CN101423807A - Method for preparing polycyclic aromatic hydrocarbon degrading bacteria - Google Patents
Method for preparing polycyclic aromatic hydrocarbon degrading bacteria Download PDFInfo
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- CN101423807A CN101423807A CNA200710157905XA CN200710157905A CN101423807A CN 101423807 A CN101423807 A CN 101423807A CN A200710157905X A CNA200710157905X A CN A200710157905XA CN 200710157905 A CN200710157905 A CN 200710157905A CN 101423807 A CN101423807 A CN 101423807A
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- polycyclic aromatic
- aromatic hydrocarbon
- degrading bacteria
- hydrocarbon degrading
- preparation
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Abstract
The invention relates to bioremediation of polycyclic aromatic hydrocarbon in an contaminated environment, in particular to a method for preparing a polycyclic aromatic hydrocarbon degrading bacteria. The zymotic fluid of a strain Mycobacterium sp.SN12 is used as an effective composition; the effective composition is adsorbed on a corncob particle carrier and is ventilated and dried at a temperature of between 20 and 50 DEG C to prepare the solid granular polycyclic aromatic hydrocarbon degrading bacteria; finally, the content of the carrier in the degrading bacteria is between 97 and 98.5 percent; the strain Mycobacterium sp.SN12 is preserved in China Center for Type Culture Collection; and the CCTCC NO is M207110. The product obtained through the method can effectively reduce the contents of pyrene, phenanthrene, fluorine and other polycyclic aromatic hydrocarbons in the contaminated environment.
Description
Technical field
The present invention relates to polycyclic aromatic hydrocarbons biological restoration in the contaminate environment, specifically a kind of preparation method of polycyclic aromatic hydrocarbon degrading bacteria.
Background technology
Polycyclic aromatic hydrocarbons is the organic pollutant that is prevalent in land and the water surrounding, has potential carcinogenic, teratogenesis, mutagenesis, is considered to the objectionable impurities of human health.In general, be less than and equal three rings for the lower molecular weight polycyclic aromatic hydrocarbons, have the above title polycyclic aromatic hydrocarbon with high molecular weight in Fourth Ring or Fourth Ring.The number of rings that polycyclic aromatic hydrocarbons had is many more, and its toxicity is big more, also difficult degradation more.In recent years, with the pyrene be the concern that the biological degradation of the polycyclic aromatic hydrocarbon with high molecular weight of representative is subjected to more and more Chinese scholars.Pyrene is a polycyclc aromatic compound, is not dissolved in water, and volatility is poor, it is colourless to be, prismatic crystal (impurity is for yellow), another name: pyrene, its molecular formula: C
16H
10Can be with soil, water etc. as carrier, have genotoxic potential, carinogenicity and teratogenesis mutagenesis, pyrene itself is not had a genetoxic, but its ketone metabolite is bigger and mutagenicity is arranged than maternal toxicity, so pyrene often is used as the polycyclic aromatic hydrocarbons contaminated indicator of monitoring and other polycyclic aromatic hydrocarbons photochemical degradation, biodegradable model molecule.
Summary of the invention
The object of the present invention is to provide that a kind of cost is low, the preparation method of the polycyclic aromatic hydrocarbon degrading bacteria of good degrading effect.
For achieving the above object, the technical solution used in the present invention is:
A kind of preparation method of polycyclic aromatic hydrocarbon degrading bacteria, fermented liquid with bacterial strain Mycobacterium sp.SN12 is an effective ingredient, effective ingredient is adsorbed on the corn cob granule carrier, makes the solid granular polycyclic aromatic hydrocarbon degrading bacteria after the 20-50 ℃ of air seasoning; The content content of carrier is 97-99% in the eventual degradation microbial inoculum.
It is a starting strain with bacterial strain SN12, this bacterium is molecule Bacillaceae (Mycobacterium sp.), its biological characteristics is that thalli morphology is a rod-short, the blunt circle in two ends, Gram-positive, single bacterium colony is golden yellow on beef extract-peptone (NR) flat board, and circular cushion, neat in edge, smooth surface combine insecure with substratum.This bacteria growing is slow, and through 30 ℃, cultivation in average 7 days can form visible single bacterium colony.Acetyl methyl carbinol (V.P.) test, methyl red (M.R.) test, starch hydrolysis experiment are negative, and it is all positive to produce ammonia test, nitrate reduction test, gelatin liquification test, catalase test, hydrogen sulfide production test.The Genbank number of landing of bacterial strain 16S rDNA is DQ512892.
