CN110358696A - The microbial bacterial agent of atrazine pesticide residue in a kind of degradation soil - Google Patents
The microbial bacterial agent of atrazine pesticide residue in a kind of degradation soil Download PDFInfo
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Abstract
The invention discloses a kind of microbial bacterial agents of atrazine pesticide residue in degradation soil, are related to microorganisms technical field, the constituent and its mass percent of microbial bacterial agent are as follows: microbial bacteria 25-30%, humic acid 30-40%, bentonite 25-35%, auxiliary agent 1-2%；Microbial bacteria includes bacillus subtilis and Methylotrophic bacillus.The present invention also provides a kind of preparation method and application of the microbial bacterial agent of atrazine pesticide residue in degradation soil, and the preparation of microbial bacterial agent includes bacillus subtilis microbial agent and the preparation of Methylotrophic gemma bacillus agent and the preparation of microbial bacterial agent.Microbial bacterial agent provided by the invention is applied to the degradation process of atrazine pesticide residue in soil, and the pesticide residue of atrazine, reduces the generation of crop disease, improve crop anti-adversity, promote crop growing state, improve crop quality in soil of effectively degrading.
Description
Technical field
The present invention relates to microorganisms technical field, in particular to the microorganism of atrazine pesticide residue in a kind of degradation soil
Microbial inoculum.
Background technique
China's cultivated area is wide, and frequency occurs for geographical environment and weather conditions multiplicity, crops a great variety, disease pest and weed
It is numerous, it causes and seriously threatens to agricultural production.People are using pesticide as weapon, in the practice with disease pest and weed long-term struggle, product
Experience abundant, significant effect are tired out.But pesticide is a large amount of using on the one hand the drug resistance of pest is continued to increase, on the other hand
Also it wrecks pest natural enemy greatly, and then increases agricultural production to the dependence of pesticide, cause a series of agricultural production
The problem of product and food safety.
With being continuously increased for Pesticide use amount, the residual quantity of Pesticide Residue in Soil is also being continuously increased, and causes the hair of crop
Sick rate rises, and large dosage applies pesticide repeatedly, will lead to vicious circle, aggravates soil pollution situation, endangers ecological environment security.
Summary of the invention
According to technological deficiency existing in the prior art, the technical problems to be solved by the present invention are: providing a kind of degradation
The microbial bacterial agent of atrazine pesticide residue in soil solves being continuously increased because of Pesticide use amount, causes Pesticide Residue in Soil
Residual quantity also the problem of being continuously increased, reduces the disease incidence of crop, reduces soil pollution situation, safety of preserving the ecological environment.
In order to realize technical purpose of the invention, the microbial bacteria of atrazine pesticide residue in a kind of degradation soil of the present invention
The technical solution of agent is:
The microbial bacterial agent of atrazine pesticide residue in a kind of degradation soil, the constituent of the microbial bacterial agent and its
Mass percent are as follows: microbial bacteria 25-30%, humic acid 30-40%, bentonite 25-35%, auxiliary agent 1-2%.
As a further improvement of the present invention, the microbial bacteria includes bacillus subtilis and Methylotrophic gemma bar
Bacterium, the bacillus subtilis (Bacillus subtilis), abbreviation JQ-001 bacterial strain are preserved on November 09th, 2015
China Committee for Culture Collection of Microorganisms's common micro-organisms center, depositary institution address: BeiChen West Road, Chaoyang District, BeiJing City 1
Number institute, deposit number are CGMCC No.11632；Methylotrophic bacillus (the Bacillus
Methylotrophicus), abbreviation JQ-018 bacterial strain is preserved in Chinese microorganism strain preservation pipe on December 07th, 2015
Reason committee common micro-organisms center, depositary institution address: Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, deposit number CGMCC
No.11838。
As a further improvement of the present invention, the living bacteria count of the microbial bacteria >=200,000,000/g, the content of organic matter >=
40%.
