CN105198548B - Biological organic fertilizer and its preparation method and application with degradation Acetochlor effect - Google Patents
Biological organic fertilizer and its preparation method and application with degradation Acetochlor effect Download PDFInfo
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Abstract
The invention belongs to biological fertilizer fields, and in particular to a kind of biological organic fertilizer with degradation Acetochlor effect further relates to the preparation method and application of above-mentioned biological organic fertilizer.The biological organic fertilizer of the present invention includes following major ingredient：Microbial bacterial agent, fixation support；The microbial bacterial agent is Rhodococcus sp, Rhodococcus sp（Rhodococcussp.）AC 1 is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, and deposit number is CGMCC NO.10778, and the deposit date is on April 30th, 2015；Fixation support is the alga fertilizer using ferment-fermented mistake.The biological organic fertilizer fertilizer efficiency of the present invention is high, and compared with common biological organic fertilizer, the organic fertilizer of the present invention uses Rhodococcus sp as microbial inoculum, energy efficient degradation alachlor, Acetochlor and butachlor, it is composed with wider degradation, degradation character excellent strains A C 1 is that it provides the foundation applied to the environmental organism reparation of chloro-acetyl amine pollution of herbicide from now on.
Description
Technical field
The invention belongs to biological fertilizer fields, and in particular to a kind of biological organic fertilizer with degradation Acetochlor effect, also
It is related to the preparation method and application of above-mentioned biological organic fertilizer.
Background technology
Organic bio-fertilizer be mainly derived from plant and（Or）Animal imposes on soil to provide plant nutrient as its major function
Carbonaceous material.Through biological substance, animals and plants waste, plant residue processing, poisonous and harmful substance therein is eliminated,
Rich in a large amount of benefit materials, the machine bio-fertilizer of common are is typically only crop and provides nutrient, rarely seen to have certain in degradation soil
The bio-fertilizer of pesticide appears on the market, the especially rarely seen biological organic fertilizer with degradation Acetochlor effect.
Acetochlor also known as resistance to this of standing grain are pacified with the grass that disappears, entitled -6 methyl-N- ethoxyl methyls of the 2- ethyls-alpha-chloro acetyl of chemistry
It is acetamide-group herbicides before a kind of internal-suction type of Monsanto Chemicals' exploitation, selective bud for aniline.Acetochlor has height
The advantages that effect, wide spectrum, low toxicity, has become one of three big herbicide of China at present, is widely used in corn, peanut, cotton and big
The prevention of beans field 1 year raw grassy weed and part broad leaved weed.Due to largely applying so that Acetochlor in the soil residual
Increase is stayed, the safety of the ecosystem is seriously threatened.Existing research reports that a large amount of uses of Acetochlor can cause agro-ecosystem
Middle-size and small-size animal, the quantity of microbial population and the abundance of uniting reduce.Although Acetochlor is less toxic, but still is good for animals and humans
Health has large effect, Environmental Protection Agency that Acetochlor is classified as B-2 class carcinogenic substances.In addition, the excessive application of Acetochlor
Can also serious poisoning be generated to succession crop, huge loss is brought to agricultural production.
The Major degradation pathways of Acetochlor include physical method, chemical method and biological method in environment at present, and with micro-
Biological prosthetic based on biodegradation, due to its economical and efficient, the features such as being not likely to produce secondary pollution, has been acknowledged as most having
The method of future.But due to the factors such as microbe density is small, is easily washed away by flowing water, is not easy to survive in complicated soil environment
Cause the degradation efficiency in field soil environmental applications low.And emerging technology-immobilization technology can effectively solve above-mentioned ask
Topic.Microorganism high density is fixed on suitable micro-organisms thalli growth and is kept on active carrier, Complex Soil can be effectively overcome
The influence of earth environment efficiently plays its degradation.
In recent years, it is protection agricultural production, stable food volume increase, the usage amount of chloroamides class herbicide is promoted to increase year by year
Add, it has also become one of three big herbicide of China.Since half-life period is longer, not volatile and photodissociation easily remains, and endangers people and animals
Greatly, excessive frequently to lead to serious environmental risk using this kind of herbicide the features such as potential carcinogenicity.Therefore, chlorine is studied
A big feature of academia is had become for the microbial degradation of acetyl herbicide.
Invention content
In order to solve the above technical problems, the present invention provides a kind of biological organics with degradation Acetochlor effect
Fertilizer；
The present invention also provides the preparation method and application of above-mentioned biological organic fertilizer.
