CN108728386A - A kind of preparation method of glyphosate solid-state degradation bacterial agent - Google Patents
A kind of preparation method of glyphosate solid-state degradation bacterial agent Download PDFInfo
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- CN108728386A CN108728386A CN201810657630.4A CN201810657630A CN108728386A CN 108728386 A CN108728386 A CN 108728386A CN 201810657630 A CN201810657630 A CN 201810657630A CN 108728386 A CN108728386 A CN 108728386A
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- glyphosate
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
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Abstract
It is to produce charcoal using rice husk thermal decomposition the present invention relates to a kind of preparation method of the fixed glyphosate efficient degrading bacteria of charcoal, is prepared with one plant of glyphosate efficient degrading bacteria co-cultivation.This method includes the activation of (1) strain；(2) prepared by charcoal and semisolid culturemedium, pH is reconciled and sterilizing；(3) degradation bacteria constant temperature incubation in semisolid culturemedium obtains purpose product.The glyphosate solid-state degradation bacterial agent of this method production has the characteristics that production cost is low, environmental-friendly, can remaining glyphosate pesticide in efficient degradation soil, contaminated soil return it is cultivated, improve Crop yield and quality in terms of with preferable potentiality.
Description
Technical field
The invention belongs to field of environmental biotechnology, and in particular to a kind of preparation method of glyphosate solid-state degradation bacterial agent.
Background technology
Glyphosate is that a kind of wide spectrum goes out raw type herbicide, has good lethal effect to various agricultural weeds, is current
Most one of extensive, the maximum pesticide of usage amount are used in the world.Its stable in physicochemical property, degradation cycle is more unrestrained in the environment
Long, to agricultural product security, there are larger threats.2015, confirm through the World Health Organization and announce, glyphosate is deposited with cancer
In certain association, use may cause human lymphocytes' gland cancer and lung cancer.Therefore, it is necessary to carry out relevant research to repair
Again by glyphosate contaminated soil.
Currently, common pesticide contaminated soil restorative procedure is based on physics, chemical method, including stove burns, heat is de-
Chemical treatment etc. is echoed, but these methods are often with high costs and easily cause secondary pollution.In recent years, emerging microorganism is repaiied
Recovering technology makes some progress, and dawn is brought for pesticide contaminated soil reparation.However, since insecticide pollution is in solid phase
The problems such as migration velocity is slow in medium, degradation of pesticide engineering bacteria is difficult to long distance transportation with liquid condition, microorganism remediation technology
Application be still subject to certain restrictions.Therefore, the development and application of solid pesticide degradation bacterial agent, the synthesis in pollution by pesticides place
There is important application value in improvement.
Invention content
The purpose of the present invention is intended to provide a kind of preparation method of glyphosate solid-state degradation bacterial agent, specifically total using charcoal
The fixed glyphosate degradation bacterium of culture.
In order to achieve the above-mentioned object of the invention, the technical solution adopted by the present invention is as follows：
(1) actication of culture.The strain for being stored in -80 DEG C is taken out and is thawed, takes and is coated on glyphosate containing 400mg/L in right amount
For on solid MSM (Minimal Salt Media) culture medium of sole carbon source, 28 DEG C of constant temperature incubations are for 24 hours.Picking single bacterium colony connects
For kind in 5mL LB liquid mediums, 180rpm shake cultures are spare for 24 hours.
(2) prepared by charcoal.By rice husk after washing twice, drying crushes, in tubular type under 250-650 DEG C of limited oxygen condition
It is pyrolyzed 2h in stove, is cooled to room temperature, ground 0.45mm sieves obtain charcoal.
(3) prepared by semisolid culturemedium.Charcoal, LB culture mediums, distilled water are with mass ratio 1:2:2-4 is uniformly mixed, with
It is 7.0 that 1M NaOH, which adjust pH, autoclave sterilization.
(4) co-cultivation prepares glyphosate solid-state degradation bacterial agent.The bacterium solution for taking step (1) to activate is inoculated in the half of step (3)
In solid medium, mixing.28 DEG C of constant temperature, 180rpm shake culture 24-48h, until thalline number reaches in semisolid culturemedium
108-109The order of magnitude.It air-dries to microbial inoculum water content and is less than 30%, obtain glyphosate solid-state degradation bacterial agent.
The microbial inoculum can directly be spread fertilizer over the fields in by glyphosate contaminated soil surface or before proportion of crop planting, be spreaded manuer in holes in contaminated
In soil, usage amount about 25-50kg/667m2。
Bacterial strain of the present invention is Aquamicrobiumahrensii, is isolated from Hubei Province Qi Zhouzhen and begs agrochemicals
In plant area of Co., Ltd surrounding soil.The bacterium can be raw in the solid and fluid nutrient medium that glyphosate maximum concentration is 1000mg/L
It is long, and 100% can reach in 48h to the degradation rate of glyphosate contained therein, final catabolite is water, carbon dioxide etc.
Inorganic component.
Glyphosate solid-state degradation bacterial agent produced by the invention has the characteristics that cheap, portable, efficient, environmental-friendly.The production
Remaining organic agricultural chemicals in the adsorbable fixation of product, efficient degradation soil, and provide good habitat for microorganism.It is raw simultaneously
Object charcoal has been demonstrated to have the function of improveing soil physico-chemical property, reduces leaching loss of nutrient, increases soil fertility, therefore, this product
There are preferable potentiality in terms of contaminated soil returns cultivated, improvement Crop yield and quality.
Description of the drawings
Fig. 1 is degradation capability curve graph of the glyphosate solid-state degradation bacterial agent to glyphosate under different applied amounts.
