CN109852604A - The preparation method and application of the net unit cell category bacteria immobilization bacterium of efficient degradation sclerotium - Google Patents
The preparation method and application of the net unit cell category bacteria immobilization bacterium of efficient degradation sclerotium Download PDFInfo
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- 230000015556 catabolic process Effects 0.000 title claims abstract description 31
- 238000006731 degradation reaction Methods 0.000 title claims abstract description 31
- 241001558929 Sclerotium <basidiomycota> Species 0.000 title claims abstract description 16
- 238000002360 preparation method Methods 0.000 title claims abstract description 9
- 235000017166 Bambusa arundinacea Nutrition 0.000 claims abstract description 16
- 235000017491 Bambusa tulda Nutrition 0.000 claims abstract description 16
- 241001330002 Bambuseae Species 0.000 claims abstract description 16
- 235000015334 Phyllostachys viridis Nutrition 0.000 claims abstract description 16
- 239000011425 bamboo Substances 0.000 claims abstract description 16
- IXPNQXFRVYWDDI-UHFFFAOYSA-N 1-methyl-2,4-dioxo-1,3-diazinane-5-carboximidamide Chemical compound CN1CC(C(N)=N)C(=O)NC1=O IXPNQXFRVYWDDI-UHFFFAOYSA-N 0.000 claims abstract description 15
- 235000010413 sodium alginate Nutrition 0.000 claims abstract description 15
- 239000000661 sodium alginate Substances 0.000 claims abstract description 15
- 229940005550 sodium alginate Drugs 0.000 claims abstract description 15
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- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 8
- 239000000463 material Substances 0.000 claims abstract description 6
- 241001658767 Brevundimonas naejangsanensis Species 0.000 claims abstract description 4
- FSCWZHGZWWDELK-UHFFFAOYSA-N 3-(3,5-dichlorophenyl)-5-ethenyl-5-methyl-2,4-oxazolidinedione Chemical compound O=C1C(C)(C=C)OC(=O)N1C1=CC(Cl)=CC(Cl)=C1 FSCWZHGZWWDELK-UHFFFAOYSA-N 0.000 claims abstract description 3
- 239000005867 Iprodione Substances 0.000 claims abstract description 3
- ONUFESLQCSAYKA-UHFFFAOYSA-N iprodione Chemical compound O=C1N(C(=O)NC(C)C)CC(=O)N1C1=CC(Cl)=CC(Cl)=C1 ONUFESLQCSAYKA-UHFFFAOYSA-N 0.000 claims abstract description 3
- 239000000725 suspension Substances 0.000 claims description 13
- 238000000034 method Methods 0.000 claims description 8
- 239000008363 phosphate buffer Substances 0.000 claims description 8
- 238000000855 fermentation Methods 0.000 claims description 7
- 230000004151 fermentation Effects 0.000 claims description 7
- 239000007788 liquid Substances 0.000 claims description 7
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 6
- 244000005700 microbiome Species 0.000 claims description 6
- UXVMQQNJUSDDNG-UHFFFAOYSA-L Calcium chloride Chemical compound [Cl-].[Cl-].[Ca+2] UXVMQQNJUSDDNG-UHFFFAOYSA-L 0.000 claims description 5
- OWEGMIWEEQEYGQ-UHFFFAOYSA-N 100676-05-9 Natural products OC1C(O)C(O)C(CO)OC1OCC1C(O)C(O)C(O)C(OC2C(OC(O)C(O)C2O)CO)O1 OWEGMIWEEQEYGQ-UHFFFAOYSA-N 0.000 claims description 3
- FRXSZNDVFUDTIR-UHFFFAOYSA-N 6-methoxy-1,2,3,4-tetrahydroquinoline Chemical compound N1CCCC2=CC(OC)=CC=C21 FRXSZNDVFUDTIR-UHFFFAOYSA-N 0.000 claims description 3
- GUBGYTABKSRVRQ-PICCSMPSSA-N Maltose Natural products O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@@H]1O[C@@H]1[C@@H](CO)OC(O)[C@H](O)[C@H]1O GUBGYTABKSRVRQ-PICCSMPSSA-N 0.000 claims description 3
- 229940041514 candida albicans extract Drugs 0.000 claims description 3
- 238000004132 cross linking Methods 0.000 claims description 3
- 238000011534 incubation Methods 0.000 claims description 3
- 238000003760 magnetic stirring Methods 0.000 claims description 3
- 239000004005 microsphere Substances 0.000 claims description 3
- 230000010355 oscillation Effects 0.000 claims description 3