As a further improvement of the present invention, the auxiliary agent includes sodium humate, potassium fulvate, potassium dihydrogen phosphate and sulfuric acid
Ammonium etc..
As a further improvement of the present invention, the preparation method of the microbial bacterial agent includes the following steps:
(1) actication of culture: by the bacillus subtilis of cryo-conservation and the Methylotrophic Bacillus strain
It is inoculated on beef-protein medium respectively, cultivates 24-36h under 36-38 DEG C of constant temperature；
(2) preparation of primary seed solution: preparing beef extract-peptone Shake flask medium, is dispensed using 500mL triangular flask,
Liquid amount 200mL, the bacillus subtilis and the Methylotrophic bacillus after high pressure sterilization after access activation, temperature
36-38 DEG C is spent, cultivates 20-24h under the conditions of revolving speed 200-260rpm/min；
(3) preparation of secondary seed solution: preparing seed tank culture base, and the primary seed solution that step (2) is obtained is pressed
The inoculum concentration of 3-5% is respectively connected to 500L seeding tank, and 36-38 DEG C of temperature, revolving speed 180-260rpm/min, ventilatory capacity 0.6-
18-24h is cultivated under the conditions of 1.2V/Vmin；
(4) large-scale industry fermented and cultured: the secondary seed solution that step (3) is obtained is fermented by large-scale industry
The 5-10% inoculum concentration of tank used medium volume ratio is respectively connected to large-scale industry fermentor, and 36-38 DEG C of temperature, revolving speed 180-
24-30h is cultivated under the conditions of 260rpm/min, ventilatory capacity 0.6-1.2V/ Vmin, completes fermentation, obtains bacillus subtilis hair
Zymotic fluid and Methylotrophic fermentation of bacillus liquid；
(5) preparation of bacillus subtilis microbial agent and Methylotrophic gemma bacillus agent: the institute that step (4) is obtained
Bacillus subtilis fermentation liquor and the Methylotrophic fermentation of bacillus liquid are stated, is 1:1 by fermentor dry matter weight ratio
Addition diatomite is adsorbed, and filters pressing, concentration, drying are then carried out；
(6) it the detection of the bacillus subtilis microbial agent and the Methylotrophic gemma bacillus agent bacteria containing amount: uses
The bacteria containing amount of filter cake after dilution-plate method detection is dry；
(7) preparation of the microbial bacterial agent: by mass percentage, the bacillus subtilis that step (5) is obtained
Microbial inoculum and the Methylotrophic gemma bacillus agent are proportionally added into diatomite and are diluted to spore concentration concentration >=5,000,000,000/g,
Then it is uniformly mixed with the humic acid 30-40%, the bentonite 25-35% and the auxiliary agent 1-2%, it is mixed
Airslide disintegrating mill is added in material, and fineness controls 325-400 mesh, the microbial bacterial agent is obtained after crushing.
As a further improvement of the present invention, filters pressing uses plate and frame filter press in the step (5).
As a further improvement of the present invention, dry in the step (5) to use boiling-bed drying, the drying temperature is
65-80℃。
As a further improvement of the present invention, atrazine residual quantity inspection in the microbial bacterial agent treated pedotheque
Survey method is as follows:
(1) instrument and parameter setting: Shimadzu LC-20A, chromatographic column C18；Mobile phase: methanol/water=65/35；Column temperature: 40
℃；Wavelength: 231nm；Flow velocity: 1mL/min；Sample volume: 10 μ L；
(2) atrazine standard substance standard curve is drawn；
(3) 20g is taken to air-dry soil sample in 250mL triangular flask, after a small amount of distilled water is wet plus 35mL methanol, oscillation 1h are filtered
Paper filtering, residue are washed for 2 times with 40mL methanol point, merge solution, remove methanol (40 DEG C) in decompression on revolving instrument；
(4) residue is dissolved with 0.1mol/L HCL solution 15mL, NaOH adjusts PH8-10, uses 50mL bis- on separatory funnel
3 extractions of chloromethanes point, collect lower layer's organic phase and 0.5g anhydrous magnesium sulfate are added and go moisture removal, anhydrous slufuric acid magnesium precipitate mistake
Filter, is evaporated methylene chloride in 40 DEG C again；
(5) 2mL methanol is added when being soon evaporated completely and continues to rotate for solution, finally uses 2mL methanol constant volume, membrane filtration,
Sample detection obtains result.
