period was almost the same as the period in which the amount of acetic acid in the container decreased. It matches. Therefore, it is considered that methane produced in a period of up to about 60 days is mainly derived from decomposition of volatile fatty acids and the like.
　一方、実施例１において、約６０日以降に、約２ｍＭの濃度増加に相当するメタンがさらに生成されている。図４に示されるように、生成メタン中の炭素の安定同位体比は約６０日以降徐々に増加し、約２８０日以降には約２ａｔｍ％に達していることから、約６０日以降に生成されたメタンは、主に原油中のトルエン、その他原油成分の分解由来と考えられる。 On the other hand, in Example 1, methane corresponding to an increase in concentration of about 2 mM was further generated after about 60 days. As shown in FIG. 4, the stable isotope ratio of carbon in the produced methane gradually increases after about 60 days and reaches about 2 atm% after about 280 days. The generated methane is thought to originate mainly from the decomposition of toluene in crude oil and other crude oil components.
　（実施例２）
　実施例２では、活性化剤の量を変更し、微生物群を含む培養液を追加で添加したこと以外は、実施例１と同様に実験を行った。 (Example 2)
In Example 2, the experiment was performed in the same manner as in Example 1 except that the amount of the activator was changed and a culture solution containing a microorganism group was additionally added.
　すなわち、原油１００ｍｌ、油層水３００ｍｌ、活性化剤２６０ｍｌ、微生物群を含む培養液４０ｍｌ、及び構成炭素の全てを安定同位体１３Ｃで標識化したトルエン５μｌを、海砂２５０ｍｌを入れておいた１Ｌステンレス容器に入れた。嫌気性雰囲気中で、容器内の圧力及び温度をそれぞれ５ＭＰａ、５５℃に保って、メタンの生成量、容器中の酢酸量、及び生成メタン中の炭素の安定同位体比を測定した。 That is, 1L stainless steel in which 250 ml of sea sand was placed in 100 ml of crude oil, 300 ml of oil reservoir water, 260 ml of activator, 40 ml of culture solution containing microorganisms, and 5 μl of toluene in which all the constituent carbons were labeled with stable isotopes 13C. Placed in a container. In an anaerobic atmosphere, the pressure and temperature in the container were maintained at 5 MPa and 55 ° C., respectively, and the amount of methane produced, the amount of acetic acid in the container, and the stable isotope ratio of carbon in the produced methane were measured.
　実施例２において使用した原油及び油層水は、実施例１で使用したものと同じであり、いずれも同じ油田から採取したものである。また、活性化剤は、実施例１で使用した活性化剤と同じものであり、ビタミン群（Biotin、p-ABA（p-aminobenzoic acid）、Pantothenate, Ca salt、Pyridoxine-HCl、Nicotinic acid、Thiamine-HCl・2H2O、Lipoic（Thioctic） acid、Folic acid、B12、Riboflavin）及び金属塩群（FeCl2、CoCl2、MnCl2・4H2O、ZnCl2、H3BO3、NiCl2、AlCl3、Na2MoO4・2H2O、CuCl2）を含む溶液である。活性化剤には、上記したビタミン群及び金属塩群に加えて、NH4Cl、KH2PO4、MgCl2・6H2O、CaCl2・2H2O、NaClを添加したものである。 The crude oil and oil reservoir water used in Example 2 are the same as those used in Example 1, both of which were collected from the same oil field. The activator is the same as the activator used in Example 1, including vitamins (Biotin, p-ABA (p-aminobenzoic acid), Pantothenate, Ca salt, Pyridoxine-HCl, Nicotinic acid, Thiamine). -HCl · 2H 2 O, Lipoic (Thioctic) acid, Folic acid, B12, Riboflavin) and metal salts (FeCl 2 , CoCl 2 , MnCl 2 · 4H 2 O, ZnCl 2 , H 3 BO 3 , NiCl 2 , AlCl 3 , Na 2 MoO 4 .2H 2 O, CuCl 2 ). In addition to the vitamin group and metal salt group described above, NH 4 Cl, KH 2 PO 4 , MgCl 2 .6H 2 O, CaCl 2 .2H 2 O, and NaCl are added to the activator.