The biological organic fertilizer with degradation Acetochlor effect of the present invention, including following major ingredients：
Microbial bacterial agent, fixation support；
Above-mentioned microbial bacterial agent is Rhodococcus sp, Rhodococcus sp（Rhodococcussp.）AC-1 is preserved in China Microbiological
Culture presevation administration committee common micro-organisms center, preservation address：Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3；Preservation is compiled
Number be CGMCC NO.10778, the deposit date is on April 30th, 2015；
Fixation support is the alga fertilizer using ferment-fermented mistake；
The mass ratio of microbial bacterial agent and fixation support is：1~2：2~3, microbial bacterial agent a concentration of 1 × 109
Cfu/ml~1 × 1010 cfu/ml。。
The preparation method of above-mentioned fixation support is as follows：
（1）Seaweed is taken, is dried to its moisture to 35 ~ 55%；
（2）Leavening is added in above-mentioned seaweed, the leavening is aspergillus oryzae, Trichoderma viride, bacillus subtilis
Bacterium, Bacillus cercus composite bacterium powder, addition accounts for raw material seaweed weight 2-4 ‰, ferments 10-15 days at 37-45 DEG C；
Aspergillus oryzae, Trichoderma viride, bacillus subtilis, Bacillus cercus weight ratio be：1-2：1-4：1-3：1-
2；
（3）By step（2）The middle seaweed drying through everfermentation is crushed to 4 ~ 10 mesh until its moisture is 15 ~ 20%；
（4）In step（3）Material in alkaline matter is added, adjust the pH to 6.5 ~ 7.5 of material, stir evenly, must consolidate
Surely change carrier.
Preferably, above-mentioned alkaline matter is plant ash；
Step（2）In, the aspergillus oryzae, Trichoderma viride, bacillus subtilis, Bacillus cercus weight ratio
For：1：3：2：1, addition accounts for the 3 ‰ of raw material seaweed weight, ferments 12 days at 40 DEG C.
Step（4）In, adjust pH to 7.5.
The preparation method of biological organic fertilizer with degradation Acetochlor effect, including following steps：
The making of fixation support：
（1）Seaweed is taken, is dried to its moisture to 35 ~ 55%；
（2）Leavening is added in above-mentioned seaweed, the leavening is aspergillus oryzae, Trichoderma viride, bacillus subtilis
Bacterium, Bacillus cercus composite bacteria agent, addition accounts for raw material seaweed weight 2-4 ‰, ferments 10-15 days at 37-45 DEG C；
Aspergillus oryzae, Trichoderma viride, bacillus subtilis, Bacillus cercus weight ratio be 1-2：1-4：1-3：1-2；
（3）By step（2）The middle seaweed drying through everfermentation is crushed to 4 ~ 10 mesh until its moisture is 15 ~ 20%；
（4）In step（3）Material in alkaline matter is added, adjust the pH to 6.5 ~ 7.5 of material, stir evenly, must consolidate
Surely change carrier；
The preparation of biological organic fertilizer：
By Rhodococcus sp（Rhodococcus sp.）Planting site soil 1-5 after AC-1 microbial inoculums, fixation support, drying：1-
4：The weight ratio of 10-40 is uniformly mixed, its moisture of planting soil after drying is 1-4%, is placed 2-3 days at room temperature,
Up to biological organic fertilizer.
Preferably, there is the preparation method of the biological organic fertilizer of degradation Acetochlor effect, including following steps：
The making of fixation support：
（1）Seaweed is taken, is dried to its moisture to 40%；
（2）Leavening is added in above-mentioned seaweed, the leavening is aspergillus oryzae, Trichoderma viride, bacillus subtilis
Bacterium, Bacillus cercus composite bacteria agent, addition accounts for raw material seaweed and weighs 2.5 ‰, ferment 10-15 days at 40 DEG C；
Aspergillus oryzae, Trichoderma viride, bacillus subtilis, Bacillus cercus weight ratio be 1：3：2：1；
（3）By step（2）The middle seaweed drying through everfermentation is crushed to 5 mesh until its moisture is 20%；
（4）In step（3）Material in alkaline matter is added, adjust the pH to 6.5 ~ 7.5 of material, stir evenly, must consolidate
Surely change carrier；
The preparation of biological organic fertilizer：
By Rhodococcus sp（Rhodococcus sp.）Planting site soil 3 after AC-1 microbial inoculums, fixation support, drying：2：30
Weight ratio be uniformly mixed, it is dry after planting soil its moisture be 1-4%, place 2-3 days at room temperature to get life
Organic fertilizer.
Rhodococcus sp of the present invention（Rhodococcus sp.）AC-1 is gram positive bacterium；
Rhodococcus sp（Rhodococcussp.）AC-1 is in pale pink bacterium colony, bacterium colony projection, surface wettability light on LB tablets
Sliding, neat in edge is opaque, and strains A C-1 is in rod-shaped under transmission electron microscope（0.5~0.9 μ m, 3.5~4.5 μ
m）, atrichia；
Rhodococcus sp（Rhodococcus sp.）The physiological and biochemical property of AC-1 is：It is aerobic, it cannot move, catalase,
Urase is the positive；Oxidizing ferment, amylolytic enzyme and nitrate reductase are feminine gender；
By Rhodococcus sp（Rhodococcussp.）The 16S rRNA gene orders of AC-1 compare in database EzBilCloud
To analysis, as a result show strains A C-1 andRhodococcusIt is nearest to belong to affiliation, wherein withR. erythropolis DSM
43066TSimilitude up to 99.93%, withR. jialingiae djl-6-2TSimilitude is up to 99.49%；
By Rhodococcus sp（Rhodococcus sp.）AC-1 is in acetochlor（A concentration of 300 mg L-1）It is formed on tablet
Transparent degradation circle；
Rhodococcus sp（Rhodococcus sp.）The optimum temperature of AC-1, Acetochlor of degrading are 30 DEG C, and optimal pH is 7.5,
The Cu of 0.1 mM2+And Hg2+It is inhibited to strains A C-1 degradation Acetochlors, and the Fe of 0.1 mM2+Then strains A C-1 is dropped
Solving Acetochlor has facilitation.
