CN106318990B - Preparation for accelerating methane oxidation of refuse landfill and preparation method and application thereof - Google Patents
Preparation for accelerating methane oxidation of refuse landfill and preparation method and application thereof Download PDFInfo
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- CN106318990B CN106318990B CN201610697445.9A CN201610697445A CN106318990B CN 106318990 B CN106318990 B CN 106318990B CN 201610697445 A CN201610697445 A CN 201610697445A CN 106318990 B CN106318990 B CN 106318990B
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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
- C12P17/00—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms
- C12P17/02—Oxygen as only ring hetero atoms
- C12P17/04—Oxygen as only ring hetero atoms containing a five-membered hetero ring, e.g. griseofulvin, vitamin C
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B09—DISPOSAL OF SOLID WASTE; RECLAMATION OF CONTAMINATED SOIL
- B09B—DISPOSAL OF SOLID WASTE
- B09B1/00—Dumping solid waste
- B09B1/004—Covering of dumping sites
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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
- C12P39/00—Processes involving microorganisms of different genera in the same process, simultaneously
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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
Abstract
The invention discloses a preparation for accelerating methane oxidation of a refuse landfill and a preparation method and application thereof, wherein the preparation comprises signal molecules AHLs and AI-2, wherein the AHLs comprise C4-HSL, C6-HSL, C8-HSL, 3-oxo-C6-HSL and 3-oxo-C8-HSL, and the preparation method comprises the following steps: respectively culturing different bacteria secreting corresponding signal molecules to logarithmic phase, respectively extracting the signal molecules in the corresponding culture solution from the obtained culture solution, and then mixing the obtained signal molecules according to the proportion. The invention respectively injects the methane oxidation accelerating preparations to different heights of the landfill layer. The invention utilizes the exogenously added microbial signal molecules as a methane oxidation preparation, improves the content of the signal molecules at the landfill position, and thus reduces the methane content in the landfill.
Description
Technical Field
The invention relates to the field of soil pollution treatment, in particular to a preparation method and application of a methane oxidation preparation for accelerating landfill, which utilize exogenous added microbial signal molecules as the methane oxidation preparation to improve the content of the signal molecules at the landfill so as to reduce the content of methane in the landfill.
Background
Global warming caused by greenhouse gases emitted from human activities has become one of the major environmental problems in the world. Among the greenhouse gases, CO2、CH4And chlorofluorocarbons constitute the most dominant greenhouse effect. The results of the study show that CH4The accelerated rising trend is shown in the last 200 years, and if not controlled, the atmospheric CH is predicted to be in 20304Will reach 2.34. mu.L.L-1May become a main factor of greenhouse effect. Due to CH4Has a greenhouse effect of CO221 times higher, it can be seen that if methane is oxidized as much as possible and converted to CO2The heating potential of the total released gas can be greatly reduced, and the effect of reducing the emission of greenhouse gas is achieved. Anaerobic landfill technology is adopted in most of landfill sites in China, the methane production rate of the technology is high, and CH in landfill gas in a stable state4The content is about 45-60%. As a contracted state of the Kyoto protocol, an economic and efficient landfill CH reduction method is found4The method of discharge is necessary, and landfill methane oxidation is an economic and efficient means for controlling methane release
Methane oxidation is primarily accomplished by methane-oxidizing bacteria. The methane-oxidizing bacteria are a branch of methyl-oxidizing bacteria, and the unique point is that the methane-oxidizing bacteria can utilize methane as the only carbon source and energy source to oxidize methane into carbon dioxide. In addition, methane-oxidizing bacteria can also participate in other important ecological processes. However, because the activity of methane-oxidizing bacteria in the landfill is low, the amount of methane capable of being oxidized is small, so that the effect of methane oxidation in the existing landfill is not obvious.
