CN102071161B - Chryseobacterium sp. capable of using methane and application thereof - Google Patents
Chryseobacterium sp. capable of using methane and application thereof Download PDFInfo
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- CN102071161B CN102071161B CN2010102601141A CN201010260114A CN102071161B CN 102071161 B CN102071161 B CN 102071161B CN 2010102601141 A CN2010102601141 A CN 2010102601141A CN 201010260114 A CN201010260114 A CN 201010260114A CN 102071161 B CN102071161 B CN 102071161B
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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
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/20—Air quality improvement or preservation, e.g. vehicle emission control or emission reduction by using catalytic converters
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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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- 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
- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P20/00—Technologies relating to chemical industry
- Y02P20/50—Improvements relating to the production of bulk chemicals
- Y02P20/59—Biological synthesis; Biological purification
Abstract
The invention discloses chryseobacterium sp. JT03 capable of using methane. The preservation number of a strain is CCTCC No.M2010100. The chryseobacterium sp. can be used for preparing a methane biological absorbent, a garbage treating agent and a biological catalyst. The chryseobacterium sp. overcomes the application defects of low bacteria density of methane-oxidizing bacteria, difficulty in amplification culture of bacteria and the like, and has high affinity to methane. The chryseobacterium sp. is particularly suitable for methane artificial sources such as household garbage landfill sites, paddy fields and the like, and can be widely popularized.
Description
Technical field
The invention belongs to the Environmental Biotechnology field, relate to a kind of golden yellow bacillus and application of methane capable of using specifically.
Background technology
Methane-oxidizing bacteria be a kind of special, be the methylotrophy type mikrobe of the sole carbon source and the energy with methane, widely dispersed finds that all methane-oxidizing bacteria exists under acid, alkali, salt, high temperature, low temperature, a lot of environment such as barren.Metabolic process and meta-bolites that it is special have great effect to global environment, have caused widely to pay close attention to.Methane-oxidizing bacteria not only plays an important role in global methane consumption, and in carbon, nitrogen, oxygen cycle, is also bringing into play important effect.
Methane-oxidizing bacteria is a kind of Gram-negative bacteria, strict oligotrophic type.Since 1906 isolate methane-oxidizing bacteria first; So far the methane-oxidizing bacteria that has been found that has 8 genus, comprises that mainly methylomonas (Methylomonas), methyl bacterium belong to (Methylobacter), methyloccccus (Methylococcus), methyl sporangiocyst Pseudomonas (Methylocytis), methyl Campylobacter (Methylosinus), methyl germ genus (Methylomicrobium), Methylocaldum and Methylospera.According to pathways metabolism, membrane structure, the main lipoid fatty acid serial characteristic that becomes to grade, can methane-oxidizing bacteria be divided into I type, II type and X type three major types type.Methane-oxidizing bacteria has two to the methane oxidation approach, and one is through by the approach of oxoethanoic acid to Serine, and another is that formaldehyde is combined in the approach that generates 6-phosphoric acid psicose on the ribose phosphoric acid.These bacterium can utilize any approach to assimilate net reaction:
CH
4→CH
3OH→HCHO→HCOOH→CO
2
The characteristic enzyme of methane-oxidizing bacteria be catalysis the first step reaction methane monooxygenase (methanemonooxygenase, MMO), the MMO highly stable c h bond that can rupture, in a Sauerstoffatom insertion C-H in the molecular oxygen, another Sauerstoffatom then generates water.It has two kinds of different types: particulate state or film combine methane monooxygenase (particulatemethane monooxygenase, pMMO) with the solubility methane monooxygenase (soluble methane monooxygenase, sMMO).
Methane-oxidizing bacteria research excessive velocities has caused at present that the appellation to this bacterioid exists disunity and ambiguity in the research report both at home and abroad.With the bibliographical information is foundation; Methane-oxidizing bacteria introduced all-sidely the earliest can trace back to 1958; Leadbetter has summed up the achievement in research since 1906 find methane-oxidizing bacteria first; Why article mentions methane-utilizing bacteria rather than methane-oxidizing bacteria, mainly is because the limitation of study condition and the research degree of depth and range at that time, can not provide the common trait of this type bacterium.In the outstanding bacterium handbook of uncle the 8th edition (1984), this type bacterium has been belonged to Methylomonadaceae, and defined this type bacterium and only utilize single carbon organic cpds as carbon source, belong to Gram-negative bacteria.1996, Hanson unified the appellation of methane-oxidizing bacteria, and promptly Methane-oxidation bacteria and Methane-utilizingbacteria can be described as methanotrophs.
