CN117089355A - Soil heavy metal pollution restoration agent and preparation method thereof - Google Patents
Soil heavy metal pollution restoration agent and preparation method thereof Download PDFInfo
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Classifications
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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
- C12N11/00—Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof
- C12N11/02—Enzymes or microbial cells immobilised on or in an organic carrier
- C12N11/04—Enzymes or microbial cells immobilised on or in an organic carrier entrapped within the carrier, e.g. gel or hollow fibres
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- 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
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- C12N11/00—Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof
- C12N11/02—Enzymes or microbial cells immobilised on or in an organic carrier
- C12N11/08—Enzymes or microbial cells immobilised on or in an organic carrier the carrier being a synthetic polymer
- C12N11/082—Enzymes or microbial cells immobilised on or in an organic carrier the carrier being a synthetic polymer obtained by reactions only involving carbon-to-carbon unsaturated bonds
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- C12N11/00—Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof
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Abstract
The invention discloses a soil heavy metal pollution restoration agent and a preparation method thereof, and belongs to the technical field of microbial soil restoration. The repairing agent disclosed by the invention is prepared from the following raw materials in parts by weight: 5-10 parts of microbial passivating agent, 15-25 parts of sepiolite, 3-6 parts of biochar and 1-3 parts of chitosan. The soil heavy metal pollution repairing agent disclosed by the invention can effectively realize the adsorption and removal of main heavy metal ions in soil by utilizing a means of combining chemical repairing and biological repairing, effectively reduces the effective state content of heavy metal elements, and has the effect of regulating the soil structure, so that the social benefit and the economic benefit are remarkable.
Description
Technical Field
The invention belongs to the technical field of microbial soil remediation, and particularly relates to a soil heavy metal pollution remediation agent and a preparation method thereof.
Background
Along with the continuous acceleration of the industrialization process, various pollutants containing heavy metals continuously enter surrounding farmland soil through various ways such as industrial waste gas, pesticide and chemical fertilizer, sewage irrigation, accumulation and transfer of wastes and the like, and serious soil heavy metal pollution (Cd, pb, as, cr and other pollution) is caused. Because the soil heavy metals have irreversible and undegraded characteristics, the heavy metals can bring huge harm after entering farmland soil, not only have negative effects on the quantity and population structure of soil microorganisms, but also interfere with normal metabolism of crops, so that the crop yield is reduced and the quality is reduced. In addition, after the heavy metals are absorbed and enriched in the soil through crops, the heavy metals are over-standard in content and finally enter the human body through a food chain, and are enriched in different organs of the human body and cannot be discharged, so that irreversible harm is caused to various physiological functions of the human body, and serious threat is brought to life and health of the human body. Therefore, repair and remediation of the soil environment has been eager.
The existing methods for heavy metals in soil mainly comprise a leaching method, a biological method and a solidifying method. The leaching method is to transfer heavy metals in soil into soil solution for leaching and removing by using certain liquid including acid, alkali, salt, surfactant, complexing or chelating agent and other solutions, so that secondary pollution is easy to cause, and the leaching solution needs to be treated again, but has the defects of large liquid consumption, high cost and the like. The biological method mainly utilizes bacteria such as thiobacillus ferrooxidans to produce acid and consume sulfur elements through metabolism, so that heavy metals in sludge are leached out, however, the method is time-consuming, and the bacteria are difficult to culture and have high requirements on equipment. The solidification technology is a waste drilling mud treatment technology developed in recent years. The principle is that curing agent with consolidation property is added into waste drilling mud to be converted into concrete-like curing body, and harmful components in the curing body are consolidated. However, in the solidification method, the soluble state of the heavy metal element is not changed. Over time, heavy metal ions can escape along with solidified body exudates, soil and groundwater are polluted, and environmental pollution risks are still high.
At present, a biological repair technology is adopted to treat and repair heavy metal contaminated soil, for example, chinese patent application 202010096834.2 discloses a repair agent manganese ion and manganese oxidizing bacteria, the provided repair agent can oxidize or adsorb and passivate heavy metals in the soil, so that the heavy metal contaminated soil is repaired in situ, and the repair agent disclosed by the invention is simple in operation, low in repair cost and free of secondary pollution, and can be widely applied to repair of various heavy metal contaminated soil. But the effect is unstable, the durability is poor, the function is single, and the method is not suitable for practical application and popularization.
Therefore, a process capable of thoroughly removing heavy metals in soil is developed, the risk caused by the fact that heavy metal ions still exist in the soil after the existing solidification method is avoided, and the process has very practical significance for the existing soil heavy metal treatment process.
Disclosure of Invention
Aiming at the problems in the prior art, the invention provides the soil restoration agent capable of continuously and stably restoring the heavy metal pollution of the soil, and simultaneously, the soil structure can be adjusted, the soil environment can be optimized, the heavy metal accumulation of crops can be reduced, and the soil restoration agent is suitable for various soil environments.
