CN111155975A - Method for improving yield of biological coal bed gas through feed supplement fermentation - Google Patents
Method for improving yield of biological coal bed gas through feed supplement fermentation Download PDFInfo
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- 239000003245 coal Substances 0.000 title claims abstract description 96
- 238000000034 method Methods 0.000 title claims abstract description 40
- 238000000855 fermentation Methods 0.000 title claims abstract description 23
- 230000004151 fermentation Effects 0.000 title claims abstract description 23
- 239000006052 feed supplement Substances 0.000 title abstract description 4
- VNWKTOKETHGBQD-UHFFFAOYSA-N methane Chemical compound C VNWKTOKETHGBQD-UHFFFAOYSA-N 0.000 claims abstract description 80
- 238000004519 manufacturing process Methods 0.000 claims abstract description 19
- 235000015097 nutrients Nutrition 0.000 claims abstract description 9
- 239000000126 substance Substances 0.000 claims abstract description 8
- 230000000813 microbial effect Effects 0.000 claims abstract description 7
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 claims description 52
- 239000007789 gas Substances 0.000 claims description 43
- 229910052757 nitrogen Inorganic materials 0.000 claims description 26
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- 229910052698 phosphorus Inorganic materials 0.000 claims description 19
- 239000011574 phosphorus Substances 0.000 claims description 19
- 239000000843 powder Substances 0.000 claims description 17
- NLXLAEXVIDQMFP-UHFFFAOYSA-N Ammonia chloride Chemical compound [NH4+].[Cl-] NLXLAEXVIDQMFP-UHFFFAOYSA-N 0.000 claims description 16
- LWIHDJKSTIGBAC-UHFFFAOYSA-K tripotassium phosphate Chemical compound [K+].[K+].[K+].[O-]P([O-])([O-])=O LWIHDJKSTIGBAC-UHFFFAOYSA-K 0.000 claims description 16
- 239000011573 trace mineral Substances 0.000 claims description 14
- 235000013619 trace mineral Nutrition 0.000 claims description 14
- 241000203069 Archaea Species 0.000 claims description 11
- 238000003935 denaturing gradient gel electrophoresis Methods 0.000 claims description 11
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- 238000004458 analytical method Methods 0.000 claims description 10
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- 239000002817 coal dust Substances 0.000 claims description 9
- 125000001477 organic nitrogen group Chemical group 0.000 claims description 8
- FGIUAXJPYTZDNR-UHFFFAOYSA-N potassium nitrate Chemical compound [K+].[O-][N+]([O-])=O FGIUAXJPYTZDNR-UHFFFAOYSA-N 0.000 claims description 8
- 229910021580 Cobalt(II) chloride Inorganic materials 0.000 claims description 6
- 239000007836 KH2PO4 Substances 0.000 claims description 6
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- 239000001110 calcium chloride Substances 0.000 claims description 6
- 229910001628 calcium chloride Inorganic materials 0.000 claims description 6
- 229940041514 candida albicans extract Drugs 0.000 claims description 6
- 229910000366 copper(II) sulfate Inorganic materials 0.000 claims description 6
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- 229910000357 manganese(II) sulfate Inorganic materials 0.000 claims description 6
- MGFYIUFZLHCRTH-UHFFFAOYSA-N nitrilotriacetic acid Chemical compound OC(=O)CN(CC(O)=O)CC(O)=O MGFYIUFZLHCRTH-UHFFFAOYSA-N 0.000 claims description 6
- 239000012138 yeast extract Substances 0.000 claims description 6
- 229910000368 zinc sulfate Inorganic materials 0.000 claims description 6
- 239000011686 zinc sulphate Substances 0.000 claims description 6
- PAWQVTBBRAZDMG-UHFFFAOYSA-N 2-(3-bromo-2-fluorophenyl)acetic acid Chemical compound OC(=O)CC1=CC=CC(Br)=C1F PAWQVTBBRAZDMG-UHFFFAOYSA-N 0.000 claims description 5
- 229910019142 PO4 Inorganic materials 0.000 claims description 5
- 239000001888 Peptone Substances 0.000 claims description 5
- 108010080698 Peptones Proteins 0.000 claims description 5
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- 240000004808 Saccharomyces cerevisiae Species 0.000 claims description 5
- 240000008042 Zea mays Species 0.000 claims description 5
- 235000005824 Zea mays ssp. parviglumis Nutrition 0.000 claims description 5
- 235000002017 Zea mays subsp mays Nutrition 0.000 claims description 5
- 235000019270 ammonium chloride Nutrition 0.000 claims description 5
- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical compound N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 claims description 5