Compared with prior art, the beneficial effects of the present invention are:
Microbial bacterial agent provided by the invention has the advantages that with short production cycle, active high, at low cost, is conducive to industrialize
Production, two plants of strains of application it is adaptable it is strong, stability is good, growth rate is fast and it is strong to pathogen Antagonism the features such as,
And synergy is significant, and applied to the degradation process of atrazine pesticide residue in soil, the agriculture of atrazine in soil of effectively degrading
Medicine residual, reduces the generation of crop disease, improves crop anti-adversity, promotes crop growing state, improves crop quality, while promoting to make
Object growth, increases crop yield, maintaining ecological balance.The microbial bacterial agent is applied to the degradation of atrazine pesticide residue in soil
Process can effectively reduce the use of chemical pesticide, reduce pollution of the chemical pesticide to soil；It is effectively reduced simultaneously because of chemical pesticide
The enhancing of pathogen drug resistance caused by effect, soil protection microecosystem balance improve crop living environment and improve
Its disease resistance improves important in inhibiting to crop yield and quality.
Detailed description of the invention
Fig. 1 is atrazine standard curve；
Fig. 2 is atrazine standard sample spectrogram；
Fig. 3 is atrazine content spectrogram after the soil pattern of addition atrazine is handled 11 days；
Fig. 4 be add atrazine and 200,000,000 microbial bacterial agent particles soil pattern handle 11 days after atrazine content spectrogram；
Fig. 5 is atrazine content spectrogram after the soil pattern of addition atrazine is handled 43 days；
Fig. 6 be add atrazine and 200,000,000 microbial bacterial agent particles soil pattern handle 43 days after atrazine content spectrogram；
Fig. 7 is residue degrading situation of the microbial bacterial agent processing to atrazine in corn field.
Specific embodiment
Bacillus subtilis (Bacillus subtilis) provided by the invention, abbreviation JQ-001 bacterial strain is in 2015
November 09 was preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, depositary institution address: Beijing
The institute of Chaoyang District North Star West Road 1, deposit number are CGMCC No.11632；Methylotrophic bacillus (Bacillus
Methylotrophicus), abbreviation JQ-018 bacterial strain is preserved in Chinese microorganism strain preservation pipe on December 07th, 2015
Reason committee common micro-organisms center, depositary institution address: Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, deposit number CGMCC
No.11838。
To be further elaborated to beneficial effect of the present invention, following specific embodiments have been carried out, special instruction, below
Specific embodiment is intended to better understand the present invention, is not restricted to the scope of the present invention.Experimental method in following embodiments,
It unless otherwise specified, is conventional method.Experimental material used in following embodiments is unless otherwise specified shop purchase
Routine biochemistry preparation.Quantitative test in following embodiment is respectively provided with and repeats to test three times, and results are averaged.
Embodiment 1: the preparation of microbial bacterial agent
1, it is a kind of degradation soil in atrazine pesticide residue microbial bacterial agent, the constituent and its matter of microbial bacterial agent
Measure percentage are as follows: microbial bacteria 25-30%, humic acid 30-40%, bentonite 25-35%, auxiliary agent 1-2%.
2, microbial bacteria includes bacillus subtilis and Methylotrophic bacillus, bacillus subtilis (Bacillus
), subtilis abbreviation JQ-001 bacterial strain is preserved in China Committee for Culture Collection of Microorganisms on November 09th, 2015
Common micro-organisms center, depositary institution address: Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, deposit number CGMCC
No.11632；Methylotrophic bacillus (Bacillus methylotrophicus), abbreviation JQ-018 bacterial strain is in 2015
On December 07, in is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, depositary institution address: Beijing
The institute of city, North Star West Road, Chaoyang District 1, deposit number are CGMCC No.11838.
