CN117089502B - Immobilized methane-oxidizing bacteria and immobilization method and application thereof - Google Patents
Immobilized methane-oxidizing bacteria and immobilization method and application thereof Download PDFInfo
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- CN117089502B CN117089502B CN202311314744.6A CN202311314744A CN117089502B CN 117089502 B CN117089502 B CN 117089502B CN 202311314744 A CN202311314744 A CN 202311314744A CN 117089502 B CN117089502 B CN 117089502B
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- 241000894006 Bacteria Species 0.000 title claims abstract description 116
- 238000000034 method Methods 0.000 title claims abstract description 25
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- 229920001817 Agar Polymers 0.000 claims abstract description 13
- 239000008272 agar Substances 0.000 claims abstract description 13
- 230000003100 immobilizing effect Effects 0.000 claims abstract description 5
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- 239000000243 solution Substances 0.000 claims description 20
- 230000001580 bacterial effect Effects 0.000 claims description 19
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- OXBLHERUFWYNTN-UHFFFAOYSA-M copper(I) chloride Chemical compound [Cu]Cl OXBLHERUFWYNTN-UHFFFAOYSA-M 0.000 claims description 7
- 229910001657 ferrierite group Inorganic materials 0.000 claims description 7
- 244000005700 microbiome Species 0.000 claims description 7
- 239000008055 phosphate buffer solution Substances 0.000 claims description 7
- 125000002496 methyl group Chemical group [H]C([H])([H])* 0.000 claims description 6
- 241000589342 Methylomonas sp. Species 0.000 claims description 3
- 230000003647 oxidation Effects 0.000 abstract description 33
- 238000007254 oxidation reaction Methods 0.000 abstract description 33
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- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 15
- 239000002002 slurry Substances 0.000 description 11
- 229930182555 Penicillin Natural products 0.000 description 9
- JGSARLDLIJGVTE-MBNYWOFBSA-N Penicillin G Chemical compound N([C@H]1[C@H]2SC([C@@H](N2C1=O)C(O)=O)(C)C)C(=O)CC1=CC=CC=C1 JGSARLDLIJGVTE-MBNYWOFBSA-N 0.000 description 9
- XAGFODPZIPBFFR-UHFFFAOYSA-N aluminium Chemical compound [Al] XAGFODPZIPBFFR-UHFFFAOYSA-N 0.000 description 9
- 229910052782 aluminium Inorganic materials 0.000 description 9
- 229920005549 butyl rubber Polymers 0.000 description 9
- IJGRMHOSHXDMSA-UHFFFAOYSA-N nitrogen Substances N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 9
- 229940049954 penicillin Drugs 0.000 description 9
- 239000008367 deionised water Substances 0.000 description 8
- 239000000306 component Substances 0.000 description 7
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- 241000589344 Methylomonas Species 0.000 description 6
- 229910052757 nitrogen Inorganic materials 0.000 description 6
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- 238000002360 preparation method Methods 0.000 description 5
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- 230000010718 Oxidation Activity Effects 0.000 description 4
- 239000012141 concentrate Substances 0.000 description 4
- 238000012258 culturing Methods 0.000 description 4
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 3
- NLOAOXIUYAGBGO-UHFFFAOYSA-N C.[O] Chemical compound C.[O] NLOAOXIUYAGBGO-UHFFFAOYSA-N 0.000 description 3
- 229910002651 NO3 Inorganic materials 0.000 description 3
- NHNBFGGVMKEFGY-UHFFFAOYSA-N Nitrate Chemical compound [O-][N+]([O-])=O NHNBFGGVMKEFGY-UHFFFAOYSA-N 0.000 description 3
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 3
- 238000004519 manufacturing process Methods 0.000 description 3
- QJGQUHMNIGDVPM-UHFFFAOYSA-N nitrogen group Chemical group [N] QJGQUHMNIGDVPM-UHFFFAOYSA-N 0.000 description 3
- 239000001301 oxygen Substances 0.000 description 3
- 229910052760 oxygen Inorganic materials 0.000 description 3
- 239000002904 solvent Substances 0.000 description 3
- 239000008223 sterile water Substances 0.000 description 3
- 239000006228 supernatant Substances 0.000 description 3
- 229910000859 α-Fe Inorganic materials 0.000 description 3
- 108020004465 16S ribosomal RNA Proteins 0.000 description 2
- 206010021143 Hypoxia Diseases 0.000 description 2
- 238000006243 chemical reaction Methods 0.000 description 2
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- 238000004817 gas chromatography Methods 0.000 description 2
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- 239000002068 microbial inoculum Substances 0.000 description 2
- 239000011259 mixed solution Substances 0.000 description 2
- 239000006911 nms medium Substances 0.000 description 2
- WZJVQUUBEVDURL-UHFFFAOYSA-N pentanedial;phosphoric acid Chemical compound OP(O)(O)=O.O=CCCCC=O WZJVQUUBEVDURL-UHFFFAOYSA-N 0.000 description 2
- 230000009467 reduction Effects 0.000 description 2
- 238000012163 sequencing technique Methods 0.000 description 2
- LPXPTNMVRIOKMN-UHFFFAOYSA-M sodium nitrite Chemical compound [Na+].[O-]N=O LPXPTNMVRIOKMN-UHFFFAOYSA-M 0.000 description 2
- 238000005303 weighing Methods 0.000 description 2
- BHPQYMZQTOCNFJ-UHFFFAOYSA-N Calcium cation Chemical compound [Ca+2] BHPQYMZQTOCNFJ-UHFFFAOYSA-N 0.000 description 1
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 1
- UFHFLCQGNIYNRP-UHFFFAOYSA-N Hydrogen Chemical compound [H][H] UFHFLCQGNIYNRP-UHFFFAOYSA-N 0.000 description 1
- JLVVSXFLKOJNIY-UHFFFAOYSA-N Magnesium ion Chemical compound [Mg+2] JLVVSXFLKOJNIY-UHFFFAOYSA-N 0.000 description 1
- 238000012408 PCR amplification Methods 0.000 description 1
- DKGAVHZHDRPRBM-UHFFFAOYSA-N Tert-Butanol Chemical compound CC(C)(C)O DKGAVHZHDRPRBM-UHFFFAOYSA-N 0.000 description 1
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- 230000004151 fermentation Effects 0.000 description 1
- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical compound [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 description 1
- 239000010931 gold Substances 0.000 description 1
- 229910052737 gold Inorganic materials 0.000 description 1
- 239000008187 granular material Substances 0.000 description 1
- 239000001257 hydrogen Substances 0.000 description 1
- 229910052739 hydrogen Inorganic materials 0.000 description 1
- 230000003301 hydrolyzing effect Effects 0.000 description 1
- 230000006872 improvement Effects 0.000 description 1
- 238000011534 incubation Methods 0.000 description 1
- 229910017053 inorganic salt Inorganic materials 0.000 description 1
- 230000006799 invasive growth in response to glucose limitation Effects 0.000 description 1
- 238000004255 ion exchange chromatography Methods 0.000 description 1
- 238000009630 liquid culture Methods 0.000 description 1
- 230000007774 longterm Effects 0.000 description 1
- 239000011777 magnesium Substances 0.000 description 1
- 229910001425 magnesium ion Inorganic materials 0.000 description 1
- 238000011177 media preparation Methods 0.000 description 1
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Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
-
- C—CHEMISTRY; METALLURGY
- C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F11/00—Treatment of sludge; Devices therefor
- C02F11/02—Biological treatment
-
- C—CHEMISTRY; METALLURGY
- C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F3/00—Biological treatment of water, waste water, or sewage
- C02F3/34—Biological treatment of water, waste water, or sewage characterised by the microorganisms used
-
- C—CHEMISTRY; METALLURGY
- C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F2101/00—Nature of the contaminant
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
- C12R2001/26—Methylomonas
Abstract
The invention discloses an immobilized methane-oxidizing bacterium, an immobilization method and application thereof. The immobilized methane-oxidizing bacteria are obtained by immobilizing the methane-oxidizing bacteria in agar containing nutrient solution. The method has the advantages of low cost, simple operation and the like, and is easy to realize large-scale industrial application. The immobilized methane-oxidizing bacteria obtained by the method has strong stress resistance, can be suitable for treating methane in different sludge, has good methane oxidation effect and is easy to store.