- 229910052921 ammonium sulfate Inorganic materials 0.000 claims description 5
- 235000011130 ammonium sulphate Nutrition 0.000 claims description 5
- 235000005822 corn Nutrition 0.000 claims description 5
- 239000002609 medium Substances 0.000 claims description 5
- 235000019796 monopotassium phosphate Nutrition 0.000 claims description 5
- 235000019319 peptone Nutrition 0.000 claims description 5
- 229910052700 potassium Inorganic materials 0.000 claims description 5
- 239000011591 potassium Substances 0.000 claims description 5
- 229910000160 potassium phosphate Inorganic materials 0.000 claims description 5
- 235000011009 potassium phosphates Nutrition 0.000 claims description 5
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 claims description 4
- 244000068988 Glycine max Species 0.000 claims description 4
- 235000010469 Glycine max Nutrition 0.000 claims description 4
- 241000202974 Methanobacterium Species 0.000 claims description 4
- 229910052799 carbon Inorganic materials 0.000 claims description 4
- MNNHAPBLZZVQHP-UHFFFAOYSA-N diammonium hydrogen phosphate Chemical compound [NH4+].[NH4+].OP([O-])([O-])=O MNNHAPBLZZVQHP-UHFFFAOYSA-N 0.000 claims description 4
- ZPWVASYFFYYZEW-UHFFFAOYSA-L dipotassium hydrogen phosphate Chemical compound [K+].[K+].OP([O-])([O-])=O ZPWVASYFFYYZEW-UHFFFAOYSA-L 0.000 claims description 4
- 239000000413 hydrolysate Substances 0.000 claims description 4
- 239000003077 lignite Substances 0.000 claims description 4
- 230000000696 methanogenic effect Effects 0.000 claims description 4
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- 239000004323 potassium nitrate Substances 0.000 claims description 4
- 241000894007 species Species 0.000 claims description 4
- RHZUVFJBSILHOK-UHFFFAOYSA-N anthracen-1-ylmethanolate Chemical compound C1=CC=C2C=C3C(C[O-])=CC=CC3=CC2=C1 RHZUVFJBSILHOK-UHFFFAOYSA-N 0.000 claims description 3
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- 239000002802 bituminous coal Substances 0.000 claims description 3
- 239000013587 production medium Substances 0.000 claims description 3
- 238000012216 screening Methods 0.000 claims description 3
- 241000205276 Methanosarcina Species 0.000 claims description 2
- XAGFODPZIPBFFR-UHFFFAOYSA-N aluminium Chemical compound [Al] XAGFODPZIPBFFR-UHFFFAOYSA-N 0.000 claims description 2
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- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 claims description 2
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- 238000012258 culturing Methods 0.000 claims description 2
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- 239000011888 foil Substances 0.000 claims description 2
- 239000007788 liquid Substances 0.000 claims description 2
- 125000000449 nitro group Chemical group [O-][N+](*)=O 0.000 claims description 2
- 125000000018 nitroso group Chemical group N(=O)* 0.000 claims description 2
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- 229910052760 oxygen Inorganic materials 0.000 claims description 2
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 claims description 2
- 239000010452 phosphate Substances 0.000 claims description 2
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 claims description 2
- 230000009467 reduction Effects 0.000 claims description 2
- 241000894006 Bacteria Species 0.000 abstract description 11
- 230000008859 change Effects 0.000 abstract description 2
- 230000002035 prolonged effect Effects 0.000 abstract description 2
- XUJNEKJLAYXESH-REOHCLBHSA-N L-Cysteine Chemical compound SC[C@H](N)C(O)=O XUJNEKJLAYXESH-REOHCLBHSA-N 0.000 description 13
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 10
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- 150000002500 ions Chemical class 0.000 description 9
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- 238000004817 gas chromatography Methods 0.000 description 4
- 238000004451 qualitative analysis Methods 0.000 description 4
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- ALYNCZNDIQEVRV-UHFFFAOYSA-N 4-aminobenzoic acid Chemical compound NC1=CC=C(C(O)=O)C=C1 ALYNCZNDIQEVRV-UHFFFAOYSA-N 0.000 description 2
- PVNIIMVLHYAWGP-UHFFFAOYSA-N Niacin Chemical compound OC(=O)C1=CC=CN=C1 PVNIIMVLHYAWGP-UHFFFAOYSA-N 0.000 description 2
- GRYLNZFGIOXLOG-UHFFFAOYSA-N Nitric acid Chemical compound O[N+]([O-])=O GRYLNZFGIOXLOG-UHFFFAOYSA-N 0.000 description 2