The identification related content of bacillus subtilis JQ-001 bacterial strain and Methylotrophic bacillus JQ-018 bacterial strain is such as
Under:
The identification (CGMCC No.11632) of bacillus subtilis JQ-001 bacterial strain
(1) Microbiological Characteristics: bacterial strain is under NA culture medium, 37 DEG C of environment, after cultivating 12-18h, bacterium colony rough surface
Opaque, dirty white or yellowish, flat dry matt, edge is irregular, not chromogenesis.Bacterial strain is rod-shaped, has Zhousheng whip
Hair, Gram's staining is positive, aerobic.
(2) molecular biological characteristic: the 16SrDNA extension increasing sequence of JQ-001 bacterial strain is compared in NCBI database, with
Bacillus subtilis homology is 99.38%, and morphological feature, physiological and biochemical property in conjunction with JQ-001 bacterial strain determine
JQ-001 bacterial strain belongs to bacillus subtilis Bacillus subtilis.
The identification (CGMCC No.11838) of Methylotrophic bacillus JQ-018 bacterial strain:
(1) Microbiological Characteristics: JQ-018 thallus is in rod-short, and peritrichous can produce spore, Gram-positive.Bacterium
Strain is in PDA culture medium after 34 DEG C of culture 12h, and bacterium colony is round, and translucent white, surface is smooth, and intermediate projections do not generate color
Element, toughness.Bacterium colony is smaller on NA culture medium or does not grow.Liquid Culture forms mycoderm, produces spore afterwards for 24 hours, brown color is muddy
It is turbid, there is precipitating.Strains expressed aerobic growth.
(2) bio-chemical characteristics:
The physiological and biochemical property of Methylotrophic bacillus JQ-018
Note: "+" indicates positive reaction, and "-" indicates negative reaction
(3) molecular biology identification:
The base sequence of 1414bp long is obtained after the sequencing of JQ-018 bacterial strain, which is compared in NCBI database, with
Bacillus methylotrophicus homology reaches 99.38%, morphological feature, Physiology and biochemistry in conjunction with JQ-018 bacterial strain
Characteristic determines that JQ-018 bacterial strain detected belongs to Methylotrophic bacillus.
3, the living bacteria count of microbial bacteria >=200,000,000/g, the content of organic matter >=40%.
4, auxiliary agent includes sodium humate, potassium fulvate, potassium dihydrogen phosphate and ammonium sulfate etc..
5, the preparation method of microbial bacterial agent includes the following steps:
(1) actication of culture: the bacillus subtilis of cryo-conservation and Methylotrophic Bacillus strain are inoculated with respectively
In on beef-protein medium, 24-36h is cultivated under 36-38 DEG C of constant temperature；
(2) preparation of primary seed solution: preparing beef extract-peptone Shake flask medium, is dispensed using 500mL triangular flask,
Liquid amount 200mL, bacillus subtilis and Methylotrophic bacillus after high pressure sterilization after access activation, temperature 36-38
DEG C, 20-24h is cultivated under the conditions of revolving speed 200-260rpm/min；
(3) seed tank culture base, the primary seed solution that step (2) is obtained, by 3- the preparation of secondary seed solution: are prepared
5% inoculum concentration is respectively connected to 500L seeding tank, and 36-38 DEG C of temperature, revolving speed 180-260rpm/min, ventilatory capacity 0.6-1.2V/
18-24h is cultivated under the conditions of Vmin；
(4) large-scale industry fermented and cultured: the secondary seed solution that step (3) is obtained, by large-scale industry fermentor institute
Large-scale industry fermentor is respectively connected to the 5-10% inoculum concentration of culture volume ratio, 36-38 DEG C of temperature, revolving speed 180-
24-30h is cultivated under the conditions of 260rpm/min, ventilatory capacity 0.6-1.2V/Vmin, completes fermentation, obtains bacillus subtilis hair