The invention patent of China with application number 201310521595.0 provides a preparation method of methane-oxidizing bacteria liquid, a refuse landfill covering material and a refuse landfill methane emission reduction method, and points out that mineralized refuse is used as a methane-oxidizing bacteria strain source, and leachate is used as a culture medium to culture methane-oxidizing bacteria in a mixed manner, so that the methane-oxidizing bacteria liquid is obtained to oxidize methane. However, the method is difficult to use in large scale due to the complex operation, low success rate and high cost. The Chinese patent with application number of 201010157441.4 discloses a method for enhancing methane oxidation of a covering layer of a landfill site, and relates to a method capable of enriching methanotrophic microorganisms in mineralized refuse. Although the cost is lower and the methane oxidation efficiency is higher than that of the common covering material of the landfill, the methane oxidation amount is limited and the material is not suitable for large-scale use.
Disclosure of Invention
The invention provides a method for accelerating methane oxidation of a refuse landfill, which adopts signal molecules secreted by different microorganisms to improve the content of the signal molecules in the refuse, enhance the activity of methane-oxidizing bacteria in the refuse and obviously reduce the content of the generated methane.
An agent for accelerating the oxidation of methane in a landfill site, which comprises signal molecules AHLs and an Autoinducer AI-2(Autoinducer-2), wherein the AHLs comprise N-butanoyl-homoserine lacton (C4-HSL), N-hexanoyl-homoserine lacton (C6-HSL), N-octanoyl-homoserine (C8-HSL), N-3-oxo-hexanoyl-homoserine lacton (3-oxo-C6-HSL) and N-3-oxo-octanoyl-homoserine (3-oxo-C8-HSL), and the AHLs comprise the following components in percentage by mass:
the sum of the mass percentages of the components is 100 percent.
Further preferably, the composition in percentage by mass is as follows:
the sum of the mass percentages of the components is 100 percent.
The preparation prepared in the preferable range can reduce the methane content of the refuse landfill by more than 55 percent.
Still more preferably, the composition in mass percent is as follows:
the sum of the mass percentages of the components is 100 percent.
The preparation prepared in the preferable range can reduce the methane content of the refuse landfill by about 80 percent.
Most preferably, the composition in mass percent is as follows:
the formulations prepared within the preferred ranges described above can reduce the methane content of a landfill by up to 85%.
The invention also provides a preparation method of the preparation for accelerating the methane oxidation of the refuse landfill, which comprises the following steps:
respectively culturing different bacteria secreting corresponding signal molecules to logarithmic phase, respectively extracting the signal molecules in the corresponding culture solution from the obtained culture solution, and then mixing the obtained signal molecules according to the proportion.
Preferably, the bacterium secreting the signal molecule is purchased from China general microbiological culture Collection center, and comprises pseudomonas aeruginosa (secreting C4-HSL), and the preservation number is CGMCC NO. 1.2421; pseudomonas putida (secreting 3-oxo-C6-HSL and 3-oxo-C8-HSL) with the preservation number of CGMCC NO. 1.2309; pseudomonas fluorescens (secreting C6-HSL) with the preservation number of CGMCC NO. 1.3202; solanaceae Ralstonia (secreting C8-HSL) with preservation number of CGMCCNO.1.2839; escherichia coli (AI-2 secretion) with a preservation number of CGMCC NO. 1.4244.
Preferably, the culture medium for culturing the bacteria is LB culture medium or casein culture medium; more preferably, a casein medium. The culture conditions of the bacteria were 37 ℃ and 250 rpm.
The LB culture medium comprises the following components: 10g/L of peptone, 5g/L, NaCl 5g/L of yeast extract,
pH 7.0。
casein culture medium: NaCl 1g/L, MgSO40.2g/L、KH2PO40.5g/L、Na2HPO41.5g/L、NH4NO31g/L and 10g/L, pH 7.2.2 of casein.
Preferably, the solvent used for extraction is ethyl acetate, methanol or acetonitrile. Further preferably, the solvent used is ethyl acetate.
Preferably, the solvent is acidified, and the acidification reagent is glacial acetic acid, hydrochloric acid or formic acid. In the invention, different strains are cultured in respective culture media to logarithmic phase, then signal molecules are respectively extracted from corresponding culture solutions, and finally the extracted signal molecules are mixed according to the proportion.