- 238000012408 PCR amplification Methods 0.000 description 2
- 230000000035 biogenic effect Effects 0.000 description 2
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- 239000000203 mixture Substances 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 239000003345 natural gas Substances 0.000 description 2
- 229910017604 nitric acid Inorganic materials 0.000 description 2
- 229920002401 polyacrylamide Polymers 0.000 description 2
- 230000008569 process Effects 0.000 description 2
- 229940088594 vitamin Drugs 0.000 description 2
- 235000013343 vitamin Nutrition 0.000 description 2
- 239000011782 vitamin Substances 0.000 description 2
- 229930003231 vitamin Natural products 0.000 description 2
- 150000003722 vitamin derivatives Chemical class 0.000 description 2
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 2
- 108020004465 16S ribosomal RNA Proteins 0.000 description 1
- 241000233866 Fungi Species 0.000 description 1
- 241000193751 Methanoculleus Species 0.000 description 1
- NBIIXXVUZAFLBC-UHFFFAOYSA-L Phosphate ion(2-) Chemical compound OP([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-L 0.000 description 1
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- 238000006243 chemical reaction Methods 0.000 description 1
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- 238000004925 denaturation Methods 0.000 description 1
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- AGBQKNBQESQNJD-UHFFFAOYSA-M lipoate Chemical compound [O-]C(=O)CCCCC1CCSS1 AGBQKNBQESQNJD-UHFFFAOYSA-M 0.000 description 1
- 235000019136 lipoic acid Nutrition 0.000 description 1
- 229920002521 macromolecule Polymers 0.000 description 1
- 230000002503 metabolic effect Effects 0.000 description 1
- 229960003512 nicotinic acid Drugs 0.000 description 1
- 235000001968 nicotinic acid Nutrition 0.000 description 1
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- 108020004707 nucleic acids Proteins 0.000 description 1
- 150000007523 nucleic acids Chemical class 0.000 description 1
- 102000039446 nucleic acids Human genes 0.000 description 1
- PJNZPQUBCPKICU-UHFFFAOYSA-N phosphoric acid;potassium Chemical compound [K].OP(O)(O)=O PJNZPQUBCPKICU-UHFFFAOYSA-N 0.000 description 1
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- 239000013589 supplement Substances 0.000 description 1
- 229960002663 thioctic acid Drugs 0.000 description 1
Images
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- E—FIXED CONSTRUCTIONS
- E21—EARTH OR ROCK DRILLING; MINING
- E21B—EARTH OR ROCK DRILLING; OBTAINING OIL, GAS, WATER, SOLUBLE OR MELTABLE MATERIALS OR A SLURRY OF MINERALS FROM WELLS
- E21B43/00—Methods or apparatus for obtaining oil, gas, water, soluble or meltable materials or a slurry of minerals from wells
- E21B43/16—Enhanced recovery methods for obtaining hydrocarbons
-
- 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
- C12P5/023—Methane
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- E—FIXED CONSTRUCTIONS
- E21—EARTH OR ROCK DRILLING; MINING
- E21B—EARTH OR ROCK DRILLING; OBTAINING OIL, GAS, WATER, SOLUBLE OR MELTABLE MATERIALS OR A SLURRY OF MINERALS FROM WELLS
- E21B43/00—Methods or apparatus for obtaining oil, gas, water, soluble or meltable materials or a slurry of minerals from wells
- E21B43/006—Production of coal-bed methane
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- 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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Abstract
The invention belongs to the technical field of coal bed gas yield increase, and particularly relates to a method for improving the yield of biological coal bed gas through feed supplement fermentation. According to the invention, by collecting heterotopic enrichment culture of microbial floras of coal bed gas production in a coal bed, nutrients are supplemented in the middle or later period of generation of biological coal bed gas, so that the yield of the coal bed gas is increased. The invention selects the change of nutrient substances or flora as the time point of adding the nutrient substances, and has very strong pertinence. Compared with the method for directly producing methane by using coalbed indigenous bacteria, the method for improving the yield of the biological coalbed methane by the fed-batch fermentation has the advantages of high gas production amount and prolonged gas production time.
