CN109762776A - Promote the microbial bacterial agent of soil quality, preparation method and application - Google Patents
Promote the microbial bacterial agent of soil quality, preparation method and application Download PDFInfo
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- CN109762776A CN109762776A CN201910216060.XA CN201910216060A CN109762776A CN 109762776 A CN109762776 A CN 109762776A CN 201910216060 A CN201910216060 A CN 201910216060A CN 109762776 A CN109762776 A CN 109762776A
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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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- B—PERFORMING OPERATIONS; TRANSPORTING
- B09—DISPOSAL OF SOLID WASTE; RECLAMATION OF CONTAMINATED SOIL
- B09C—RECLAMATION OF CONTAMINATED SOIL
- B09C1/00—Reclamation of contaminated soil
- B09C1/10—Reclamation of contaminated soil microbiologically, biologically or by using enzymes
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B09—DISPOSAL OF SOLID WASTE; RECLAMATION OF CONTAMINATED SOIL
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- B09C1/00—Reclamation of contaminated soil
- B09C1/10—Reclamation of contaminated soil microbiologically, biologically or by using enzymes
- B09C1/105—Reclamation of contaminated soil microbiologically, biologically or by using enzymes using fungi or plants
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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
- C12N1/14—Fungi; Culture media therefor
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Abstract
The present invention relates to microbial bacterial agents; it is characterized in that including function bacterium and protection bacterium; wherein the function bacterium is made of pseudomonas putrefaciens (Shewanella putrefaciens), metal reduction ground bacillus (Geobacter metallireducens), Trichoderma viride (Trichoderma viride) and Trichoderma harzianum (Trichoderma harzianum), and the protection bacterium is bacillus subtilis (Bacillus subtilis).Further relate to a kind of method for promoting soil quality.The preparation method includes the following steps: that function bacterium, protection bacterium and bioactive substance are mixed to get composite bacteria agent by (1) in proportion；(2) composite bacteria agent obtained by step (1) is fixed on charcoal.Microbial bacterial agent produced by the present invention has the effect of very excellent in terms of promoting soil organic matter content, and method is simple, at low cost, without secondary pollution, can Synergistic degradation organic pollution of soil, have a vast market value.
Description
Technical field
The invention belongs to field of environment engineering, more particularly to a kind of microbial bacterial agent for promoting soil quality, preparation
Method and application.
Background technique
Currently, the high fertilising for increasing situation, especially industrialized agriculture is presented in the degree and area of China's organic pollution
Amount and continuous cropping planting patterns, cause the organic pollutants content such as phthalate ester, polycyclic aromatic hydrocarbon to be accumulated year by year, become influence soil quality
And an important factor for quality of agricultural product.Microorganism remediation technology can utilize the vital movement of microorganism itself, adjust or control is given birth to
Domestic biochemical reaction, and then realize the degradation and removal of organic pollutant, it is at low cost, without secondary pollution, there is biggish answer
Use potentiality.However, the features such as biological prosthetic more demanding, repairing efficiency is long for microenvironment, still restrict soil quality
Fast lifting.
Summary of the invention
The present invention relates to the following aspects:
1. microbial bacterial agent, it is characterised in that include compound bacteria I, the compound bacteria I is made of function bacterium and protection bacterium, institute
It states function bacterium and ground bacillus (Geobacter is restored by pseudomonas putrefaciens (Shewanella putrefaciens), metal
Metallireducens), Trichoderma viride (Trichoderma viride) and Trichoderma harzianum (Trichoderma
Harzianum it) forms, the protection bacterium is bacillus subtilis (Bacillus subtilis), it is preferable that corruption vacation unit cell
Bacterium (S.putrefaciens), metal restore ground bacillus (G.metallireducens), Trichoderma viride (T.viride), Ha Ci
The mass ratio of trichoderma (T.harzianum) and bacillus subtilis (B.subtilis) is 4: 3: 2: 2: 4.