Description
Technical Field
The invention belongs to the field of treatment of water, wastewater, sewage or sludge, and particularly relates to an immobilized methane-oxidizing bacterium, an immobilization method and application thereof.
Background
With the improvement of the popularity of sewage treatment, the sewage treatment plant in China basically realizes full coverage, and the sewage treatment rate is as high as 93.4 percent. Anaerobic biological treatment is an important link of sewage treatment, and the anaerobic biological treatment process can be divided into 4 stages of hydrolytic fermentation, hydrogen production, acetic acid production, homoacetogenesis and methane production. Thus, a large amount of activated sludge containing methane gas is produced after the anaerobic biological treatment is completed.
Methane-oxidizing bacteria are ubiquitous in nature, and can utilize methane as the only carbon source and energy source, thereby playing an important role of a methane filter. In recent years, the microbial inoculum prepared from methane-oxidizing bacteria liquid also has multiple applications in the aspects of treating landfill leachate methane gas, preventing and controlling coal mine local gas and guaranteeing the safety of natural gas pipelines. However, due to the influence of microbial activity, the microbial inoculum has the obvious defects of unstable effect, incapability of recycling and the like.
Disclosure of Invention
The primary aim of the invention is to overcome the defects and shortcomings of the prior art and provide a methane-oxidizing bacteria immobilization method.
Another object of the present invention is to provide an immobilized methane-oxidizing bacterium obtained by the above immobilization method.
It is still another object of the present invention to provide the use of the above-mentioned immobilization method or the above-mentioned immobilized methane-oxidizing bacteria.
The aim of the invention is achieved by the following technical scheme: an immobilization method of methane-oxidizing bacteria, comprising the following steps:
(1) Uniformly mixing a solid culture medium in a liquid state with methane-oxidizing bacteria to obtain a bacterial suspension;
(2) And (3) solidifying the bacterial suspension obtained in the step (1) to obtain the immobilized methane-oxidizing bacteria.
The solid medium in the liquid state in the step (1) means a solid medium in the liquid state at a temperature of not higher than 40 ℃.
The solid medium described in step (1) refers to a nutrient solution containing agar, preferably a nutrient solution containing agar and ferrihydrite.
The concentration of the agar in the solid culture medium is 15-20 g/L.
The concentration of the ferrierite in the solid culture medium is 0.1-2 mM.
The nutrient solution is a culture medium which is favorable for survival or reproduction of methane-oxidizing bacteria, generally contains nitrate, trace elements, magnesium ions and calcium ions, and has the pH value of 6.5-7.2.
The group of solid culture mediaThe following are preferred: 0.1-0.3 g/L MgSO 4 ·7H 2 O、0.10～0.18 g CaCl 2 ·6H 2 O、0.5～1.5 g KNO 3 45 to 55 percent mL phosphate buffer solution, 1.5 to 2.5 percent mL microelement solution, 0 to 2 percent mM ferrihydrite and 15 to 20 percent g/L agar, and the pH value is 6.8 to 7.2; more preferably as follows: 0.1-0.3 g/L MgSO 4 ·7H 2 O、0.10～0.18 g CaCl 2 ·6H 2 O、0.5～1.5 g KNO 3 45-55 mL phosphate buffer solution, 1.5-2.5 mL microelement solution, 0.1-2 mM ferrihydrite and 15-20 g/L agar, and the pH value is 6.8-7.2; most preferably as follows: 0.2 g/L MgSO 4 ·7H 2 O、0.14 g CaCl 2 ·6H 2 O、1.0 g KNO 3 50 mL phosphate buffer solution, 2.0 mL microelement liquid, 0.1-2 mM ferrihydrite and 15-20 g/L agar, and the pH value is 6.8-7.0;
the composition of the phosphate buffer was as follows: 5.44 g/L KH 2 PO 4 And 5.68 g/L Na 2 HPO 4 ；
The trace element liquid comprises the following components: 1.0 g/L Na 2 -EDTA、2.0 g/L FeSO 4 •7H 2 O、0.8 g/L ZnSO 4 •7H 2 O、0.03 g/L MnCl 2 •4H 2 O、0.03 g/L H 3 BO 3 、0.2 g/L CoCl 2 •6H 2 O、0.6 g/L CuCl 2 •2H 2 O、0.02 g/L NiCl 2 •6H 2 O and 0.05 g/L Na 2 MoO 4 •2H 2 O。
The culture medium, the phosphate buffer solution and the trace element liquid are water as solvents; preferably deionized or distilled water.