Described bacterial strain on July 20th, 2007 in China's typical culture collection center preservation, deposit number is CCTCC NO:M207110; Diagnostic characteristics is Mycobacterium gilvum SN12;
The concrete operations step is as follows:
(1) single colony inoculation of picking Mycobacterium sp.SN12 is in being equipped with the shaking in the bottle of seed culture medium, 28-30 ℃ of aerated culture 36 hours;
Described seed culture based formulas is: in every premium on currency, and extractum carnis 0.5g, peptone 1g, NaCl 0.5g, pH are 7.2～7.4;
(2) with the culture in the step (1), the inoculum size enlarged culturing of pressing 5-10% once, culture condition is with (1);
(3) with the culture in the step (2), be seeded in the fermentor tank by the inoculum size of 5-10%, 28-30 ℃ of aerobic fermentation cultivated 56 hours;
Described fermentative medium formula is: in every premium on currency, and glucose 5g, NH
4NO
310g, K
2HPO
42g, MgSO
47H
2O 0.5g, Na
2HPO
42g, CaCO
33g, pH-7.2～7.4;
Described fermentor tank air flow is 1:0.8-1.0, and stirring velocity is 150-180 rev/min;
(4) back of having fermented is extremely saturated with commercial corn cob granule absorption fermented liquid, makes the solid particulate formulation after the 20-50 ℃ of air seasoning and is distributed into bag.
The specification of described commercialization corn cob granule is diameter 2-4mm;
When described fermented liquid was made product, corn cob and fermented liquid proportioning were 150-200g:100ml;
The product that method of the present invention obtains can effectively reduce polycyclic aromatic hydrocarbon contents such as pyrene in the contaminate environment, phenanthrene, acenaphthene, fluorenes
The present invention has following advantage:
1. cost is low.The present invention utilizes cheap plant vector corn cob to make product for carrier, and corn cob is as agricultural by-products, and output is huge, and is cheap, wide material sources.Corn cob mainly is made up of Mierocrystalline cellulose and xylogen, has stability and physical strength preferably, because corn cob has vesicular structure, thereby adsorptive power is strong, is easy to make solution to penetrate into corn cob inside.Be widely used in the decontamination polishing of water cleaning, health-care pillow, machinery etc.Also be the good carrier of edible mushrooms, soilless culture green organic fertilizer simultaneously.
2. effect is good, the degradation efficiency height.The present invention utilizes cheap nontoxic corn cob to prepare microbiobacterial agent; be implemented in the contaminate environment and can not bring secondary pollution; corn cob after playing effectiveness is easily utilized by other microorganism; for preserving the ecological environment; the content of the organic pollutant of reduction contaminate environment is significant, and especially the polycyclic aromatic hydrocarbon with high molecular weight for difficult degradation has better degradation effect.Can big area promote the use of.Be implemented on luxuriant and rich with fragrance and pyrene polluted-water and soil, can effectively remove phenanthrene and pyrene, the clearance of pollutent is and reaches more than 90% in the water body, and the clearance of pollutant in soil can reach more than 70%.
Embodiment
Embodiment 1
(1) single colony inoculation of picking Mycobacterium sp.SN12 shakes in the bottle in the 50ml that seed culture medium is housed, 28-30 ℃ of aerated culture 36 hours;
Described seed culture based formulas is: in every premium on currency, and extractum carnis 0.5g, peptone 1g, NaCl 0.5g, pH are 7.3;
(2) with the culture in the step (1), inoculum size (inoculum size) enlarged culturing of pressing 5-10% once, culture condition is with (1);
(3) with the culture in the step (2), be seeded in the 30L fermentor tank by the inoculum size (inoculum size) of 5-10%, 28-30 ℃ of aerobic fermentation cultivated 56 hours;
Described fermentative medium formula is: in every premium on currency, and glucose 5g, NH
4NO
310g, K
2HPO
42g, MgSO
47H
2O 0.5g, Na
2HPO
42g, CaCO
33g, pH-7.3;
Described fermentor tank air flow is 1:0.9, and stirring velocity is 180 rev/mins;
(4) back of having fermented is extremely saturated with commercial corn cob granule absorption fermented liquid, and the specification of commercialization corn cob granule is diameter 2-4mm; Corn cob and fermented liquid proportioning are 200g:1L; Make the solid particulate formulation after 50 ℃ of air seasonings, and be distributed into bag, get polycyclic aromatic hydrocarbon degrading bacteria.
