CN103343095B - Bacterium capable of degrading herbicide acetochlor and application thereof - Google Patents
Bacterium capable of degrading herbicide acetochlor and application thereof Download PDFInfo
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- CN103343095B CN103343095B CN201310271167.7A CN201310271167A CN103343095B CN 103343095 B CN103343095 B CN 103343095B CN 201310271167 A CN201310271167 A CN 201310271167A CN 103343095 B CN103343095 B CN 103343095B
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- mycobacterium
- acetochlor
- yca11
- bacterium
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- VTNQPKFIQCLBDU-UHFFFAOYSA-N Acetochlor Chemical compound CCOCN(C(=O)CCl)C1=C(C)C=CC=C1CC VTNQPKFIQCLBDU-UHFFFAOYSA-N 0.000 title claims abstract description 75
- 241000894006 Bacteria Species 0.000 title abstract description 45
- 239000004009 herbicide Substances 0.000 title abstract description 15
- 230000002363 herbicidal effect Effects 0.000 title abstract description 13
- 230000000593 degrading effect Effects 0.000 title abstract description 8
- 241000187488 Mycobacterium sp. Species 0.000 claims abstract description 31
- 239000002689 soil Substances 0.000 claims abstract description 13
- 241000186359 Mycobacterium Species 0.000 claims description 31
- 239000002068 microbial inoculum Substances 0.000 claims description 19
- 238000000034 method Methods 0.000 claims description 9
- 244000005700 microbiome Species 0.000 claims description 5
- 238000002360 preparation method Methods 0.000 claims description 2
- 230000015556 catabolic process Effects 0.000 abstract description 28
- 238000006731 degradation reaction Methods 0.000 abstract description 28
- 229910017053 inorganic salt Inorganic materials 0.000 abstract description 7
- 238000004321 preservation Methods 0.000 abstract description 6
- 239000001963 growth medium Substances 0.000 abstract 1
- 238000009629 microbiological culture Methods 0.000 abstract 1
- 238000005067 remediation Methods 0.000 abstract 1
- 239000007788 liquid Substances 0.000 description 12
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 9
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 9
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- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 2
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- FFBHFFJDDLITSX-UHFFFAOYSA-N benzyl N-[2-hydroxy-4-(3-oxomorpholin-4-yl)phenyl]carbamate Chemical compound OC1=C(NC(=O)OCC2=CC=CC=C2)C=CC(=C1)N1CCOCC1=O FFBHFFJDDLITSX-UHFFFAOYSA-N 0.000 description 1
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- 238000001514 detection method Methods 0.000 description 1
- GUJOJGAPFQRJSV-UHFFFAOYSA-N dialuminum;dioxosilane;oxygen(2-);hydrate Chemical compound O.[O-2].[O-2].[O-2].[Al+3].[Al+3].O=[Si]=O.O=[Si]=O.O=[Si]=O.O=[Si]=O GUJOJGAPFQRJSV-UHFFFAOYSA-N 0.000 description 1
- HNPSIPDUKPIQMN-UHFFFAOYSA-N dioxosilane;oxo(oxoalumanyloxy)alumane Chemical compound O=[Si]=O.O=[Al]O[Al]=O HNPSIPDUKPIQMN-UHFFFAOYSA-N 0.000 description 1
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- NLYAJNPCOHFWQQ-UHFFFAOYSA-N kaolin Chemical compound O.O.O=[Al]O[Si](=O)O[Si](=O)O[Al]=O NLYAJNPCOHFWQQ-UHFFFAOYSA-N 0.000 description 1
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- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention discloses a bacterium capable of degrading herbicide acetochlor and an application of the bacterium. The bacterium is mycobacterium sp. YCA11 and the preservation number of the bacterium is CGMCC (China General Microbiological Culture Collection Center) No. 7771. The mycobacterium sp. YCA11 with the preservation of CGMCC No.7771, disclosed by the invention, has the advantage that the degradation rate of the bacterium to 45mg/L acetochlor is up to 51.86% in 7 days in an inorganic salt culture medium, which indicates that the bacterium is capable of efficiently degrading the acetochlor and has a broad application prospect in the remediation of soil polluted by the acetochlor.
Description
Technical field
The present invention relates to bacterium of a strain degrading herbicide acetochlor and uses thereof.
Background technology
Acetochlor, english common name acetochlor, chemical name 2-ethyl-6 methyl--N-ethoxyl methyl-alpha-chloro acetanilide N, structural formula as shown in Equation 1:
Formula 1.
