CN109852567A - Accelerate the microbial bacterial agent and its preparation method and application of heavy metal pollution of soil reparation - Google Patents
Accelerate the microbial bacterial agent and its preparation method and application of heavy metal pollution of soil reparation Download PDFInfo
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- CN109852567A CN109852567A CN201910212083.3A CN201910212083A CN109852567A CN 109852567 A CN109852567 A CN 109852567A CN 201910212083 A CN201910212083 A CN 201910212083A CN 109852567 A CN109852567 A CN 109852567A
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- composite bacteria
- bacteria agent
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- charcoal
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Abstract
The present invention relates to microbial bacterial agents, it is characterized in that including composite bacteria agent I, the composite bacteria agent I by humus respiration bacterium and probiotic group at, wherein the humus respiration bacterium is made of pseudomonas putrefaciens (Shewanella putrefaciens), metal reduction ground bacillus (Geobacter metallireducens) and Shewanella alga (Shewanella alga), and the probiotics is made of Anammox Broca moral Salmonella (Brocadia anammoxidans) and bacillus subtilis (Bacillus subtilis).The invention further relates to the preparation method and its usages of the microbial bacterial agent.
Description
Technical field
The invention belongs to field of environment engineering, more particularly to a kind of microbial bacteria for accelerating heavy metal pollution of soil reparation
Agent and preparation method thereof.
Background technique
China's farmland quality is totally on a declining curve, and heavy metal pollution is the main problem of current soil pollution, based on soil
There are a large amount of electron transmission mediators in earth can using electron transmission and circulation ability in extracellular breathing microorganism enhancing soil
The deoxidization, degradation of multiple pollutant is effectively facilitated, the environmental effect with highly significant.
Summary of the invention
The present invention relates to following one aspect:
1. microbial bacterial agent, it is characterised in that include composite bacteria agent I, the composite bacteria agent I is by humus respiration bacterium and benefit
Raw bacterium composition, wherein the humus respiration bacterium is by pseudomonas putrefaciens (Shewanella putrefaciens), metal
Ground bacillus (Geobacter metallireducens) and Shewanella alga (Shewanella alga) composition are restored, it is described
Probiotics is by Anammox Broca moral Salmonella (Brocadia anammoxidans) and bacillus subtilis (Bacillus
Subtilis it) forms, it is preferable that pseudomonas putrefaciens (S.putrefaciens), metal restore ground bacillus
(G.metallireducens), Shewanella alga (S.alga), Anammox Broca moral Salmonella
(B.anammoxidans) and the mass ratio of bacillus subtilis (B.subtilis) is A: B: C: D: E, and wherein A is selected from 2-4's
Number, B are selected from the number of 2-4, and C is selected from the number of 1-3, and D is selected from the number of 1-3, and E is selected from the number of 3-5.
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, sugared and amino acid mass ratio is a: b: c, and wherein a is selected from the number of 60-80
Word, b are selected from the number of 15-40, and c is selected from the number of 10-30.
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, pine needle is more preferably come from, more preferably it is to cross 150~200 meshes by 400 DEG C of pyrolysis 4~6h of pine needle and make
, it is further preferred that the mass percent that compound bacteria II accounts for charcoal is 6~12%.
4. the method for preparing above-mentioned 3 microbial bacterial agents, it is characterised in that include the following steps:
(1) humus respiration bacterium and probiotic's culture are uniformly mixed afterwards to logarithmic growth phase, obtain composite bacteria agent I.
(2) it will be mixed after the freeze-drying of composite bacteria agent I obtained by step (1) with bioactive substance, obtain 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 the training of humus respiration bacterium and Anammox Broca moral Salmonella
The condition of supporting is 35~38 DEG C, anaerobic condition；The condition of culture of bacillus subtilis is 35~38 DEG C, aerobic condition.
6. the above-mentioned described in any item preparation methods of 4-5, it is characterised in that the logarithmic growth phase refers to bacterium solution in 600nm
OD value (OD at wavelength600) range be 0.5~0.9.
7. the above-mentioned described in any item preparation methods of 4-5, it is characterised in that the charcoal the preparation method comprises the following steps: 400 DEG C
It is pyrolyzed 4~6h of pine needle, crosses 150~200 meshes.
8. the above-mentioned described in any item preparation methods of 4-5, it is characterised in that the fixing means are as follows:
100mL Immobilized culture base is added in charcoal (preferably 1~1.2g), after sterilizing, it is 6~12% that mass ratio, which is added,
Composite bacteria agent II, temperature be 35~38 DEG C, the set time be 16~20h.
9. the above-mentioned described in any item microbial bacterial agents of 1-3 are promoting heavy metal pollution of soil reparation (preferably reduction soil
Content of beary metal) in application.