The methane-oxidizing bacteria in the step (1) include, but are not limited to, methane-oxidizing bacteriaMethylomonassp.) HYX-M1 and methane-oxidizing bacteriaMethylomonassp. YDR-M2 and methane-oxidizing bacteriaMethylomonas sp.）LW 13。
The methane oxidizing bacteria are [ ]Methylomonassp.) HYX-M1, accession number GDMCC No:63477 and deposited on day 18 of 2023 under 5 of 5 th edition on Guangdong province of the university of 100 th Hirship, 59 th floor, 5 th floor, guangdong province of the institute of microorganisms, inc. of the university of Guangdong provinceThe collection center.
The methane oxidizing bacteria are [ ]Methylomonassp.) YDR-M2 deposited 24 months 2021 at 30 th Hirship No. 100 college, no. 59 building No. 5, guangdong province institute of microorganisms and species, under accession number GDMCC No. 61952. The strain is disclosed in a national invention patent CN 202111291004.6-a methane oxidizing bacterium with denitrification function and hypoxia stress resistance and application thereof.
When the methane-oxidizing bacteria are methane-oxidizing bacteria HYX-M1, the concentration of the ferrierite in the solid culture medium is preferably 0.1-2 mM.
When the methane-oxidizing bacteria are the methane-oxidizing bacteria YDR-M2, the concentration of the ferrierite in the solid culture medium is preferably 0.1. 0.1 mM.
When the methane-oxidizing bacteria are methane-oxidizing bacteria LW13, the concentration of the ferrierite in the solid culture medium is preferably 2 mM.
An immobilized methane-oxidizing bacterium obtained by the above immobilization method.
The immobilized method or the immobilized methane-oxidizing bacteria are applied to methane treatment.
The above application preferably comprises the steps of: the immobilized methane-oxidizing bacteria or the immobilized methane-oxidizing bacteria obtained by the immobilization method are placed in an environment containing methane.
The methane gas treatment is to remove methane by using methane oxidizing bacteria.
The environment includes sludge, water or gas.
Compared with the prior art, the invention has the following advantages and effects:
1) The immobilized methane-oxidizing bacteria particles have the advantages of simple culture components, low cost, easy operation of the immobilization process, good methane oxidation effect, one-time use and long-term effectiveness.
2) The immobilized methane-oxidizing bacteria particles can further improve the methane oxidation effect after the ferrihydrite is added, the raw materials are low in price, and the immobilization process is easy to operate.
3) The immobilized methane-oxidizing bacteria particles have strong stress resistance, can be suitable for treating methane in different sludge, have good methane oxidation effect, and are produced into products which are easy to store and transport.
4) According to the invention, the methane concentration in the sludge is reduced by immobilizing methane-oxidizing bacteria particles, so that potential safety hazards such as fire hazards caused by methane combustion are reduced, and the occurrence of the fire hazards is effectively controlled.
Drawings
FIG. 1 is a graph showing the effect of methane-oxidizing bacteria HYX-M1 on methane oxidation in different states (left graph) and a graph showing the statistical result of the total methane oxidation amount in different states of methane-oxidizing bacteria HYX-M1 (right graph); wherein P <0.05, P <0.01, P <0.001.
FIG. 2 is a graph showing the methane-oxidizing effect of immobilized particles of methane-oxidizing bacteria HYX-M1.
FIG. 3 is a graph showing the statistical result of the total methane oxidation amount of immobilized particles of different methane-oxidizing bacteria; wherein the values on the bar graph represent the ratio of the increase in methane oxidation for the different treatments compared to the control group; p <0.05, P <0.01, P <0.001.
FIG. 4 is a photograph showing the cell morphology of methane-oxidizing bacteria.
FIG. 5 is a graph of nitrite versus Methylomonas at various concentrationsMethylomonassp, effect result graph of methane oxidation; where a is the result of a nitrate concentration of 4 mM and b is the result of a nitrite concentration of 6 mM.
FIG. 6 is Methylomonas spMethylomonassp. a graph of the detection results of the utilization of nitrite; where a is the result of nitrite concentration of 4 mM and b is the result of nitrite concentration of 6 mM.
FIG. 7 is Methylomonas spMethylomonassp. graph of growth under different nitrite conditions.
Detailed Description
The present invention will be described in further detail with reference to examples and drawings, but embodiments of the present invention are not limited thereto.
Example 1
1) Preparation of slurry:
100g of the soil of the Zhujiang river mouth wetland is weighed by an electronic balance, placed into a beaker, 100mL of sterile water is added for uniform mixing, 15 mL slurry is split charging into 125mL penicillin bottles, the butyl rubber plug is sealed, and the bottle is sealed by an aluminum cover.
2) Preparation of nutrient medium:
0.2 g MgSO was weighed separately by an electronic balance 4 ·7H 2 O、0.14 g CaCl 2 ·6H 2 O、1.0 g KNO 3 50 mL phosphate buffer (5.44 g/L KH) was then added by syringe 2 PO 4 、5.68 g/L Na 2 HPO 4 ) And 2 mL trace element liquid (1.0 g/L Na) 2 -EDTA、2.0 g/L FeSO 4 ·7H 2 O、0.8 g/L ZnSO 4 ·7H 2 O、0.03 g/L MnCl 2 ·4H 2 O、0.03 g/L H 3 BO 3 、0.2 g/L CoCl 2 ·6H 2 O、0.6 g/L CuCl 2 ·2H 2 O、0.02 g/L NiCl 2 ·6H 2 O and 0.05 g/L Na 2 MoO 4 ·2H 2 O) adding the mixture into deionized water, uniformly mixing, then fixing the volume to 1L, and adding sodium hydroxide to adjust the pH value to 6.8 to obtain the liquid nutrient medium. Subpackaging a part of liquid nutrient medium into 125mL penicillin bottles, sealing with butyl rubber plug, sealing with aluminum cap, sterilizing at 121deg.C for 20 min, and culturing methane-oxidizing bacteria; and adding 100mL nutrient medium into a conical flask, adding 2g of agar, sealing with a sealing film, and sterilizing at 121deg.C for 20 min to obtain solid nutrient medium.
3) Culture of methane-oxidizing bacteria
And (3) performing enrichment culture on the methyl monad HYX-M1 by using an NMS culture solution, and performing stationary culture under the condition of a darkroom at 30 ℃ until the concentration of methane-oxidizing bacteria is 0.05-0.06 mg/mL, thereby obtaining a methane-oxidizing bacteria solution in a logarithmic phase.