- 239000011780 sodium chloride Substances 0.000 claims description 3
- 239000012138 yeast extract Substances 0.000 claims description 3
- 239000001110 calcium chloride Substances 0.000 claims description 2
- 229910001628 calcium chloride Inorganic materials 0.000 claims description 2
- IGLKELDWPZFFKF-UHFFFAOYSA-N OC(C1=CC=CC=C1C(O)=O)=O.OC(C1=CC=CC=C1C(O)=O)=O.OC(C1=CC=CC=C1C(O)=O)=O.P.P Chemical compound OC(C1=CC=CC=C1C(O)=O)=O.OC(C1=CC=CC=C1C(O)=O)=O.OC(C1=CC=CC=C1C(O)=O)=O.P.P IGLKELDWPZFFKF-UHFFFAOYSA-N 0.000 claims 1
- 239000000872 buffer Substances 0.000 claims 1
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- 238000004321 preservation Methods 0.000 claims 1
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- 238000001179 sorption measurement Methods 0.000 claims 1
- CFZLNRGUBAVQNO-UHFFFAOYSA-N N-(3,5-Dichlorophenyl)succinimide Chemical compound ClC1=CC(Cl)=CC(N2C(CCC2=O)=O)=C1 CFZLNRGUBAVQNO-UHFFFAOYSA-N 0.000 abstract description 27
- 230000000694 effects Effects 0.000 abstract description 13
- 230000002045 lasting effect Effects 0.000 abstract description 3
- 210000004027 cell Anatomy 0.000 description 22
- 102000004190 Enzymes Human genes 0.000 description 3
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- 230000001580 bacterial effect Effects 0.000 description 3
- 239000003344 environmental pollutant Substances 0.000 description 3
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- 231100000719 pollutant Toxicity 0.000 description 3
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 2
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 2
- 229910052799 carbon Inorganic materials 0.000 description 2
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- 239000000126 substance Substances 0.000 description 2
- 238000012360 testing method Methods 0.000 description 2
- -1 3,5- dichlorophenyl Chemical group 0.000 description 1
- 235000005254 Allium ampeloprasum Nutrition 0.000 description 1
- 240000006108 Allium ampeloprasum Species 0.000 description 1
- 241001530056 Athelia rolfsii Species 0.000 description 1
- 244000106835 Bindesalat Species 0.000 description 1
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- 241000499436 Brassica rapa subsp. pekinensis Species 0.000 description 1
- 235000002566 Capsicum Nutrition 0.000 description 1
- 240000008574 Capsicum frutescens Species 0.000 description 1
- 240000008067 Cucumis sativus Species 0.000 description 1
- 235000010799 Cucumis sativus var sativus Nutrition 0.000 description 1
- 235000007688 Lycopersicon esculentum Nutrition 0.000 description 1
- 241000124008 Mammalia Species 0.000 description 1
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- 241000607479 Yersinia pestis Species 0.000 description 1
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Landscapes
- Immobilizing And Processing Of Enzymes And Microorganisms (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention discloses a kind of preparation method and applications of unit cell category bacteria immobilization bacterium that efficient degradation sclerotium is net, it is with unit cell category bacterium (Brevundimonas naejangsanensis) CCTCC M 2017739 for strain, using 4% sodium alginate and bamboo charcoal as immobilization material, unit cell category bacterium is effectively embedded and immobilized bacterium is made.The immobilized bacterium can be applied to efficiently thoroughly degradation soil, the dimethachlon in water body, vinclozolin and iprodione.Has many advantages, such as good lasting effect, environmental-friendly, significant effect, low in cost and easy to spread.
Description
Technical field
The present invention relates to biotechnology degrading pesticide residues field, especially a kind of unit cell category bacterium that efficient degradation sclerotium is net
The preparation method and application of immobilized bacterium.