Application of the biological organic fertilizer with degradation Acetochlor effect in planting grain class crop.
Application of the biological organic fertilizer with degradation Acetochlor effect in maize planting, peanut ground is also that the present invention is wanted
The range of protection.
The beneficial effects of the present invention are, biological organic fertilizers of the invention, and fertilizer efficiency is high, and with common biological organic fertilizer phase
Use Rhodococcus sp as microbial inoculum than, organic fertilizer of the invention, can efficient degradation alachlor, Acetochlor and butachlor, have compared with
Wide degradation spectrum, degradation character excellent strains A C-1 are that it will be applied to the environment life of chloro-acetyl amine pollution of herbicide from now on
Object reparation provides the foundation.
Description of the drawings
Fig. 1 is the Rhodococcus sp of the present invention（Rhodococcus sp.）Drop of the AC-1 bacterial strains to chloro-acetyl amine herbicide
Solution；
Fig. 2 is the Rhodococcus sp of the present invention（Rhodococcus sp.）The bacterium colony photo of AC-1 bacterial strains（A）And electromicroscopic photograph
（B）；
Fig. 3 is temperature and pH of the invention to Rhodococcus sp（Rhodococcus sp.）The shadow of AC-1 strains for degrading Acetochlors
It rings；
Fig. 4 is the metal ion of the present invention to Rhodococcus sp（Rhodococcus sp.）The shadow of AC-1 strains for degrading Acetochlors
It rings；
Fig. 5 is the initial inoculation amount of the present invention to Rhodococcus sp（Rhodococcus sp.）AC-1 strains for degrading Acetochlors
It influences；
Fig. 6 is the Rhodococcus sp of the present invention（Rhodococcus sp.）The metabolite HPLC- of AC-1 strains for degrading Acetochlors
MS qualification figures；A. the liquid chromatogram of metabolite；B. retention time is the corresponding mass spectrogram of 13.49 min substances；C. it protects
It is the corresponding mass spectrogram of 7.56 min substances to stay the time；
Fig. 7 is peanut field Test Drawing.
Specific implementation mode
The present invention is further described with reference to the accompanying drawings and detailed description, so as to the technology of this field
Personnel know more about the present invention, but do not limit the present invention with this.
Embodiment 1
The preparation method of biological organic fertilizer with degradation Acetochlor effect, including following steps：
The making of fixation support：
（1）Seaweed is taken, is dried to its moisture to 40%；
（2）It is added leavening in above-mentioned seaweed, leavening is aspergillus oryzae, Trichoderma viride, bacillus subtilis, wax-like
The composite bacteria agent of bacillus, addition account for raw material seaweed and weigh 2.5 ‰, ferment 12 days at 40 DEG C；
Aspergillus oryzae, Trichoderma viride, bacillus subtilis, Bacillus cercus weight ratio be 1：3：2：1；
（3）By step（2）The middle seaweed drying through everfermentation is crushed to 5 mesh until its moisture is 20%；
（4）In step（3）Material in alkaline matter is added, adjust the pH to 6.5 of material, stir evenly, obtain immobilization
Carrier；
The preparation of biological organic fertilizer：
By Rhodococcus sp（Rhodococcus sp.）Planting site soil 3 after AC-1 microbial inoculums, fixation support, drying：2：30
Ratio be uniformly mixed, at room temperature place 2-3 days to get biological organic fertilizer.
The ingredient of 1 biological organic fertilizer of table
Embodiment 2
The preparation method of biological organic fertilizer with degradation Acetochlor effect, including following steps：
The making of fixation support：
（1）Seaweed is taken, is dried to its moisture to 35%；
（2）It is added leavening in above-mentioned seaweed, leavening is aspergillus oryzae, Trichoderma viride, bacillus subtilis, wax-like
The composite bacteria agent of bacillus, addition account for raw material seaweed and weigh 2 ‰, ferment 10 days at 37 DEG C；
Aspergillus oryzae, Trichoderma viride, bacillus subtilis, Bacillus cercus weight ratio be 1：3：2：1；
（3）By step（2）The middle seaweed drying through everfermentation is crushed to 4 mesh until its moisture is 15%；
（4）In step（3）Material in alkaline matter is added, adjust the pH to 7.0 of material, stir evenly, obtain immobilization
Carrier；
The preparation of biological organic fertilizer：
By Rhodococcus sp（Rhodococcus sp.）Planting site soil 1 after AC-1 microbial inoculums, fixation support, drying：1：10
Ratio be uniformly mixed, at room temperature place 2 days to get biological organic fertilizer.