2. above-mentioned 1 microbial bacterial agent, it is characterised in that include also bioactive substance, the bioactive substance is by sugar
(such as glucose, fructose, lactose, maltose, preferably glucose) and amino acid composition, the composite bacteria agent I, sugar and amino acid
Composite bacteria agent II, the preferably described composite bacteria agent I are formed, the mass ratio of sugar and amino acid is 75: 10: 15.
3. the above-mentioned described in any item microbial bacterial agents of 1-2, it is characterised in that also include charcoal, the preferably described charcoal
From plant, wheat stalk is more preferably come from, more preferably it is 120 meshes to be crossed, into one by 450 DEG C of pyrolysis wheat stalk 7h
Preferably, the mass percent that compound bacteria II accounts for charcoal is 10% to step.
4. the method for microbial bacterial agent described in preparing above-mentioned 3, it is characterised in that include the following steps:
(1) by function bacterium and protection bacterium cultivate to logarithmic growth phase, after be uniformly mixed so as to obtain composite bacteria agent I；
(2) composite bacteria agent I obtained by step (1) is mixed with bioactive substance, obtains composite bacteria agent II；
(3) by composite bacteria agent II obtained by step (2), fixation is mixed with charcoal.
5. above-mentioned 4 preparation method, it is characterised in that in step (1), the condition of culture are as follows: pseudomonas putrefaciens
(S.putrefaciens) and metal restores ground bacillus (G.metallireducens), and 37 DEG C, anaerobic condition；Trichoderma viride
(T.viride) and Trichoderma harzianum (T.harzianum), PDA culture medium, 30 DEG C, 200r.min-1, aerobic condition；Withered grass gemma
Bacillus (B.subtilis), 37 DEG C, aerobic condition.
6. the above-mentioned described in any item preparation methods of 4-5, in step (1), the logarithmic growth phase refers to corrupt false unit cell
Bacterium (S.putrefaciens), metal restore ground bacillus (G.metallireducens), bacillus subtilis (B.subtilis)
By CMC model 12h described in claim 5, Trichoderma viride (T.viride) and Trichoderma harzianum (T.harzianum) claim 5
The CMC model 108h.
7. the above-mentioned described in any item preparation methods of 4-5, in step (3), the fixing means are as follows: by charcoal mass ratio
1g.100mL is added in 10% composite bacteria agent II-1Immobilized culture base, temperature is to 30 DEG C, set time 18h.
8. the above-mentioned described in any item microbial bacterial agents of 1-3 are improving soil organic matter content or are promoting soil organic matter dirty
Application in dye reparation.
9. application described in above-mentioned 8, wherein the organic matter is selected from polycyclic aromatic hydrocarbon, Polychlorinated biphenyls, phthalate ester, organochlorine agriculture
Medicine, petroleum hydrocarbon or its composition, it is preferable that the Polychlorinated biphenyls be selected from PCB28 (2,4,4 '-trichloro biphenyl), PCB52 (2,2 ', 5,
5 '-tetrachloro biphenyls), PCB101 (2,2 ', 4,5,5 '-pentachlorodiphenyl), PCB118 (2,3 ', 4,4 ', 5- pentachlorodiphenyl), PCB138
(2,2 ', 3,4,4 ', 5 '-chlordene biphenyl), PCB153 (2,2 ', 4,4 ', 5,5 '-chlordene biphenyl) and/or PCB180 (2,2 ', 3,
4,4 ', 5,5 '-heptachlor biphenyl), the phthalate ester is selected from repefral, dibutyl phthalate, O-phthalic
Acid butyl benzyl ester, diethyl phthalate, (2- ethylhexyl) ester of phthalic acid two and/or phthalic acid are just pungent
Ester.
In specific embodiments of the present invention, the composition of Immobilized culture base are as follows: sucrose 10g, beef extract 6g, yeast leaching
Cream 1.5g, distilled water 1000mL adjust pH 7.0~7.5.