The NMS culture solution comprises the following components: 0.2 g MgSO 4 •7H 2 O、0.14 g CaCl 2 ·6H 2 O, 50. 50 mL phosphate buffer (5.44 g/L KH) 2 PO 4 、5.68 g/L Na 2 HPO 4 ) And 2 mL trace element liquid (solute: 1.0 g/L Na 2 -EDTA、2.0 g/L FeSO 4 •7H 2 O、0.8 g/L ZnSO 4 •7H 2 O、0.03 g/L MnCl 2 •4H 2 O、0.03 g/L H 3 BO 3 、0.2 g/L CoCl 2 •6H 2 O、0.6 g/L CuCl 2 •2H 2 O、0.02 g/L NiCl 2 •6H 2 O and 0.05 g/L Na 2 MoO 4 •2H 2 O, deionized water as solvent), adding into deionized water, mixing, fixing volume to 1L, adding sodium hydroxide, and adjusting pH to 6.8 to obtain NMS culture medium. Subpackaging 25mL culture medium into 125mL penicillin bottles, sealing with butyl rubber plug, sealing with aluminum cap, and sterilizing at 121deg.C for 20 min.
4) Methane-oxidizing bacteria immobilization
Taking 100mL step 3) to culture methane-oxidizing bacteria liquid in logarithmic phase, centrifuging at 6000 r/min for 5 min, fully suspending in a liquid nutrient medium again, centrifuging again, repeating for 2 times, then re-suspending methane-oxidizing bacteria in 100mL solid nutrient medium (the solid nutrient medium is pre-melted, and methane-oxidizing bacteria is added when cooling to 40 ℃), and finally introducing 100mL uniformly mixed bacterial suspension into an immobilization mould to prepare 100 immobilized particles of 1cm multiplied by 1 cm.
5) Methane-oxidizing bacteria immobilized particle activity test
In an ultra-clean workbench, 5 methane-oxidizing bacteria HYX-M1 immobilized particles are taken as an experimental group to be inoculated into the 15 mL slurry to be used as an experimental group, and a control group is arranged at the same time, wherein only the 15 mL slurry is used as one control group, and the step 3) of adding 5mL into the 15 mL slurry is used as another control group, and the culture is carried out until the methane-oxidizing bacteria liquid in the logarithmic phase is used as another control group, and each treatment is repeated for 3 times. 15 mL methane was added for the first time in 15% (v/v) (methane volume/headspace volume) per group; after each time the methane was substantially completely consumed, the flask was replaced with nitrogen, 20 was then evacuated mL, and replaced with 20 mL oxygen-methane mixed gas (oxygen: methane: 3:1 by volume), and the flask was stationary cultured at 30℃in a dark room for 40 days, during which time the methane concentration was measured by means of a high performance gas chromatograph (Agilent 7820A, USA) to determine methane oxidation activity (see FIG. 1). Subtracting the methane amount at the end of the cycle from the initial amount of methane initially added per cycle to obtain the methane oxidation amount per cycle; and adding the methane oxidation amount of each cycle to obtain the final total methane oxidation amount.
As can be seen from fig. 1, the immobilized methane-oxidizing bacteria HYX-M1 particles have similar methane oxidation effects with the methane-oxidizing bacteria liquid in the first three additional experiments, and still maintain a higher methane degradation rate after the fourth additional methane, so that the methane oxidation effect is obviously better than that of the two groups of controls; the immobilized methane-oxidizing bacteria co-oxidize methane by 2.19 mmol and methane-oxidizing bacteria liquid co-oxidize methane by 1.28 mmol in the whole period, and compared with the immobilized methane-oxidizing bacteria, the immobilized methane-oxidizing bacteria improves the total methane oxidation by 71.09%. The immobilized methane-oxidizing bacteria can well play a role in stably and efficiently reducing the methane concentration for a long time, and the culture medium components are easy to obtain, the use amount is small, and the cost is low. Therefore, the immobilized methane-oxidizing bacteria HYX-M1 particles are considered to have the characteristic of efficiently and stably reducing the methane concentration for a long time, and the purpose of the invention can be achieved.
Example 2
1) Preparation of slurry:
weighing 100g by electronic balance, placing the soil from the Zhujiang river mouth wetland into a beaker, adding 100mL sterile water, mixing, subpackaging 15 mL slurry into 125mL penicillin bottles, sealing by a butyl rubber plug, and sealing by an aluminum cover.
2) And (3) detecting the activity of methane-oxidizing bacteria immobilized particles:
in an ultra clean bench, 5 immobilized particles of methane-oxidizing bacteria HYX-M1 prepared according to example 1, which were stored in a methane-free atmosphere at ambient temperature for 30 days in a dark place, were inoculated into the 15 mL slurry, and each treatment was repeated 3 times. 15 mL methane was added for the first time in 15% (v/v) (methane volume/headspace volume) per group; after each time the methane was substantially completely consumed, the flask was replaced with nitrogen, 20 was then evacuated mL, and replaced with 20 mL oxygen-methane mixed gas (3:1 oxygen: methane) and the flask was incubated at 30℃in a dark room for 15 days, during which time the methane concentration was measured by means of an efficient gas chromatograph (Agilent 7820A, USA) to determine methane oxidation activity.
As can be seen from fig. 2, the immobilized methane-oxidizing bacteria particles still have good methane oxidation effect in four periods of the experiment after being stored for 30 days in a methane-free atmosphere at normal temperature and in a dark place. And the immobilized strain has good stability, strong controllability, strong stress resistance, easy storage and easy recovery, and can be widely applied.
Example 3
1) Preparation of slurry:
weighing 100g soil by an electronic balance, placing into a beaker, adding 100mL sterile water, mixing uniformly, taking 15 mL slurry, subpackaging into 125mL penicillin bottles, sealing by a butyl rubber plug, and sealing by an aluminum cover.