Acetochlor is interior absorption acetamide-group herbicides, is one of several herbicides of consumption maximum in agriculture production.Can be absorbed by weeds young shoot and young root, suppress weeds protein synthesis, and make weeds dead.For the various vegetables fields such as corn, cotton, soybean, peanut, rape, potato, sugarcane, sesame, Sunflower Receptacle and pulse family, Cruciferae, Solanaceae, composite family, Carrot family and orchard, prevent and kill off annual gramineous weed and part broadleaf weeds, Soybean Dodder is had to good preventive effect, invalid to perennial weeds, in soil, the lasting period can reach 2 months, and a dispenser can be controlled the whole crop growth phase and endanger without weeds.
Due to the raising of labor cost, agriculture production more and more relies on weedicide.Labor cost has greatly been saved in a large amount of uses of weedicide, has improved agricultural production efficiency, but has also brought the ecological problems such as some agriculture productions and environmental pollution.The acetochlor transformation period is shorter, is considered to a kind of environmentally friendly agricultural chemicals of low toxicity always, but frequent, excessive or improper use has in recent years caused pollution in various degree to agricultural land soil and underground water etc., also more and more serious to the harm of succession crop.Research shows, acetochlor can the residual several months in soil, can the residual several years in underground water, surface water, soil microorganisms is had to restraining effect, and fish are had to stronger toxicity, can bring out cancer, by EPA, be decided to be carcinogens.
In order to tackle these problems, lot of domestic and international scientific worker has carried out a large amount of research, attempt to accelerate the degraded of herbicide residue in soil by screening, application efficient degrading bacteria, repairing polluted soil, is one of agroecological environment study hotspot to the degraded of residual weedicide.
Summary of the invention
The object of this invention is to provide the bacterium that a strain can efficient degradation Determination of Herbicide Acetochlor.
Bacterium provided by the present invention is mycobacterium (Mycobacterium sp.) YCA11, this bacterial strain is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center on June 20th, 2013 and (is called for short CGMCC, address is: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City), deposit number is CGMCC No.7771.
Another object of the present invention is to provide a kind of microbial inoculum, and the activeconstituents of this microbial inoculum is described mycobacterium (Mycobacterium sp.) YCA11CGMCC No.7771.
Described microbial inoculum is following 1) or 2) described microbial inoculum:
1) for the microbial inoculum of degrading herbicide acetochlor;
2) microbial inoculum polluting for rehabilitating soil Determination of Herbicide Acetochlor.
Described microbial inoculum also can comprise carrier.Described carrier can be solid carrier or liquid vehicle.Described solid carrier is mineral material, vegetable material or macromolecular compound; Described mineral material is at least one in clay, talcum, kaolin, montmorillonite, white carbon, zeolite, silica and diatomite; Described vegetable material is at least one in Semen Maydis powder, bean powder and starch; Described macromolecular compound is polyvinyl alcohol and/or polyglycol.Described liquid vehicle is organic solvent, vegetables oil, mineral oil or water; Described organic solvent is decane and/or dodecane.In described microbial inoculum, described activeconstituents can exist with viable cell, the fermented liquid of viable cell, the form of the mixture of the filtrate of cell culture or cell and filtrate of being cultivated.The formulation of described composition can be multiple formulation, as liquor, emulsion, suspension agent, pulvis, granule, wettable powder or water dispersible granules.
As required, in described microbial inoculum, also can add tensio-active agent (as polysorbas20, tween 80 etc.), tackiness agent, stablizer (as antioxidant), pH adjusting agent etc.
The application of the microbial inoculum that described mycobacterium (Mycobacterium sp.) YCA11CGMCC No.7771 or mycobacterium (Mycobacterium sp.) the YCA11CGMCC No.7771 of take are activeconstituents in degraded acetochlor also belongs to protection scope of the present invention.
The application of the microbial inoculum that described mycobacterium (Mycobacterium sp.) YCA11CGMCC No.7771 or mycobacterium (Mycobacterium sp.) the YCA11CGMCC No.7771 of take are activeconstituents in rehabilitating soil acetochlor pollutes also belongs to protection scope of the present invention.
A further object of the present invention is to provide the method for cultivation mycobacterium (Mycobacterium sp.) YCA11CGMCC No.7771 a kind of, and the method comprises mycobacterium (Mycobacterium sp.) YCA11CGMCC No.7771 in the step of cultivating for cultivating the substratum of mycobacterium.