Zymotic fluid and Methylotrophic fermentation of bacillus liquid；
(5) preparation of bacillus subtilis microbial agent and Methylotrophic gemma bacillus agent: step (4) is obtained withered
Careless fermentation of bacillus liquid and Methylotrophic fermentation of bacillus liquid are that 1:1 adds diatom by fermentor dry matter weight ratio
Soil is adsorbed, and filters pressing, concentration, drying are then carried out；
(6) detection of bacillus subtilis microbial agent and Methylotrophic gemma bacillus agent bacteria containing amount: dilution plate is used
The bacteria containing amount of filter cake after method detection is dry；
(7) preparation of microbial bacterial agent: by mass percentage, the bacillus subtilis microbial agent and first that step (5) is obtained
Base auxotype gemma bacillus agent is proportionally added into diatomite and is diluted to spore concentration concentration >=5,000,000,000/g, then with humic acid
30-40%, bentonite 25-35% and auxiliary agent 1-2% are uniformly mixed, and airslide disintegrating mill, fineness control is added in mixed material
325-400 mesh processed, obtains microbial bacterial agent after crushing.
6, filters pressing uses plate and frame filter press in step (5).
7, dry in step (5) to use boiling-bed drying, drying temperature is 65-80 DEG C.
The research of embodiment 2, microbial bacterial agent to atrazine chemical residual degradation situation
Soil sample source: plastic shed soil sample is tested by Shandong Jing Qing agricultural science and technology Co., Ltd
Test site: Shandong Jing Qing agricultural science and technology Co., Ltd researches and develops laboratory
1, atrazine residues detection method is as follows in microbial bacterial agent treated pedotheque:
(1) instrument and parameter setting: Shimadzu LC-20A, chromatographic column C18；Mobile phase: methanol/water=65/35；Column temperature: 40
℃；Wavelength: 231nm；Flow velocity: 1mL/min；Sample volume: 10 μ L；
(2) atrazine standard substance standard curve is drawn, as shown in Figure 1 and Figure 2；
(3) 20g is taken to air-dry soil sample in 250mL triangular flask, after a small amount of distilled water is wet plus 35mL methanol, oscillation 1h are filtered
Paper filtering, residue are washed for 2 times with 40mL methanol point, merge solution, remove methanol (40 DEG C) in decompression on revolving instrument；
(4) residue is dissolved with 0.1mol/L HCL solution 15mL, NaOH adjusts PH8-10, uses 50mL on separatory funnel
3 extractions of methylene chloride point, collect lower layer's organic phase and 0.5g anhydrous magnesium sulfate are added and go moisture removal, anhydrous slufuric acid magnesium precipitate mistake
Filter, is evaporated methylene chloride in 40 DEG C again；
(5) 2mL methanol is added when being soon evaporated completely and continues to rotate for solution, finally uses 2mL methanol constant volume, membrane filtration,
Sample detection obtains result.
2, research of the microbial bacterial agent to atrazine chemical residual degradation situation
(1) 100mg/mL atrazine mark experimental design: is added in the soil pattern without containing atrazine through detecting to 100g
Quasi- substance 1mL, setting control and parallel test, simulated rice field are added pure water and there were not soil surface about 5cm, are placed in experimental bench
On, avoid direct sunlight, timing adding water, atrazine content of the difference after detection processing 11 days and after 43 days in soil pattern.
(2) sample pre-treatments: the processed soil pattern after taking 20g to air-dry is placed in 250mL triangular flask, with a small amount of
After distilled water is wet plus 35mL methanol, oscillation 1h, filter paper filtering, residue 2 cleanings of 40mL methanol point merge solution, Yu Xuan
It steams decompression on instrument and removes methanol (40 DEG C).