2) Preparation of nutrient medium:
0.2 g MgSO was weighed separately by an electronic balance 4 ·7H 2 O、0.14 g CaCl 2 ·6H 2 O、1.0 g KNO 3 50 mL phosphate buffer (5.44 g/L KH) was then added by syringe 2 PO 4 、5.68 g/L Na 2 HPO 4 ) And 2 mL trace element liquid (1.0 g/L Na) 2 -EDTA、2.0 g/L FeSO 4 ·7H 2 O、0.8 g/L ZnSO 4 ·7H 2 O、0.03 g/L MnCl 2 ·4H 2 O、0.03 g/L H 3 BO 3 、0.2 g/L CoCl 2 ·6H 2 O、0.6 g/L CuCl 2 ·2H 2 O、0.02 g/L NiCl 2 ·6H 2 O and 0.05 g/L Na 2 MoO 4 ·2H 2 O) adding the mixture into deionized water, uniformly mixing, then fixing the volume to 1L, and adding NaOH to adjust the pH value to 6.8 to obtain the liquid nutrient medium. Subpackaging a part of liquid nutrient medium into 125mL penicillin bottles, sealing with butyl rubber plug, sealing with aluminum cap, sterilizing at 121deg.C for 20 min, and culturing methane-oxidizing bacteria; and adding 100mL liquid nutrient medium into a conical flask, adding 2g of agar, sealing with a sealing film, and sterilizing at 121deg.C for 20 min to obtain solid nutrient medium.
3) Culture of methane-oxidizing bacteria
Methane-oxidizing bacteriaMethylomonas sp, YDR-M2, deposited 24.9.2021 on floor 5, guangdong province of the scientific microbiological institute of Guangdong, mitsui 100, guangzhou City, first of allThe collection of microorganism strains is GDMCC No. 61952. The strain is disclosed in a national invention patent CN 202111291004.6-a methane oxidizing bacterium with denitrification function and hypoxia stress resistance and application thereof.
Methane-oxidizing bacteriaMethylomonas sp, LW13, number DSM 24493, purchased from German collection of microorganisms and cell cultures, website https:// www.dsmz.de.
Methane-oxidizing bacteriaMethylomonassp. HYX-M1, accession number GDMCC No:63477, 5 months 18 of 2023, was deposited with the Guangdong province microorganism strain collection center located at Guangzhou Kogyo No. 100, no. 59 building 5, guangdong university of Kogyo Proc.
The three strains were cultured in accordance with step 3) of example 1 to obtain a methane-oxidizing bacteria YDR-M2 bacterial solution, a methane-oxidizing bacteria LW13 bacterial solution and a methane-oxidizing bacteria HYX-M1 bacterial solution, which were cultured to the logarithmic phase.
4) Three methane-oxidizing bacteria are respectively immobilized
A. And respectively taking 150 mL to culture methane-oxidizing bacteria YDR-M2 bacterial liquid, methane-oxidizing bacteria LW13 bacterial liquid and methane-oxidizing bacteria HYX-M1 bacterial liquid in logarithmic phase, centrifuging at 6000 r/min for 5 min, fully suspending in a culture medium again, centrifuging again, repeating for 2 times, and then respectively suspending the methane-oxidizing bacteria in 150 mL of solid nutrient medium at 40 ℃ for uniform mixing to obtain bacterial suspension.
B. The bacterial suspension obtained in step A of 50 mL was introduced into an immobilization mold to prepare 50 immobilized particles of 1 cm. Times.1 cm as a control group.
C. And C, uniformly mixing the bacterial suspension obtained in the step A with ferrihydrite so that the final concentration of ferrihydrite in the solution is 0.1 mM, uniformly introducing the 50 mL mixed solution into an immobilization mold, and preparing 50 immobilized particles with the size of 1cm multiplied by 1cm to obtain the experimental group 1 (immobilized+0.1 mM ferrihydrite).
D. And C, uniformly mixing the bacterial suspension obtained in the step A with ferrihydrite so that the final concentration of ferrihydrite in the solution is 2 mM, uniformly introducing the 50 mL mixed solution into an immobilization mould to prepare 50 immobilized particles with the size of 1cm multiplied by 1cm, and taking the immobilized particles as an experimental group 2 (immobilized + mM ferrihydrite).
4) Methane-oxidizing bacteria immobilized particle activity test
In an ultra-clean workbench, 5 methane-oxidizing bacteria are respectively takenMethylomonas sp, YDR-M2 immobilized particles and methane-oxidizing bacteriaMethylomonas sp, LW13 immobilized particles and methane-oxidizing bacteriaMethylomonas sp. HYX-M1 immobilized particles were inoculated as an experimental group into the above 15 mL slurry as a control group, respectively, while "immobilized +0.1 mM ferrihydrite" and "immobilized +2 mM ferrihydrite" were used as the experimental groups, and 3 replicates were set for each treatment. 15 mL methane was added for the first time in 15% (v/v) (methane volume/headspace volume) per group; after each time the methane was substantially completely consumed, the flask was replaced with nitrogen, 20 was then evacuated mL, and replaced with 20 mL oxygen-methane mixed gas (3:1 oxygen: methane) and the mixture was stationary cultured at 30℃in a darkroom for 24 days, during which time the methane concentration was measured by means of an efficient gas chromatograph (Agilent 7820A, USA) to determine methane oxidation activity.
As can be seen from fig. 3:
immobilized methane-oxidizing bacteria added with ferrihydrite with different concentrationsMethylomonas The sp, HYX-M1 granule methane oxidation effect is obviously stronger than that of the control group. The methane oxidation effect of the experimental group 'immobilized +0.1 mM ferrites' is improved, and compared with a control, the total methane oxidation amount in the experimental period is improved by 18.5%. The methane oxidation effect of the experimental group 'immobilized + mM ferrites' is improved, and compared with a control, the methane oxidation amount in the experimental period is improved by 22.56%. The addition of the ferrihydrite with different concentrations can promote the methane oxidation effect of the immobilized particles, promote the methane oxidation amount and achieve the effect of accelerating the methane oxidation.
Compared with the control group, the experimental group methane-oxidizing bacteriaMethylomonas The methane oxidation effect of sp, LW13 'immobilized + mM ferrites' is improved, and compared with a control, the total methane oxidation amount in the experimental period is improved by 15.15%. The addition of the high-concentration ferrihydrite (2 mM) can improve the oxidation amount of methane and achieve the effect of accelerating the oxidation of methane.
Compared with the control group, the experimental group methane-oxidizing bacteriaMethylomonas The sp, YDR-M2 'immobilization+0.1 mM ferrihydrite' methane oxidation effect is relatively goodThe irradiation group is obviously improved, and the total methane oxidation amount in the experimental period is improved by 24.05 percent. The addition of the low-concentration ferrihydrite (0.1 mM) can improve the oxidation amount of methane, and the effect of accelerating the oxidation of methane is achieved.
