CN102586149B - Methyl bacterium capable of degrading dichloromethane - Google Patents
Methyl bacterium capable of degrading dichloromethane Download PDFInfo
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- CN102586149B CN102586149B CN 201210051551 CN201210051551A CN102586149B CN 102586149 B CN102586149 B CN 102586149B CN 201210051551 CN201210051551 CN 201210051551 CN 201210051551 A CN201210051551 A CN 201210051551A CN 102586149 B CN102586149 B CN 102586149B
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- dichloromethane
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Abstract
The invention provides a methyl bacterium capable of degrading dichloromethane, relating to the methyl bacterium which can degrade dichloromethane. The methyl bacterium provided by the invention can efficiently degrade the dichloromethane and can degrade the dichloromethane under aerobic condition, and important basis is provided for researching a biological degradation mechanism of halogenated hydrocarbon organic pollutant in which dichloromethane is taken as representative. The methyl bacterium capable of degrading dichloromethane is the methyl bacterium Met26 and is preserved in China General Microbiological Culture Collection Center in the data of January 4th, 2012 with the preservation number of CGMCC No.5693. The methyl bacterium Met26 provided by the invention can effectively degrade dichloromethane, after the methyl bacterium Met26 is cultured for ten days in a culture medium which takes dichloromethane as unique carbon source and has final concentration of dichloromethane of 30mmol/L, concentration of the dichloromethane is reduced by 63.5%, and the concentration of the dichloromethane after the methyl bacterium Met26 is cultured for fifteen days is reduced by 78.6%.
Description
Technical field
The present invention relates to the methyl bacterium of strain degraded methylene dichloride.
Background technology
Methylene dichloride, CH
2Cl
2, be colourless liquid, in pharmaceutical industry, do reaction medium, for the preparation of penbritin, carbenicillin and cephalosporin etc.; Solvent, grease-removing agent in also producing as film, gas smog propellant, polyurethane foams etc.Methylene dichloride accounts for 50% of aggregate consumption at the consumption that China is used for film production, and medical aspect accounts for 20% of aggregate consumption, and clean-out system and chemical industry consumption account for 20% of aggregate consumption, and other aspects account for 10%.
Methylene dichloride has anesthetic action, mainly damages nervus centralis and respiratory system.The main path of human contact is to suck.According to estimates, in the world wide production of methylene dichloride, about 80% is released in the atmosphere and goes, but because the speed of this compound photodissociation is very fast, makes it and can not accumulate in atmosphere.Its initial degraded product is phosgene and carbon monoxide, so methylene dichloride is considered to the bigger halogenated hydrocarbon material of topsoil toxic, and it is extensive use of and brings a series of problem of environmental pollutions, serious crisis human health, and damage the ozone layer.
Can comprise up to now that as the methyl bacillus (Methylobacterium sp.) of sole carbon source and the energy pseudomonas, the strain more than 10 of silk germ decompose in the bacterium of methylene dichloride with methylene dichloride since Rittman in 1980 etc. isolate from trade effluent first, (Methylobacter sp.) do not appear in the newspapers as the biological degradation of carbon source and the energy is domestic with methylene dichloride about the methyl bacterium.
Summary of the invention
The invention provides strain methyl bacterium (Methylobacter sp.) bacterial strain, but efficient degradation methylene dichloride, the methylene dichloride of can degrading under aerobic conditions is for being that the research of biological degradation mechanism of the halogenated hydrocarbon organic pollutant of representative provides important foundation with the methylene dichloride.
The degrade methyl bacterium of methylene dichloride of the present invention is methyl bacterium (Methylobacter sp.) Met26, be deposited in China Committee for Culture Collection of Microorganisms common micro-organisms center (CGMCC), the preservation address is No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, preservation date is on January 4th, 2012, and preserving number is CGMCC No.5693; Methyl bacterium Met26 of the present invention is Gram-negative bacteria, rod-short, and the cell size is (0.33 μ m～0.4 μ m) * (0.39 μ m～0.46 μ m), no gemma, atrichia; On the MNMS substratum, cultivate 10d for 28 ℃, colony diameter 0.5-1mm, circle, transparent, neat in edge, smooth surface.
Methyl bacterium Met26 of the present invention can grow at the MNMS substratum, and optimum growth temperature is 28 ℃, and the suitableeest growing environment pH is 6.8.