10. application described in above-mentioned 9, wherein the heavy metal is selected from Cd, Cr, U, Cu, Pb, Hg, Zn and combinations thereof.
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 be a kind of large specific surface area, it is loose porous, containing the strong object of carbon rich, height aromatization, antidecomposition ability
Matter has preferable adsorption effect to multiple pollutant, and soil can be improved containing hydroxyl, carboxyl, carbonyl isoreactivity functional group
PH value reduces nitrogen loss and improves Microorganism Characteristics, has been widely used in soil improvement.Microbial technique reparation
It is the vital movement using microorganism, adjusts or control the biochemical reaction in habitat, the process of degradable organic pollutant, because of cost
It is low, quick and have biggish application potential.
In R&D process of the invention, the inventors found that this natural redox system in soil
In, using humus as the humus respiration bacterium of electron acceptor, under the conditions of can be existing for the humus, pass through electron transmission mistake
Journey, by the pollutant of macromolecular, gradually Degradation and Transformation is non-toxic or low-toxic small-molecule substance, to realize the removal of pollutant.This
Outside, charcoal can be used as the source of height aromatization structural constituent in agron, is conducive to the rotten degree for improving compost, increases
Add the content of organic matter in soil.Practical proof by a large amount of Experimental Research and repeatedly, the present inventor send out finally
Existing, of the invention microbial bacterial agent has excellent ideal effect in terms of accelerating heavy metal conversion, improving,
So as to complete the present invention.
On the basis of common knowledge of the art, above-mentioned each optimum condition, can any combination to get each preferable reality of the present invention
Example.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 economical and efficient handled pine needle, stalk etc. and may cause the solid of problem of environmental pollution
Waste is allowed to turn waste into wealth, and realizes the recycling in environmental protection as beneficial agents, while avoiding secondary pollution and asking
Topic.In addition, microbial bacterial agent produced by the present invention has both adsorption capacity and deoxidization, degradation ability, in degradation of contaminant, soil is promoted
All have the effect of in terms of the earth content of organic matter very excellent.
Specific embodiment
To make the objectives, technical solutions, and advantages of the present invention clearer, below in conjunction with specific embodiment, to this hair
It is bright to be described in further detail.It will be understood by those skilled in the art that following embodiment was merely illustrative
Purpose, protection of the invention are subject to documented by appended claims.
Some specific embodiments are set forth below, to do further description to implementation of the invention and technical effect.Under
It states in each embodiment, unless otherwise instructed, the percentage refers both to mass percent.
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, the preparation method of microbial bacterial agent:
(1) by pseudomonas putrefaciens, (S.putrefaciens, such as Chinese microorganism strain inquire net, number bio-
097659), (G.metallireducens, such as Chinese microorganism strain inquire net, number bio- to metal reduction ground bacillus
091064), Shewanella alga (S.alga, such as Chinese microorganism strain inquire net, number bio-74287), anaerobism ammonia oxygen
Change Broca moral Salmonella (B.anammoxidans, for example, anaerobic ammonia oxidizing bacteria metabolic pathway and its key enzyme progress,
Zhou Yingjie, Wang Shumei, Zhang Zhaoji, Chen Shaohua, " ecological magazine ", 2012,31 (3)；738-744) in LB liquid medium,
37 DEG C, anaerobic environment culture to OD600It is 0.6, by bacillus subtilis (B.subtilis) in LB liquid medium, 37 DEG C,
Aerobic environment culture is to OD600It is 0.5.
(2) pseudomonas putrefaciens (S.putrefaciens) is collected, metal restores ground bacillus
(G.metallireducens), Shewanella alga (S.alga), Anammox Broca moral Salmonella
(B.anammoxidans) and bacillus subtilis (B.subtilis) thallus, according to the ratio of 2: 2: 1: 1: 3 mass ratioes after freeze-drying
Example mixing, obtains composite bacteria agent I.
(3) composite bacteria agent is mixed with bioactive substance, obtains composite bacteria agent II.Specific proportion are as follows: composite bacteria agent I:
Glucose: amino acid masses ratio is 60: 20: 20.
(4) composite bacteria agent II is fixed on charcoal (400 DEG C of pyrolysis pine needle 6h cross 150 meshes and are made).1.2g is raw
100mL Immobilized culture base is added in object charcoal, and (sucrose 10g, beef extract 6g, yeast extract 1.5g, distilled water 1000mL adjust pH
7.0~7.5), cooling after 121 DEG C of sterilizing 20min, the composite bacteria agent II that mass ratio is 12% is added, adjust revolving speed to
170r.min-1, temperature is to 38 DEG C, set time 16h, the precipitating after centrifugation, is cleaned 2 times, that is, obtained using sterile saline
Obtain microbial bacterial agent of the present invention.