The invention also provides a method for accelerating the methane oxidation of the refuse landfill, which comprises the following steps: the methane oxidation accelerating preparation for the landfill site is injected to different heights of the landfill layer, preferably to the top, the middle and the bottom.
Specifically, the preparation for accelerating methane oxidation is added into the garbage by a deep injection method, and the injection depth is three positions, namely the top, the middle and the bottom of the garbage. The specific operation is as follows: dividing areas on the refuse landfill site, and respectively injecting reagents into the refuse by extending to different depths by using steel needles or by well drilling. The using amount of the methane injection agent is 100-1000 mg per ton of garbage, the methane injection agent is injected once every 2-4 days for 10-15 times, and the methane content generated in a garbage landfill can be obviously removed.
Most preferably, the methane oxidation agent is added at the middle of the landfill.
Quorum sensing is used as an exchange regulation mechanism widely existing among cells, has the functions of regulating and inducing the directional growth of cells and synthesizing extracellular metabolites, and simultaneously finds that the addition of signal molecules can enable methane-oxidizing bacteria to generate a biological membrane and enhance the capability of resisting external interference, thereby having an important effect on improving the activity of the methane-oxidizing bacteria. The method takes different types of quorum-sensing signal molecules as the preparation for accelerating the methane oxidation of the refuse landfill, can obviously improve the activity of methane-oxidizing bacteria in the landfill, obviously remove the methane content, has simple operation and lower cost, and is a better method for realizing the methane oxidation of the refuse landfill.
In order to reduce the cost on the premise of ensuring a certain methane removal rate, the dosage of the added preparation is 300-700 mg per ton of garbage.
The invention optimizes the addition amount and the preparation process of the preparation, can achieve higher methane removal effect under the condition of less preparation use amount, and can act on different receptor bacteria (including gram-negative bacteria and gram-positive bacteria) by applying different types of signal molecules, so that the methane removal efficiency is higher.
Detailed Description
Example 1
(1) Culturing the bacteria secreting the signal molecules by adopting an LB culture medium, wherein the culture medium comprises the following components: 10g/L of peptone and 5g/L, NaCl 5g/L, pH 7.0.0 of yeast extract, and different strains are cultured in respective culture media to logarithmic phase.
(2) The bacteria secreting the signal molecules are cultured in casein medium: NaCl 1g/L, MgSO40.2g/L、KH2PO40.5g/L、Na2HPO41.5g/L、NH4NO31g/L, 10g/L, pH 7.2.2 casein, different strains were cultured to log phase in respective media.
(3) By comparison, the effect of the casein culture medium is better than that of the LB culture medium, and the signal molecular weight generated by the casein culture medium is improved by 30 percent compared with that of the LB culture medium.
Example 2
(1) First, a bacterium secreting a signal molecule is cultured using a casein medium: NaCl 1g/L, MgSO40.2g/L、KH2PO40.5g/L、Na2HPO41.5g/L、NH4NO31g/L of casein and 10g/L, pH 7.2.2 of pseudomonas aeruginosa,Pseudomonas putida, Pseudomonas fluorescens, Ralstonia solanacearum, and Escherichia coli were cultured in the respective media to logarithmic phase, and each bacterium was cultured in three groups.
(2) The signal molecule was extracted, the first group was extracted with ethyl acetate as solvent, and the ethyl acetate was acidified with glacial acetic acid before extraction.
(3) The signal molecules were extracted, the second group was extracted with methanol as solvent, which was acidified with glacial acetic acid before extraction.
(4) Extracting the signal molecule, extracting the third group with acetonitrile as solvent, and acidifying the acetonitrile with glacial acetic acid before extraction.
(5) By comparison, the ethyl acetate is found to have better extraction effect when used as a solvent, and the amount of the extracted product is 20 percent higher than that of other solvents.