　本実施例で用いた、微生物群を含む培養液中の微生物群は、上記原油及び上記油層水を採取した油田とは異なる油田から採取し、その原油（基質）、水、及び微生物活性化物質を含む液において、圧力５ＭＰａ、温度５５℃の嫌気性環境下で培養したものを用いた。微生物群を含む培養液に含まれる微生物群を解析した結果、Firmicutes門の細菌、Ca.Atribacteria門の細菌、Ca.Cloacimonetes門の細菌、Methanothermobacter属の古細菌、及びMethanosaeta属の古細菌が含まれていた。 The microorganism group in the culture solution containing the microorganism group used in this example was collected from an oil field different from the oil field from which the crude oil and the oil reservoir water were collected, and the crude oil (substrate), water, and microorganism activation substance. In the liquid containing the lysate, one cultured in an anaerobic environment at a pressure of 5 MPa and a temperature of 55 ° C was used. As a result of analysis of the microorganism group contained in the culture solution containing the microorganism group, the bacteria include the bacteria of the Firmicutes, Ca.Atribacteria, Ca.Cloacimonetes, Methanothermobacter, and Methanosaeta It was.
　（比較例２）
　油層水及び活性化剤の量を変更し、微生物群を含む培養液及び安定同位体で標識化したトルエンを添加しなかったこと以外は、実施例２と同様に実験を行った。 (Comparative Example 2)
Experiments were conducted in the same manner as in Example 2 except that the amounts of the oil reservoir water and the activator were changed and the culture solution containing the microorganism group and toluene labeled with a stable isotope were not added.
　すなわち、原油１００ｍｌ、油層水４００ｍｌ、活性化剤３００ｍｌを１Ｌステンレス容器に入れ、容器内の圧力及び温度をそれぞれ５ＭＰａ、５５℃に保って、メタン生成量及び容器中の酢酸量を測定した。 That is, 100 ml of crude oil, 400 ml of oil reservoir water, and 300 ml of activator were placed in a 1 L stainless steel container, and the pressure and temperature in the container were maintained at 5 MPa and 55 ° C., respectively, and the amount of methane produced and the amount of acetic acid in the container were measured.
　なお、比較例２において使用した原油、油層水、及び活性化剤は、実施例２において使用した原油、油層水、及び活性化剤と同じものである。 The crude oil, oil reservoir water, and activator used in Comparative Example 2 are the same as the crude oil, oil reservoir water, and activator used in Example 2.
　図５は、実施例２及び比較例２における、メタンの生成量及び酢酸量の変化を示す図である。図５に示されるグラフでは、横軸が測定開始からの経過日数、縦軸がメタンの濃度及び酢酸の濃度（ｍＭ）である。 FIG. 5 is a graph showing changes in the amount of methane produced and the amount of acetic acid in Example 2 and Comparative Example 2. In the graph shown in FIG. 5, the horizontal axis represents the number of days elapsed from the start of measurement, and the vertical axis represents the methane concentration and the acetic acid concentration (mM).
　図６は、実施例２における生成メタン中の炭素の安定同位体比の測定結果を示す図である。図６に示されるグラフでは、横軸が測定開始からの経過日数、縦軸が生成メタン中の炭素の安定同位体比である。 FIG. 6 is a graph showing the measurement results of the stable isotope ratio of carbon in the produced methane in Example 2. In the graph shown in FIG. 6, the horizontal axis represents the number of days elapsed from the start of measurement, and the vertical axis represents the stable isotope ratio of carbon in the produced methane.
　また、実施例２については、実験前後、すなわち、０日経過時点（反応開始前）及び７５０日経過時点での脂肪族及び芳香族の各炭化水素の量を、ガスクロマトグラフを用いて測定した。図７は、炭素数９～２５の直鎖のアルカンについての結果を示し、図８は、単環芳香族炭化水素についての結果を示し、図９は、多環芳香族炭化水素についての結果を示す。 For Example 2, the amounts of aliphatic and aromatic hydrocarbons were measured before and after the experiment, that is, at the time when 0 day had elapsed (before the start of the reaction) and at the time when 750 days had elapsed, using a gas chromatograph. FIG. 7 shows the results for straight chain alkanes having 9 to 25 carbon atoms, FIG. 8 shows the results for monocyclic aromatic hydrocarbons, and FIG. 9 shows the results for polycyclic aromatic hydrocarbons. Show.