Description
Technical Field
The invention belongs to the technical field of coal bed gas yield increase, and particularly relates to a method for improving the yield of biological coal bed gas through feed supplement fermentation.
Background
Coal Bed Methane (CBM) is a methane gas that is present in coal seams and releases only half of the CO when burning coal compared to burning coal2The emission of CO and NOx is reduced by 80%. As a clean energy source, coal bed gas has attracted attention and attention in many countries. Because the natural gas content in our country is low, the exploitation of coal bed gas is particularly important as a substitute energy of natural gas. Geological data accumulated over the past three decades indicate that biogenic gas is an important source of coal bed methane. The generation of secondary biogenic gas from coal, which typically occurs in shallow layers at temperatures below 100 ℃, is a result of microbial community activity after coal coalification. However, the biogenesis of coal bed gas is slow, resulting in its slow formationThe application is restricted. Therefore, how to improve the efficiency of producing coal bed gas by microorganisms is a problem to be solved at present.
Coal, under certain conditions, can produce new methane during the degradation process of microorganisms. Because coal is a very complex heterocyclic macromolecule, the coal needs to be gradually degraded under the combined action of various coal bed microorganisms to finally generate coal bed gas, and on the basis, a method for degrading coal by microorganisms in an anaerobic manner is generally adopted at present, and the characteristic of methane production by methanogens in an anaerobic manner is combined to realize the microbial increase of the coal bed gas. For example, CN201210035682.0 discloses a method for increasing the yield of coal bed gas by using exogenous microorganisms, which specifically comprises activating indigenous bacteria in a coal bed by using exogenous bacteria, and degrading organic matters on the surface of coal to generate methane; CN201610710769.1 discloses a method for improving the yield of coal bed gas by using indigenous bacteria; CN201710721266.9 discloses a method for improving the yield of biological coal bed gas by using indigenous fungi.
Disclosure of Invention
Aiming at the problems, the invention provides a method for improving the yield of biological coal bed gas by feed fermentation.
In order to achieve the purpose, the invention adopts the following technical scheme:
a method for improving the yield of biological coal bed gas by feed fermentation comprises the following steps:
step 1, collecting coal dust and coal seam effluent samples of a plurality of coal seam gas wells, filling nitrogen to isolate oxygen and wrapping the coal seam effluent samples with aluminum foil paper for later use;
step 2, adding the collected coal dust and coal bed effluent samples into an enrichment medium, acclimating, culturing and screening the floras with the highest coal bed gas yield of coal dust per unit mass as a bacterial source for biological coal bed gas production;
step 3, adding the flora screened in the step 2 into 900mL of coal bed methane production medium according to the volume ratio of 10% for fermentation, and adding 0.05-0.2% w/w of nutrient substances for biological production of coal bed methane when the nitrogen content or phosphorus content in the biological coal bed methane fermentation liquid is reduced to the basic level nitrogen content or phosphorus content or the consumption is finished, or the content of methanogenic archaea is reduced;
and 4, extracting microbial DNA from the collected coal dust and coal seam effluent samples and the flora in the step 2, and performing microbial flora structure analysis by a PCR-DGGE method.
Further, the enrichment medium in the step 2 is composed of 5% of pulverized coal as a unique carbon source, 0.2% of a nitrogen source, 0.4% of phosphorus and potassium and 1% of a trace element solution.
Further, the pulverized coal is any one of anthracite, bituminous coal or lignite; the granularity of the coal powder is 60-160 meshes, and the coal powder needs to be dried for 6 months;
the nitrogen source is an organic nitrogen source or an inorganic nitrogen source;
the organic nitrogen source is any one of yeast powder, peptone, soybean hydrolysate or corn steep liquor dry powder;
the inorganic nitrogen source is any one of ammonium sulfate, ammonium chloride, ammonium nitrate or ammonium hydrogen phosphate;
the phosphorus and potassium are any one of potassium phosphate, monopotassium phosphate, dipotassium hydrogen phosphate or potassium nitrate.
Still further, the trace elements are selected from nitrilotriacetic acid 1.5g and CaCl20.1g、MgSO4·7H2O 3.0g、H3BO30.05g、FeSO40.1g、NaCl 1.0g、CoCl20.1g、MnSO40.5g、ZnSO40.1g、NaMO40.05g、A1K(SO4)20.01g、NiCl20.1g、CuSO40.01 g.