Example 3
(1) First, a bacterium secreting a signal molecule is cultured using a casein medium: NaCl 1g/L, MgSO40.2g/L、KH2PO40.5g/L、Na2HPO41.5g/L、NH4NO31g/L, 10g/L, pH 7.2.2 casein, Pseudomonas aeruginosa, Pseudomonas putida, Pseudomonas fluorescens, Ralstonia solanacearum and Escherichia coli were cultured in the respective media to logarithmic phase, and three groups were cultured for each bacterium.
(2) The signal molecule was extracted, the first group was extracted with ethyl acetate as solvent, and the ethyl acetate was acidified with glacial acetic acid before extraction.
(3) Extracting the signal molecule, extracting the second group with ethyl acetate as solvent, and acidifying the ethyl acetate with hydrochloric acid before extraction.
(4) The signal molecule was extracted, the third group was extracted with ethyl acetate as solvent, and ethyl acetate was acidified with formic acid before extraction.
(5) By comparison, the signal molecule persists for 10 hours after acidification with hydrochloric acid, 12 hours after acidification with formic acid and 20 hours after acidification with glacial acetic acid, so the acidification effect with glacial acetic acid is better.
Example 4
(1) Firstly, toThe bacteria secreting the signal molecule are cultured using casein medium: NaCl 1g/L, MgSO40.2g/L、KH2PO40.5g/L、Na2HPO41.5g/L、NH4NO31g/L, 10g/L, pH 7.2.2 casein, Pseudomonas aeruginosa, Pseudomonas putida, Pseudomonas fluorescens, Ralstonia solanacearum and Escherichia coli were cultured in the respective media to logarithmic phase.
(2) Respectively extracting signal molecules in each bacterial liquid, extracting by using ethyl acetate as a solvent, acidifying the ethyl acetate by using glacial acetic acid before extraction, and specifically comprising the following steps:
1) 3ml of the cell suspension was taken into a 15ml glass centrifuge tube, 3ml of ethyl acetate containing 1 ‰ glacial acetic acid was added, and vortexed for 30s (10 times shaking vigorously).
2) Standing for 10min to separate two phases, if not layering 10000rpm 3min to separate two phases, and if layering is not obvious, adding 3ml ethyl acetate containing 1 ‰ glacial acetic acid to layer.
3) The organic layer was pipetted into a clean 10ml vial of penicillin using a pipette gun.
4) Repeat steps 1-3 (final vial total 9 ml).
5) And (3) blowing the final sample to be completely dry by nitrogen, and detecting the content of the corresponding signal molecules by adopting an HPLC (high performance liquid chromatography) or a report strain method, wherein the following method can be specifically adopted:
AHLs were measured by HPLC: the ethyl acetate containing the signal molecule was rotary evaporated at 35 ℃ at 40r/min and the solvent was evaporated. Acetonitrile filtered through a 0.22um filter membrane and 2ml of water (1:1, v/v) dissolve the signal molecule, and the signal molecule is stored at-80 ℃ for later use and then detected by HPLC.
HPLC detection conditions are Waters X bridgeTMThe column temperature of the C18 column (5um D, 4.6 x 250mm) was 35 ℃, the mobile phase was acetonitrile (A) -water (D) (A; D ═ 50:50, v/v), the flow rate was 0.7mL/min, and the detection wavelength was 210 nm.
AI-2 can be obtained from the colony reporter Vibrio harveyi BB170(luxN:: Tn5: sensor 1)-，sensor1-) The assay was performed (TAGA M E, XAVIER K B. methods for Analysis of bacterial Autoinducer-2 Production [ J ]].Current Protocols in Microbiology，2005:1C.1.1-1C.1.15.)
6) Quantitatively detecting, and storing at-20 deg.C.
(3) Mixing the signal molecules extracted from each bacterial liquid according to the following mass ratio (namely the mass percentage of each component after mixing is as follows):
the method comprises the following specific steps: the amount of each signal molecule to be added is calculated separately from the total amount of preparation to be injected, and the mixture containing the corresponding amount of signal molecule is mixed to produce the desired preparation. (4) The accelerated methane oxidation preparation is added to the waste by means of deep injection: the addition is carried out by using a steel needle injector, the injection position is the top of the garbage, and the using amount is 500mg per ton of the garbage.