Residue is dissolved with the HCL solution 15mL of 0.1mol/L, PH to 8-10 is adjusted with NaOH, is used on separatory funnel
Points of 3 times extractions of 50mL methylene chloride, collect lower layer's organic phase and 0.5g anhydrous magnesium sulfate are added and remove moisture removal, with filter paper by nothing
Water magnesium sulfate precipitating filtering, is evaporated methylene chloride in 40 DEG C again.
2mL methanol is added when solution is soon evaporated completely to continue to rotate, 2mL methanol constant volume is finally used, on membrane filtration
Sample detection.
(3) testing result such as Fig. 3, Fig. 4, Fig. 5, shown in Fig. 6:
Can be seen that the microbial bacterial agent from Fig. 3, Fig. 4, Fig. 5 and Fig. 6 has preferable degradation effect to atrazine in soil
Fruit.
The application of embodiment 3, microbial bacterial agent field experiment
1, test medicine
The microbial bacterial agent of atrazine pesticide residue in degradation soil prepared by the present invention.
2, trial crops and kind
Trial crops: corn；
Crop varieties: first jade 335.
3, test site
Test site: Jilin Agriculture University experimental plot in the school.
4, field experiment
(1) test medicine is arranged
(Jilin Province eight reaches pesticide for microbial bacterial agent (Shandong Jing Qing agricultural science and technology Co., Ltd), 38% atrazine suspending agent
Co., Ltd), microbial bacterial agent 600kg/hm2；38% atrazine suspending agent using highest recommend application dosage (2280ga.i./
It hm2 is) low dosage of final residue test, 1.5 times of highest recommended doses (3420ga.i./hm2) are tested as final residue
High dose, it is specific as described in Table 1.
Table 1, the setting of test medicine concentration
(2) experimental design and arrangement
Cell arrangement: setting experimental plot by pesticide residue test rule requirement, by the low to high sequence random alignment of concentration,
Minizone sets protection band, and plot area 30m2 is repeated 3 times, separately sets check plot, amounts to 24 cells.
(3) test method
Residue test design: imposing on microbial bacterial agent granule as base fertilizer ditch in corn field soil, then sows,
Before corn broadcasts rear seedling, with 38% atrazine suspending agent 2280ga.i./hm2 and 3420ga.i./hm2, it is administered respectively, with bacterial manure
Untreated application herbicide is to compare, 40L/ mus of the amount of being watered.Before applying herbicide (taking 5 parts of samples at random), application after 30d,
60d and 90d are separately sampled, totally 77 processing.6-12 sampled point acquisition 0-15cm pedotheque 2kg of random selection and soil
Earth control sample removes the sundries such as rubble, weeds and plant roots and stems in soil, is kept sample 500g after mixing using quartering, dress
Enter in sample container, glue label, field sample after acquisition, transports laboratory back in 8h, and laboratory sample storage is made immediately
It is stored in -20 DEG C of refrigerator-freezers and saves.
The preparation of soil water experiment room sample is carried out by the regulation of SOP FT-04-01.
Influence to crop yield: carrying out cauline leaf to all processing in one heart stage of three leaves and be sprayed, and concentration is 50g/ mus, is watered
40L/ mu of amount surveys production when harvest.
(4) test result
The measurement of residual quantity of the microbial bacterial agent of the present invention in corn field to atrazine in the soil, as a result such as table 2, table 3,
Fig. 7.