Background technique
Pesticide plays an important role in agricultural pest integrative improvement, but pesticide residue and its metabolite have become
Severe pollutant constitutes a threat to human health and the ecosystem.Dimethachlon (Dimetachlone), also known as dimethachlon, molecule
Formula C10H7Cl2NO2, relative molecular mass 244.07, chemical name are as follows: N- (3,5- dichlorophenyl) succimide.It is wide in China
The general sclerotiniose on the crops such as rape, Chinese cabbage, Zicaitai, romaine lettuce, leek, cucumber, tomato, capsicum, potato, tobacco,
The prevention and treatment of the fungal diseases such as banded sclerotial blight, rust.Although the disinfection vitality promise well of dimethachlon, due to health problem, mesh
It is preceding to be prohibited from using in US and European, but be still widely used in some developing countries (such as China).In addition, previous grinds
Study carefully and show that the dimethachlon of 0.4mmol/L is rat acute renal toxicity substance, and it has interference effect to mammal endocrine,
To human kidney also toxic effect.In conclusion a large amount of uses of dimethachlon can generate the exposure of dimethachlon in agricultural, this is right
Human health and Environmental security constitute potential threat, eliminate dimethachlon pollutant ecology is friendly and Low-Cost Strategy there is an urgent need to
It establishes.
Sclerotium is solved in many kinds of measures such as many countries, including legislation, agricultural standardization, Farmers ' Education and recovery technique
Net residue problem, although these measures assist in removing pesticide residue, dimethachlon cannot be effectively removed.It is biological prosthetic
It (Bioremediation) is a kind of using living microorganism or the enzyme from microorganism is detoxified and degraded to pollutant,
Have many advantages, such as easy to operate, economical and practical, environmental-friendly environment and cost-effective method.However, free cell
It is easy to be influenced by a variety of environmental factors (such as temperature, pH value) with enzyme, and its unstability, high production cost and separation
Difficulty, which greatly limits practical applications.Fortunately, immobilization technology provides the substitution for solving these challenges
Scheme, and have become the perfect selection for improving microorganism and enzyme stability, operability, durability and commercial viability.
So far, research work there is no to carry out the biodegrade of dimethachlon.
Summary of the invention
The present invention lacks for dimethachlon degradation technique, it is desirable to provide a kind of quick-acting that efficient degradation sclerotium is net is good, ring
The unit cell category bacteria immobilization bacterium that border is friendly, significant effect and low-cost efficient degradation sclerotium are net.
Technical scheme is as follows:
The deposit number of unit cell category bacterium (Brevundimonas naejangsanensis) bacterial strain is CCTCC M
2017739, it is existing bacterial strain.The technical scheme is that with unit cell category bacterium (Brevundimonas
Naejangsanensis) CCTCC M 2017739 is strain, using 4% sodium alginate and bamboo charcoal as immobilization material, by unit cell
Belong to bacterium effectively to embed and immobilized bacterium is made.
Wherein, the net unit cell category bacteria immobilization bacterium preparation method of the efficient degradation sclerotium includes the following steps:
1) prepared by bacteria suspension: 2mL sterile phosphate buffer being added under aseptic condition in the Spawn incubation ware of culture 48h
Parallel oscillation, and gently scrape with oese the bacterium colony of media surface, crude bacteria suspension, be transferred into liquid fermentation
In culture medium (maltose 7g/L, yeast extract 6g/L, NaCl 3g/L) pH be 7.5~8,30~35 DEG C, 150~200rpm item
48~56h is cultivated under part.The fermentation liquid of taking-up is centrifuged under 8 000~10000r/min (3~4 DEG C) to 10~15min immediately,
Wet thallus is collected, and is rinsed 3 times with sterile phosphate buffer.Then sterile phosphate buffer and wet thallus are mixed, control
Thalline quantity processed is >=6 × 1010cfu/mL.3~4 DEG C of bacteria suspension obtained save backup.
2) prepared by immobilized bacterium: bacteria suspension being added in 0.1g (0.1g/1mL) bamboo charcoal, shaken cultivation makes microorganism in bamboo
Carbon surface adsorbs biofilm；After the sodium alginate soln of the bamboo charcoal for being adsorbed with thallus and 4% is mixed well again, it is added dropwise with syringe
Enter and be stirred continuously 10~15h of crosslinking using magnetic stirring apparatus into 4% calcium chloride solution, makes microballoon, microsphere diameter
Substantially 2~3mm, as unit cell category bacteria immobilization bacterium.
Further, the bamboo charcoal partial size is 0.60~0.80mm, and sodium alginate and calcium chloride are that chemistry is pure.
Unit cell category bacteria immobilization bacterium of the invention can be applied to efficient degradation soil, the dimethachlon in water body.Unit cell category bacterium
It is 60~100kg/ha that immobilized bacterium recommends dosage in the soil, and recommending dosage in water body is 20~40mg/L.