(5) The injection is carried out once every 3 days for 10-15 times, and the methane content of the refuse landfill is reduced by about 55%.
Example 5
(1) First, a bacterium secreting a signal molecule is cultured using a casein medium: NaCl 1g/L, MgSO40.2g/L、KH2PO40.5g/L、Na2HPO41.5g/L、NH4NO31g/L, 10g/L, pH 7.2.2 casein, Pseudomonas aeruginosa, Pseudomonas putida, Pseudomonas fluorescens, Ralstonia solanacearum and Escherichia coli were cultured in the respective media to logarithmic phase.
(2) Respectively extracting signal molecules in each bacterial liquid, extracting by using ethyl acetate as a solvent, acidifying the ethyl acetate by using glacial acetic acid before extraction, and specifically comprising the following steps:
1) 3ml of the cell suspension was taken into a 15ml glass centrifuge tube, 3ml of ethyl acetate containing 1 ‰ glacial acetic acid was added, and vortexed for 30s (10 times shaking vigorously).
2) Standing for 10min to separate two phases, if not layering 10000rpm 3min to separate two phases, and if layering is not obvious, adding 3ml ethyl acetate containing 1 ‰ glacial acetic acid to layer.
3) The organic layer was pipetted into a clean 10ml vial of penicillin using a pipette gun.
4) Repeat steps 1-3 (final vial total 9 ml).
5) The final sample was nitrogen purged to full dryness.
6) Storing at-20 deg.C.
(3) Mixing the signal molecules extracted from each bacterial liquid according to the following mass ratio (namely the mass percentage of each component after mixing is as follows):
(4) the accelerated methane oxidation preparation is added to the waste by means of deep injection: the addition is carried out by using a steel needle injector, the injection position is the bottom of the garbage, and the using amount is 500mg per ton of the garbage.
(5) The injection is carried out once every 3 days for 10-15 times, and the methane content of the refuse landfill is reduced by about 65%.
Example 6
(1) First, a bacterium secreting a signal molecule is cultured using a casein medium: NaCl 1g/L, MgSO40.2g/L、KH2PO40.5g/L、Na2HPO41.5g/L、NH4NO31g/L, 10g/L, pH 7.2.2 casein, Pseudomonas aeruginosa, Pseudomonas putida, Pseudomonas fluorescens, Ralstonia solanacearum and Escherichia coli were cultured in the respective media to logarithmic phase.
(2) Respectively extracting signal molecules in each bacterial liquid, extracting by using ethyl acetate as a solvent, acidifying the ethyl acetate by using glacial acetic acid before extraction, and specifically comprising the following steps:
1) 3ml of the cell suspension was taken into a 15ml glass centrifuge tube, 3ml of ethyl acetate containing 1 ‰ glacial acetic acid was added, and vortexed for 30s (10 times shaking vigorously).
2) Standing for 10min to separate two phases, if not layering 10000rpm 3min to separate two phases, and if layering is not obvious, adding 3ml ethyl acetate containing 1 ‰ glacial acetic acid to layer.
3) The organic layer was pipetted into a clean 10ml vial of penicillin using a pipette gun.
4) Repeat steps 1-3 (final vial total 9 ml).
5) The final sample was nitrogen purged to full dryness.
6) Storing at-20 deg.C.
(3) Mixing the signal molecules extracted from each bacterial liquid according to the following mass ratio (namely the mass percentage of each component after mixing is as follows):
(4) the accelerated methane oxidation preparation is added to the waste by means of deep injection: the garbage is added by a steel needle injector, the injection position is the middle part of the garbage, and the using amount is 500mg per ton of the garbage.
(5) The injection is carried out once every 3 days for 10-15 times, and the methane content of the refuse landfill is reduced by about 70%.
Example 7
(1) First, a bacterium secreting a signal molecule is cultured using a casein medium: NaCl 1g/L, MgSO40.2g/L、KH2PO40.5g/L、Na2HPO41.5g/L、NH4NO31g/L, 10g/L, pH 7.2.2 casein, Pseudomonas aeruginosa, Pseudomonas putida, Pseudomonas fluorescens, Ralstonia solanacearum and Escherichia coli were cultured in the respective media to logarithmic phase.