Embodiment 3
The preparation method of biological organic fertilizer with degradation Acetochlor effect, including following steps：
The making of fixation support：
（1）Seaweed is taken, is dried to its moisture to 55%；
（2）It is added leavening in above-mentioned seaweed, leavening is aspergillus oryzae, Trichoderma viride, bacillus subtilis, wax-like
The composite bacteria agent of bacillus, addition account for raw material seaweed and weigh 4 ‰, ferment 15 days at 45 DEG C；
Aspergillus oryzae, Trichoderma viride, bacillus subtilis, Bacillus cercus weight ratio be 1：3：2：1；
（3）By step（2）The middle seaweed drying through everfermentation is crushed to 10 mesh until its moisture is 20%；
（4）In step（3）Material in alkaline matter is added, adjust the pH to 7.5 of material, stir evenly, obtain immobilization
Carrier；
The preparation of biological organic fertilizer：
By Rhodococcus sp（Rhodococcus sp.）Planting site soil 3 after AC-1 microbial inoculums, fixation support, drying：2：30
Ratio be uniformly mixed, at room temperature place 3 days to get biological organic fertilizer.
Embodiment 4
Degradation bacteria strains Rhodococcus sp employed in 1-3 of the embodiment of the present invention（Rhodococcussp.）Separation screening with
Purifying
Rhodococcus sp（Rhodococcus sp.）The culture medium and reagent of AC-1
LB culture mediums：10.0 g/L of peptone, 5.0 g L of yeast extract-1, 5.0 g L of NaCl-1, pH 7.0-7.4, solid
It needs that 2% agar is added in culture medium.
Minimal medium（MM）：NH4NO3 1.0 g L-1, K2HPO4 1.5 g L-1, KH2PO4 0.5 g L-1, MgSO4
0.2 g L-1, 1.0 g L of NaCl-1, pH 7.0~7.4 needs in solid medium that 2% agar is added.
Acetochlor tc is purchased from lark prestige Science and Technology Ltd.（Purity>98%）, acetochlor tc is dissolved in methanol in experiment
（Chromatographically pure）It is configured to the mother liquor of a concentration of 20 mM（About 5 400 mg L-1）, used after filtration sterilization.Conventional reagent is purchased from Ji
Nan Pengyuan Bioisystech Co., Ltd.Molecular biology reagents are purchased from Shanghai life work and Dalian treasured bioengineering Co., Ltd
（TaKaRa）.
Using the method for continuous enrichment secondary culture, one is separated in by Acetochlor contaminated soil sample from for a long time
The bacterial strain of high-efficiency degradation Acetochlor, is named as AC-1.The bacterial strain can in 48 h degradable 0.2 mM Acetochlor（Attached drawing
1）.The alachlor and butachlor similar with Acetochlor structure in addition, strains A C-1 can also degrade, and imitated with good degradation
Fruit（Attached drawing 1）.But strains A C-1 is very weak to the degradation effect of isopropyl methoxalamine.
The specific method is as follows：
Acquire contaminated soil from the Shandong pesticide plant area periphery of long-term production Acetochlor, for degradation bacteria strains enrichment and
Separation.Contain the liquid inorganic salt culture medium of 0.2 mM Acetochlors in 100 mL（MM）5 g pedotheques of middle addition, 30 DEG C, 150
7 d of rpm shaking table cultures, with 5% inoculum concentration（v/v）It is forwarded in fresh same culture medium, continuous enrichment culture four times.By
Five generation pregnant solutions dilution spread on the MM solid mediums containing 0.2 mM Acetochlors, 30 DEG C of 5 d of culture, on picking tablet
Single bacterium colony is forwarded in 3 mL LB test tubes and cultivates, and thalline were collected by centrifugation, is forwarded to the MM trainings that 20 mL contain 0.2 mM Acetochlors
It supports in base, 30 DEG C of 5 d of culture are molten with 5 mL methanol weight after being freeze-dried culture solution, use UV scanning and high-efficient liquid phase color
Spectrum（HPLC）The degradation effect for detecting single bacterium colony preserves the single bacterium colony for having degradation effect with for further study.
Rhodococcus sp（Rhodococcus sp.）The taxonomic identification of AC-1
Degradation bacteria strains morphological feature and physio-biochemical characteristics identification according to《Common bacteria system identification handbook》With
《Bergey’s Manual of Determinative Bacteriology》.The transmission electron microscope observation of thalline：It selects
The thalline of exponential phase is fixed with 2.5% glutaraldehyde, then is dyed with 0.1 M di(2-ethylhexyl)phosphate tungsten, and transmission electron microscope is finally used
（H-7650； Hitachi）Observation.
The extraction of thalline total DNA expands the 16S rRNA gene sequences of degradation bacteria strains using method with high salt using total DNA as template
Row.Amplimer is a pair of of universal primer, 5 ' end primer 5 '-AGAGTTTGATCCTGGCTCAG-3 '（E. coli bases 8
to 27）, 3 ' end 5 '-TACCTTGTTACGACT T-3 ' of primer（E. colibases 1 507 to 1 492）.PCR amplification
Product AXYGEN Nucleic acid purification kits purify recycling.It is pure that the DNA fragmentation of recycling by agarose gel electrophoresis is detected its
Degree and size（About 1.5 kb）, TA clone after be sequenced.TA clones system（5 μL）For：0.5 mL of pMD18-T, DNA fragmentation
2 μ L, Solution I（TaKaRa）2.5 μ L, 16 °C of enzymes are even stayed overnight.Sequencing result uses prokaryotes appraising datum library
EzTaxon carries out online compare and analyzes.
