CN103865853B - One strain quinclorac efficient degrading bacteria and uses thereof and using method - Google Patents
One strain quinclorac efficient degrading bacteria and uses thereof and using method Download PDFInfo
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- CN103865853B CN103865853B CN201410095770.9A CN201410095770A CN103865853B CN 103865853 B CN103865853 B CN 103865853B CN 201410095770 A CN201410095770 A CN 201410095770A CN 103865853 B CN103865853 B CN 103865853B
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- quinclorac
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Abstract
Patent of the present invention belongs to environmental protection technical field, is specifically related to strain quinclorac efficient degrading bacteria and uses thereof.Quinclorac efficient degrading bacteria code name of the present invention is MC-10, through being accredited as nicotianae Arthrobacter nicotianae, on November 19th, 2013 in Chinese culture presevation management committee common micro-organisms center preservation, preserving number is: CGMCC No.8489; This bacterium can be used for the improvement in quinclorac contaminated soil and waters, has good degradation effect.This bacterium has good colonization ability in quinclorac contaminated soil and waters, quinclorac residual in can degrade fully soil and water, and can not produce the advantages such as secondary pollution, has no adverse effects to the microorganism in original ecotope.
Description
Technical field
The present invention relates to a strain nicotianae (
arthrobacter nicotianae) MC-10, and the application in microbiological deterioration quinclorac pesticide residue.
Background technology
Quinclorac is a kind of hormone-type quinoline acids weedicide that BASF Aktiengesellschaft starts to promote for 1984, ISO common name: Quinclorac, molecular formula: C
10h
5c
l2nO
2, chemical name: 3,7-bis-chloroquine beautiful jade-8-carboxylic acid, its common trade(brand)name has: god's hoe (1 generation, 2 generations), quinclorac, kill that barnyard grass spirit, barnyard grass are clean, gram barnyard grass star and barnyard grass king etc.Two chloroquine beautiful jades acid are stablized the ambient conditions such as light, temperature, in soil the disappearance of quinclorac and soil humidity linear.Under the state of nature of field, dichloroquinoline degraded slowly, microorganism in main dependence soil and relevant degraded enzyme, Li Jing newly waits the research of people to show that rice field is by under recommend use amount, Acid soil, after 269d, just the growth wide to tobacco leaf has no significant effect, after 342d, just eliminate to Tobacco Root being grown to the scope had no significant effect.Because south is all much rice-cigarette crop rotation field, many rice fields are used quinclorac and are prevented and treated the multiple weeds such as barnyard grass, and and then lower stubble plants tobacco again, and because its transformation period is longer, its remaining in soil to be brought to the growth of tobacco and had a strong impact on.But at present for quinclorac soil contamination problem, not effectively improvement and solution, the material that can only have an adsorption activity by gac etc. adsorbs quinclorac residual in soil; Or after there is poisoning, use some biological regulators such as brassinolide to alleviate the damage symptoms of cigarette strain; These methods all fundamentally can not solve the pollution of quinclorac for topic, but also easily cause secondary pollution.So seek the new efficient administering method of dichloroquinoline acid pollution to seem very urgent and important.
Microbial technology has that removal efficiency is high, processing costs is low, can fundamentally solve quinclorac pollution problem, and can not produce secondary pollution problem.But adopt one of key of microbial technology to be the efficient degrading bacterial strain obtaining quinclorac.At present, Chinese scholars has carried out large quantifier elimination to quinclorac microbiological deterioration, but contains quinoline ring due to quinclorac, comparatively stablize and not easily degrade, and content is lower in soil, the degradation bacteria be separated to up to now is also more limited, mainly comprise Ochrobactrum (
ochrobactrum sp.), Burkholderia cepecia, Alcaligenes sp., Bordetella sp., Pantoea sp., Bordetella sp.deng, these bacterial strains be separated to need to improve further to the quinclorac degradation effect under lower concentration.Large quantifier elimination shows the efficient quinclorac degradation bacteria of separation screening from environment, is the Important Action fundamentally solving dichloroquinoline acid pollution.And there is not yet the research report of relevant nicotianae genus degraded quinclorac at present both at home and abroad.