　図５に示されるように、実施例２では、メタンの濃度は、測定開始から約４０日までの間に約１０ｍＭまで上昇し、約４０日から約２５０日までの間はほぼ変化がなかった。また、メタン濃度は、約２５０日から約７５０日までの間に、さらに約４０ｍＭ上昇した。酢酸の濃度は、測定開始時から約２０日までの間に約５ｍＭ減少し、その後は定量下限値以下となった。 As shown in FIG. 5, in Example 2, the methane concentration increased to about 10 mM from the start of measurement to about 40 days, and remained almost unchanged from about 40 days to about 250 days. . The methane concentration further increased by about 40 mM between about 250 days and about 750 days. The concentration of acetic acid decreased by about 5 mM between the start of measurement and about 20 days, and thereafter became lower than the lower limit of quantification.
　これに対して、比較例２では、メタンは、測定開始から約５０日までの間ではほぼ生成されず、約５０日以降から約２００日までの間で約１０ｍＭの濃度増加が見られた。その後、約２００日から約７５０日までの間では、メタンの濃度には変化がなかった。比較例２において、酢酸濃度は、測定開始時から約２０日までの間に約１０ｍＭまで増加し、約２０日から約２００日までの間に減少した。酢酸濃度は、約２００日以降には定量下限値以下となった。 On the other hand, in Comparative Example 2, almost no methane was produced between about 50 days from the start of measurement and an increase in concentration of about 10 mM was observed from about 50 days to about 200 days. Thereafter, there was no change in the concentration of methane between about 200 days and about 750 days. In Comparative Example 2, the acetic acid concentration increased to about 10 mM from the start of measurement to about 20 days and decreased from about 20 days to about 200 days. The acetic acid concentration was below the lower limit of quantification after about 200 days.
　図８に示されるように、実施例２において、所定期間経過後、トルエンが約４５ｍＭから約７ｍＭに減少したが、他の成分にはほとんど変化がなかった。 As shown in FIG. 8, in Example 2, toluene decreased from about 45 mM to about 7 mM after a predetermined period, but there was almost no change in other components.
　このように、実施例２では、微生物群及び活性化剤を供給したことで、活性化剤により活性化した微生物群による原油からのメタン生成が行われ、短期間でメタン生成量が増大していることが分かる。 Thus, in Example 2, by supplying the microorganism group and the activator, methane generation from crude oil by the microorganism group activated by the activator is performed, and the amount of methane generated increases in a short period of time. I understand that.
　上述のように、実施例２において、測定開始から約４０日までの期間に約１０ｍＭの濃度増加に相当するメタンが生成されており、この期間は、容器中の酢酸量が減少した期間とほぼ合致している。そのため、約４０日までの期間に生成されたメタンは、主に揮発性脂肪酸等の分解由来であると考えられる。 As described above, in Example 2, methane corresponding to an increase in concentration of about 10 mM was generated in the period from the start of measurement to about 40 days, and this period was almost the same as the period in which the amount of acetic acid in the container decreased. It matches. Therefore, it is considered that methane produced in a period of up to about 40 days is mainly derived from decomposition of volatile fatty acids and the like.
　一方、実施例２において、約２５０日から約７５０日までの間に約４０ｍＭの濃度増加に相当するメタンがさらに生成されている。約２９０日以降に生成メタン中の炭素の安定同位体比が増加して約１．３ａｔｍ％まで達していること（図６）、実験前後で原油成分中のトルエンの減少が観測されていること（図８）、またトルエンの減少量から化学量論的に計算されるメタン生成量とほぼ合致することから、約２５０日以降に生成されたメタンは、主に原油中のトルエン分解由来と考えられる。 On the other hand, in Example 2, methane corresponding to a concentration increase of about 40 mM was further generated between about 250 days and about 750 days. The stable isotope ratio of carbon in the generated methane has increased to about 1.3 atm% after about 290 days (Fig. 6), and a decrease in toluene in crude oil components has been observed before and after the experiment. (Fig. 8) In addition, methane produced after about 250 days is considered to originate mainly from the decomposition of toluene in crude oil because it almost matches the methane production calculated stoichiometrically from the reduction in toluene. It is done.