Example 4
1) Enrichment culture of methane-oxidizing bacteria
And (5) pre-incubating and culturing the soil of the Zhujiang delta wetland. 10 g fresh soil was taken in 125mL penicillin bottles and sealed with butyl rubber and aluminum caps. About 25% (v/v) methane (25 mL) was added as a substrate and cultured statically in a dark room at 30 ℃. During the period, 0.2 bottle of mL bottle of gas is taken by a gas injection needle, the concentration of methane is detected by utilizing high-efficiency gas chromatography (Agilent 7820A, USA), after the methane in the bottle is not consumed any more, 3 mL incubation soil is taken and inoculated into 25mL nitrate inorganic salt (NMS) culture medium, and the process is repeated three times.
NMS medium preparation process is as follows: 0.2 g MgSO was weighed separately by an electronic balance 4 ·7H 2 O、0.14 g CaCl 2 ·6H 2 O and 1.0 g KNO 3 50 mL phosphate buffer (5.44 g/L KH) was then added by syringe 2 PO 4 、5.68 g/L Na 2 HPO 4 ) And 2 mL trace element liquid (1.0 g Na/liter) 2 -EDTA、2.0 g FeSO 4 ·7H 2 O、0.8 g ZnSO 4 ·7H 2 O、0.03 g MnCl 2 ·4H 2 O、0.03 g H 3 BO 3 、0.2 g CoCl 2 ·6H 2 O、0.6 g CuCl 2 ·2H 2 O、0.02 g NiCl 2 ·6H 2 O and 0.05 g Na 2 MoO 4 ·2H 2 O) adding into deionized water, mixing, constant volume to 1L, adding sodium hydroxide to adjust pH to 7.0, taking 25mL culture medium, subpackaging into 125mL penicillin bottles, sealing with butyl rubber plug, sealing with aluminum cap, and sterilizing at 121deg.C for 20 min to obtain NMS culture medium.
2) And (3) separating and identifying methane-oxidizing bacteria:
methane-oxidizing bacteria were isolated using the Hungate roller tube technique, and the 0.1 mL methane-oxidizing bacteria concentrate was transferred to 5mL agar NMS medium containing 2% (w/v) in a 25mL anaerobic tube, and approximately 25% methane (5 mL) was added thereto, and the culture was stopped at 30 ℃ under dark conditions for 5 days. The single colony is picked up to 5mL liquid culture medium, the steps of rolling tube and picking up the single colony are repeated for 3 times, and the purified strain is obtained and named HYX-M1. During the process, 0.2 mL tube of gas is taken by a gas injection needle, and the methane concentration is detected by using high-performance gas chromatography (Agilent 7820A, USA) to determine the methane removal effect.
And taking the bacterial liquid as a DNA template for PCR amplification. PCR reaction system composition (50. Mu.L): 10×ExTaq buffer (containing Mg 2+ ) 5.0 [ mu ] L, dNTPs (2.5 mM) 12.5 [ mu ] L, primers 27F/1492R (10 [ mu ] M) 1.0 [ mu ] L respectively, a DNA template 1.0 [ mu ] L and sterile ultra-pure water 9.5 [ mu ] L. The PCR reaction conditions were: 94. pre-denaturing at the temperature for 2 min; 94. denaturation at 30℃ 30 s, annealing at 55℃30 s, extension at 72℃1 min,30 cycles; 72. finally, the temperature is increased for 10 min.
27F：5’-AGAGTTTGATCCTGGCTCAG-3’；
1492R：5’-GGYTACCTTGTTACGACTT-3’。
The 16S rDNA sequence obtained by sequencing is as follows:
TGCACCATGGCGGGATGCTTAACACATGCAAGTCGAACGCTGATAAGGTGCTTGCACCTTTGATGAGTGGCGGACGGGTGAGTAATGCATAGGAATCTGCCTATTAGTGGGGGATAACGTGGGGAAACTCACGCTAATACCGCATACGATCTACGGATGAAAGCAGGGGACCTTTTGGCCTTGCGCTAATAGATGAGCCTATGTCGGATTAGCTAGTTGGTAGGGTAAAGGCCTACCAAGGCGACGATCCGTAGCTGGTCTGAGAGGATGATCAGCCACACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTGGGGAATATTGGACAATGGGCGCAAGCCTGATCCAGCAATACCGCGTGTGTGAAGAAGGCCTGAGGGTTGTAAAGCACTTTCAATGGGAAGGAATACCTATCGGTTAATACCCGGTAGACTGACATTACCCATACAAGAAGCACCGGCTAACTCCGTGCCAGCAGCCGCGGTAATACGGAGGGTGCAAGCGTTAATCGGAATTACTGGGCGTAAAGCGTGCGTAGGCGGTTTTTTAAGTCAGATGTGAAAGCCCTGGGCTTAACCTGGGAACTGCATTTGATACTGGAAAACTAGAGTTGAGTAGAGGAGAGTGGAATTTCAGGTGTAGCGGTGAAATGCGTAGAGATCTGAAGGAACACCAGTGGCGAAGGCGGCTCTCTGGACTCAAACTGACGCTGAGGTACGAAAGCGTGGGTAGCAAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGATGTCAACTAACCGTTGGGCGCTTTAAGTGCTTAGTGGTGGAGCTAACGTATTAAGTTGACCGCCTGGGGAGTACGGCCGCAAGGCTAAAACTCAAATGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGATGCAACGCGAAGAACCTTACCTACCCTTGACATCCAGAGAATCTGTTAGAGATAGCGGAGTGCCTTCGGGAACTCTGAGACAGGTGCTGCATGGCTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGTAACGAGCGCAACCCTTATCCTTAGTTGCCAGCGGTTCGGCCGGGAACTCTAGGGAGACTGCCGGTGATAAACCGGAGGAAGGTGGGGACGACGTCAAGTCATCATGGCCCTTATGGGTAGGGCTACACACGTGCTACAATGGCCGGTACAGAGGGTTGCGATCTCGCGAGAGCAAGCTAATCCCAAAAAGCCGGTCTTAGTCCGGATTGCAGTCTGCAACTCGACTGCATGAAGTCGGAATCGCTAGTAATCGCAGATCAGAATGCTGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCATGGGAGTGGGTTGCAAAAGAAGTAGGTAGTTTAACCTTCGGGAGGGCGCTACCACTTTGATTCCGG。
3) Morphological observation of methane-oxidizing bacteria:
scanning electron microscope observation: taking 2 mL bacterial liquid, centrifuging at 5000 r/min for 3 min, and discarding the supernatant; adding 2 ml of 0.1 mol/L PBS buffer solution, centrifuging for 3 min at 5000 r/min after re-suspending, and repeatedly cleaning for 3 times; 1 mL of 2.5% glutaraldehyde phosphate buffer (pH 7.2) was added thereto, and the mixture was fixed at 4℃overnight; the next day, wash with 0.15% glutaraldehyde phosphate buffer; then sequentially carrying out gradient dehydration by 30%, 50%, 70%, 90% and absolute ethyl alcohol, and centrifuging at 5000 r/min for 3 min after 15 min of each dehydration; adding 2 mL tertiary butanol to replace ethanol, centrifuging at 5000 r/min for 3 min, removing most of supernatant, leaving a small amount of supernatant for resuspension of thallus, sucking 0.1 mL bacteria, dripping onto clean cover glass, and blow-drying on an ultra-clean workbench; after gold plating, the morphology of the cells was observed under a scanning electron microscope.