Further, the coal bed methane culture medium in the step 3 is: adding yeast extract 0.5g and K into 1L deionized water2HPO42.9g、KH2PO41.5g、NH4Cl 1.8g、MgCl20.4g, 3g of cysteine, 2mL of 0.2% resazurin, and 10mL of a trace element solution, and the pH was 7.0.
Further, the base level of nitrogen content or phosphorus content in step 3: the nitrogen content refers to the content of amino, nitro and nitroso salts in the coal seam, and the phosphorus content refers to the content of phosphate in the coal seam.
Further, the methanogenic archaea is Methanosarcina, Methanobacterium or Methanoculleus.
Further, the content of the archaeological methane-producing bacteria in the step 3 is reduced in a production period, the content of the archaeological methane-producing bacteria is increased and then reduced, and the content reduction is compared with the highest content.
Further, the nutrient substance in the step 3 is one or two of organic nitrogen, inorganic nitrogen and inorganic phosphorus, including a nitrogen source and a phosphorus source;
the organic nitrogen is any one of yeast powder, peptone, soybean hydrolysate or corn steep liquor dry powder;
the inorganic nitrogen is any one of ammonium sulfate, ammonium chloride, ammonium nitrate or ammonium hydrogen phosphate;
the inorganic phosphorus is any one of potassium phosphate, monopotassium phosphate, dipotassium phosphate or potassium nitrate.
Further, the PCR-DGGE method in the step 4 is a PCR-DGGE analysis method with species standard introduced during electrophoresis analysis.
Compared with the prior art, the invention has the following advantages:
the coal geological microorganisms are in a severe environment and are in an inactive state, and can be stimulated by some outside world to perform metabolic activity better. In terms of biological coal bed gas production, the process is a process for producing methane by mixed bacteria fermentation, and in comparison, a carbon source, namely coal is excessive, and other nutrient substances, such as nitrogen sources, phosphorus, potassium and the like, are insufficient, so that the carbon source, namely the coal is required to be supplemented in time to be beneficial to the growth of microorganisms, but the selection time of the supplement is very critical, and the growth and the gas production are greatly influenced by early, late and insufficient amount. The invention selects the change of nutrient substances or flora as the adding time point, and has very strong pertinence. Compared with the method for directly producing methane by using coalbed indigenous bacteria, the method for improving the yield of the biological coalbed methane by the fed-batch fermentation has the advantages of high gas production amount and prolonged gas production time.
Drawings
FIG. 1 is a diagram showing the condition of coal bed methane produced by microorganisms in a coal bed after nitric acid is supplemented under the experimental conditions of the invention.
Detailed Description
Example 1
In an anaerobic glove box, 50mL of a coal bed output water sample and 2g of a coal sample are added into a 500mL anaerobic bottle, 200mL of a sterilization enrichment culture medium is added, then 0.04% of L-cysteine and 20g of lignite powder which are introduced with nitrogen are added, the mixture is gently mixed, the anaerobic bottle is rapidly sealed, and the mixture is subjected to standing culture at the temperature of 25 ℃. During the period, a sterile syringe is inserted from the top of the anaerobic bottle every week for automatic collection of gas, 0.5mL of the collected gas is taken for gas chromatography analysis, and the relative content of methane generation is calculated. (enrichment culture screening bacteria)
Enrichment medium
2g of yeast extract and K are added into 1L of deionized water2HPO42.9g、KH2PO41.5g、NH4Cl 1.8g、MgCl20.4g, 3g of cysteine, 2mL of resazurin (0.2%), 10mL of a trace element solution with a pH of 7.0, and 5mL of a vitamin solution.
The formula of the trace element solution is as follows: nitrilotriacetic acid 1.5g, CaCl20.1g、MgSO4·7H2O 3.0g、H3BO30.05g、FeSO40.1g、NaCl 1.0g、CoCl20.1g、MnSO40.5g、ZnSO40.1g、NaMO40.05g、A1K(SO4)20.01g、NiCl20.1g、CuSO40.01g。
1L vitamin solution comprises: biotin 2mg, folic acid 2mg, B610 mg, B25 mg, B15 mg, nicotinic acid 5mg, B120.1mg, lipoic acid 5mg, p-aminobenzoic acid 5 mg.