In of the description of the outstanding bacterium handbook of uncle, found the information that another is important, " many bacteriums are to utilize many carbon cpds and single carbon cpd, rather than specially rely on methane or methyl alcohol as the carbon source and the energy " about Methylomonadaceae.Distinguish mutually with the methane-oxidizing bacteria that on the traditional sense only with methane is the sole carbon source and the energy, this bacterioid is referred to as amphitrophy methane-oxidizing bacteria (facultative methanotrophs).Classical biological study less relevance in medium optimization is carbon source with methane, at present, more existing bibliographical informations non-methane-oxidizing bacteria can consume methane.Wolf has just found to utilize the yeast of methane as far back as 1979, have many methane to utilize the separated and purifying of yeast at present, comprising Pichiapastoris, and Hansensulapolymorpha, Candida spp., and Trichsporon spp..
The amphitrophy methane-oxidizing bacteria can utilize other carbon source, therefore more is prone to realize enlarged culturing.Domestic refuse has been rich in abundant mikrobe in very long degradation process; Early-stage Study shows that mineralized waste has good degraded methane ability; From household refuse landfill sites, extract the amphitrophy methane-oxidizing bacteria and be expected to obtaining new breakthrough aspect the through engineering approaches of artificial source methane emission reductions such as household refuse landfill sites, and new platform is provided for the practical applications of methane oxidation aspect greenhouse gases.
Summary of the invention
The present invention is directed to the deficiency that the biological methane of methane in the prior art can't the through engineering approaches aspect exists; A kind of golden yellow bacillus JT03 and purposes of methane capable of using are provided; Said golden yellow bacillus JT03 can utilize economical carbon sources such as starch to grow fast; Realize methane oxidation efficiently on this basis, the through engineering approaches of strengthening landfill yard methane emission reduction for methane biologic oxidation provides very important biological basis.。
Technical scheme of the present invention is:
The golden yellow bacillus of methane capable of using, its preserving number are CCTCC No.M2010100, and its 16S rDNA sequence is shown in SEQ ID NO:1.
The colony diameter 0.8-1.2mm of the golden yellow bacillus of methane capable of using, circular protrusions or flat, yellow or white or orange, transparent glossy; Thalline is shaft-like or oval, does not move, aerobic, chmosynthetic heterotrophs bacterium; Wide 0.4～0.6 μ m of cell, long 1.0～1.3 μ m; Gram-negative.
The golden yellow bacillus of methane capable of using according to the invention can be used for preparing the agent of methane bio-absorbable, refuse treatment or preparation biological catalyst.
Bacterial strain of the present invention is called golden yellow bacillus (Chryseobacterium sp.) JT03, and (preserving number is CCTCCNo.M2010100; Depositary institution is Chinese typical culture collection center-Wuhan University preservation center; Preservation date is on April 25th, 2010), separation obtains from household refuse landfill sites.This bacterium can efficiently utilize methane, has high using value.The order-checking of bacterial strain DNA entrusts the precious biotech firm in Dalian to accomplish; The 16S rDNA part base sequence length of golden yellow bacillus JT03 is 664bp (SEQ ID NO:1), and base sequence compares the homology found with 3 bacterial strains of golden yellow Bacillaceae (Chryseobacterium sp.) more than 99% in GenBank nucleic acid sequence data storehouse.A series of Physiology and biochemistries and process optimization test through this bacterial strain is carried out show; The golden yellow bacillus of methane capable of using provided by the invention (Chryseobacterium sp.) JT03CCTCC NO.M2010100; Can effectively solve the reduction of discharging of artificial source methane, principal feature is following:
(1) to have overcome single-minded nutrition methane-oxidizing bacteria cell density low in the discovery of golden yellow bacillus (Chryseobacterium sp.) JT03, and thalline is difficult to enlarged culturing etc. and uses a difficult problem.
(2) with the intact cell of golden yellow bacillus (Chryseobacterium sp.) JT03 as biological catalyst, break away from that still can to keep higher methane oxidation behind the environmental system active; Be applicable to the high density methane oxidation, concentration is that the methane of 40% (v/v) can be degraded fully in the serum bottle.