The bacterial colony morphology of the strain HYX-M1 on the anaerobic pipe wall is as shown in FIG. 4, is round with neat edges, and has a smoother bacterial colony surface and a pink opaque shape. Strain HYX-M1 was in the form of a short rod under scanning electron microscope.
4) And (3) separating and identifying methane-oxidizing bacteria:
comparing the sequencing result of the 16S rDNA of the screened strain with the Nucleotide BLAST sequence of NCBI database, and displaying the result that the strain is matched with the strainMethylomonas rhizoryzaeThe strain GJ1 has 99.86 percent sequence similarity, and is preliminarily judged to be methane oxidizing bacteria by combining physical and chemical properties and bacteria form characteristicsMethylomonas) Named as methyl monad [ ]Methylomonas sp.) HYX-M1, accession number GDMCC No:63477 it was deposited on day 18 of 2023 at 5 of 5 to 5 of 5 th China on a building 59 of 100 th Hirship in Guangzhou City, guangdong province of China institute of microorganismsThe collection of strains.
Example 5
1) Preparing a nutrient solution:
firstly, culturing methyl monad enrichment culture with NMS culture solution until the concentration of methane-oxidizing bacteria is 0.05-0.06 mg/mL, and obtaining methane-oxidizing bacteria seed solution.
The NMS culture solution comprises the following components: 0.2 g MgSO 4 •7H 2 O、0.14 g CaCl 2 ·6H 2 O, 50. 50 mL phosphate buffer (5.44 g/L KH) 2 PO 4 、5.68 g/L Na 2 HPO 4 ) And 2 mL trace element liquid (solute: 1.0 g/L Na 2 -EDTA、2.0 g/L FeSO 4 •7H 2 O、0.8 g/L ZnSO 4 •7H 2 O、0.03 g/L MnCl 2 •4H 2 O、0.03 g/L H 3 BO 3 、0.2 g/L CoCl 2 •6H 2 O、0.6 g/L CuCl 2 •2H 2 O、0.02 g/L NiCl 2 •6H 2 O and 0.05 g/L Na 2 MoO 4 •2H 2 O, deionized water as solvent), adding into deionized water, mixing, fixing volume to 1L, adding sodium hydroxide, and adjusting pH to 6.8 to obtain NMS culture medium. Subpackaging 25mL culture medium into 125mL penicillin bottles, sealing with butyl rubber plug, sealing with aluminum cap, and sterilizing at 121deg.C for 20 min.
2) And (3) detecting the activity of methane-oxidizing bacteria:
in an ultra-clean workbench, 1 mL methane-oxidizing bacteria are takenMethylomonas sp, HYX-M1 and 1 mL methane oxidizing bacteriaMethylomonas sp.LW13 was inoculated into the above 25mL medium, respectively, and nitrite concentrate (100 mM sodium nitrite) was added so that the final concentration of nitrite in the medium was 4 mM and 6mM, respectively, and 25mL methane was added per group in 25% (v/v) (methane volume/headspace volume) and 3 replicates were set per treatment, and 96 h was stationary cultured under dark conditions at 30℃during which methane concentration was measured by means of a high performance gas chromatograph (Agilent 7820A, USA) to determine methane oxidation activity.
3) And (3) detecting nitrite reduction of methane-oxidizing bacteria:
in an ultra clean bench, 1 mL methane-oxidizing bacteria HYX-M1 were inoculated into the above 25mL medium while adding nitrite concentrate to give final nitrite concentrations of 4 mM and 6mM, respectively, and 25mL methane was added in 25% (v/v) (methane volume/headspace volume) per group, 3 replicates were set per treatment, and 96 h was statically cultured at 30℃under dark conditions, during which the nitrite concentration was detected by ion chromatography (ICS-90, USA).
4) And (3) detecting the activity of methane-oxidizing bacteria:
in an ultra-clean workbench, 1 mL methane-oxidizing bacteria are takenMethylomonas sp, HYX-M1/methane-oxidizing bacteriaMethylomonas sp, LW13 and 1 mL nitrite concentrates were inoculated into the above 25mL medium to give final nitrite concentrations of 4 mM and 6mM, respectively, and 25mL methane was added in 25% (v/v) (methane volume/headspace volume) per group, 3 replicates were set for each treatment, and 96 h was statically cultured at 30℃under dark conditions, during which time the growth of the cells was examined using an ultraviolet spectrophotometer.
The results of the methane-oxidizing bacteria activity test are shown in FIG. 5. As can be seen from FIG. 5, under the conditions of 4 mM and 6mM nitrite as nitrogen source, with methane-oxidizing bacteriaMethylomonas In contrast to the sp, LW13,Methylomonas the sp, HYX-M1 strain can rapidly degrade methane, the methane oxidation amount of 96 h is 6.21 and 5.52 mM respectively, and the effect of reducing the methane concentration can be well achieved. And the culture medium components are easy to obtain, the use amount is small, and the cost is low. The strain has good stability, simple culture and strong controllability.
The results of the methane-oxidizing bacteria nitrite reduction test are shown in FIG. 6. As can be seen from FIG. 6, under the conditions of 4 mM and 6mM nitrite as nitrogen source, with methane-oxidizing bacteriaMethylomonas In contrast to the sp, LW13,Methylomonas the sp, HYX-M1 strain can rapidly consume nitrite, the nitrite removal amount of 96 h is 2.76 and 1.31 mM respectively, and the effect of reducing nitrite in water environment can be well achieved. And the culture medium components are easy to obtain, the use amount is small, and the cost is low. The strain has good stability, simple culture and strong controllability.