Strains A C-1 is in pale pink bacterium colony on LB tablets, and bacterium colony projection, surface wettability is smooth, neat in edge, opaque,
In acetochlor（A concentration of 300 mg L-1）Transparent degradation circle can be formed on tablet（Attached drawing 2A）.In transmission electron microscopy
Strains A C-1 is in rod-shaped under mirror（0.5~0.9 3.5~4.5 μm of μ m）, atrichia（2 B of attached drawing）.The physiology of strains A C-1 is given birth to
Change is characterized as：It is aerobic, it cannot move, Gram's staining is positive, and catalase, urase are the positive, oxidizing ferment, Starch Hydrolysis
Enzyme and nitrate reductase are feminine gender.The 16S rRNA gene orders of strains A C-1 are compared in database EzBilCloud and are divided
Analysis, as a result show strains A C-1 andRhodococcusIt is nearest to belong to affiliation, wherein withR. erythropolis DSM
43066TSimilitude up to 99.93%, withR. jialingiae djl-6-2TSimilitude is up to 99.49%.In conjunction with colonial morphology characteristic,
Physiological and biochemical property and 16S rRNA genes compare analysis, and strains A C-1 is initially identified asRhodococcusBelong to.
Embodiment 5
Acetochlor degradation characteristic
By Rhodococcus sp（Rhodococcus sp.）In 100 mL LB culture mediums of the AC-1 accesses containing 0.2 mM Acetochlors, 30
DEG C, 150 rpm shaking table cultures, thalline were collected by centrifugation by 6 000 rpm after 24 h, twice using MM washing thallines, finally with 10 mL
MM is resuspended, spare as seed liquor.
In the basal salt media of addition final concentration of 0.2 mM of Acetochlor, strains A C-1 seeds are accessed by 5% inoculum concentration
Liquid, under the conditions of 15,20,25,30,35 and 40 DEG C, 150 rpm shaking table cultures, sample detection Acetochlor is residual after 24 hours
Allowance calculates degradation rate, influence of the temperature to strains A C-1 degradation Acetochlors.
In the MM culture mediums that initial pH is respectively 6.0,6.5,7.0,7.5,8.0,8.5 and 9.0, the second of 0.2 mM is added
Careless amine, by the seed liquor of 5% inoculum concentration access strains A C-1, in 30 DEG C, 150 rpm shaking table cultures, sample detection after 24 hours
Residual Determination of Acetochlor calculates degradation rate, determines influences of the pH to strains A C-1 degradation Acetochlors.It is pair not to be inoculated with degradation bacteria strains
According to.
In the MM culture mediums of addition final concentration of 0.2 mM of Acetochlor, the metal ion of 0.1 mM is added（Mg2+、Co2+、
Cu2+、Li+、Hg2+、Ni2+、Ca2+、Mn2+、Fe2+And Zn2+）, access strains A C-1 seed liquors, while control is set by 5% inoculum concentration
（It connects bacterium but does not add any metal ion）, in 30 DEG C, 150 rpm shaking table cultures, sample detection Acetochlor remains after 24 hours
Amount calculates degradation rate, determines influence of the metal ion to strains A C-1 degradation Acetochlors.
Temperature and pH are to Rhodococcus sp（Rhodococcus sp.）The influence of AC-1 degradation Acetochlors
Attached drawing 3（A）It is shown, strains A C-1 at 30 DEG C to Acetochlor degradation rate highest, within the scope of 25~35 DEG C,
Can be degraded Acetochlor well, and degradation rate is 80% or more；When temperature is less than 15 DEG C, degradations of the strains A C-1 to Acetochlor
Rate drops to 40% or less.By Fig. 3（B）Shown, strains A C-1 is best to the degradation effect of Acetochlor in pH 7.5；In pH
Can preferably degrade Acetochlor in the range of 6.5-8.5；And when pH is less than 6 or is more than 9, under degradation capability is apparent
Drop, degradation rate are less than 45%.
Metal ion is to Rhodococcus sp（Rhodococcus sp.）The influence of AC-1 degradation Acetochlors
Shown in attached drawing 4, compared with the control, the Cu of 0.1 mM2+And Hg2+Have to strains A C-1 degradation Acetochlors very strong
Inhibiting effect, degradation rate are less than 20% compared with the control；The Co of 0.1 mM2+、Ni2+、Mn2+、Ca2+And Li+It degrades to strains A C-1
Acetochlor has partial inhibition；The Mg of 0.1 mM2+And Zn2+Strains A C-1 degradation Acetochlors are not influenced；And 0.1 mM
Fe2+Then there is faint facilitation to strains A C-1 degradation Acetochlors.