(2) Respectively extracting signal molecules in each bacterial liquid, extracting by using ethyl acetate as a solvent, acidifying the ethyl acetate by using glacial acetic acid before extraction, and specifically comprising the following steps:
1) 3ml of the cell suspension was taken into a 15ml glass centrifuge tube, 3ml of ethyl acetate containing 1 ‰ glacial acetic acid was added, and vortexed for 30s (10 times shaking vigorously).
2) Standing for 10min to separate two phases, if not layering 10000rpm 3min to separate two phases, and if layering is not obvious, adding 3ml ethyl acetate containing 1 ‰ glacial acetic acid to layer.
3) The organic layer was pipetted into a clean 10ml vial of penicillin using a pipette gun.
4) Repeat steps 1-3 (final vial total 9 ml).
5) The final sample was nitrogen purged to full dryness.
6) Storing at-20 deg.C.
(3) Mixing the signal molecules extracted from each bacterial liquid according to the following mass ratio (namely the mass percentage of each component after mixing is as follows):
(4) the accelerated methane oxidation preparation is added to the waste by means of deep injection: the feeding is carried out by a steel needle injector, the injection position is the middle part of the garbage, and the using amount is 700mg per ton of the garbage.
(5) The injection is carried out once every 3 days for 10-15 times, and the methane content of the refuse landfill is reduced by about 80%.
Example 8
(1) First, a bacterium secreting a signal molecule is cultured using a casein medium: NaCl 1g/L, MgSO40.2g/L、KH2PO40.5g/L、Na2HPO41.5g/L、NH4NO31g/L, 10g/L, pH 7.2.2 casein, Pseudomonas aeruginosa, Pseudomonas putida, Pseudomonas fluorescens, Ralstonia solanacearum and Escherichia coli were cultured in the respective media to logarithmic phase.
(2) Respectively extracting signal molecules in each bacterial liquid, extracting by using ethyl acetate as a solvent, acidifying the ethyl acetate by using glacial acetic acid before extraction, and specifically comprising the following steps:
1) 3ml of the cell suspension was taken into a 15ml glass centrifuge tube, 3ml of ethyl acetate containing 1 ‰ glacial acetic acid was added, and vortexed for 30s (10 times shaking vigorously).
2) Standing for 10min to separate two phases, if not layering 10000rpm 3min to separate two phases, and if layering is not obvious, adding 3ml ethyl acetate containing 1 ‰ glacial acetic acid to layer.
3) The organic layer was pipetted into a clean 10ml vial of penicillin using a pipette gun.
4) Repeat steps 1-3 (final vial total 9 ml).
5) The final sample was nitrogen purged to full dryness.
6) Storing at-20 deg.C.
(3) Mixing the signal molecules extracted from each bacterial liquid according to the following mass ratio (namely the mass percentage of each component after mixing is as follows):
(4) the accelerated methane oxidation preparation is added to the waste by means of deep injection: the feeding is carried out by a steel needle injector, the injection position is the middle part of the garbage, and the using amount is 700mg per ton of the garbage.
(5) The injection is carried out once every 3 days for 10-15 times, and the methane content of the refuse landfill is reduced by about 80%.
Example 9
(1) First, a bacterium secreting a signal molecule is cultured using a casein medium: NaCl 1g/L, MgSO40.2g/L、KH2PO40.5g/L、Na2HPO41.5g/L、NH4NO31g/L, 10g/L, pH 7.2.2 casein, Pseudomonas aeruginosa, Pseudomonas putida, Pseudomonas fluorescens, Ralstonia solanacearum and Escherichia coli were cultured in the respective media to logarithmic phase.
(2) Respectively extracting signal molecules in each bacterial liquid, extracting by using ethyl acetate as a solvent, acidifying the ethyl acetate by using glacial acetic acid before extraction, and specifically comprising the following steps:
1) 3ml of the cell suspension was taken into a 15ml glass centrifuge tube, 3ml of ethyl acetate containing 1 ‰ glacial acetic acid was added, and vortexed for 30s (10 times shaking vigorously).