Another object of the present invention is to provide a kind of preparation method of take the microbial inoculum that mycobacterium (Mycobacterium sp.) YCA11CGMCC No.7771 is activeconstituents, comprise the steps: described mycobacterium (Mycobacterium sp.) YCA11CGMCC No.7771 as activeconstituents, to obtain described microbial inoculum.
The present invention is the farmland collection soil sample of polluting from being subject to for a long time Determination of Herbicide Acetochlor, and Determination of Herbicide Acetochlor degradation bacteria mycobacterium (Mycobacterium sp.) the YCA11CGMCC No.7771 that therefrom separated, screening obtains.Experiment showed, that this bacterial strain reaches 51.86% to the degradation rate of 45mg/L acetochlor in 7 days in minimal medium.This shows, this bacterial strain energy efficient degradation Determination of Herbicide Acetochlor, is having broad application prospects aspect the pollution of rehabilitating soil Determination of Herbicide Acetochlor.
preservation explanation
Strain name: mycobacterium
Latin name: (Mycobacterium sp.)
Strain number: YCA11
Preservation mechanism: China Committee for Culture Collection of Microorganisms's common micro-organisms center
Preservation mechanism is called for short: CGMCC
Address: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City
Preservation date: on June 20th, 2013
The preservation center numbering of registering on the books: CGMCC No.7771
Embodiment
The experimental technique using in following embodiment if no special instructions, is ordinary method.
In following embodiment, material used, reagent etc., if no special instructions, all can obtain from commercial channels.
Substratum used in following embodiment is as follows:
Enrichment culture liquid: K
2hPO
47g/L, KH
2pO
42g/L, (NH
4)
2sO
42g/L, MgSO
47H
2o0.1g/L, MnSO
40.05g/L, FeSO
40.05g/L, CaCl
20.003g/L, is settled to 1L with distilled water, pH7.0; (amount of acetochlor is according to below embodiment 1 interpolation).
Isolation medium: be 2% to adding agar to its mass concentration in enrichment culture liquid.
Inorganic salt liquid substratum: NH
4cl1.0g/L, KH
2pO
40.5g/L, K
2hPO
41.5g/L, MgSO
40.2g/L, NaCl0.5g/L, is settled to 1L with distilled water, pH7.0.
Inorganic salt solid medium: be 2% to adding agar to its mass concentration in inorganic salt liquid substratum.
Beef-protein medium: extractum carnis 5g/L, peptone 10g/L, NaCl5g/L, agar 20g/L, is settled to 1L with distilled water, pH7.2.
Separation and the evaluation of embodiment 1, mycobacterium (Mycobacterium sp.) YCA11CGMCC No.7771
One, the separation of acetochlor degradation bacteria YCA11
In the enrichment culture liquid that is 300mg/L in 50mL acetochlor concentration, add 5g soil sample (pick up from BeiJing, China and be subject to the farmland that Determination of Herbicide Acetochlor pollutes), 28 ℃, 200r/min shaking culture 7d, it is in 500mg/L enrichment culture liquid that absorption 5mL nutrient solution is forwarded to acetochlor concentration, continue to cultivate 7d, so continuously enrichment, cultivate 5 times, acetochlor concentration is followed successively by 300,500,700,900 and 1100mg/L.After enrichment, the pregnant solution of getting acetochlor concentration 1100mg/L carries out serial dilution, evenly coat on the inorganic salt solid medium of same concentrations acetochlor, 28 ℃ of cultivations, until dull and stereotyped upper appearance after single bacterium colony, the larger single bacterium colony of picking is forwarded on beef-protein medium, and 28 ℃ of constant temperature culture are purified to after pure culture standby repeatedly.
Through a large amount of enrichment culture, separation, screening, bacterial strain 45 strains that acquisition can degrading herbicide acetochlor, under pure culture condition, these bacterial strains 0.43%-51.86% that the acetochlor of starting point concentration 45mg/L can be degraded in 7d.By the stronger bacterium called after acetochlor degradation bacteria YCA11 of a strain degraded acetochlor ability screening.
Two, the evaluation of acetochlor degradation bacteria YCA11
The acetochlor degradation bacteria YCA11 separated from the following aspects authentication step one and purifying obtains:
1, Morphological Identification
Will be in logarithmic phase, and bacterium colony size is stable, above-mentioned steps one acetochlor degradation bacteria YCA11 separated and that purifying obtains carries out single bacterium colony state observation, mainly comprises size, color, transparency, wettability, bacterium colony condition of surface (whether smooth, projection, fold, depression etc.), the colony edge state (whether neat, irregular, radial etc.) of bacterium colony.