　また、比較例２においては、測定開始から約２０日まで、容器中の酢酸量が増加している。これは、易分解性の揮発性脂肪酸等の低炭素鎖を持つ成分が、活性化した微生物群によって酢酸及び／又はその誘導体に分解されたために生じたものと考えられる。また、比較例２において、約５０日から約２００日までの期間に、約１０ｍＭの濃度増加に相当するメタンが生成しており、この期間は、容器中の酢酸量が減少した期間とほぼ合致している。そのため、約５０日から約２００日までの期間に生成したメタンは、主に揮発性脂肪酸等の分解由来であると考えられる。 In Comparative Example 2, the amount of acetic acid in the container is increased from the start of measurement to about 20 days. This is considered to be caused by the fact that a component having a low carbon chain such as an easily decomposable volatile fatty acid was decomposed into acetic acid and / or a derivative thereof by an activated microorganism group. Further, in Comparative Example 2, methane corresponding to an increase in concentration of about 10 mM was generated in the period from about 50 days to about 200 days, and this period substantially coincides with the period in which the amount of acetic acid in the container decreased. I'm doing it. Therefore, it is considered that methane produced in a period from about 50 days to about 200 days is mainly derived from decomposition of volatile fatty acids and the like.
　なお、実施例２では、微生物群と共に活性化剤を原油に供給したが、地中に微生物を活性化させる物質が存在する場合は、その存在量を差し引いて活性化剤を調製して、供給してもよい。あるいは、活性化剤を与えずに微生物群のみを供給してもよい。この場合にも、供給した微生物群が原油からメタンを生成することで、短期間でメタン生成量が増大する。 In Example 2, the activator was supplied to the crude oil together with the microorganism group. However, if there is a substance that activates the microorganism in the ground, the activator is prepared by subtracting the existing amount and supplied. May be. Or you may supply only a microorganism group, without giving an activator. Also in this case, the supplied microorganism group generates methane from crude oil, so that the amount of methane generated increases in a short period of time.
　（実施例３）
　油層水の量を変更し、活性化剤の組成及び量を変更し、安定同位体で標識化したトルエンを添加しなかったこと以外は実施例１と同様にして、実験を行った。 (Example 3)
The experiment was performed in the same manner as in Example 1 except that the amount of oil layer water was changed, the composition and amount of the activator were changed, and toluene labeled with a stable isotope was not added.
　すなわち、原油１００ｍｌ、油層水５００ｍｌ、活性化剤６ｍｌを、海砂２５０ｍｌを入れておいた１Ｌステンレス容器に入れ、容器内を嫌気性雰囲気中、圧力及び温度をそれぞれ５ＭＰａ、５５℃に保った。 That is, 100 ml of crude oil, 500 ml of oil layer water and 6 ml of activator were placed in a 1 L stainless steel container in which 250 ml of sea sand had been placed, and the inside of the container was maintained at 5 MPa and 55 ° C. in an anaerobic atmosphere.
　活性化剤としては、ビタミン群（Biotin、p-ABA（p-aminobenzoic acid）、Pantothenate, Ca salt、Pyridoxine-HCl、Nicotinic acid、Thiamine-HCl・2H2O、Lipoic（Thioctic） acid、Folic acid、B12、Riboflavin）及び金属塩群（FeCl2、CoCl2、MnCl2・4H2O、ZnCl2、H3BO3、NiCl2、AlCl3、Na2MoO4・2H2O、CuCl2）を含む水溶液に、NH4Cl、KH2PO4、MgCl2・6H2O、CaCl2・2H2O、NaClを添加し、酵母抽出物５ｍｌを添加したものである。 Activators include vitamins (Biotin, p-ABA (p-aminobenzoic acid), Pantothenate, Ca salt, Pyridoxine-HCl, Nicotinic acid, Thiamine-HCl · 2H 2 O, Lipoic (Thioctic) acid, Folic acid, B12, Riboflavin) and metal salts (FeCl 2 , CoCl 2 , MnCl 2 · 4H 2 O, ZnCl 2 , H 3 BO 3 , NiCl 2 , AlCl 3 , Na 2 MoO 4 · 2H 2 O, CuCl 2 ) NH 4 Cl, KH 2 PO 4 , MgCl 2 .6H 2 O, CaCl 2 .2H 2 O, and NaCl are added to the aqueous solution, and 5 ml of yeast extract is added.