Further, the unit cell category bacteria immobilization bacterium apply also for vinclozolin in efficient degradation soil and water body and
Iprodione.
The present invention provides a kind of unit cell category bacteria immobilization bacterium that efficient degradation sclerotium is net for the first time, the good, ring with lasting effect
Border is friendly, significant effect and it is low in cost many advantages, such as.
Detailed description of the invention
Fig. 1 illustrates different fixed materials to the degradation effect of dimethachlon；
Fig. 2 illustrates free bacterium and immobilized bacterium to the degradation effect of dimethachlon.
Specific embodiment
It is further specifically described below by embodiment to of the invention.Involved ingredient or material in following embodiments, such as
It is that commercial sources can get without specified otherwise.In related experimental methods unless otherwise specified be that the art is existing
Conventional method.Numerical value or number ratios therein refer both to mass figures or mass ratio such as without mark.
Embodiment 1:
1. the screening of fixation support:
Pre-stage test shows to fix in suspension free cell in 1%, 2%, 3%, 4%, 5% sodium alginate (SA),
4%SA shows preferable effect.Further, measuring free cell, bamboo charcoal (BC)+free cell, 4%SA+ trip
From cell, 4%SA+ bamboo charcoal (BC)+degradation effect of the free cell to the dimethachlon of 75mg/L, the result is shown in Figure 1.
2. prepared by immobilized bacterium:
1) prepared by bacteria suspension: 2mL sterile phosphate buffer being added under aseptic condition in the Spawn incubation ware of culture 48h
Parallel oscillation, and gently scrape with oese the bacterium colony of media surface, crude bacteria suspension, be transferred into liquid fermentation
In culture medium (maltose 7g/L, yeast extract 6g/L, NaCl 3g/L) pH be 7.5~8,30~35 DEG C, 150~200rpm item
48~56h is cultivated under part.The fermentation liquid of taking-up is centrifuged 10min under 10000r/min (3~4 DEG C) immediately, collects wet thallus,
And it is rinsed 3 times with sterile phosphate buffer.Then sterile phosphate buffer and wet thallus are mixed, control thalline quantity is
≥6×1010cfu/mL.3~4 DEG C of bacteria suspension obtained save backup.
2) prepared by immobilized bacterium: bacteria suspension being added in 0.1g (0.1g/1mL) bamboo charcoal, shaken cultivation makes microorganism in bamboo
Carbon surface adsorbs biofilm；After the sodium alginate soln of the bamboo charcoal for being adsorbed with thallus and 4% is mixed well again, it is added dropwise with syringe
Enter and be stirred continuously 10~15h of crosslinking using magnetic stirring apparatus into 4% calcium chloride solution, makes microballoon, microsphere diameter
Substantially 2mm, as unit cell category bacteria immobilization bacterium.
3. field trial:
It is wettable that test area sprays 40% dimethachlon that effective component is 20.25kg/ha (than recommending high dose 10 times high)
Property pulvis, the water process of control group equivalent.In field trials, fine, it is sometimes cloudy, without rainfall in 7d.Dimethachlon
After for 24 hours, each region is handled with free cell 19L/ha and immobilized cell 85kg/ha respectively, and control group is used etc.
The water process of amount carries out Soil tillage after spraying or spreading fertilizer over the fields.Every 1,3,5,7,14 and 9d random acquisition pedotheque (deep 0~
15cm, 1000g).Using dimethachlon remaining in Soil by Gas Chromatography.In order to accurately show degradation trend and effect
These values are calculated and be shown with the degradation rate of correction in rate.
4. measuring method:
Agilent6890N gas chromatograph, detector: band μ ECD；Chromatographic column: HP-5 quartz capillary column (30m ×
0.32mm×0.25μm)；Injector temperature: 260 DEG C；Detector temperature: 280 DEG C；Post case temperature: temperature-programmed mode, initially
200 DEG C of temperature, 1min is kept, rises to 270 DEG C with 10 DEG C/min, carrier gas is high pure nitrogen, flow velocity 1.2mL/min；Split ratio is
10:1；Sample volume: 1 μ L.Under the above conditions, with dimethachlon concentration of standard solution (x) for abscissa, peak area (y) is vertical sits
Plotting standard curve.Linear equation is y=991.37x+11.136, related coefficient are as follows: R2=0.9998.
5. embodiment effect:
Shown in Fig. 1,4%SA+BC+ free cell handles the degradation effect of dimethachlon well than other, therefore has selected 4%
Immobilization material of the SA and BC as bacterial strain.