(3) after this bacterium breaks away from environmental system, after cultivating 84h, can recover active, compare, shorten about 24h time of lag with the methane-oxidizing bacteria of having reported.This bacterium disengaging environmental resistance is strong, is fit to the methane oxidation under the landfill later stage oligotrophy environment, and the liquid culture concentration of this thalline is higher than the most methane-oxidizing bacterias with report, and methane is had higher affinity.
(4) the required substratum of the enlarged culturing of golden yellow bacillus (Chryseobacterium sp.) JT03 is simple, and cost is low.Be specially adapted to methane artificial sources such as household refuse landfill sites, rice terrace, can extensively promote.
Golden yellow bacillus according to the invention (Chryseobacterium sp.) JT03 separates from household refuse landfill sites to obtain; After breaking away from the refuse landfill environment, still keep higher methane oxidation activity; Can be applicable to the reduction of discharging of artificial source methane such as household refuse landfill sites, rice terrace, have broad application prospects in the reduction of greenhouse gas discharge field; Golden yellow bacillus according to the invention (Chryseobacterium sp.) JT03 also can cultivate on the NMS substratum, and many carbon cpds capable of using are the carbon source fast breeding, is easy to extension and cultivates; Said golden yellow bacillus still has the ability of efficient degradation methane after utilizing many carbon cpd propagation.
Below in conjunction with accompanying drawing and embodiment the present invention is done further explanation, but embodiment of the present invention is not limited thereto.
Description of drawings
The electron microscope photo scanning of the golden yellow bacillus Chryseobacterium of Fig. 1 sp.JT03 bacterial strain;
The golden yellow bacillus JT03 of Fig. 2 cuts glue and reclaims the segmental DNA electrophoresis of purpose, and wherein M:DL 2,000DNAMarker; The 1:JT03PCR product; +: over against photograph;-: negative contrast;
The golden yellow bacillus Chryseobacterium of Fig. 3 sp.JT03 bacterial strain is the growth curve of carbon source with methane;
The Chryseobacterium sp.JT03 bacterial strain that the different carbon sources of Fig. 4 are cultivated is to methane degradation effect figure;
Fig. 5 is the Chryseobacterium sp.JT03 bacterial strain metabolic process carbon dioxide production design sketch that different carbon sources are cultivated.
Embodiment
1. experiment material
The composition of NMS substratum is (g/L) as follows: KH
2PO
41.0gL
-1, Na
2HPO
412H
2O 2.9gL
-1, MgSO
47H
2O 0.32gL
-1, (NH
4)
2SO
43.0gL
-1, trace element solution 10mL, zero(ppm) water 990mL, pH 6.8.Trace element solution is formed (mg/L): ZnSO as follows
47H
2O 0.287; MnSO
47H
2O0.223; H
3BO
30.062; Na
2M
OO
42H
2O 0.048; COCl
26H
2O 0.048; KI 0.083; CaCl
22H
2O 3.5.
Granulated garbage is selected from the household refuse landfill sites mineralized waste; Present embodiment is chosen Shanghai Lao Gang refuse landfill (the old Gangdong of the Shanghai City Nanhui District portion) rubbish in 10 years of landfill; Get 4mm and sieve granulated garbage on following and the 2mm sieve, the rejuvenation of airtight domestication 2 all bacterial strains in methane and air Mixture earlier.
2. bacterial classification enrichment and optimization experiment
1) enrichment culture: take by weighing mineralized waste 1g and put into 100ml NMS substratum, place 30 ℃, 160rpm shaking table vibration 2h.Get bacteria suspension 2ml and inserted packing in the 100ml serum bottle of 20ml NMS substratum, add a cover rubber stopper seal as seed; With a 20ml air in the 20ml methane gas displacement bottle, one week of shaking culture under 30 ℃, 160rpm condition then.
2) pure bacterium separates: with chilled sterile distilled water bacterium liquid is carried out 10 times of serial dilutions, process the diluent that extent of dilution is 10-1,10-2.Adopt tilt-pour process to carry out the NMS culture medium culturing.Flat board is inverted in the vacuum drier, and in moisture eliminator, feeds a certain amount of methane, seal with preservative film then.Moisture eliminator is placed biochemical incubator, cultivated 4～5 days for 30 ℃.The bacterial classification that growing way is good repeatedly goes down to posterity purifying.