Summary of the invention
The object of the invention be to provide one plant height effect, colonization ability strong there is nicotianae of quinclorac degradation capability and uses thereof.
The technical solution used in the present invention is:
One strain quinclorac degradation bacteria nicotianae (
arthrobacter nicotianae) MC-10, be preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, address: China, Beijing, No. 3, No. 1, Beichen Lu, Chaoyang District institute, deposit number: CGMCC No.8489, preservation date: on November 19th, 2013.
Described quinclorac degradation bacteria MC-10 colony characteristics is as follows: bacterium colony is yellow, and neat in edge is smooth moistening; The form of observing this thalline under transmission electron microscope is spherical, does not move; Gram-positive, catalase is positive, and oxidase test, VP test, methyl red test are all negative.Can reduce nitrate, O/F test-results is to be oxidized, and can utilize glucose, ribose, lactose.
One aspect of the present invention, the application of quinclorac degradation bacteria nicotianae MC-10 in degraded quinclorac.
One aspect of the present invention, quinclorac degradation bacteria nicotianae MC-10 is administering the application in quinclorac contaminated soil.
One aspect of the present invention, a kind of microbial inoculum comprising quinclorac degradation bacteria nicotianae MC-10, described microbial inoculum can be that known to those skilled in the art, any technique means prepares any type of microbial inoculum, such as by carrying out the dry microbial inoculum of freeze-drying acquisition after fermentation culture, it also can be the concentrated liquid microbial inoculum of liquid form.
One aspect of the present invention, described in comprise quinclorac degradation bacteria nicotianae MC-10 microbial inoculum degraded quinclorac in application.
One aspect of the present invention, described in comprise quinclorac degradation bacteria nicotianae MC-10 microbial inoculum and administering the application in quinclorac contaminated soil.
Described degraded 25-35 DEG C, carry out under pH 6-8.
Preferably, described degraded 35 DEG C, carry out under pH=7.5.
Nicotianae MC-10 in the present invention is a kind of common Arthrobacter, through patent searching and other pertinent literatures, the nicotianae of quinclorac of not yet finding to degrade belongs to bacterial classification, and this bacterial classification can be degraded to quinclorac at low concentrations, and in soil, there is good colonization ability.
Accompanying drawing explanation
Fig. 1 is the transmission electron microscope photo of nicotianae MC-10 in the present invention.
Fig. 2 is CK measurement result figure.
Fig. 3 is the quinclorac degradation effect figure of nicotianae MC-10 in the present invention.
Embodiment
Below in conjunction with specific embodiment, the present invention is described further, but protection scope of the present invention is not limited to this.
Embodiment 1: the separation of nicotianae MC-10, purifying and preservation.
Described degradation bacteria is cultivated through domestication and screening purifies and separates gets, and concrete steps are as follows:
Domestication is cultivated: join in the quinclorac minimal medium of 100mg/L by the rice field soil sample 5g using quinclorac throughout the year, in 28 DEG C, shaking culture 7d on 150r/min shaking table; Then with 5% inoculum size, be transferred in new quinclorac minimal medium, the gradient that the concentration of quinclorac presses 100mg/L increases, and transfers and reaches 500mg/L to dichloroquinoline acid concentration 4 times.
Described minimal medium is: often liter of minimal medium is containing ammonium nitrate 4g, potassium primary phosphate 1.5g, dipotassium hydrogen phosphate 0.5g, iron trichloride 0.01g, calcium chloride 0.01g, micro-mother liquor (EDTA-disodium 0.01g/L, Manganous chloride tetrahydrate 39mg/L, zinc sulfate 43mg/L, key acid sodium 36mg/L) 10mL.