Inoculum concentration is to Rhodococcus sp（Rhodococcus sp.）The influence of AC-1 degradation Acetochlors
Shown in attached drawing 5, with the increase of inoculum concentration, the rate of strains A C-1 degradation Acetochlors is also accelerated, when inoculum concentration is
When 0.5%, strains A C-1 after 56 h still cannot degradable 0.2 mM Acetochlor, and when inoculum concentration reaches 10%, bacterial strain
AC-1 can degradable Acetochlor in 32 h.
Embodiment 6
The biological organic fertilizer of the present invention acts on geobiont repair
1. pressing 1000ml/ mus of Acetochlors before repairing（50%）Amount of application to peanut spray, then 0-15cm is uniform
Acetochlor in soil residual quantity is surveyed in plowed soils, sampling；
2. by Rhodococcus sp（Rhodococcus sp.）AC-1（Cell concentration is 1.04 × 109 cfu/ml）It is dropped by Acetochlor
Solve microbial inoculum：Biological organic fertilizer is with 2:1~3:2（m:m）Ratio mixing, be then aided with a certain amount of experimental field soil again, mixing is equal
It is even, room temperature（25-30℃）It places 2-3 days, in being applied experimental field by 500 ~ 600kg/ mus.Using turning over soil immediately after degradation bacterial agent
Earth so that degradation bacterial agent and soil mix well, while being also beneficial to colonizing for herbicide residue efficient degrading bacteria in microbial inoculum,
To preferably play the degradation effect of microbial inoculum.
3. from soil remediation, herbicide residue, monitoring herbicide residue drop are detected every 7 days acquisition pedotheques
Solve situation.
After 4. degradation bacterial agent and bio-fertilizer mixing are applied 14 days, acetochlor in soil degradation rate reaches 86%, degrades after 21 days
Rate reaches 99%.
2 Field information effect of table
5. sowing time under the same conditions, degradation bacterial agent apply 21 days after, processing 4 performance peanut emergence rates reach 50%
More than, and other leaf emergences are less than 10%, and the increment after emerging is also different, and increment is big after 4 emergence of processing,
Its increment is few（See attached drawing 7）.
Embodiment 7
Acetochlor degradation pathway
Resting cell is tested：Strains A C-1 is connected in the 100 mL LB liquid mediums containing 0.2 mM Acetochlors,
30 DEG C, 150 rpm cultivate to the logarithmic growth later stage, 4 DEG C, 6 000 rpm centrifuge 5 min and collect thalline, with sterile water washing bacterium
Body is twice.Thalline is resuspended in 10 mL sterile waters.Thalline is pressed into final concentration OD600It is connected in 50 mL MM culture mediums for 1.0（Contain
0.2 mM Acetochlors）, 30 DEG C, 150 rpm cultures.3 mL, dichloromethane extraction, with 1 mL methanol after volatilization are sampled every 4 h
Weight is molten, and the sample for generating metabolite is further used LC-MS detection and identifications by HPLC detections.
The analysis method of compound
High performance liquid chromatography（HPLC）Detection：Sample to be tested centrifuges 5 min through 12 000 rpm, and supernatant is taken to cross organic phase filter
Film (0.22 μm) goes up machine testing afterwards.Liquid phase chromatogram condition：Instrument, Shimadzu RID-10A；Liquid-phase chromatographic column is C18Reversed-phase column, specification
250 mm × 4.6 mm；Mobile phase is methanol:Water（80:20, V:V）；Column temperature is 40 DEG C；1.0 mL min of flow velocity-1；Detect wave
Long 220 nm.
HPLC-MS is detected：For detecting Acetochlor and its metabolite.LC-MS analyses use LCQ OECAXP plus
(Thermo Finnigan), detector are SURVEYOR PDA plus Detector, ion source ESI, liquid-phase chromatographic column C18
Reversed-phase column（250 mm × 4.6 mm）, mobile phase is methanol:Water（80:20, V:V）, column temperature is 20 DEG C, and flow velocity is 1 mL/
Min, 220 nm of Detection wavelength, sample size are 6 μ L, scanning of the mass spectrum range：50~1 000 m/z.
The metabolite of strains A C-1 degradation Acetochlors is subjected to HPLC-MS identifications.Such as attached drawing 6（A）Shown, sample is in liquid
Occur two peaks, retention time in phase chromatogram（RT）Respectively 13.49 min and 7.56 min；Fig. 6（B）Indicate retention time
It is the mass spectrogram of the substance of 13.49 min, M/Z is 270.13 [M]+, it is consistent with Acetochlor molecular weight, and Acetochlor molecule
One chlorine atom of upper band, therefore will appear the peak 272.18 [M] of isotope+；Attached drawing 6（C）It is 7.56 to show retention time
The mass spectrogram of the substance of min, M/Z are 212.23 [M]+, chloro- with 2-N-（2- ethyl -6- aminomethyl phenyls）The molecule of acetamide
Amount is consistent.It is chloro- with 2-N-（2- ethyl -6- aminomethyl phenyls）Acetamide is that substrate continues to cultivate strains A C-1, it is found that bacterial strain cannot
Continue the substrate of degrading.It could therefore be concluded that going out the metabolic pathway of strains A C-1 degradation Acetochlors：I.e. Acetochlor sloughs methylamino ethoxy
Alkyl in base branch, it is chloro- to be converted into 2-N-（2- ethyl -6- aminomethyl phenyls）Acetamide.