The residual quantity of original atrazine in table 2, soil
Residual quantity of the atrazine in corn field soil after table 3, microbial bacterial agent processing
From table 2, table 3 can be seen that the residual quantity of 30d, 60d and 90d after atrazine is applied in corn field soil not
With on a declining curve, 30d after processing in processing, the atrazine processing residual quantity of high concentration is higher than low concentration processing, wherein micro-
Bacteria agent+low concentration atrazine processing residual quantity is minimum, is 0.052mg/kg, residual quantity sequence are as follows: and atrazine high concentration >
Atrazine high concentration+microbial bacterial agent > atrazine low concentration > atrazine low concentration+microbial bacterial agent；60d after processing, it is highly concentrated
The atrazine processing residual quantity of degree is still higher than low concentration processing, and the residual quantity that wherein low concentration atrazine is individually handled is minimum, is
0.028mg/kg, atrazine high concentration residual quantity > atrazine high concentration+microbial bacterial agent > atrazine low concentration+microbial bacterial agent
> atrazine low concentration；The atrazine residual quantity of 90d after processing, all processing are below 0.031mg/kg, and microbial bacterial agent
Processed atrazine residual quantity is below untreated, and wherein the residual quantity of microbial bacterial agent+low concentration atrazine processing is most
It is low, it is 0.017mg/kg, atrazine high concentration residual quantity > atrazine low concentration > atrazine high concentration+microbial bacterial agent > green bristlegrass is gone
Saliva low concentration+microbial bacterial agent.
From figure 7 it can be seen that influence of the microbial bacterial agent to atrazine residual quantity in corn field soil, the 90d to after handling
When, it is most that microbial bacterial agent 600kg/hm2+38% atrazine suspending agent 2280ga.i./hm2, which handles atrazine residual quantity,
It is few, it was demonstrated that the atrazine residue degrading amount of low concentration, which is lower than, in bacterial manure treated corn field soil does not apply bacterial manure processing.
The above is only preferable embodiment of the invention, is not intended to limit the present invention in any form, Ren Hewei
It is detached from technical solution of the present invention content, according to the technical essence of the invention to any simple modification, transformation made by above example
Material equivalent variations and modification, all of which are still within the scope of the technical scheme of the invention.
Claims (8)
1. the microbial bacterial agent of atrazine pesticide residue in a kind of degradation soil, it is characterised in that: the group of the microbial bacterial agent
At ingredient and its mass percent are as follows: microbial bacteria 25-30%, humic acid 30-40%, bentonite 25-35%, auxiliary agent 1-2%.
2. the microbial bacterial agent of atrazine pesticide residue in a kind of degradation soil according to claim 1, it is characterised in that:
The microbial bacteria includes bacillus subtilis and Methylotrophic bacillus, the bacillus subtilis (Bacillus
), subtilis abbreviation JQ-001 bacterial strain is preserved in China Committee for Culture Collection of Microorganisms on November 09th, 2015
Common micro-organisms center, depositary institution address: Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, deposit number CGMCC
No.11632；The Methylotrophic bacillus (Bacillus methylotrophicus), abbreviation JQ-018 bacterial strain in
On December 07th, 2015 is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, depositary institution address:
Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, deposit number are CGMCC No.11838.
3. the microbial bacterial agent of atrazine pesticide residue in a kind of degradation soil according to claim 2, it is characterised in that:
The living bacteria count of the microbial bacteria >=200,000,000/g, the content of organic matter >=40%.
4. the microbial bacterial agent of atrazine pesticide residue in a kind of degradation soil according to claim 1, it is characterised in that:
The auxiliary agent includes sodium humate, potassium fulvate, potassium dihydrogen phosphate and ammonium sulfate etc..