　酵母抽出物は、アラニン（Alanine）、アルギニン（Arginine）、アスパラギン酸（Aspartic Acid）、シスチン（Cystine）、グルタミン酸（Glutamic Acid）、グリシン（Glycine）、ヒスチジン（Histidine）、イソロイシン（Isoleucine）、ロイシン（Leucine）、リジン（Lysine）、メチオニン（Methionine）、フェニルアラニン（Phenylalanine）、プロリン（Proline）、セリン（Serine）、トレオニン（Threonine）、トリプトファン（Tryptophan）、チロシン（Tyrosine）、バリン（Valine）、ビオチン（Biotin）、コリン（Choline）、葉酸（Folic acid）、イノシトール（Inositol）、ニコチン酸（Nicotinic Acid）、４－アミノ安息香酸（p-aminobenzoic acid）、パントテン酸（Pantothenic acid）、ピリドキシン（Pyridoxine）、リボフラビン（Riboflavin）、及びチアミン（Thiamine）を含むものであった。 Yeast extracts include Alanine, Arginine, Aspartic Acid, Cystine, Glutamic Acid, Glycine, Histidine, Isoleucine, Leucine (Isoleucine) Leucine, Lysine, Methionine, Phenylalanine, Proline, Serine, Threonine, Tryptophan, Tyrosine, Valine, Biotin ( Biotin), Choline, Folic acid, Inositol, Nicotinic acid, 4-Aminobenzoic acid, Pantothenic acid, Pyridoxine, It contained riboflavin and Thiamine.
　実施例１と同様に、容器内のメタンの濃度及び酢酸の濃度を、約７５０日にわたり、数日から数十日の任意の時間間隔で測定した。培養初日（０日目）の段階では、メタン濃度 ０ｍＭであったが、培養１２４日経過後では、メタン濃度１９０ｍＭとなっていることが記録された。 In the same manner as in Example 1, the concentration of methane and the concentration of acetic acid in the container were measured at arbitrary time intervals of several days to several tens of days over about 750 days. At the stage of the first day of culture (day 0), the methane concentration was 0 mM, but after 124 days of culture, it was recorded that the methane concentration was 190 mM.
　さらに、実験前後、すなわち０日経過時点（反応開始前）及び７５０日経過時点での、複数の脂肪族炭化水素について、実施例２と同様にして容器内の濃度を測定した。測定結果を図１０に示す。図１０のグラフは、炭素数９～３４の直鎖アルカン（ｎ－アルカン）についての結果である。実験後（７５０日経過後）、測定対象である全ての直鎖アルカンの濃度が、実験前（０日）の濃度に対して低下していることが分かった。 Furthermore, the concentration in the container was measured in the same manner as in Example 2 for a plurality of aliphatic hydrocarbons before and after the experiment, that is, at the time when 0 day had elapsed (before the start of reaction) and at the time when 750 days had elapsed. The measurement results are shown in FIG. The graph in FIG. 10 shows the results for a linear alkane (n-alkane) having 9 to 34 carbon atoms. After the experiment (after 750 days had elapsed), it was found that the concentration of all linear alkanes to be measured was lower than the concentration before the experiment (0 days).
　このように、実施例３では、酵母抽出物を含む活性化剤を用いることによって、微生物群が、脂肪族炭化水素を基質として短期間でメタンを生成できることが分かった。 Thus, in Example 3, it was found that by using an activator containing a yeast extract, the microorganism group can produce methane in a short period of time using an aliphatic hydrocarbon as a substrate.
　以上、実施形態に係る微生物を用いた地層内メタン生成方法について説明したが、本発明は上記実施形態に限定されるものではなく、本発明の範囲内で種々の変形及び改良が可能である。 As described above, the in-situ methane production method using the microorganism according to the embodiment has been described. However, the present invention is not limited to the above embodiment, and various modifications and improvements can be made within the scope of the present invention.
　本出願は、２０１６年３月４日に日本国特許庁に出願された特願２０１６－０４２７３８号に基づく優先権を主張するものであり、特願２０１６－０４２７３８号の全内容を本国際出願に援用する。 This application claims priority based on Japanese Patent Application No. 2016-042738 filed with the Japan Patent Office on March 4, 2016. The entire contents of Japanese Patent Application No. 2016-042738 are incorporated herein by reference. Incorporate.