Before experiment, the primary deposit amount of detection display pesticide in the soil is 16.11mg/kg.Fig. 2 shows that free bacterium exists
Good compared with immobilized bacterium to the degradation effect of dimethachlon in 1d, this may be the reason of free bacterium Rapid contact dimethachlon.After 1d, Gu
Surely it is good compared with free bacterium to the degradation effect of dimethachlon to change bacterium.After 9d, to 20.25kg/ha (than recommending high dose 5 times high
With 10 times) 40% dimethachlon wettable powder degradation rate respectively up to 95.78%, soil dimethachlon residual quantity is only 0.368mg/
Kg, and the degradation rate of free bacterium processing is only 78.81%, shows that unit cell category bacteria immobilization bacterium provided by the invention has dimethachlon
There is efficient degradation, and lasting effect is good compared with free bacterium.
Certainly, above is specific application example of the invention, and there are other embodiments of the invention, all using equivalent
The technical solution that replacement or equivalent transformation are formed, all falls within protection scope of the presently claimed invention.
Claims (5)
1. a kind of preparation method for the unit cell category bacteria immobilization bacterium that efficient degradation sclerotium is net, it is characterised in that: it is with unit cell category bacterium
Brevundimonas naejangsanensis is strain, using 4% sodium alginate and bamboo charcoal as immobilization material, by unit cell category
Bacterium effectively embeds and immobilized bacterium is made.
2. the preparation method of the net unit cell category bacteria immobilization bacterium of efficient degradation sclerotium according to claim 1, feature exist
In: include the following steps:
1) prepared by bacteria suspension: sterile phosphate buffer parallel oscillation in Spawn incubation ware being added under aseptic condition, and with connecing
Kind of ring gently scrapes the bacterium colony of media surface, crude bacteria suspension, be transferred into liquid fermentation medium and be in pH
7.5~8,30~35 DEG C, 48~56h is cultivated under the conditions of 150~200rpm；By the fermentation liquid of taking-up immediately 8000~
It is centrifuged 10~15min under 10000r/min, collects wet thallus, and rinsed 3 times with sterile phosphate buffer；Then by sterile phosphorus
Phthalate buffer and wet thallus mix, and control thalline quantity is >=6 × 1010cfu/mL；3~4 DEG C of preservations of bacteria suspension obtained are standby
With；
2) prepared by immobilized bacterium: bacteria suspension being added in bamboo charcoal, shaken cultivation makes microorganism in bamboo charcoal adsorption biofilm；Again will
It is adsorbed with the bamboo charcoal of thallus and after 4% sodium alginate soln mixes well, is added dropwise to the calcium chloride solution to 4% with syringe
It is middle to be stirred continuously 10~15h of crosslinking using magnetic stirring apparatus, make microballoon, microsphere diameter substantially 2~3mm, as singly
Born of the same parents belong to bacteria immobilization bacterium.
3. the preparation method of the net unit cell category bacteria immobilization bacterium of efficient degradation sclerotium according to claim 2, feature
Be: the liquid fermentation medium is made in proportion by following raw material: maltose 7g/L, yeast extract 6g/L, NaCl 3g/L.
4. the preparation method of the net unit cell category bacteria immobilization bacterium of efficient degradation sclerotium according to claim 2, feature
Be: the bamboo charcoal partial size is 0.60~0.80mm, and sodium alginate and calcium chloride are that chemistry is pure.
5. a kind of application for the unit cell category bacteria immobilization bacterium that efficient degradation sclerotium prepared by the method such as claims 1 or 2 is net,
Be characterized in that: the net unit cell category bacteria immobilization bacterium of the efficient degradation sclerotium is applied to the sclerotium in efficient degradation soil, water body
Only or vinclozolin and iprodione in efficient degradation soil and water body.
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Cited By (3)
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|---|---|---|---|---|
| CN109971679A (en) * | 2019-03-28 | 2019-07-05 | 贵州大学 | The preparation method and application of the net unit cell category bacterium enzyme agent of efficient degradation sclerotium |
| CN113249263A (en) * | 2021-06-01 | 2021-08-13 | 江苏大学 | Bacillus subtilis UJSQL 01 and application thereof |
| CN114717144A (en) * | 2022-03-21 | 2022-07-08 | 浙江归野生物科技有限公司 | Achromobacter and application thereof, and soil improvement microbial inoculum containing same |
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