Methyl bacterium of the present invention (Methylobacter sp.) Met26 analyzes by 16S rDNA sequence alignment, and the sibship that belongs to (Methylobacter sp.) with the methyl bacterium is the most approaching, and the conserved regions similarity is 99%.By determining that in conjunction with morphological features, growth conditions methyl bacterium Met26 is the new bacterial strain of a strain of methyl bacterium genus (Methylobacter sp.), called after methyl bacterium Met26.
Methyl bacterium Met26 of the present invention can the efficient degradation methylene dichloride, methyl bacterium Met26 is being that sole carbon source, methylene dichloride final concentration are in the substratum of 30mmol/L with the methylene dichloride, concentration dichloromethane decline 78.6% behind concentration dichloromethane decline 63.5%, the 15d behind the cultivation 10d.
Methyl bacterium of the present invention (Methylobacter sp.) Met26, belong to the methyl bacterium and belong to (Methylobacter sp.), be deposited in China Committee for Culture Collection of Microorganisms common micro-organisms center (CGMCC), the preservation address is No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, preservation date is on January 4th, 2012, and preserving number is CGMCC No.5693.
Description of drawings
Fig. 1 carries out the phylogeny tree graph that the homology comparison makes up for the 16S rDNA sequence of methyl bacterium Met26 and close bacterial strain.
Embodiment
Technical solution of the present invention is not limited to following cited embodiment, also comprises the arbitrary combination between each embodiment.
Embodiment one: the methyl bacterium of present embodiment degraded methylene dichloride is methyl bacterium (Methylobacter sp.) Met26, be deposited in China Committee for Culture Collection of Microorganisms common micro-organisms center (CGMCC), the preservation address is No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, preservation date is on January 4th, 2012, and preserving number is CGMCC No.5693.
Present embodiment methyl bacterium Met26 is Gram-negative bacteria, rod-short, and the cell size is (0.33 μ m～0.4 μ m) * (0.39 μ m～0.46 μ m), no gemma, atrichia; On the MNMS substratum, cultivate 10d for 28 ℃, colony diameter 0.5-1mm, circle, transparent, neat in edge, smooth surface.
Present embodiment methyl bacterium Met26 can grow at the MNMS substratum, and optimum growth temperature is 28 ℃, and the suitableeest growing environment pH is 6.8.
Embodiment two: present embodiment methyl bacterium (Methylobacter sp.) Met26 obtains by screening in the sandwich rock in 600m coal seam that is collected in new positive colliery, Baoshan District, Shuangyashan in August, 2010.Screening is carried out according to the following steps: take by weighing the sandwich rock sample product 10.0g that dries, crushes, pack into and be added with in the triangular flask of granulated glass sphere sterilized water, after soaking 30min, at the vibrator of the 180r/min 30mim that vibrates, static then, get suspension liquid at the centrifugal 10-15min of 10000r/min, static reserve.Get the 1ml supernatant liquor with the 2.5ml injector for medical purpose and join in the 100ml MNMS liquid nutrient medium, feed the 100ml methane gas again, added one time methane gas every 2-3 days, each 50-100ml.Shaking table is cultivated 120r/min and was cultivated 9-15 days for 28 ℃, with the fermented liquid muddiness, for the first time enrichment culture finish.Get 1ml the enrichment culture liquid from the first time again and carry out the enrichment culture second time, method is the same.Enrichment culture is no less than 3 times continuously.Obtain preservation behind the single pure bacterium, the primary dcreening operation bacterial strain is carried out methylene dichloride utilizes ability to measure, on the MNMS solid medium, be sole carbon source and the energy with the methylene dichloride, providing the pure air method to carry out selectivity cultivates, select single bacterium colony, switching MNMS substratum, last shaking table carries out multiple sieve by detecting degradation efficiency, obtains that methylene dichloride is had efficient degradation ability bacterial strain methyl bacterium Met26.
The MNMS substratum is made up of the potassium primary phosphate of 1.0g/L, the disodium hydrogen phosphate of 2.9g/L, the magnesium sulfate heptahydrate of 0.32g/L, sulfuric acid two ammoniums, 10mL trace element solution and the 990mL distilled water of 3.0g/L, and the pH value is 6.8.It is as follows that wherein trace element solution is formed (mg/L): 0.287mg/L Zinc Sulphate Heptahydrate, 0.223mg/L seven water manganous sulfates, 0.062mg/L boric acid, 0.048mg/L Sodium Molybdate Dihydrate, 0.048mg/L six water cobaltrichlorides, 0.083mg/L potassiumiodide, 3.5mg/L Calcium dichloride dihydrate and distilled water are formed.