Application examples 1
Take by weighing 1 gram microbial inoculum and be inoculated in that (culture medium prescription is: glucose 0.05%, NH in the 100ml degraded substratum
4NO
31%, K
2HPO
40.2%, MgSO
47H
2O 0.05%, Na
2HPO
40.2%, CaCO
30.3%, pyrene 0.01%, luxuriant and rich with fragrance 0.01%, pH7.2～7.4), 30 ℃ of lucifuges were cultivated 7 days, measured residually, and phenanthrene and pyrene clearance are respectively 95.2% and 91.5%.
Application examples 2
Take by weighing 0.1g phenanthrene and pyrene respectively and be dissolved in 50ml acetone, evenly admix in the 1000g soil, treat that the acetone volatilization is clean, add the 300ml sterilized water and make mud, inoculation 10g microbial inoculum and mixing, lucifuge cultivation 10 days, every day, stirred slurry was 2 times.The soil of handling with quadrat method with the 50ml acetone of not Jia Fei and pyrene is contrast.Get the 5g soil sample air-dry, get 2g after grinding and place the 100ml centrifuge tube. add the 20ml methylene dichloride. centrifuge tube is built. put into ultrasonic water bath case extraction 2h. water-bath process temperature and be controlled at below 40 ℃, get supernatant and irritate and put into thickener. use N
2Dry up, move into chromatogram sample introduction bottle after the methanol constant volume. use the residual of high-performance liquid chromatogram determination phenanthrene and pyrene.The result shows luxuriant and rich with fragrance and the pyrene degradation rate is respectively 72.2% and 81.5%.
Claims (7)
1. the preparation method of a polycyclic aromatic hydrocarbon degrading bacteria is characterized in that:
Fermented liquid with bacterial strain Mycobacterium sp.SN12 is an effective ingredient, and effective ingredient is adsorbed on the corn cob granule carrier, makes the solid granular polycyclic aromatic hydrocarbon degrading bacteria after the 20-50 ℃ of air seasoning; The content of carrier is 97-99% in the eventual degradation microbial inoculum;
Described bacterial strain Mycobacterium sp.SN12 is preserved in Chinese typical culture collection center, and deposit number is CCTCCNO:M207110.
2. according to the preparation method of the described polycyclic aromatic hydrocarbon degrading bacteria of claim 1, it is characterized in that:
Can operate as follows,
(1) single colony inoculation of picking Mycobacterium sp.SN12 is in being equipped with the shaking in the bottle of seed culture medium, 28-30 ℃ of aerated culture 36 hours;
(2) with the culture in the step (1), the inoculum size enlarged culturing of pressing 5-10% once, culture condition is with (1);
(3) with the culture in the step (2), be seeded in the fermentor tank by the inoculum size of 5-10%, 28-30 ℃ of aerobic fermentation cultivated 56 hours;
(4) back of having fermented is extremely saturated with commercial corn cob granule absorption fermented liquid, makes the solid particulate formulation after the 20-50 ℃ of air seasoning and is distributed into bag.
3. according to the preparation method of the described polycyclic aromatic hydrocarbon degrading bacteria of claim 2, it is characterized in that:
Described fermentative medium formula is: in every premium on currency, and glucose 5g, NH
4NO
310g, K
2HPO
42g, MgSO
47H
2O 0.5g, Na
2HPO
42g, CaCO
33g, pH-7.2～7.4;
Described seed culture based formulas is: in every premium on currency, and extractum carnis 0.5g, peptone 1g, NaCl0.5g, pH are 7.2～7.4.
4. according to the preparation method of the described polycyclic aromatic hydrocarbon degrading bacteria of claim 2, it is characterized in that: described fermentor tank air flow is 1:0.8-1.0, and stirring velocity is 150-180 rev/min.