Specific embodiment
Below in conjunction with specific embodiment, technical scheme of the present invention is described in further detail.Involved embodiment party
Case is only used for the present invention, not as the restriction of the scope of the present invention.
Embodiment 1:
A kind of preparation method of glyphosate solid-state degradation bacterial agent, includes the following steps：
(1) actication of culture.The strain for being stored in -80 DEG C is taken out and is thawed, takes and is coated on glyphosate containing 400mg/L in right amount
For the solid MSM of sole carbon source, (Minimal Salt Media, ingredient are：NH4NO31.0g/L, MgSO4·7H2O 0.5g/
L, (NH4)2SO40.5g/L, KH2PO40.5g/L, NaCl 0.5g/L, K2HPO41.5g/L, pH=7) on culture medium, 28 DEG C
Constant temperature incubation is for 24 hours.Picking single bacterium colony is inoculated in (medium component in 5mL liquid LB culture mediums：Tryptone 10g/L, yeast
Extract 1g/L, NaCl 1g/L, agar 20g/L, pH=7), 180rpm shake cultures are spare for 24 hours.
(2) prepared by charcoal.By rice husk after washing twice, drying crushes, in tubular type under 250-650 DEG C of limited oxygen condition
It is pyrolyzed 2h in stove, is cooled to room temperature, ground 0.45mm sieves obtain charcoal.
(3) prepared by semisolid culturemedium.By charcoal, LB culture mediums, distilled water with mass ratio 1:2:2-4 is uniformly mixed,
It is 7.0,121 DEG C of sterilizing 20min to adjust pH with 1M NaOH.
(4) co-cultivation prepares glyphosate solid-state degradation bacterial agent.The bacterium solution for taking step (1) to activate is inoculated in the half of step (3)
In solid medium, mixing.28 DEG C of constant temperature, 180rpm shake culture 24-48h, until thalline in final every gram of semisolid culturemedium
Number is up to 108-109The order of magnitude.It air-dries to microbial inoculum water content and is less than 30%, packaging is deposited at shady and cool drying, the term of validity
12 months.
Embodiment 2:
Degradation effect of the glyphosate solid-state degradation bacterial agent to pedo relict glyphosate pesticide：
It is tested using experimental plot cell, 3 identical hillslope processes of condition is set, each plot area is 10m2。
In upper soll layer by the maximum dose (200g/667m indicated in glyphosate packaging bag2) sprinkling glyphosate aqueous solution, respectively with
0kg/667m2(control), 25kg/667m2And 50kg/667m2Amount apply invention glyphosate solid-state degradation bacterial agent.After processing
It samples for the first time immediately, hereafter uses the daily timing sampling of five point sampling, grass is measured with rp-hplc analysis method
Sweet phosphine degradation rate.After applying microbial inoculum of the present invention, processing group glyphosate degradation rate is apparently higher than control group, at the 4th day
50kg/667m2Processing group degradation rate is more than 90%.It is efficient that the experimental result illustrates that the microbial bacterial agent has glyphosate
Degradation capability.
Claims (5)
1. a kind of preparation method of glyphosate solid-state degradation bacterial agent, which is characterized in that include the following steps：
(1) actication of culture.The strain for being stored in -80 DEG C is taken out and is thawed, takes and is coated in right amount using glyphosate as sole carbon source
On solid MSM (Minimal Salt Media) culture medium, 28 DEG C of constant temperature incubations are for 24 hours.Picking single bacterium colony is inoculated in 5mL liquid LB
In culture medium, 180rpm shake cultures are spare for 24 hours.
(2) prepared by charcoal.By rice husk after washing twice, drying crushes, under 250-650 DEG C of limited oxygen condition in tube furnace
Pyrolysis, is cooled to room temperature, and ground 0.45mm sieves obtain charcoal.
(3) prepared by semisolid culturemedium.Charcoal, LB culture mediums, distilled water mix in proportion, and it is 7.0 to adjust pH, high temperature
High pressure sterilization.
(4) co-cultivation prepares glyphosate solid-state degradation bacterial agent.The bacterium solution for taking step (1) to activate, is inoculated in the semisolid of step (3)
In culture medium, mixing.Then isothermal vibration culture air-dries to microbial inoculum water content and is less than 30%, obtain the degradation of glyphosate solid-state
Microbial inoculum.
2. the preparation method of glyphosate solid-state degradation bacterial agent according to claim 1, which is characterized in that MSM in step (1)
A concentration of 400mg/L of culture medium glyphosate.
3. the preparation method of glyphosate solid-state degradation bacterial agent according to claim 1, which is characterized in that step (2) middle pipe
Pyrolysis time is 2h in formula stove.
4. the preparation method of glyphosate solid-state degradation bacterial agent according to claim 1, which is characterized in that raw in step (3)
Object charcoal, LB culture mediums, distilled water ratio are 1:2:2-4, it is 1M NaOH to adjust solution used in pH.
5. the preparation method of glyphosate solid-state degradation bacterial agent according to claim 1, which is characterized in that permanent in step (4)
Warm shake culture refers to 28 DEG C, 180rpm shake culture 24-48h, until thalline number is up to 10 in semisolid culturemedium8-109Quantity
Grade.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN109536173A (en) * | 2018-12-04 | 2019-03-29 | 苏州科技大学 | A kind of composite material and preparation method of while remediating heavy metal and glyphosate |
| CN112280717A (en) * | 2020-11-09 | 2021-01-29 | 上海市农业科学院 | Compound microbial agent, preparation method thereof and method for improving soil biological diversity |
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| CN112280717A (en) * | 2020-11-09 | 2021-01-29 | 上海市农业科学院 | Compound microbial agent, preparation method thereof and method for improving soil biological diversity |
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Application publication date: 20181102 |