2, microbial bacterial agent obtained is applied to Cd polluted farmland soil (52.69mg/kg) near the mining area of Xiangtan City
Repairing test, to detect the microbial bacterial agent to the reducing power of Cd.The results show that 15% present invention of addition by mass percentage
The Cd contaminated soil of the microbial bacterial agent is handled 10 days, and more un-added sample improves 32% to the removal rate of Cd, this table
The bright microbial bacterial agent has remarkable efficacy in terms of promoting heavy metal pollution reparation.
Embodiment 2
1, the preparation method of microbial bacterial agent:
(1) by pseudomonas putrefaciens (S.putrefaciens), metal reduction ground bacillus (G.metallireducens),
Shewanella alga (S.alga), Anammox Broca moral Salmonella (B.anammoxidans) in LB liquid medium,
35 DEG C, anaerobic environment culture to OD600It is 0.8, by bacillus subtilis (B.subtilis) in LB liquid medium, 35 DEG C,
Aerobic environment culture is to OD600It is 0.6.
(2) pseudomonas putrefaciens (S.putrefaciens) is collected, metal restores ground bacillus
(G.metallireducens), Shewanella alga (S.alga), Anammox Broca moral Salmonella
(B.anammoxidans) and bacillus subtilis (B.subtilis) thallus, according to the ratio of 4: 4: 3: 3: 5 mass ratioes after freeze-drying
Example mixing, obtains composite bacteria agent I.
(3) composite bacteria agent is mixed with bioactive substance, obtains composite bacteria agent II.Specific proportion are as follows: composite bacteria agent I:
Glucose: amino acid masses ratio is 65: 20: 15.
(4) composite bacteria agent II is fixed on charcoal (400 DEG C of pyrolysis pine needle 5h cross 180 meshes and are made).1.2g is raw
100mL Immobilized culture base is added in object charcoal, and (sucrose 10g, beef extract 6g, yeast extract 1.5g, distilled water 1000mL adjust pH
7.0~7.5), cooling after 121 DEG C of sterilizing 20min, the composite bacteria agent II that mass ratio is 10% is added, adjust revolving speed to
160r.min-1, temperature is to 35 DEG C, set time 20h, the precipitating after centrifugation, is cleaned 3 times, that is, obtained using sterile saline
Obtain microbial bacterial agent of the present invention.
2, microbial bacterial agent obtained is applied to Cd and Cr polluted farmland soil near the mining area of Xiangtan City (Cd content is
48.31mg/kg, Cr content are 29.23mg/kg) repairing test, to detect the microbial bacterial agent to the reducing power of Cd and Cr,
The results show that adding the contaminated soil of 15% microbial bacterial agent of the present invention by mass percentage, handles 7 days, be relatively not added with
Sample 18% and 15% has been respectively increased to the removal rate of Cd and Cr, handle 15 days, more un-added sample is to Cd's and Cr
42% and 37% has been respectively increased in removal rate, this shows that the microbial bacterial agent has in terms of promoting heavy-metal composite pollution reparation
Remarkable efficacy.
Embodiment 3
1, the preparation method of microbial bacterial agent:
(1) by pseudomonas putrefaciens (S.putrefaciens), metal reduction ground bacillus (G.metallireducens),
Shewanella alga (S.alga), Anammox Broca moral Salmonella (B.anammoxidans) in LB liquid medium,
37 DEG C, anaerobic environment culture to OD600It is 0.9, by bacillus subtilis (B.subtilis) in LB liquid medium, 35 DEG C,
Aerobic environment culture is to OD600It is 0.7.
(2) pseudomonas putrefaciens (S.putrefaciens) is collected, metal restores ground bacillus
(G.metallireducens), Shewanella alga (S.alga), Anammox Broca moral Salmonella
(B.anammoxidans) and bacillus subtilis (B.subtilis) thallus, according to the ratio of 3: 3: 2: 2: 4 mass ratioes after freeze-drying
Example mixing, obtains composite bacteria agent I.
(3) composite bacteria agent is mixed with bioactive substance, obtains composite bacteria agent II.Specific proportion are as follows: composite bacteria agent I:
Glucose: amino acid masses ratio is 70: 15: 15.
(4) composite bacteria agent II is fixed on charcoal (400 DEG C of pyrolysis pine needle 4h cross 200 meshes and are made).By 1g charcoal
Addition 100mL Immobilized culture base (sucrose 10g, beef extract 6g, yeast extract 1.5g, distilled water 1000mL, adjusting pH 7.0~
7.5), cooling after 121 DEG C of sterilizing 20min, the composite bacteria agent II that mass ratio is 6% is added, adjusts revolving speed to 170r.min-1, temperature
Degree is to 37 DEG C, set time 19h, the precipitating after centrifugation, is cleaned 3 times, that is, obtained of the present invention using sterile saline
Microbial bacterial agent.