5. the preparation method of the microbial bacterial agent of atrazine pesticide residue in a kind of degradation soil according to claim 1,
It is characterized by: the preparation method of the microbial bacterial agent includes the following steps:
(1) actication of culture: the bacillus subtilis of cryo-conservation and the Methylotrophic Bacillus strain are distinguished
It is inoculated on beef-protein medium, cultivates 24-36h under 36-38 DEG C of constant temperature；
(2) preparation of primary seed solution: preparing beef extract-peptone Shake flask medium, is dispensed using 500mL triangular flask, liquid amount
200mL, the bacillus subtilis and the Methylotrophic bacillus after high pressure sterilization after access activation, temperature 36-
38 DEG C, 20-24h is cultivated under the conditions of revolving speed 200-260rpm/min；
(3) seed tank culture base, the primary seed solution that step (2) is obtained, by 3-5% the preparation of secondary seed solution: are prepared
Inoculum concentration be respectively connected to 500L seeding tank, 36-38 DEG C of temperature, revolving speed 180-260rpm/min, ventilatory capacity 0.6-1.2V/V
18-24h is cultivated under the conditions of min；
(4) large-scale industry fermented and cultured: the secondary seed solution that step (3) is obtained, by large-scale industry fermentor institute
Large-scale industry fermentor is respectively connected to the 5-10% inoculum concentration of culture volume ratio, 36-38 DEG C of temperature, revolving speed 180-
24-30h is cultivated under the conditions of 260rpm/min, ventilatory capacity 0.6-1.2V/Vmin, completes fermentation, obtains bacillus subtilis hair
Zymotic fluid and Methylotrophic fermentation of bacillus liquid；
(5) preparation of bacillus subtilis microbial agent and Methylotrophic gemma bacillus agent: the withered grass that step (4) is obtained
Fermentation of bacillus liquid and the Methylotrophic fermentation of bacillus liquid are that 1:1 adds silicon by fermentor dry matter weight ratio
Diatomaceous earth is adsorbed, and filters pressing, concentration, drying are then carried out；
(6) detection of the bacillus subtilis microbial agent and the Methylotrophic gemma bacillus agent bacteria containing amount: using dilution
The bacteria containing amount of filter cake after flat band method detection is dry；
(7) preparation of the microbial bacterial agent: by mass percentage, the bacillus subtilis microbial agent that step (5) is obtained
Diatomite is proportionally added into the Methylotrophic gemma bacillus agent and is diluted to spore concentration concentration >=5,000,000,000/g, then
It is uniformly mixed with the humic acid 30-40%, the bentonite 25-35% and the auxiliary agent 1-2%, mixed material is added
Airslide disintegrating mill, fineness control 325-400 mesh, the microbial bacterial agent are obtained after crushing.
6. the preparation method of the microbial bacterial agent of atrazine pesticide residue in a kind of degradation soil according to claim 5,
It is characterized by: filters pressing uses plate and frame filter press in the step (5).
7. the preparation method of the microbial bacterial agent of atrazine pesticide residue in a kind of degradation soil according to claim 5,
It is characterized by: dry in the step (5) use boiling-bed drying, the drying temperature is 65-80 DEG C.
8. the application of the microbial bacterial agent of atrazine pesticide residue, special in a kind of degradation soil according to claim 1
Sign is: atrazine residues detection method is as follows in the microbial bacterial agent treated pedotheque:
(1) instrument and parameter setting: Shimadzu LC-20A, chromatographic column C18；Mobile phase: methanol/water=65/35；Column temperature: 40 DEG C；Wave
It is long: 231nm；Flow velocity: 1mL/min；Sample volume: 10 μ L；
(2) atrazine standard substance standard curve is drawn；
(3) it takes 20g to air-dry soil sample in 250mL triangular flask, adds 35mL methanol after a small amount of distilled water is wet, vibrate 1h, filter paper mistake
Filter, residue are washed for 2 times with 40mL methanol point, merge solution, remove methanol (40 DEG C) in decompression on revolving instrument；
(4) residue is dissolved with 0.1mol/L HCL solution 15mL, NaOH adjusts PH8-10, uses 50mL dichloromethane on separatory funnel
3 extractions of alkane point collect lower layer's organic phase and 0.5g anhydrous magnesium sulfate are added and remove moisture removal, and anhydrous slufuric acid magnesium precipitate filters, then
Methylene chloride is evaporated inferior to 40 DEG C；
(5) 2mL methanol is added when solution is soon evaporated completely to continue to rotate, finally uses 2mL methanol constant volume, membrane filtration, loading
Detection obtains result.
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Application publication date: 20191022 |