Last domestication nutrient solution is diluted to 10 by concentration gradient
-9, respectively at coated plate on the quinclorac minimal medium of 100mg/L, in 34 DEG C of incubators, 3d is cultivated in inversion, and the bacterium colony that picking growing way is vigorous expands in LB liquid nutrient medium, purifying is cultivated.
The thalline portion obtained by purifying is inoculated in LB slant medium, 4 DEG C of preservations; At being stored in-20 DEG C in glycerine simultaneously.
Embodiment 2: the qualification of nicotianae MC-10.
By 16S rDNA sequential analysis and bio-chemical characteristics qualification, determine that bacterial strain MC-10 is that nicotianae belongs to bacterial classification.Concrete steps are as follows:
Adopt the DNA of DNA extraction kit (Beijing full formula gold biotechnology (TransGen Biotech) company limited) Isolation and purification bacterial strain, 4 DEG C of preservations.The 16s universal primer of bacterium is selected to be synthesized by Hua Da genome company:
Upstream primer is 5 '-AGAGTTTGATCMTGGCTCAG-3 ';
Downstream primer is 5 '-TACGGCTACCTTGTTACGACTT-3 '.
Amplification reaction system: 10XBuffer (Mg
2+) 2 μ L, dNTPs2 μ L, each 1 μ L of primer, thallus DNA 1 μ L, Taq archaeal dna polymerase 0.5 μ L, adds deionized water to 20 μ L.
PCR response procedures is set as: 94 ° of C denaturation 4min; Then 94 ° of C sex change lmin, 55 ° of C annealing lmin, 72 ° of C extend 1.5min, circulate 35 cycles; Then 72 ° of C extend IOmin; Last 4 ° of C keep lOmin.PCR primer is carried out check order (Hua Da genome company).
Sequencing result and Genbank database are compared, the Arthrobacter nicotianae that finds that it belongs to
.fig. 1 is the electromicroscopic photograph of MC-10 bacterium.
Based on sequencing result and physiological and biochemical test result, determine that MC-10 belongs to
arthrobacter nicotianae.Therefore, by this bacterium called after
arthrobacter nicotianaestrain MC-10, be preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center and (be called for short CGMCC: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, Institute of Microorganism, Academia Sinica, postcode: 100101), preservation date is on November 19th, 2013, and deposit number is CGMCCNo.8489.
The degradation effect qualification of embodiment 3: degradation bacteria MC-10.
MC-10 bacterium is inoculated in liquid LB in 35 DEG C, 180r/min shaking culture is to logarithmic phase.By 5ml nutrient solution in the centrifugal 2min of 10000r/min, be inoculated in 50mg/L quinclorac minimal medium with after the resuspended thalline of isopyknic 0.01PBS damping fluid after abandoning supernatant, in 30 DEG C, 150r/min, after cultivating 7d, carry out with liquid chromatograph the degradation rate detecting thalline.Degrading experiment establishes contrast, three repetitions.
Liquid chromatographic detection condition: chromatographic column: Waters Sun FireTM-C18,5.0 μm, 4.6 mm × 250 mm.Moving phase: methyl alcohol/0.01% phosphate aqueous solution (3:1, V/V), flow velocity 0.6 mL/min sample size 10 μ L, column temperature 30 DEG C, determined wavelength is 240 nm.
Relative degradation rate=(after CK content-degradation bacteria degraded content)/CK content * 100%
The degradation rate being gone out quinclorac degradation bacteria MC-10 by above-mentioned formulae discovery is 91.30%.
Embodiment 4: outside environmental elements is on the impact of degradation bacteria degradation effect.
Quinclorac starting point concentration is on the impact of degradation bacteria MC-10 degradation rate: each 100ml of quinclorac minimal medium sterilizing in triangular flask of preparing 10ppm, 20ppm, 40ppm, 50ppm is respectively stand-by.Respectively to the good MC-10 bacteria suspension of activation inoculating 5% in above-mentioned quinclorac inorganic salt, in 30 DEG C, the constant-temperature table of 150r/min cultivates 7d after, measure the degradation efficiency of each process.