For the described acetochlor degradation bacteria YCA11 in logarithmic phase, after smear staining, adopt the form of observation by light microscope thalline.
Result shows, above-mentioned steps one acetochlor degradation bacteria YCA11 bacterium colony circular protrusion, yellow, smooth surface separated and that purifying obtains is moistening, neat in edge; Thalline is shaft-like, 0.5 * 2.0 μ m, Gram-positive.
2, analysis of physio biochemical characteristics
With reference to < < common bacteria system identification handbook > > (eastern elegant pearl, Cai Miaoying. common bacteria system identification handbook. Beijing: Science Press, 2011.) and < < Microbiology Experiment > > (Shen Ping, Fan Xiurong, Li Guangwu. Microbiology Experiment (third edition). Beijing: Higher Education Publishing House, 1999.) measure the physiological and biochemical property of above-mentioned acetochlor degradation bacteria YCA11.
The physiological and biochemical property measurement result of described acetochlor degradation bacteria YCA11 is as shown in table 1:
The physiological and biochemical property of table 1 acetochlor degradation bacteria YCA11
Note: "+" represents positive, "-" represents negative.
3,16s rDNA sequence homology analysis
Ordinary method is cultivated the acetochlor degradation bacteria YCA11 that above-mentioned steps one separation and purification obtains, extract total DNA of bacterial strain as gene amplification template, with bacterium 16s rDNA universal primer, 27f:5 '-AGAGTTTGATCCTGGCTCAG-3 ', 1492r:5 '-TACCTTGTTACGACTT-3 ' carries out PCR reaction.Reaction system adopts Shanghai biotechnology company limited pcr amplification test kit.Response procedures is: 95 ℃ of sex change 30s, 55 ℃ of annealing 1min, 72 ℃ of extensions 2min, totally 30 circulations.DNA sequencing is completed by Beijing San Bo polygala root biotech company, sequence assembly and similarity analysis are used DNAStar software to complete, and gene comparison completes online by American National biotechnology information center ncbi database (http://www.ncbi.nlm.nih.gov) and EzTaxon.
Pcr gene amplification obtains the about 1.5kb of 16S rDNA gene fragment of acetochlor degradation bacteria YCA11, measure after sequence with NCBI and EzTaxon database in published 16S rDNA sequence carry out online sequence analysis, result shows YCA11 and pale yellow mycobacterium Mycobacterium gilvum ATCC43909
tthe homology of (GenBank accession number X81996.1) is the highest, reaches 99.482%.
The 16s rDNA sequence of acetochlor degradation bacteria YCA11 refers to sequence 1 in sequence table.
4, growth characteristics analysis
Optimum temperuture and the optimal pH growth experiment of acetochlor degradation bacteria YCA11 have been carried out.Adopt inorganic salt liquid substratum, respectively at 4 ℃, 28 ℃, 37 ℃, the 60 ℃ thermal adaptabilities of cultivating, observing, record bacterial strain, each is processed 3 times and repeats.Adjust pH and be respectively 4,5,6,7,8,9,10, each is processed 3 times and repeats, and cultivates, observes, records the optimal pH of strain growth.
Result shows, the optimum growth temperature of described acetochlor degradation bacteria YCA11 is 28 ℃, and the most suitable growth pH is 7～8.
In view of above-mentioned form, analysis of physio biochemical characteristics and 16s rDNA sequence homology analysis result, the acetochlor degradation bacteria YCA11 that step 1 separation and purification is obtained is accredited as bacterium territory actinomycete group mycobacteriaceae (Mycobacteriaceae) Mycobacterium (Mycobacterium sp.).This acetochlor degradation bacteria YCA11 is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center on June 20th, 2013 and (is called for short CGMCC, address is: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City), deposit number is CGMCC No.7771.
The quantitative assay of embodiment 2, mycobacterium (Mycobacterium sp.) YCA11CGMCC No.7771 degraded acetochlor ability
One, acetochlor bioassay standard curve plotting
With methyl alcohol, acetochlor standard substance (purchased from Fluka company) are mixed with to series concentration 10,20,40,60,80,100mg/L standardized solution, adopt high performance liquid chromatography (HPLC) to measure the peak area of different concns acetochlor standard substance, repeat for 3 times.The concentration of acetochlor of take is X-coordinate, and peak area is ordinate zou, draws acetochlor typical curve.