Strains A C-1 energy efficient degradations alachlor, Acetochlor and the butachlor of separation screening of the present invention, have wider drop
Spectrum unscrambling.Degradation character excellent strains A C-1 is that it will be applied to the environmental organism reparation of chloro-acetyl amine pollution of herbicide from now on
It provides the foundation.
The structure of compound determines biodegradability of the microorganism to this kind of compound.Chloro-acetyl amine herbicide
Two classes can be divided into according to the difference of substituent group on its aniline Amide N, one kind is that substituent group is alkoxy methyl on amide N, such as
Alachlor, Acetochlor and butachlor；Another kind of is that substituent group is alkoxyethyl on amide N, such as pretilachlor, propisochlor and different
Methoxalamine.Strains A C-1 has good degradation effect to alachlor, Acetochlor and butachlor, and to isopropyl methoxalamine
Degradation effect is very weak, shows that the unit structure that the amide N atoms of chloro-acetyl amine herbicide are connected determines such
The biodegradability of herbicide.
The bacterial strain of a high-efficiency degradation Acetochlor is separated from insecticide factory's contaminated soil of long-term production Acetochlor
AC-1 identifies strains A C-1 according to morphological feature, physiological and biochemical property and 16S rRNA gene order similarity analysis
ForRhodococcusBelong to.
The bacterial strain can in 48 h degradable 0.2 mM Acetochlor（Attached drawing 1）.In addition, strains A C-1 can also degrade with
Acetochlor structure similar alachlor and butachlor, and there is good degradation effect（Attached drawing 1）.But C-1 pairs of strains A
The degradation effect of isopropyl methoxalamine is very weak.
Rhodococcus sp（Rhodococcus sp.）The optimum temperature of AC-1 strains for degrading Acetochlors is 30 DEG C, and optimal pH is
7.5, degradation effect is proportionate with inoculum concentration.The Cu of 0.1 mM2+And Hg2+Have to strains A C-1 degradation Acetochlors very strong
Inhibiting effect, and the Fe of 0.1 mM2+Then there is faint facilitation to strains A C-1 degradation Acetochlors.
It is analyzed by HPLC-MS, is inferred to the degradation pathway of strains A C-1 degradation Acetochlors, i.e., it is de- to carry out N- to Acetochlor
Alkyl, and then it is converted into the chloro- N- of 2- (2- ethyl -6- aminomethyl phenyls) acetamide.
Claims (9)
1. the biological organic fertilizer with degradation Acetochlor effect, which is characterized in that the biological organic fertilizer includes following master
Material：
Microbial bacterial agent, fixation support；
The microbial bacterial agent is Rhodococcus sp（Rhodococcussp.）AC-1 is preserved in Chinese microorganism strain preservation management
Committee's common micro-organisms center, deposit number are CGMCC NO.10778, and the deposit date is on April 30th, 2015；
The fixation support is the alga fertilizer using ferment-fermented mistake；
The mass ratio of the microbial bacterial agent and fixation support is：1~2：2~3；The microbial bacterial agent is a concentration of
1×109Cfu/ml~1 × 1010 cfu/ml；
The preparation method of the fixation support is as follows：
（1）Raw material seaweed is taken, is dried to its moisture to 35 ~ 55%；
（2）In step（1）It is added leavening in the seaweed that middle moisture is 35 ~ 55%, the leavening is aspergillus oryzae, green
Color trichoderma, bacillus subtilis, Bacillus cercus composite bacterium powder, the addition of composite bacterium powder accounts for raw material seaweed weight 2-4 ‰,
It ferments 10-15 days at 37-45 DEG C；
The aspergillus oryzae, Trichoderma viride, bacillus subtilis, Bacillus cercus weight ratio be：1-2：1-4：1-3：
1-2；
（3）By step（2）It is 15 ~ 20% that the middle seaweed through everfermentation, which is dried to its moisture, is crushed to 4 ~ 10 mesh；
（4）In step（3）Alkaline matter is added in material after middle crushing, adjusts the pH to 6.5 ~ 7.5 of material, stirs evenly,
Obtain fixation support.
2. the biological organic fertilizer with degradation Acetochlor effect as described in claim 1, which is characterized in that the basic species
Matter is plant ash.
3. the biological organic fertilizer with degradation Acetochlor effect as claimed in claim 2, which is characterized in that the step
（2）In, aspergillus oryzae, Trichoderma viride, bacillus subtilis, Bacillus cercus weight ratio be：1：3：2：1, addition
The 3 ‰ of raw material seaweed weight are accounted for, are fermented 12 days at 40 DEG C.
4. the biological organic fertilizer with degradation Acetochlor effect as claimed in claim 2, which is characterized in that the step
（4）In, adjust pH to 7.5.