The MNMS solid medium is made up of the potassium primary phosphate of 1.0g/L, the disodium hydrogen phosphate of 2.9g/L, the magnesium sulfate heptahydrate of 0.32g/L, sulfuric acid two ammoniums, 10mL trace element solution and the 990mL distilled water of 3.0g/L, and the pH value is 6.8.It is as follows that wherein trace element solution is formed (mg/L): 0.287mg/L Zinc Sulphate Heptahydrate, 0.223mg/L seven water manganous sulfates, 0.062mg/L boric acid, 0.048mg/L Sodium Molybdate Dihydrate, 0.048mg/L six water cobaltrichlorides, 0.083mg/L potassiumiodide, 3.5mg/L Calcium dichloride dihydrate, agar and distilled water are formed, wherein the mass concentration of agar is 2%.
The methyl bacterium Met26 that screening is obtained carries out Molecular Identification, carries out according to the following steps: extracting total DNA of bacterial strain, adopt the 16S rDNA universal primer of bacterium, is that template is carried out pcr amplification with the genomic dna.Utilize glue to reclaim test kit (available from Dalian treasured biotechnology company limited) then and reclaim purified pcr product, clone afterwards, transform, the screening positive clone daughter colony entrusts Shanghai to give birth to the order-checking of worker Bioisystech Co., Ltd after enlarged culturing.
The 16SrDNA sequence length of methyl bacterium (Methylobacter sp.) Met26 is 1498bp, its sequence is shown in SEQ ID NO:1,16S rDNA sequence among sequencing result and the GenBank is carried out the homology comparison, use software building phylogenetic tree (as shown in Figure 1) then, to determine the race relation of bacterial strain.Homology analysis is the result show, the sibship of this sequence and methyl bacterium genus (Methylobacter sp.) is the most approaching, and the conserved regions similarity is 99%.Called after methyl bacterium Met26.By determining that in conjunction with morphological features, growth conditions methyl bacterium Met26 is the new bacterial strain of a strain of methyl bacterium genus (Methylobacter sp.), called after methyl bacterium Met26.
Degradation rate to present embodiment methyl bacterium Met26 degraded methylene dichloride is measured, and concrete grammar is as follows:
Get 10
6The bacteria suspension 1ml of cfu/ml adds shaking table cultivation in the 100mlMNMS substratum, is the substratum of sole carbon source with the methylene dichloride, namely adds methylene dichloride in the MNMS substratum, and the methylene dichloride final concentration is 30mmol/L, divides 4 addings.120r/min, 28 ℃ of cultivations.Detection of biological amount, pH, concentration dichloromethane change, and every 12h detects once.Result: concentration dichloromethane decline 78.6% behind concentration dichloromethane decline 63.5%, the 15d behind the 10d.
Claims (1)
1. the methyl bacterium of strain degraded methylene dichloride, the methyl bacterium of methylene dichloride of it is characterized in that degrading is methyl bacterium (Methylobacter sp.) Met26, be deposited in China Committee for Culture Collection of Microorganisms common micro-organisms center, the preservation address is No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, preservation date is on January 4th, 2012, and preserving number is CGMCC No.5693.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101200697A (en) * | 2007-03-02 | 2008-06-18 | 浙江工业大学 | Bacillus circulans WZ-12 and its application in microorganism resolving treatment of dichloromethane |
| CN101993839A (en) * | 2010-07-23 | 2011-03-30 | 浙江工业大学 | Methylobacterium rhodesianum H13 capable of efficiently degrading dichloromethane and application thereof |
| WO2011124521A2 (en) * | 2010-04-09 | 2011-10-13 | Omya Development Ag | Process to preserve aqueous preparations of mineral materials, preserved aqueous preparations of mineral materials and use of preservative compounds in aqueous preparations of mineral materials |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN101200697A (en) * | 2007-03-02 | 2008-06-18 | 浙江工业大学 | Bacillus circulans WZ-12 and its application in microorganism resolving treatment of dichloromethane |
| WO2011124521A2 (en) * | 2010-04-09 | 2011-10-13 | Omya Development Ag | Process to preserve aqueous preparations of mineral materials, preserved aqueous preparations of mineral materials and use of preservative compounds in aqueous preparations of mineral materials |
| CN101993839A (en) * | 2010-07-23 | 2011-03-30 | 浙江工业大学 | Methylobacterium rhodesianum H13 capable of efficiently degrading dichloromethane and application thereof |
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