In the present invention, bioactive substance is used to for microorganism provide nutrition, used in sugar be that this field is conventional
For the sugar of microculture, include, but are not limited to glucose, fructose, lactose, maltose.Used amino acid is also this
Amino acid of the field conventionally used for microculture.
Charcoal is bio-organic materials (biomass) in anoxic or anoxybiotic environment, and what is generated after high temperature pyrolysis consolidates
State product, can not only be used for the high-quality energy, soil conditioner, also can be used as reducing agent, fertilizer slow release carrier and carbon dioxide
It seals agent etc. up for safekeeping, is widely used to carbon fixation and emission reduction, water source purifying, heavy metal adsorption and soil improvement etc., it can be to a certain extent
Solution is provided for the whole world such as climate change, environmental pollution and soil function degeneration hot issue deeply concerned.The present invention is directed to
Straw under high temperature is carbonized preparation biology by the problems such as crop material yield is big, processing disposal efficiency is low and resource utilization degree is low
Material, had not only avoided the waste of stalk resource, but also provided new and effective biomass environment material.
In R&D process of the invention, the inventors found that utilizing microorganism remediation soil organic contamination technology
There is a problem of universality difference, microorganism is fixed on charcoal, the active area of the microbial inoculum of microorganism is greatly improved, mentions
Degradation efficiency of the height to pollutant.In addition, charcoal can be used as the source of height aromatization structural constituent in agron, it is rotten
The effect of the presence meeting enhancement microbiological microbial inoculum of matter is grown, while increasing the content of organic matter in soil.By largely testing spy
Study carefully and practical proof repeatedly, it was found by the inventors of the present invention that by microbial bacterial agent of the invention, in degradation organic contamination
Object, raising soil organic matter content, improvement soil nutrient structure, crumb structure etc. have excellent ideal effect, thus
Complete the present invention.
The reagents and materials used in the present invention are commercially available.
Compared with prior art, the positive effect of the present invention is that:
The advantage of the invention is that microbial bacterial agent of the invention is in terms of degradation of contaminant, promotion soil organic matter content
All have the effect of very excellent.In addition, stalk processing disposition can be realized to economical and efficient, secondary pollution is avoided.
Specific embodiment
To be more clear the object, technical solutions and advantages of the present invention, below in conjunction with specific embodiment, the present invention is made
It is further to be described in detail.
Some specific embodiments are set forth below, to do further description to implementation of the invention and technical effect.
Bacterial strain used in following embodiment is that the public can obtain, such as protect purchased from China General Microbiological strain
Administrative center is hidden, Chinese microorganism strain inquires net, and biology is received in north, Chinese agriculture Microbiological Culture Collection administrative center, or hair
For table in technical journal, other reagents include that amino acid is the conventional reagent for being purchased from company.
Embodiment 1
(1) preparation method of microbial bacterial agent:
By pseudomonas putrefaciens, (Shewanella putrefaciens, such as Chinese microorganism strain inquire net, number
Bio-097659) and metal restores ground bacillus (Geobacter metallireducens, such as Chinese microorganism strain inquiry
Net, number bio-091064) activation, 37 DEG C, 170r.min in LB liquid medium-1, anaerobic condition culture 11h, green wood
Mould (Trichoderma viride, such as Chinese microorganism strain inquire net, number: bio-098339) and Trichoderma harzianum
(Trichoderma harzianum, such as Chinese microorganism strain inquire net, number: bio-102235) cultivates in liquid PDA
30 DEG C, 200r.min in base-1, aerobic condition culture 108h, and bacillus subtilis (Bacillus subtilis, for example, it is Chinese micro-
Biological inoculum inquires net, number: bio-103318) 37 DEG C, 180r.min in LB liquid medium-1, aerobic condition culture
12h.It will be lyophilized after above-mentioned microorganism collection, pseudomonas putrefaciens (S.putrefaciens): metal restored into ground bacillus
(G.metallireducens): Trichoderma viride (T.viride): Trichoderma harzianum (T.harzianum): bacillus subtilis
(B.subtilis) it is mixed by 4: 3: 2: 2: 4 mass ratioes, obtains composite bacteria agent I, by composite bacteria agent I: glucose: amino acid 75
: the mixing of 10: 15 mass ratioes obtains composite bacteria agent II
(2) preparation method of charcoal:
By wheat stalk in being pyrolyzed 7h in 450 DEG C of Muffle furnaces, 120 meshes are crossed.