3) optimization experiment: utilize pure bacterial strain to prepare certain density bacteria suspension, join in the serum bottle that a certain amount of NMS substratum is housed; With air in the methane displacement bottle, the plug of having afterwards sealing.Shaking culture under 30 ℃, 160rpm condition.Certain interval of time detects bacterial concentration and biogas concentration.Along with the consumption of methane, need the nitrogen of regular replenishment certain volume, to eliminate negative pressure in the bottle.
The experiment of 3 strain identification
Use TaKaRa 16S rDNA Bacterial Identification PCR Kit (Code No.D310) to carry out pcr amplification purpose fragment.Get 5 μ l and carry out 3% agarose gel electrophoresis, use TaKaRaAgarose GelDNAPurification Kit Ver.2.0 (Code No.DV805A) to cut glue recovery purpose fragment and carry out dna sequencing.The order-checking of DNA entrusts the precious biotech firm in Dalian to accomplish.With Seq Forward, Seq Reverse, SeqInternal is that primer carries out dna sequencing.
4 detection methods
The OD value of bacterium liquid adopts the UV2000 spectrophotometer to detect, and wavelength is 560nm.Viable cell concentrations adopts the method for plate culture count to confirm.Dry cell weight is dried to constant weight under 80 ℃ through the bacterium liquid of certain volume, uses the precise electronic balance weighing.Each tests the minimum 2 groups of parallel tests of doing, and guarantees that RSD is less than 5%.
Gc (Agilent 6890N) is adopted in the detection of methane.(10m * 2mm), injector temperature, column temperature and detector (TCD) temperature is respectively 80,50,120 ℃ to the stainless steel column of chromatographic condition: GDX, and hydrogen is carrier gas, and flow velocity is 25ml/min, sample size 0.5ml.
Purifying and the evaluation of embodiment 1. golden yellow bacillus Chryseobacterium sp.JT03
This bacterial strain belongs to Gram-negative, bacterium colony circular protrusions or flat, yellow, white or orange, and the edge is smooth, and is transparent glossy, about colony diameter 1mm; Thalline is shaft-like or oval, do not move aerobic, chmosynthetic heterotrophs bacterium.Wide about 0.4～0.6 μ m of cell is about 1.0～1.3 μ m.Stereoscan photograph is seen Fig. 1.
PCR product to bacterial strain JT03 carries out 3% agarose gel electrophoresis (Fig. 2), can see that the gel band of PCR product is very clear.The order-checking of bacterial strain DNA entrusts the precious biotech firm in Dalian to accomplish; The length that the 16S rDNA base of golden yellow bacillus JT03 is measured is 664bp, and base sequence compares the homology found with 3 bacterial strains of golden yellow Bacillaceae (Chryseobacterium sp.) more than 99% in GenBank nucleic acid sequence data storehouse.
The physicochemical property of table 1Chryseobacterium sp.JT03
| Physiological and biochemical index | Characteristic | Physiological and biochemical index | Characteristic |
| PD | + | Gelatin hydrolysis | + |
| Katalase | + | Trehalose utilizes | + |
| β 2 tilactases | + | Citrate trianion utilizes | - |
| Lysine decarboxylase | - | Acetate utilizes | - |
| Ornithine decarboxylase | - | Nitrate salt | - |
| Amylolytic enzyme | + | Nitrite | - |
Annotate :+the positive ,-feminine gender
Growth curve and the physicochemical property of embodiment 2. bacterial strain Chryseobacterium sp.JT03
Can know that by Fig. 3 be 84h the lag period of golden yellow bacillus JT03 growth, compare with the methane-oxidizing bacteria of having reported; Shortened about 24h time of lag; (84～156h) bacterium liquid are obviously muddy, and OD value has appreciated about 0.65 from 0.05, and this cell concentration is higher than the most methane-oxidizing bacterias to report for logarithmic phase.