2) Standing for 10min to separate two phases, if not layering 10000rpm 3min to separate two phases, and if layering is not obvious, adding 3ml ethyl acetate containing 1 ‰ glacial acetic acid to layer.
3) The organic layer was pipetted into a clean 10ml vial of penicillin using a pipette gun.
4) Repeat steps 1-3 (final vial total 9 ml).
5) The final sample was nitrogen purged to full dryness.
6) Storing at-20 deg.C.
(3) Mixing the signal molecules extracted from each bacterial liquid according to the following mass ratio (namely the mass percentage of each component after mixing is as follows):
(4) the accelerated methane oxidation preparation is added to the waste by means of deep injection: the feeding is carried out by a steel needle injector, the injection position is the middle part of the garbage, and the using amount is 700mg per ton of the garbage.
(5) The injection is carried out once every 3 days for 10-15 times, and the methane content of the refuse landfill is reduced by about 80%.
Example 10
(1) First, a bacterium secreting a signal molecule is cultured using a casein medium: NaCl 1g/L, MgSO40.2g/L、KH2PO40.5g/L、Na2HPO41.5g/L、NH4NO31g/L, 10g/L, pH 7.2.2 casein, Pseudomonas aeruginosa, Pseudomonas putida, Pseudomonas fluorescens, Ralstonia solanacearum and Escherichia coli were cultured in the respective media to logarithmic phase.
(2) Respectively extracting signal molecules in each bacterial liquid, extracting by using ethyl acetate as a solvent, acidifying the ethyl acetate by using glacial acetic acid before extraction, and specifically comprising the following steps:
1) 3ml of the cell suspension was taken into a 15ml glass centrifuge tube, 3ml of ethyl acetate containing 1 ‰ glacial acetic acid was added, and vortexed for 30s (10 times shaking vigorously).
2) Standing for 10min to separate two phases, if not layering 10000rpm 3min to separate two phases, and if layering is not obvious, adding 3ml ethyl acetate containing 1 ‰ glacial acetic acid to layer.
3) The organic layer was pipetted into a clean 10ml vial of penicillin using a pipette gun.
4) Repeat steps 1-3 (final vial total 9 ml).
5) The final sample was nitrogen purged to full dryness.
6) Storing at-20 deg.C.
(3) Mixing the signal molecules extracted from each bacterial liquid according to the following mass ratio (namely the mass percentage of each component after mixing is as follows):
(4) the accelerated methane oxidation preparation is added to the waste by means of deep injection: the feeding is carried out by a steel needle injector, the injection position is the middle part of the garbage, and the using amount is 700mg per ton of the garbage.
(5) The injection is carried out once every 3 days for 10-15 times, and the methane content of the refuse landfill is reduced by about 80%.
Example 11
(1) First, a bacterium secreting a signal molecule is cultured using a casein medium: NaCl 1g/L, MgSO40.2g/L、KH2PO40.5g/L、Na2HPO41.5g/L、NH4NO31g/L, 10g/L, pH 7.2.2 casein, Pseudomonas aeruginosa, Pseudomonas putida, Pseudomonas fluorescens, Ralstonia solanacearum and Escherichia coli were cultured in the respective media to logarithmic phase.
(2) Respectively extracting signal molecules in each bacterial liquid, extracting by using ethyl acetate as a solvent, acidifying the ethyl acetate by using glacial acetic acid before extraction, and specifically comprising the following steps:
1) 3ml of the cell suspension was taken into a 15ml glass centrifuge tube, 3ml of ethyl acetate containing 1 ‰ glacial acetic acid was added, and vortexed for 30s (10 times shaking vigorously).
2) Standing for 10min to separate two phases, if not layering 10000rpm 3min to separate two phases, and if layering is not obvious, adding 3ml ethyl acetate containing 1 ‰ glacial acetic acid to layer.