Testing conditions is as follows:
Detection system: Agilent1100Series.Chromatographic column: C18Diamosil
tMreversed-phase column, 250mm * 4.6mm, particle diameter 5 μ m.Chromatographic condition: moving phase: methyl alcohol: water=80:20(v/v), water is adjusted to pH=3 with Glacial acetic acid; Detect wavelength, 215nm; Flow velocity, 1.0mL/min; Sampling volume, 20 μ L; 30 ℃ of column temperatures.
Measurement result is as shown in table 2:
Table 2 different concns acetochlor HPLC measures peak area
According to table 2 data, obtain acetochlor bioassay standard curve: y=51.44x+1615 (R
2=0.999).Wherein, y is peak area, and x is acetochlor concentration.
Two, mycobacterium (Mycobacterium sp.) YCA11CGMCC No.7771 degraded acetochlor ability quantitative assay
50mL containing the triangular flask (volume is 250ml) of the minimal medium of the about 50mg/L of acetochlor (being the substratum that about 50mg/L obtains to adding acetochlor to make the concentration of acetochlor in inorganic salt liquid substratum) in mycobacterium (Mycobacterium sp.) the YCA11CGMCC No.7771 of access OD600=1.0 or mycobacterium (Mycobacterium sp.) DJL22CGMCC No.5041(in Granted publication day, be that on November 07th, 2012, Granted publication are open in number for the Chinese invention patent in CN102260641B) bacterium liquid (is 1 * 10
9cfu/ml) 2mL, 30 ℃, 200r/min shaking table is cultivated 7 days, measures Residual Determination of Acetochlor.Method is: get 3mL degradation solution to 50mL centrifuge tube, the centrifugal 5min of 8000r/min collects supernatant liquor, add 3mL methylene dichloride, thermal agitation 5min, standing 10min, treat water and organic phase layering, add a small amount of anhydrous sodium sulphate to dewater to organic phase, accurately draw 800 μ L organic phases in the centrifuge tube of 1.5mL, with Nitrogen evaporator, dry up, add 400 μ L methyl alcohol, ultrasonic 10min in ultrasonic cleaner, with the organic phase aculeus type filter of 0.22 μ m, be filtered to liquid chromatography sample bottle, according to above-mentioned HPLC method, measure acetochlor.Meanwhile, using do not access bacterium the above-mentioned minimal medium containing the about 50mg/L of acetochlor as blank, measure according to the method described above the concentration of acetochlor.Acetochlor degradation rate: X=(1-A/B) * 100%, in formula, X is degradation rate (%), and A connects acetochlor concentration residual in bacterium treatment solution, and B is not for connecing acetochlor concentration residual in bacterium blank treatment solution.3 repetitions are established in experiment, repeat each at every turn and process 1 triangular flask of inoculation.
Result shows, it is as shown in table 3 that described mycobacterium after 7 days (Mycobacterium sp.) YCA11CGMCC No.7771 reaches 51.86%(to the degradation rate of acetochlor), mycobacterium (Mycobacterium sp.) DJL22CGMCCNo.5041 only has 21.76% to the degradation rate of acetochlor.This result shows, mycobacterium provided by the present invention (Mycobacterium sp.) YCA11CGMCC No.7771 can efficient degradation acetochlor.
Table 3 mycobacterium (Mycobacterium sp.) YCA11CGMCC No.7771 degraded acetochlor effect
Claims (6)
1. mycobacterium (Mycobacterium sp.) YCA11, the deposit number at Qi China Committee for Culture Collection of Microorganisms common micro-organisms center is CGMCC No.7771.
2. a microbial inoculum, is characterized in that: the activeconstituents of described microbial inoculum is mycobacterium claimed in claim 1 (Mycobacterium sp.) YCA11.
3. mycobacterium claimed in claim 1 (Mycobacterium sp.) YCA11 or microbial inoculum claimed in claim 2 application in degraded acetochlor.
4. mycobacterium claimed in claim 1 (Mycobacterium sp.) YCA11 or microbial inoculum claimed in claim 2 application in rehabilitating soil acetochlor pollutes.
5. cultivate the method for mycobacterium claimed in claim 1 (Mycobacterium sp.) YCA11, comprise described mycobacterium (Mycobacterium sp.) YCA11 in the step of cultivating for cultivating the substratum of mycobacterium.
6. the preparation method of microbial inoculum described in claim 2, comprises the steps: mycobacterium claimed in claim 1 (Mycobacterium sp.) YCA11 as activeconstituents, to obtain described microbial inoculum.
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