(3) fixing means of microbial bacterial agent:
With the composite bacteria agent II of charcoal mass ratio 10%, 1g.100mL is added-1Immobilized culture base (sucrose 10g, ox
Meat extract 6g, yeast extract 1.5g, distilled water 1000mL adjust pH 7.0~7.5), revolving speed is adjusted to 170r.min-1, temperature is extremely
30 DEG C, set time 18h, the precipitating after centrifugation is cleaned 3 times using sterile saline, that is, obtains micro- life of the present invention
Object microbial inoculum.
Organic matter in the soil to be repaired mainly includes polycyclic aromatic hydrocarbon.
By microbial bacterial agent obtained, applied to polycyclic aromatic hydrocarbon (PAHs) contaminated soil near the chemical plant of Beijing, (PAHs is total
Amount is 352 μ gkg-1(218~536 μ gkg-1)) repairing test, to detect promotion of the microbial inoculum to organic pollutant degradation
Ability.The results show that 20% microbial inoculum of addition, repairing in 20 days soil, polycyclic aromatic hydrocarbon content is compared with pair for being not added with microbial inoculum
According to reducing 43%, there is apparent facilitation, accelerate the degradation of polycyclic aromatic hydrocarbon.In addition, the processing group soil organism contains
Amount increases 7.5% compared with control group.
Embodiment 2
Microbial bacterial agent made from embodiment 1 is applied to soil quality lifting test, to detect the microbial inoculum to soil matter
The improvement ability of amount.The results show that adding 20% microbial inoculum, processing 20 days according to mass percent, processing group organic matter contains
The control that amount is relatively not added with microbial inoculum improves 7% or more, increases micelle grain number amount in soil, improves soil porosity, has
Apparent improvement result.In addition, to organic matter in soil environment, (such as nearby Polychlorinated biphenyls (PCBs) pollutes agriculture in Hebei paper mill
(7 kinds of PCB28, PCB52, PCB118, PCB138, PCB153, PCB180 etc. indicative PCBs average values are 95.82 μ g/ to field soil
Kg), handle 20 days after above-mentioned Polychlorinated biphenyls content average value compared with the control for being not added with microbial inoculum reduce 51%) or heavy metal (such as
Cd polluted farmland soil, original concentration 52.69mg/kg, removal rate of the more un-added sample to Cd near the mining area of Xiangtan City
46%) improving the pollutants such as also has Synergistic degradation effect.
Embodiment 3
Microbial bacterial agent made from embodiment 1 is applied to Beijing Huairou area green house of vegetables phthalate ester contaminated soil (adjacent benzene
Dicarboxylic acid dimethyl ester, dibutyl phthalate, butyl benzyl phthalate, diethyl phthalate, O-phthalic
6 kinds of phthalate ester total concentrations such as sour two (2- ethylhexyl) esters, phthalic acid n-octyl are 0.58~3.26mg/kg), with detection
Promotion ability of the microbial inoculum to organic pollutant degradation.The results show that adding 20% microbial inoculum, reparation according to mass percent
In 15 days soil, 6 kinds of phthalate ester total concentrations reduce 48% compared with the control for being not added with microbial inoculum, have apparent facilitation,
Accelerate the degradation of phthalate ester.In addition, processing group soil organic matter content increases 7.3% compared with control group.