Embodiment 3. bacterial strain Chryseobacterium sp.JT03 utilize many carbon cpds proliferation experiment
Subsequent use with 7 kinds of sugar solns such as NMS substratum configuration 3g/L glucose, fructose, seminose, lactose, sucrose, starch and raffinoses; Getting above-mentioned 7 kinds of NMS substratum 20mL adds as in the 100mL serum bottle.Other gets 1 serum bottle adding 20mL NMS substratum but does not add carbon source as contrast.With the high-temperature steam sterilization under 121 ℃ of conditions of above-mentioned substratum, in bottle, add 1mL kind daughter bacteria liquid with micropipet after cooling, build plug and shake up, put into shaking table and be set under 30 ℃, 160rpm condition and cultivate 4d.Measure the OD of bacterium liquid then
560nmValue is confirmed the thalli growth situation, and other extracts 1.2mL left and right sides bacterium liquid high speed centrifugation, gets supernatant then and detects the consumption situation that sugared concentration is confirmed carbon source with the sulfuric acid anthrone method.The result sees table 2.All carbon sources all can be utilized by golden yellow bacillus JT03, and what consume when wherein glucose is for carbon source is the fastest, and golden yellow bacillus JT03 propagation was very fast when this explanation was carbon source with glucose.7 kinds of carbon sources all have promoter action to thalli growth, and the golden yellow bacillus JT03 bacterium liquid of 7 flask culture is carried out the carbon source of methane consumption experiment in the hope of the methane that obtains to degrade.
Table 2 is that carbon source is cultivated the thalline absorbancy of golden yellow bacillus JT03 and the variation of sugared concentration with the different sugar
Embodiment carbon cpd more than 4. propagation strain golden bacillus JT03 utilizes the methane experiment
Take the back bacterium liquid 1mL of the different many carbon cpds propagation among the embodiment 3, be diluted to 20mL with the NMS nutritive medium of no carbon source, cultivation is 22 days under 30 ℃, 160rpm condition, investigates the effect that it utilizes methane.Can know that by Fig. 4 the golden yellow bacillus JT03 that 7 kinds of carbon sources are cultivated equal effective oxidizing of methylene after through the adjustment in about 1 week wherein is the best results of carbon source with starch, at the about 42mL of 22d internal consumption methane.Can be known that by Fig. 5 carbonic acid gas generates volume and is directly proportional basically with the consumption of methane, the carbonic acid gas of every generation 1mol consumes methane 2mol, and remaining methane conversion is for thalline.
Can know that to sum up golden yellow bacillus JT03 not only can utilize economical carbon sources such as starch to grow fast, and can realize methane oxidation efficiently on this basis.This through engineering approaches of strengthening landfill yard methane emission reduction for methane biologic oxidation provides very important biological basis.
At last, it is also to be noted that what more than enumerate only is several specific embodiment of the present invention.Obviously, the invention is not restricted to above embodiment, many distortion can also be arranged.All distortion that those of ordinary skill in the art can directly derive or associate from content disclosed by the invention all should be thought protection scope of the present invention.
Claims (4)
1. the golden yellow bacillus of a methane capable of using is characterized in that, golden yellow bacillus (
Chryseobacterium sp.) JT03, its preserving number is CCTCC No.M2010100.
2. the application of the golden yellow bacillus of the described methane capable of using of claim 1 in the agent of preparation methane bio-absorbable.
3. the application of golden yellow bacillus in refuse treatment of the described methane capable of using of claim 1.
4. the application of the golden yellow bacillus of the described methane capable of using of claim 1 in the preparation biological catalyst.
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| CN116814511B (en) * | 2023-08-31 | 2023-12-12 | 广东省科学院生态环境与土壤研究所 | Methane-oxidizing bacteria with high nitrite tolerance and reducing capability and application thereof |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6458573B1 (en) * | 1993-09-23 | 2002-10-01 | New England Biolabs, Inc. | Isolation and composition of a novel glycosidase from chryseobacterium |
| CN101230326A (en) * | 2008-01-07 | 2008-07-30 | 哈尔滨工业大学 | Biological deferrization demanganization functional bacterium |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6458573B1 (en) * | 1993-09-23 | 2002-10-01 | New England Biolabs, Inc. | Isolation and composition of a novel glycosidase from chryseobacterium |
| CN101230326A (en) * | 2008-01-07 | 2008-07-30 | 哈尔滨工业大学 | Biological deferrization demanganization functional bacterium |
Non-Patent Citations (1)
| Title |
|---|
| 吴鏖.临床标本中分离出4株产吲哚金黄杆菌.《江西医学检验》.2000,(第2期),126. * |
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