Example 2
Adding 25g pulverized lignite, 350mL of production medium and 0.408mg of Resazurin in a 1L anaerobic bottle, and adding 1 × 10 parts of5Pa sterilizing for 30 min. Adding 800 microliter of sterile 20 percent L-cysteine into a 1L anaerobic bottle in an anaerobic glove box, and then continuously introducing high-purity N into the anaerobic bottle2Until the color of the culture medium is nearly colorless. In an anaerobic glove box, 50mL of the culture solution of example 1 was inoculated into the 1L anaerobic flask described above. Sealing each anaerobic bottle for gas production experiments, and performing standing culture in an anaerobic box for 1-3 months. During the culture period, samples were taken every other week to measure methane in the culture brothContent of archaea, and simultaneous determination of NH4 +、NO3 -And PO4 3-When the content of Methanosarccina and Methanobacterium in the archaeolhamus methanetus flora is reduced, or when the content of the ions in the archaeolhamus methanetus flora is reduced to a basic level (the content of coal bed ions) or the ions are completely consumed, 0.2% of yeast powder, ammonium sulfate and 0.4% of potassium phosphate are supplemented, a sterile syringe is inserted from the top of an anaerobic bottle every week in the period of time to automatically collect gas, 0.5mL of collected gas is taken to perform gas chromatography analysis, and the relative content of generated methane is calculated. Meanwhile, 5-10 mL of culture solution is taken for qualitative and quantitative analysis of flora.
Coal bed gas culture medium:
adding yeast extract 0.5g and K into 1L deionized water2HPO42.9g、KH2PO41.5g、NH4Cl 1.8g、MgCl20.4g, 3g of cysteine, 2mL of resazurin (0.2%), and 10mL of a trace element solution, with a pH of 7.0.
The formula of the trace element solution is as follows: nitrilotriacetic acid 1.5g, CaCl20.1g、MgSO4·7H2O 3.0g、H3BO30.05g、FeSO40.1g、NaCl 1.0g、CoCl20.1g、MnSO40.5g、ZnSO40.1g、NaMO40.05g、A1K(SO4)20.01g、NiCl20.1g、CuSO40.01g。
Example 3
Adding 25g bituminous coal powder, 350mL of production-period culture medium and 0.408mg of Resazurin in a 1L anaerobic bottle, and adding 1 × 105Pa sterilizing for 30 min. Adding 800 microliter of sterile 20 percent L-cysteine into a 1L anaerobic bottle in an anaerobic glove box, and then continuously introducing high-purity N into the anaerobic bottle2Until the color of the culture medium is nearly colorless. In an anaerobic glove box, 50mL of the culture solution of example 1 was inoculated into the 1L anaerobic flask described above. Sealing each anaerobic bottle for gas production experiments, and performing standing culture in an anaerobic box for 1-3 months. During the culture period, samples were taken every other week, and the content of archaea methanes in the culture broth was measured, together with NH, and4 +、NO3 -and PO4 3-When Methanosarccina, Met is present in the methanoarchaea groupWhen the content of the hanobacterium is reduced, or when the ions are reduced to a basic level (the content of the coal bed ions) or the ions are completely consumed, 0.2 percent of peptone, ammonium chloride and 0.4 percent of potassium dihydrogen phosphate are supplemented, a sterile syringe is inserted from the top of an anaerobic bottle every week during the period for automatically collecting gas, 0.5mL of the collected gas is taken for gas chromatography analysis, and the relative content of the generated methane is calculated. Meanwhile, 5-10 mL of culture solution is taken for qualitative and quantitative analysis of flora.
Coal bed gas culture medium:
adding yeast extract 0.5g and K into 1L deionized water2HPO42.9g、KH2PO41.5g、NH4Cl 1.8g、MgCl20.4g, 3g of cysteine, 2mL of resazurin (0.2%), and 10mL of a trace element solution, with a pH of 7.0.