Embodiment 4
The experiment for repeating embodiment 1, is only that the organic matter in soil is replaced with organo-chlorine pesticide or petroleum hydrocarbon etc., with inspection
The microbial inoculum is surveyed to the promotion ability of organic pollutant degradation.The results show that adding 20% microbial inoculum, reparation by mass percentage
In 20 days soil, which reduces that (wherein soil containing organo-chlorine pesticide is from Shandong compared with the control for being not added with microbial inoculum
Certain farmland, original amount are 65.37 μ g/kg, and Organochlorine Pesticide Residues content reduces 62% after adding microbial inoculum；Containing petroleum hydrocarbon
Soil comes from Heilungkiang oil recovery factory, and original total amount average value is 60.37mg/kg, and petroleum hydrocarbon content reduces after adding microbial inoculum
83%), there is apparent facilitation, accelerate the degradation of organic matter.In addition, processing group soil organic matter content relatively compares
(wherein soil organic matter content containing organo-chlorine pesticide increases 7.2% for group increase；Soil organic matter content containing petroleum hydrocarbon increases
7.0%).
According to Environmental, Science&Techology, 2017,51,3176-3186 method is measured above-mentioned
The content of organic matter (humus) in embodiment 1-4:
Humus is most active part in the soil organism, humus mass fraction number, be soil fertility height
An important symbol.Therefore, soil organic matter content is using humus content as representative in this patent, referring to international humus association
The method that can be provided is measured[1].Specifically:
1, the extraction of humus
Pedotheque is weighed in centrifuge tube, extracting solution (0.1M Na is added in 1g: 10ml ratio4P2O7: 0.1M NaOH,
1: 1) and mixing well, use N2Persistently blow 30min.Then 15h is vibrated in 60 DEG C of water bath with thermostatic control, centrifugation discards lower sediment
Object, gained filtrate is with the HCl solution tune pH to 1~2 of 6M, and water-bath 1h, stands overnight under 60 DEG C of constant temperatures, is centrifuged 15min,
Lower sediment is humic acid crude product, and supernatant liquor is fulvic acid solution.
2, the purifying of humus
Humic acid crude product is subjected to repeated precipitation by alkali extraction-acid precipitation, after being repeated 3 times, is removed wherein using electroosmose process
Metal ion, 60 DEG C reduced pressure, pure humic acid dry sample is obtained after vacuum drying；Fulvic acid solution crosses macroporous absorbent resin
(XAD-8) and hydrogen type cation exchange resin pure fulvic acid dry sample, is obtained after being then freeze-dried.Dry sample weighing is added
With obtain humus content.
Claims (9)
1. microbial bacterial agent, it is characterised in that include compound bacteria I, the compound bacteria I is made of function bacterium and protection bacterium, the function
Energy bacterium restores ground bacillus (Geobacter by pseudomonas putrefaciens (Shewanella putrefaciens), metal
Metallireducens), Trichoderma viride (Trichoderma viride) and Trichoderma harzianum (Trichoderma
Harzianum it) forms, the protection bacterium is bacillus subtilis (Bacillus subtilis), it is preferable that corruption vacation unit cell
Bacterium (S.putrefaciens), metal restore ground bacillus (G.metallireducens), Trichoderma viride (T.viride), Ha Ci
The mass ratio of trichoderma (T.harzianum) and bacillus subtilis (B.subtilis) is 4: 3: 2: 2: 4.
2. the microbial bacterial agent of claim 1, it is characterised in that include also bioactive substance, the bioactive substance is by sugar
(such as glucose, fructose, lactose, maltose, preferably glucose) and amino acid composition, the composite bacteria agent I, sugar and amino acid
Composite bacteria agent II, the preferably described composite bacteria agent I are formed, the mass ratio of sugar and amino acid is 75: 10: 15.
3. the described in any item microbial bacterial agents of claim 1-2, it is characterised in that also include charcoal, the preferably described charcoal
From plant, wheat stalk is more preferably come from, more preferably it is 120 meshes to be crossed, into one by 450 DEG C of pyrolysis wheat stalk 7h
Preferably, the mass percent that compound bacteria II accounts for charcoal is 10% to step.