１１、２１　地下資源
１２，２１０　微生物群
１３、２２　メタン
１００、２２０　活性化剤 11, 21 Underground resources 12, 210 Microbial group 13, 22 Methane 100, 220 Activator

Claims (8)

  1. 　炭化水素系の地下資源及び前記地下資源からメタンを生成する微生物群が存在する地層内に、前記微生物群を活性化させる活性化剤を供給する活性化剤供給工程を有する
    ことを特徴とする微生物を用いたメタン生成方法。     A microorganism having an activator supplying step of supplying an activator for activating the microorganism group in a formation in which a hydrocarbon-based underground resource and a microorganism group that generates methane from the underground resource exist Methane production method using
  2. 　炭化水素系の地下資源が存在する地層内に、前記地下資源からメタンを生成する微生物群を供給する微生物群供給工程を有する
    ことを特徴とする微生物を用いたメタン生成方法。     A method for producing methane using microorganisms, comprising a microorganism group supply step of supplying a microorganism group that generates methane from the underground resources in a formation in which hydrocarbon-based underground resources exist.
  3. 　前記地層内に前記微生物群を活性化させる活性化剤を供給する活性化剤供給工程を有する
    ことを特徴とする請求項２に記載の微生物を用いたメタン生成方法。     The method for producing methane using microorganisms according to claim 2, further comprising an activator supplying step of supplying an activator for activating the microorganism group in the formation.
  4. 　前記炭化水素系の地下資源と、前記地下資源からメタンを生成する微生物群と、前記供給された前記微生物群を活性化させる活性化剤とを含む混合物が、嫌気性雰囲気中、１０～１２０℃の温度で、０．１～７０ＭＰａの圧力で保持される保持工程を有することを特徴とする請求項１又は３に記載の微生物を用いたメタン生成方法。 A mixture containing the hydrocarbon-based underground resource, a microorganism group that generates methane from the underground resource, and an activator that activates the supplied microorganism group is 10 to 120 ° C. in an anaerobic atmosphere. The method for producing methane using a microorganism according to claim 1 or 3, further comprising a holding step of holding at a pressure of 0.1 to 70 MPa at a temperature of 5 ° C.
  5. 　前記微生物群を活性化させる活性化剤が、ビタミン及び／又は金属塩を含むこと
    を特徴とする請求項１又は３に記載の微生物を用いたメタン生成方法。     The method for producing methane using a microorganism according to claim 1 or 3, wherein the activator for activating the microorganism group contains vitamins and / or metal salts.
  6. 　前記微生物群を活性化させる活性化剤が、酵母抽出物を含む
    ことを特徴とする請求項１、３又は５に記載の微生物を用いたメタン生成方法。     6. The method for producing methane using a microorganism according to claim 1, wherein the activator for activating the microorganism group includes a yeast extract.
  7. 　前記微生物群が、Methanothermobacter属、Methanosaeta属、Methanoculleus属のいずれかに属するメタン生成菌のうち少なくとも１種と、Firmicutes門、Ca.Atribacteria門、Ca.Cloacimonetes門のいずれかに属する細菌のうち少なくとも１種とを含む
    ことを特徴とする請求項１から６のいずれか一項に記載の微生物を用いたメタン生成方法。     The microorganism group is at least one methanogen belonging to any of the genus Methanothermobacter, Methanosaeta, or Methanoculleus, and at least one of the bacteria belonging to any of the Firmicutes, Ca. Atribacteria, and Ca. Cloacimonetes gates. The method for producing methane using the microorganism according to any one of claims 1 to 6, wherein the method comprises a seed.
  8. 　前記地下資源が、炭素数６又は７の芳香族炭化水素、及び炭素数６～７０の脂肪族炭化水素のうち１種以上の炭化水素を含み、
    　前記微生物群によって前記１種以上の炭化水素が分解される
    ことを特徴とする請求項１から７のいずれか一項に記載の微生物を用いたメタン生成方法。     The underground resource includes an aromatic hydrocarbon having 6 or 7 carbon atoms and one or more hydrocarbons among aliphatic hydrocarbons having 6 to 70 carbon atoms;
    The method for producing methane using a microorganism according to any one of claims 1 to 7, wherein the one or more hydrocarbons are decomposed by the microorganism group.
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