The formula of the trace element solution is as follows: nitrilotriacetic acid 1.5g, CaCl20.1g、MgSO4·7H2O 3.0g、H3BO30.05g、FeSO40.1g、NaCl 1.0g、CoCl20.1g、MnSO40.5g、ZnSO40.1g、NaMO40.05g、A1K(SO4)20.01g、NiCl20.1g、CuSO40.01g。
Example 4
Adding 25g anthracite coal powder, 350mL production period culture medium and 0.408mg resazurin, 1 × 10mg into a 1L anaerobic bottle5Pa sterilizing for 30 min. Adding 800 microliter of sterile 20 percent L-cysteine into a 1L anaerobic bottle in an anaerobic glove box, and then continuously introducing high-purity N into the anaerobic bottle2Until the color of the culture medium is nearly colorless. In an anaerobic glove box, 50mL of the culture solution of example 1 was inoculated into the 1L anaerobic flask described above. Sealing each anaerobic bottle for gas production experiments, and performing standing culture in an anaerobic box for 1-3 months. During the culture period, samples were taken every other week, and the content of archaea methanes in the culture broth was measured, together with NH, and4 +、NO3 -and PO4 3-When the content of Methanosarccina and Methanobacterium in the archaeolhamus methanolica flora is reduced, or the content of the ions is reduced to a basic level (the content of coal bed ions) or the ions are completely consumed, 0.2 percent of corn steep liquor dry powder, 0.4 percent of ammonium nitrate and 0.4 percent of hydrogen phosphate are supplementedDipotassium, during which a sterile syringe was inserted from the top of the anaerobic flask every week for automatic collection of gas, 0.5mL of the collected gas was analyzed by gas chromatography, and the relative content of methanogenesis was calculated. Meanwhile, 5-10 mL of culture solution is taken for qualitative and quantitative analysis of flora. As shown in figure 1, the condition of coal bed methane produced by coal bed microorganisms after nitric acid is supplemented is shown.
Coal bed gas culture medium:
adding yeast extract 0.5g and K into 1L deionized water2HPO42.9g、KH2PO41.5g、NH4Cl 1.8g、MgCl20.4g, 3g of cysteine, 2mL of resazurin (0.2%), and 10mL of a trace element solution, with a pH of 7.0.
The formula of the trace element solution is as follows: nitrilotriacetic acid 1.5g, CaCl20.1g、MgSO4·7H2O 3.0g、H3BO30.05g、FeSO40.1g、NaCl 1.0g、CoCl20.1g、MnSO40.5g、ZnSO40.1g、NaMO40.05g、A1K(SO4)20.01g、NiCl20.1g、CuSO40.01g。
Example 5
The primers for PCR amplification of 16S rRNA genes of bacteria and archaea are BAC-338F (plus GC clamp)/BAC-518R and AR-344F (plus GC clamp)/AR-519R respectively. And (3) PCR reaction system: pre-denaturation at 95 deg.C for 5min, 30s at 95 deg.C, 30s at 55 deg.C, 30s at 72 deg.C, 30 cycles, 10min at 72 deg.C, and preservation at 10 deg.C.
The conditions for the DGGE analysis of bacteria and archaea were the same. DGGE gel is polyacrylamide gel with the concentration of 10% (v/v), and the denaturation gradient range of the gel is 40-60%. The electrophoresis condition is that the electrophoresis is carried out for 13 hours at the constant temperature of 60 ℃ and the voltage of 85V. After the electrophoresis is finished, the gel is taken down, stained in 4S red plus nucleic acid stain for 15min, then placed into pure water, and decolorized for 10min by a decolorization shaking table. And observing and photographing under a gel image analysis system to obtain a DGGE map. DGGE map result analysis adopts Quantity One software.
Species qualitative analysis: after the sample is subjected to PCR amplification, a product and the coal bed microorganism species DGGE Marker (CN 2017103488119.0, CN201710349248.2) are simultaneously loaded on polyacrylamide denatured gel, imaging is carried out after electrophoresis, and a sample DNA fragment at the same position with the Marker represents a species the same as the Marker.
The embodiments are described in detail, but the present invention is not limited to the above embodiments and examples, and various changes and modifications within the knowledge of those skilled in the art may be made without departing from the spirit of the present invention, and the changes and modifications fall within the scope of the present invention.