The results of the methane-oxidizing bacteria activity test are shown in FIG. 7. As can be seen from FIG. 7, the methane-oxidizing bacteria were cultured under the conditions of 4 mM and 6mM nitrite as nitrogen sourceMethylomonas The sp, LW13 hardly grows,Methylomonas sp, HYX-M1 strain grows faster, OD of 96 h thallus 600 Can reach about 0.5 and has better tolerance to nitrite.
The above examples are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above examples, and any other changes, modifications, substitutions, combinations, and simplifications that do not depart from the spirit and principle of the present invention should be made in the equivalent manner, and the embodiments are included in the protection scope of the present invention.
Claims (7)
1. The methane oxidizing bacteria immobilization method is characterized by comprising the following steps:
(1) Uniformly mixing a solid culture medium in a liquid state with methane-oxidizing bacteria to obtain a bacterial suspension;
(2) Solidifying the bacterial suspension obtained in the step (1) to obtain immobilized methane-oxidizing bacteria;
the composition of the solid culture medium is as follows: 0.1-0.3 g/L MgSO 4 ·7H 2 O、0.10～0.18 g/L CaCl 2 ·6H 2 O、0.5～1.5 g/L KNO 3 45-55 mL/L phosphate buffer solution, 1.5-2.5 mL/L microelement solution, 0-2 mM ferrihydrite and agar 15-20 g/L, and pH 6.8-7.2;
the composition of the phosphate buffer was as follows: 5.44 g/L KH 2 PO 4 And 5.68 g/L Na 2 HPO 4 ；
The trace element liquid comprises the following components: 1.0 g/L Na 2 -EDTA、2.0 g/L FeSO 4 •7H 2 O、0.8 g/L ZnSO 4 •7H 2 O、0.03 g/L MnCl 2 •4H 2 O、0.03 g/L H 3 BO 3 、0.2 g/L CoCl 2 •6H 2 O、0.6 g/L CuCl 2 •2H 2 O、0.02 g/L NiCl 2 •6H 2 O and 0.05 g/L Na 2 MoO 4 •2H 2 O；
The methane-oxidizing bacteria in the step (1) are methyl monad @, the methane-oxidizing bacteria are methyl monad @Methylomonassp.) HYX-M1 and methane-oxidizing bacteriaMethylomonas sp. YDR-M2 or methane-oxidizing bacteriaMethylomonas sp.）LW 13；
The methyl monad is [ ]Methylomonassp.) HYX-M1, accession number GDMCC No:63477, 5 months 18 of 2023, was deposited with the Guangdong province microorganism strain collection center of the university of Mitsui, 100 th, building 59, 5 th, guangdong university of academy of sciences of Guangdong province;
the methane oxidizing bacteria are [ ]Methylomonas sp.) YDR-M2 deposited 24 months at 2021 at 30 th edition 100 th university of Mitsui, guangdong province, building 5, guangdong province, china center for type culture collection, accession number GDMCC No. 61952;
the methane oxidizing bacteria are [ ]Methylomonas sp.) LW13, number DSM 24493, deposited in the German collection of microorganisms.
2. The method for immobilizing methane-oxidizing bacteria according to claim 1, characterized in that:
the composition of the solid culture medium is as follows: 0.1-0.3 g/L MgSO 4 ·7H 2 O、0.10～0.18 g/L CaCl 2 ·6H 2 O、0.5～1.5 g/L KNO 3 45-55 mL/L phosphate buffer solution, 1.5-2.5 mL/L microelement solution, 0.1-2 mM ferrihydrite and agar 15-20 g/L, and pH 6.8-7.2.
3. The method for immobilizing methane-oxidizing bacteria according to claim 2, characterized in that:
the composition of the solid culture medium is as follows: 0.2 g/L MgSO 4 ·7H 2 O、0.14 g/L CaCl 2 ·6H 2 O、1.0 g/L KNO 3 50 mL/L phosphate buffer solution, 2.0 mL/L microelement solution, 0.1-2 mM ferrihydrite and agar 15-20 g/L, and pH 6.8-7.0.
4. The method for immobilizing methane-oxidizing bacteria according to claim 1, characterized in that:
when the methane-oxidizing bacteria are methane-oxidizing bacteria HYX-M1, the concentration of the ferrierite in the solid culture medium is 0.1-2 mM;
when the methane-oxidizing bacteria are the methane-oxidizing bacteria YDR-M2, the concentration of the ferrierite in the solid culture medium is 0.1 and mM;
when the methane-oxidizing bacteria are methane-oxidizing bacteria LW13, the concentration of the ferrierite in the solid culture medium is 2 mM.
5. An immobilized methane-oxidizing bacterium, which is characterized in that: the immobilization method according to any one of claims 1 to 4.
6. The use of the immobilized methane-oxidizing bacteria of claim 5 in methane treatment.
7. The use according to claim 6, characterized in that: the immobilized methane-oxidizing bacteria of claim 5 are disposed in an environment comprising methane to remove methane.
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| CN105861353A (en) * | 2016-01-11 | 2016-08-17 | 吉林大学 | Methane-oxidizing bacteria separated from dairy cow feces and separation method thereof |
| CN107653209A (en) * | 2017-11-15 | 2018-02-02 | 中国环境科学研究院 | Phenol degrading microbial bacterial agent, immobilized spherule and preparation method |
| CN116790452A (en) * | 2023-08-24 | 2023-09-22 | 广东省科学院生态环境与土壤研究所 | Composite microbial agent capable of synchronously reducing emission of methane and nitrous oxide under anoxic condition and application |
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| CN105861353A (en) * | 2016-01-11 | 2016-08-17 | 吉林大学 | Methane-oxidizing bacteria separated from dairy cow feces and separation method thereof |
| CN107653209A (en) * | 2017-11-15 | 2018-02-02 | 中国环境科学研究院 | Phenol degrading microbial bacterial agent, immobilized spherule and preparation method |
| CN116790452A (en) * | 2023-08-24 | 2023-09-22 | 广东省科学院生态环境与土壤研究所 | Composite microbial agent capable of synchronously reducing emission of methane and nitrous oxide under anoxic condition and application |
Non-Patent Citations (1)
| Title |
|---|
| Efficient Immobilization of Whole Cells of Methylomonas sp. Strain GYJ3 by Sol–Gel Entrapment;Jianbo Chen等;《Journal of Molecular Catalysis B Enzymatic》;第30卷(第3期);第167-172页 * |
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