2, by microbial bacterial agent obtained, applied to U polluted farmland soil near the uranium ore of Jiangxi, (U content is 10.87mg/
Kg) repairing test, to detect the microbial bacterial agent to the reducing power of U, the results show that adding 20% hair by mass percentage
The contaminated soil of the bright microbial bacterial agent is handled 7 days, and more un-added sample improves 31% to the removal rate of U, processing 15
It, more un-added sample has been respectively increased 46% to the removal rate of U, this shows that the microbial bacterial agent is promoting heavy metal pollution
Repairing aspect has remarkable efficacy.
Embodiment 4
The experiment for repeating embodiment 1, is only that the heavy metal in soil is replaced with Cu, Pb, Hg, Zn and combination, with detection
The promotion ability of the microbial inoculum counterweight metal degradation.The results show that adding 20% microbial inoculum by mass percentage, repairing 20 days
In soil, data are referring to following table
The implementation of the various embodiments described above and the result shows that, the present invention use charcoal fixation of microbe microbial inoculum remediating heavy metal
Contaminated soil can produce synergistic effect, improves the heavy metal pollution removal efficiency in soil, shortens repair time, and nothing
Secondary pollution problem, can also large area be used for heavy-metal composite pollution place, market popularization value is high, before having fabulous application
Scape.
Particular embodiments described above has carried out further in detail the purpose of the present invention, technical scheme and beneficial effects
Describe in detail bright, it should be understood that the above is only a specific embodiment of the present invention, is not intended to restrict the invention, it is all
Within the spirit and principles in the present invention, any modification, equivalent substitution, improvement and etc. done should be included in protection of the invention
Within the scope of.
Claims (10)
1. microbial bacterial agent, it is characterised in that include composite bacteria agent I, the composite bacteria agent I is by humus respiration bacterium and probiotics
Composition, wherein the humus respiration bacterium is restored by pseudomonas putrefaciens (Shewanella putrefaciens), metal
Ground bacillus (Geobacter metallireducens) and Shewanella alga (Shewanella alga) composition, it is described prebiotic
Bacterium is by Anammox Broca moral Salmonella (Brocadia anammoxidans) and bacillus subtilis (Bacillus
Subtilis it) forms, it is preferable that pseudomonas putrefaciens (S.putrefaciens), metal restore ground bacillus
(G.metallireducens), Shewanella alga (S.alga), Anammox Broca moral Salmonella
(B.anammoxidans) and the mass ratio of bacillus subtilis (B.subtilis) is A: B: C: D: E, and wherein A is selected from 2-4's
Number, B are selected from the number of 2-4, and C is selected from the number of 1-3, and D is selected from the number of 1-3, and E is selected from the number of 3-5.
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, sugared and amino acid mass ratio is a: b: c, and wherein a is selected from the number of 60-80
Word, b are selected from the number of 15-40, and c is selected from the number of 10-30.
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, pine needle is more preferably come from, more preferably it is to cross 150~200 meshes by 400 DEG C of pyrolysis 4~6h of pine needle and make
, it is further preferred that the mass percent that compound bacteria II accounts for charcoal is 6~12%.
4. the method for preparing microbial bacterial agent described in claim 3, it is characterised in that include the following steps:
(1) humus respiration bacterium and probiotic's culture are uniformly mixed afterwards to logarithmic growth phase, obtain composite bacteria agent I.
(2) it will be mixed after the freeze-drying of composite bacteria agent I obtained by step (1) with bioactive substance, obtain 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 the training of humus respiration bacterium and Anammox Broca moral Salmonella
The condition of supporting is 35~38 DEG C, anaerobic condition；The condition of culture of bacillus subtilis is 35~38 DEG C, aerobic condition.
6. the described in any item preparation methods of claim 4-5, it is characterised in that the logarithmic growth phase refers to bacterium solution in 600nm
OD value (OD at wavelength600) range be 0.5~0.9.
7. the described in any item preparation methods of claim 4-5, it is characterised in that the charcoal the preparation method comprises the following steps: 400 DEG C
It is pyrolyzed 4~6h of pine needle, crosses 150~200 meshes.
8. the described in any item preparation methods of claim 4-5, it is characterised in that the fixing means are as follows:
100mL Immobilized culture base is added in charcoal (preferably 1~1.2g), after sterilizing, it is 6~12% to answer that mass ratio, which is added,
Combined bacteria agent II, temperature are 35~38 DEG C, and the set time is 16~20h.
9. the described in any item microbial bacterial agents of claim 1-3 are promoting heavy metal pollution of soil reparation (preferably reduction soil
Content of beary metal) in application.
10. application as claimed in claim 9, wherein the heavy metal is selected from Cd, Cr, U, Cu, Pb, Hg, Zn and combination.
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| CN109852567B (en) | 2021-03-12 |
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