Claims (10)
1. A method for improving the yield of biological coal bed gas by feed fermentation is characterized by comprising the following steps: the method comprises the following steps:
step 1, collecting coal dust and coal seam effluent samples of a plurality of coal seam gas wells, filling nitrogen to isolate oxygen and wrapping the coal seam effluent samples with aluminum foil paper for later use;
step 2, adding the collected coal dust and coal bed effluent samples into an enrichment medium, acclimating, culturing and screening the floras with the highest coal bed gas yield of coal dust per unit mass as a bacterial source for biological coal bed gas production;
step 3, adding the flora screened in the step 2 into 900mL of coal bed methane production medium according to the volume ratio of 10% for fermentation, and adding 0.05-0.2% w/w of nutrient substances for biological production of coal bed methane when the nitrogen content or phosphorus content in the biological coal bed methane fermentation liquid is reduced to the basic level nitrogen content or phosphorus content or the consumption is finished, or the content of methanogenic archaea is reduced;
and 4, extracting microbial DNA from the collected coal dust and coal seam effluent samples and the flora in the step 2, and performing microbial flora structure analysis by a PCR-DGGE method.
2. The method for improving the yield of biological coal bed methane through fed-batch fermentation according to claim 1, wherein the method comprises the following steps: the enrichment medium in the step 2 is composed of 5% of coal dust as a unique carbon source, 0.2% of nitrogen source, 0.4% of phosphorus and potassium and 1% of trace element solution.
3. The method for improving the yield of biological coal bed methane through fed-batch fermentation according to claim 2, wherein the method comprises the following steps: the pulverized coal is any one of anthracite, bituminous coal or lignite; the granularity of the coal powder is 60-160 meshes, and the coal powder needs to be dried for 6 months;
the nitrogen source is an organic nitrogen source or an inorganic nitrogen source;
the organic nitrogen source is any one of yeast powder, peptone, soybean hydrolysate or corn steep liquor dry powder;
the inorganic nitrogen source is any one of ammonium sulfate, ammonium chloride, ammonium nitrate or ammonium hydrogen phosphate;
the phosphorus and potassium are any one of potassium phosphate, monopotassium phosphate, dipotassium hydrogen phosphate or potassium nitrate.
4. The method for improving the yield of biological coal bed methane through fed-batch fermentation according to claim 2, wherein the method comprises the following steps: the trace elements comprise nitrilotriacetic acid 1.5g and CaCl20.1g、MgSO4·7H2O 3.0g、H3BO30.05g、FeSO40.1g、NaCl1.0g、CoCl20.1g、MnSO40.5g、ZnSO40.1g、NaMO40.05g、A1K(SO4)20.01g、NiCl20.1g、CuSO40.01 g.
5. The method for improving the yield of biological coal bed methane through fed-batch fermentation according to claim 1, wherein the method comprises the following steps: the coal bed gas production culture medium in the step 3 is as follows: adding yeast extract 0.5g and K into 1L deionized water2HPO42.9g、KH2PO41.5g、NH4Cl 1.8g、MgCl20.4g, 3g of cysteine, 2mL of 0.2% resazurin, and 10mL of a trace element solution, and the pH was 7.0.
6. The method for improving the yield of biological coal bed methane through fed-batch fermentation according to claim 1, wherein the method comprises the following steps: the base level of nitrogen or phosphorus content in step 3: the nitrogen content refers to the content of amino, nitro and nitroso salts in the coal seam, and the phosphorus content refers to the content of phosphate in the coal seam.
7. The method for improving the yield of biological coal bed methane through fed-batch fermentation according to claim 1, wherein the method comprises the following steps: the methanogenic archaea in the step 3 is Methanosarcina, Methanobacterium or Methanovulleus.
8. The method for improving the yield of biological coal bed methane through fed-batch fermentation according to claim 1, wherein the method comprises the following steps: the content of the archaea producing methane in the step 3 is reduced in a production period, the content of the archaea producing methane is increased firstly and then reduced, and the content reduction is compared with the highest content.
9. The method for improving the yield of biological coal bed methane through fed-batch fermentation according to claim 1, wherein the method comprises the following steps: in the step 3, the nutrient substance is one or two of organic nitrogen, inorganic nitrogen and inorganic phosphorus, including a nitrogen source and a phosphorus source;
the organic nitrogen is any one of yeast powder, peptone, soybean hydrolysate or corn steep liquor dry powder;
the inorganic nitrogen is any one of ammonium sulfate, ammonium chloride, ammonium nitrate or ammonium hydrogen phosphate;
the inorganic phosphorus is any one of potassium phosphate, monopotassium phosphate, dipotassium phosphate or potassium nitrate.
10. The method for improving the yield of biological coal bed methane through fed-batch fermentation according to claim 1, wherein the method comprises the following steps: the PCR-DGGE method in the step 4 is a PCR-DGGE analysis method with species standard introduced during electrophoretic analysis.
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