3) The organic layer was pipetted into a clean 10ml vial of penicillin using a pipette gun.
4) Repeat steps 1-3 (final vial total 9 ml).
5) The final sample was nitrogen purged to full dryness.
6) Storing at-20 deg.C.
(3) Mixing the signal molecules extracted from each bacterial liquid according to the following mass ratio (namely the mass percentage of each component after mixing is as follows):
(4) the accelerated methane oxidation preparation is added to the waste by means of deep injection: the feeding is carried out by a steel needle injector, the injection position is the middle part of the garbage, and the using amount is 700mg per ton of the garbage.
(5) The injection is carried out once every 3 days for 10-15 times, and the methane content of the refuse landfill is reduced by about 80%.
Example 12
(1) First, a bacterium secreting a signal molecule is cultured using a casein medium: NaCl 1g/L, MgSO40.2g/L、KH2PO40.5g/L、Na2HPO41.5g/L、NH4NO31g/L, 10g/L, pH 7.2.2 casein, Pseudomonas aeruginosa, Pseudomonas putida, Pseudomonas fluorescens, Ralstonia solanacearum and Escherichia coli were cultured in the respective media to logarithmic phase.
(2) Respectively extracting signal molecules in each bacterial liquid, extracting by using ethyl acetate as a solvent, acidifying the ethyl acetate by using glacial acetic acid before extraction, and specifically comprising the following steps:
1) 3ml of the cell suspension was taken into a 15ml glass centrifuge tube, 3ml of ethyl acetate containing 1 ‰ glacial acetic acid was added, and vortexed for 30s (10 times shaking vigorously).
2) Standing for 10min to separate two phases, if not layering 10000rpm 3min to separate two phases, and if layering is not obvious, adding 3ml ethyl acetate containing 1 ‰ glacial acetic acid to layer.
3) The organic layer was pipetted into a clean 10ml vial of penicillin using a pipette gun.
4) Repeat steps 1-3 (final vial total 9 ml).
5) The final sample was nitrogen purged to full dryness.
6) Storing at-20 deg.C.
(3) Mixing the signal molecules extracted from each bacterial liquid according to the following mass ratio (namely the mass percentage of each component after mixing is as follows):
(4) the accelerated methane oxidation preparation is added to the waste by means of deep injection: the feeding is carried out by a steel needle injector, the injection position is the middle part of the garbage, and the using amount is 700mg per ton of the garbage.
(5) The injection is carried out once every 3 days for 10-15 times, and the methane content of the refuse landfill is reduced by about 85%.
The above description is only an embodiment of the present invention, but the technical features of the present invention are not limited thereto, and any person skilled in the relevant art can change or modify the present invention within the scope of the present invention.
Claims (6)
1. The preparation for accelerating the methane oxidation of the refuse landfill is characterized by comprising signal molecules AHLs and AI-2, and comprises the following components in percentage by mass:
the sum of the mass percentages of the components is 100 percent.
2. The process for preparing a methane oxidation accelerating formulation for a landfill as claimed in claim 1, comprising the steps of:
respectively culturing different bacteria secreting corresponding signal molecules to logarithmic phase, respectively extracting the signal molecules in the corresponding culture solution from the obtained culture solution, and then mixing the signal molecules according to the mass percentage in claim 1.
3. The method according to claim 2, wherein the culture medium for culturing the bacteria is LB medium or casein medium.
4. The method according to claim 2, wherein the solvent used for extraction is ethyl acetate, methanol or acetonitrile.
5. The method according to claim 4, wherein the solvent is acidified before extraction, and the acidifying agent is glacial acetic acid, hydrochloric acid or formic acid.
6. A method for accelerating methane oxidation of a refuse landfill is characterized by comprising the following steps: injecting the accelerated landfill methane oxidation preparation as claimed in claim 1 into landfill layers at different heights; the dosage of the methane oxidation accelerating preparation for the refuse landfill is 100-1000 mg per ton of refuse; the methane oxidation accelerating preparation for the refuse landfill is injected once every 2-4 days for 10-15 times.
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