5. the preparation method of the biological organic fertilizer as claimed in claim 2 with degradation Acetochlor effect, including following steps
Suddenly：The making of fixation support：
（1）Seaweed is taken, is dried to its moisture to 35 ~ 55%；
（2）In step（1）It is added leavening in the seaweed that middle moisture is 35 ~ 55%, the leavening is aspergillus oryzae, green
Color trichoderma, bacillus subtilis, Bacillus cercus composite bacteria agent, addition accounts for raw material seaweed weight 2-4 ‰, in 37-45
It ferments 10-15 days at DEG C；
The aspergillus oryzae, Trichoderma viride, bacillus subtilis, Bacillus cercus weight ratio be 1-2：1-4：1-3：1-
2；
（3）By step（2）The middle seaweed drying through everfermentation is crushed to 4 ~ 10 mesh until its moisture is 15 ~ 20%；
（4）In step（3）Material in alkaline matter is added, adjust the pH to 6.5 ~ 7.5 of material, stir evenly, obtain immobilization
Carrier；
By Rhodococcus sp（Rhodococcus sp.）Planting site soil 1-5 after AC-1 microbial inoculums, fixation support, drying：1-4：
The weight ratio of 10-40 is uniformly mixed, its moisture of planting soil after drying is 1-4%, at room temperature placement 2-3 days, i.e.,
Obtain biological organic fertilizer.
6. the preparation method of the biological organic fertilizer as claimed in claim 5 with degradation Acetochlor effect, including following steps
Suddenly：
The making of fixation support：
（1）Seaweed is taken, is dried to its moisture to 40%；
（2）In step（1）Leavening is added in seaweed after middle drying, the leavening is aspergillus oryzae, Trichoderma viride, withered grass
Bacillus, Bacillus cercus composite bacteria agent, addition accounts for raw material seaweed and weighs 2.5 ‰, and ferment 10-15 at 40 DEG C
It；
The aspergillus oryzae, Trichoderma viride, bacillus subtilis, Bacillus cercus weight ratio be 1：3：2：1；
（3）By step（2）The middle seaweed drying through everfermentation is crushed to 5 mesh until its moisture is 20%；
（4）In step（3）Material in alkaline matter is added, adjust the pH to 6.5 ~ 7.5 of material, stir evenly, obtain immobilization
Carrier；
The preparation of biological organic fertilizer：
By Rhodococcus sp（Rhodococcus sp.）Planting site soil 3 after AC-1 microbial inoculums, fixation support, drying：2：30 weight
Amount ratio is uniformly mixed, its moisture of planting soil after drying is 1-4%, and place 2-3 days has to get biology at room temperature
Machine fertilizer.
7. the preparation method of the biological organic fertilizer as claimed in claim 5 with degradation Acetochlor effect, which is characterized in that institute
The Rhodococcus sp stated（Rhodococcussp.）AC-1 is gram positive bacterium；
The Rhodococcus sp（Rhodococcussp.）AC-1 is in pale pink bacterium colony, bacterium colony projection, surface wettability on LB tablets
Smooth, neat in edge is opaque, strains A C-1 is observed under transmission electron microscope in rod-shaped, and 0.5~0.9 μm × 3.5
~4.5 μm, atrichia；
Rhodococcus sp（Rhodococcussp.）The physiological and biochemical property of AC- is：It is aerobic, it cannot move, catalase, urase are
It is positive；Oxidizing ferment, amylolytic enzyme and nitrate reductase are feminine gender；
By the Rhodococcus sp（Rhodococcus sp.）The 16S rRNA gene orders of AC-1 are in database EzBilCloud
Analysis is compared, as a result shows that strains A C-1 and Rhodococcus category affiliations are nearest, wherein with R. erythropolis
DSM 43066TSimilitude is up to 99.93%, with R. jialingiae djl-6-2TSimilitude is up to 99.49%；
By the Rhodococcus sp（Rhodococcus sp.）AC-1 forms transparent degradation circle, second on acetochlor tablet
A concentration of 300 mg L of careless amine pesticide-1；
The Rhodococcus sp（Rhodococcussp.）The optimum temperature of AC-1, Acetochlor of degrading are 30 DEG C, and optimal pH is 7.5,
The Cu of 0.1 mM2+And Hg2+It is inhibited to strains A C-1 degradation Acetochlors, and the Fe of 0.1 mM2+Then strains A C-1 is dropped
Solving Acetochlor has facilitation.
8. biological organic fertilizer the answering in planting grain class crop with degradation Acetochlor effect as described in claim 1
With.
9. biological organic fertilizer the answering in maize planting, peanut ground with degradation Acetochlor effect as described in claim 1
With.
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| CN109206181B (en) * | 2018-09-28 | 2021-09-03 | 东北师范大学 | Method for degrading acetochlor in farmland soil |
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| CN102757915A (en) * | 2012-07-02 | 2012-10-31 | 南京农业大学 | Chloro acetamide herbicide degrading bacteria as well as bactericide prepared thereby and application thereof |
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| CN102757915A (en) * | 2012-07-02 | 2012-10-31 | 南京农业大学 | Chloro acetamide herbicide degrading bacteria as well as bactericide prepared thereby and application thereof |
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| CN104045442A (en) * | 2014-05-30 | 2014-09-17 | 青岛辰青信息技术有限公司 | Composite microbial fertilizer |
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