4. the method for preparing microbial bacterial agent as claimed in claim 3, it is characterised in that include the following steps:
(1) by function bacterium and protection bacterium cultivate to logarithmic growth phase, after be uniformly mixed so as to obtain composite bacteria agent I；
(2) composite bacteria agent I obtained by step (1) is mixed with bioactive substance, obtains composite bacteria agent II；
(3) by composite bacteria agent II obtained by step (2), fixation is mixed with charcoal.
5. the preparation method of claim 4, it is characterised in that in step (1), the condition of culture are as follows: pseudomonas putrefaciens
(S.putrefaciens) and metal restores ground bacillus (G.metallireducens), and 37 DEG C, anaerobic condition；Trichoderma viride
(T.viride) and Trichoderma harzianum (T.harzianum), PDA culture medium, 30 DEG C, 200r.min-1, aerobic condition；Withered grass gemma
Bacillus (B.subtilis), 37 DEG C, aerobic condition.
6. the described in any item preparation methods of claim 4-5, in step (1), the logarithmic growth phase refers to corrupt false unit cell
Bacterium (S.putrefaciens), metal restore ground bacillus (G.metallireducens), bacillus subtilis (B.subtilis)
By CMC model 12h described in claim 5, Trichoderma viride (T.viride) and Trichoderma harzianum (T.harzianum) claim 5
The CMC model 108h.
7. the described in any item preparation methods of claim 4-5, in step (3), the fixing means are as follows: by charcoal mass ratio
1g.100mL is added in 10% composite bacteria agent II-1Immobilized culture base, temperature is to 30 DEG C, set time 18h.
8. the described in any item microbial bacterial agents of claim 1-3 are improving soil with organic matter content or are promoting soil organic matter
Application in pollution amelioration.
9. application according to any one of claims 8, wherein the organic matter is selected from polycyclic aromatic hydrocarbon, Polychlorinated biphenyls, phthalate ester, organochlorine agriculture
Medicine, petroleum hydrocarbon or combinations thereof, it is preferable that the Polychlorinated biphenyls be selected from 2,4,4 '-trichloro biphenyls, 2,2 ', 5,5 '-tetrachloro biphenyls,
2,2 ', 4,5,5 '-pentachlorodiphenyls, 2,3 ', 4,4 ', 5- pentachlorodiphenyl, 2,2 ', 3,4,4 ', 5 '-chlordene biphenyl, 2,2 ', 4,4 ',
5,5 '-chlordene biphenyl and/or 2,2 ', 3,4,4 ', 5,5 '-heptachlor biphenyl, the phthalate ester are selected from repefral, neighbour
Dibatyl phithalate, butyl benzyl phthalate, diethyl phthalate, phthalic acid two (2- ethylhexyl)
Ester and/or phthalic acid n-octyl.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109868243A (en) * | 2019-01-30 | 2019-06-11 | 中国环境科学研究院 | Accelerate the microbial bacterial agent of organic pollution of soil reparation, preparation method and application |
| CN113698936A (en) * | 2020-05-22 | 2021-11-26 | 中国环境科学研究院 | Technical method for emulsifying and activating bacteria agent |
| CN113699055A (en) * | 2020-05-22 | 2021-11-26 | 中国环境科学研究院 | Technical equipment for emulsifying and activating bacteria agent |
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| CN109609417A (en) * | 2019-01-30 | 2019-04-12 | 中国环境科学研究院 | Promote the microbial bacterial agent of soil quality, preparation method and application |
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| CN113698936A (en) * | 2020-05-22 | 2021-11-26 | 中国环境科学研究院 | Technical method for emulsifying and activating bacteria agent |
| CN113699055A (en) * | 2020-05-22 | 2021-11-26 | 中国环境科学研究院 | Technical equipment for emulsifying and activating bacteria agent |
| CN113699055B (en) * | 2020-05-22 | 2023-10-13 | 中国环境科学研究院 | Technical equipment for emulsifying and activating microbial inoculum |
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| CN109762776B (en) | 2022-02-01 |
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