5. according to the preparation method of the described polycyclic aromatic hydrocarbon degrading bacteria of claim 2, it is characterized in that: the specification of described commercialization corn cob granule is diameter 2-4mm;
6. according to the preparation method of the described polycyclic aromatic hydrocarbon degrading bacteria of claim 2, it is characterized in that: when described fermented liquid was made product, corn cob and fermented liquid proportioning were 150-200g:100ml.
7. according to the preparation method of the described polycyclic aromatic hydrocarbon degrading bacteria of claim 2, it is characterized in that: described polycyclic aromatic hydrocarbons is meant pyrene, phenanthrene, acenaphthene and/or fluorenes.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN200710157905XA CN101423807B (en) | 2007-11-02 | 2007-11-02 | Method for preparing polycyclic aromatic hydrocarbon degrading bacteria |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN200710157905XA CN101423807B (en) | 2007-11-02 | 2007-11-02 | Method for preparing polycyclic aromatic hydrocarbon degrading bacteria |
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| Publication Number | Publication Date |
|---|---|
| CN101423807A true CN101423807A (en) | 2009-05-06 |
| CN101423807B CN101423807B (en) | 2010-12-08 |
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| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN200710157905XA Expired - Fee Related CN101423807B (en) | 2007-11-02 | 2007-11-02 | Method for preparing polycyclic aromatic hydrocarbon degrading bacteria |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101974445A (en) * | 2010-05-26 | 2011-02-16 | 北京师范大学 | High molecular weight polycyclic aromatic hydrocarbon degrading strains and mixed strain system thereof |
| CN102277350A (en) * | 2011-08-06 | 2011-12-14 | 浙江大学 | Single-carrier polycyclic aromatic hydrocarbon degrading bacterium and preparation method thereof |
| CN102392012A (en) * | 2011-08-08 | 2012-03-28 | 浙江大学 | Polycyclic aromatic hydrocarbon antibacterial agent of composite carrier and preparation method of polycyclic aromatic hydrocarbon antibacterial agent |
| CN103695348A (en) * | 2013-12-16 | 2014-04-02 | 广东瀚森生物药业有限公司 | Method for fermenting mycobacterium tuberculosis bacteria |
| CN111088187A (en) * | 2019-12-31 | 2020-05-01 | 华东理工大学 | Charcoal-based immobilized polycyclic aromatic hydrocarbon efficient degradation microbial inoculum and preparation method and application thereof |
-
2007
- 2007-11-02 CN CN200710157905XA patent/CN101423807B/en not_active Expired - Fee Related
Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101974445A (en) * | 2010-05-26 | 2011-02-16 | 北京师范大学 | High molecular weight polycyclic aromatic hydrocarbon degrading strains and mixed strain system thereof |
| CN101974445B (en) * | 2010-05-26 | 2012-10-24 | 北京师范大学 | High molecular weight polycyclic aromatic hydrocarbon degrading strains and mixed strain system thereof |
| CN102277350A (en) * | 2011-08-06 | 2011-12-14 | 浙江大学 | Single-carrier polycyclic aromatic hydrocarbon degrading bacterium and preparation method thereof |
| CN102392012A (en) * | 2011-08-08 | 2012-03-28 | 浙江大学 | Polycyclic aromatic hydrocarbon antibacterial agent of composite carrier and preparation method of polycyclic aromatic hydrocarbon antibacterial agent |
| CN102392012B (en) * | 2011-08-08 | 2013-05-01 | 浙江大学 | Polycyclic aromatic hydrocarbon antibacterial agent of composite carrier and preparation method of polycyclic aromatic hydrocarbon antibacterial agent |
| CN103695348A (en) * | 2013-12-16 | 2014-04-02 | 广东瀚森生物药业有限公司 | Method for fermenting mycobacterium tuberculosis bacteria |
| CN103695348B (en) * | 2013-12-16 | 2015-07-08 | 广东瀚森生物药业有限公司 | Method for fermenting mycobacterium tuberculosis bacteria |
| CN111088187A (en) * | 2019-12-31 | 2020-05-01 | 华东理工大学 | Charcoal-based immobilized polycyclic aromatic hydrocarbon efficient degradation microbial inoculum and preparation method and application thereof |
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| Publication number | Publication date |
|---|---|
| CN101423807B (en) | 2010-12-08 |
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Granted publication date: 20101208 Termination date: 20121102 |