Culture temperature is on the impact of degradation bacteria MC-10 degradation rate: in the 50ppm quinclorac minimal medium of bacterium of having gone out, inoculate the 5% MC-10PBS bacteria suspension activated, at 20 DEG C, 25 DEG C, 30 DEG C, 35 DEG C, 40 DEG C, cultivate 7d, measure the degradation rate of each process.
Medium pH is on the impact of degradation bacteria MC-10 degradation rate: configure respectively pH=6,6.5,7,7.5, the 50ppm quinclorac minimal medium of 8, the PBS bacteria suspension of the MC-10 of inoculation 5%, in 30 DEG C, the constant-temperature table of 150r/min cultivates 7d, measure the degradation effect of each process.
Incubation time is on the impact of degradation bacteria MC-10 degradation effect: in the quinclorac minimal medium of 50ppm, pH=7, inoculate the MC-10PBS bacteria suspension of 5%, in 30 DEG C, the constant-temperature table of 150r/min cultivates 7d, within each 24 hours, get a sample, measure dichloroquinoline acid content.
Result shows, and the best degradation condition of quinclorac degradation bacteria MC-10 is: 35 ° of C, pH value 7.5.Quinclorac initial mass concentration has good degradation effect at 10ppm-50ppm, and degradation rate can reach 90%.When cultivating by the 6th day, degradation bacteria is more abundant to the degraded of dichloroquinoline phosphoric acid.
Embodiment 5: the field planting effect of quinclorac degradation bacteria MC-10 in soil and the repair ability of quinclorac contaminated soil is assessed.
The quinclorac pesticide-clay mixture of 25mg/kg is configured in the mode of spraying, degradation bacteria MC-10 is cultured to logarithmic phase, get 5ml bacterium liquid centrifugal after remove LB, with the resuspended thalline of 20mlPBS damping fluid, be inoculated in the quinclorac pesticide-clay mixture configured, cultivate 7d in 35 DEG C of incubators after, measure the content of quinclorac in soil.Test arranges 3 and repeats and contrast.
Quinclorac content assaying method in pedotheque: accurately take 20.0 g pedotheques, be placed in ground triangular flask, add 60 mL methyl alcohol/0.01% phosphate aqueous solution (3:1, V/V), after Clothoid type vibrator extracts 1 h, extracting solution is transferred in 50 mL centrifuge tubes, centrifugal 5 min of 4000 r/min, filter paper filtering, gets supernatant liquor 30 mL, cross 0.22 μm of filter membrane, for measuring.Condition determination is with embodiment 3.
The degradation effect of degradation bacteria MC-10 7d at 30 DEG C can reach 74.9%.
Although above the present invention is described in detail with a general description of the specific embodiments, on basis of the present invention, can make some modifications or improvements it, this will be apparent to those skilled in the art.Therefore, these modifications or improvements without departing from theon the basis of the spirit of the present invention, all belong to the scope of protection of present invention.
Claims (6)
1. quinclorac efficient degrading bacteria MC-10, is characterized in that: Classification And Nomenclature be nicotianae (
arthrobacter nicotianae), quinclorac residual in can degrade efficiently soil and water, culture presevation is numbered: CGMCC No.8489, and on November 19th, 2013 is in Chinese culture presevation management committee common micro-organisms center preservation.
2. the application of quinclorac efficient degrading bacteria MC-10 as claimed in claim 1 in degraded quinclorac.
3. quinclorac efficient degrading bacteria MC-10 as claimed in claim 1 is administering the application in quinclorac contaminated soil.
4. one kind comprises the microbial inoculum of quinclorac efficient degrading bacteria MC-10 described in claim 1.
5. a kind of application of microbial inoculum in degraded quinclorac comprising quinclorac efficient degrading bacteria MC-10 described in claim 1 as claimed in claim 4.
6. the application in quinclorac contaminated soil administered by a kind of microbial inoculum comprising quinclorac efficient degrading bacteria MC-10 described in claim 1 as claimed in claim 4.
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