In order to achieve the technical purpose, the invention adopts the following technical scheme:
the soil heavy metal pollution repairing agent is prepared from the following raw materials in parts by weight: 5-10 parts of microbial passivating agent, 15-25 parts of sepiolite, 3-6 parts of biochar and 1-3 parts of chitosan.
Further, the sepiolite is pretreated sepiolite, and the pretreatment method comprises the following steps: sintering sepiolite at 300-500 ℃ for 30-60min, cooling, ball milling, mixing with 20-30% hydrochloric acid by mass fraction, stirring, mixing, filtering, washing, and drying.
Further, the biochar is corn stalk biochar.
Further, the preparation method of the microbial deactivation agent comprises the following steps:
(1) Preparing a microbial bacterial liquid: activating Pseudomonas stutzeri and bacillus megatherium respectively, inoculating to LB liquid culture medium, shake culturing at 30deg.C until the bacterial concentration is O.D600 apprxeq 2.0, and mixing according to the volume ratio of 1:1;
(2) Mixing 0.5g agarose and 0.5g polyacrylamide into 100mL water, fully stirring for 0.5-1h under the assistance of ultrasound, then placing the mixed solution at 95 ℃ for continuous stirring for 0.5h, and cooling the obtained solution at room temperature for 24h to obtain a mixed solution; and mixing the microbial liquid into the mixed solution under continuous stirring, fully drying and crushing to obtain the microbial deactivation agent.
The microbial strains can be purchased through the purchasing process by the China general microbiological culture collection center, and the biological collection is not needed.
Furthermore, the pseudomonas stutzeri (Pseudomonas stutzeri) is purchased from China general microbiological collection center, the collection number is CGMCC1.15316, and the original collection time is 2015, 6 and 15.
Furthermore, the bacillus megatherium (Bacillus megaterium) is purchased from China general microbiological collection center, the preservation number is CGMCC1.10466, and the original preservation time is 2010, 3 months and 18 days.
Further, the volume ratio of the microbial liquid to the mixed solution in the step (2) is 1:10.
The preparation method of the soil heavy metal pollution repairing agent comprises the following preparation steps:
(1) Pretreatment of sepiolite: sintering sepiolite at 300-500 ℃ for 30-60min, cooling, ball milling, mixing with 20-30% hydrochloric acid by mass fraction, stirring, mixing, filtering, washing, and drying;
(2) Preparing a microbial deactivation agent: activating Pseudomonas stutzeri and bacillus megatherium respectively, inoculating the activated Pseudomonas stutzeri and bacillus megatherium into an LB liquid culture medium, performing shake culture at 30 ℃ until the bacterial concentration is O.D600 apprxeq 2.0, and then mixing according to the volume ratio of 1:1 to obtain microbial bacterial liquid; mixing 0.5g agarose and 0.5g polyacrylamide into 100mL water, stirring thoroughly under the assistance of ultrasound for 0.5-1h,
then placing the mixed solution at 95 ℃ for continuous stirring for 0.5h, and cooling the obtained solution at room temperature for 24h to obtain a mixed solution;
mixing the microbial liquid into the mixed solution under continuous stirring, fully drying and crushing to obtain the microbial passivating agent;
(3) Preparing the raw materials according to the parts by weight, crushing, uniformly mixing, and granulating.
The raw materials used in the invention are all commercially available.
The dosage of the soil restoration agent is 5-10 kg/mu. The application method comprises mixing soil restoration agent and soil to be restored uniformly, spraying water until soil humidity is 60-70%, and maintaining for 5-10 days.
Advantageous effects
Firstly, the invention adopts the combination of chemical and microbiological technology to realize the efficient passivation of heavy metal elements in soil, the sepiolite is sintered and etched, the sepiolite has a unique nano rod-shaped crystal structure, and after sintering and etching, the adsorption capacity and the hydrogen bonding effect of hydroxyl and heavy metal are further enhanced, and the adsorption and fixing effect of the sepiolite on heavy metal are improved. The use of the biochar is characterized in that on one hand, the biochar is rich in nutrient substances such as NPK, and the addition of the biochar can increase the nutrients of soil, and on the other hand, the biochar has ion exchange, precipitation and static effects on heavy metals, so that the content of heavy metal elements in the soil can be effectively reduced.
In order to thoroughly and stably remove the heavy metal elements in the soil, the microbial strain with the function of adsorbing and reducing the heavy metal elements is screened and added, and the intracellular adsorption of bacillus megaterium can combine the heavy metal elements with complexin and the like to form thermostable proteins, so that the toxicity of the heavy metal elements is weakened or eliminated in the process, while pseudomonas stutzeri has good reduction effect on the heavy metal ions, and meanwhile, has strong extracellular adsorption effect, and various metabolites such as lipids, proteins and the like secreted by the pseudomonas stutzeri are polymerized with the heavy metal elements to form extracellular polymers, so that the heavy metal elements are adsorbed. The bacillus megaterium is matched with the intracellular adsorption stabilization effect, and the bacillus megaterium cooperate to realize the removal of metal elements in soil.
Finally, functional microorganisms hardly continuously and stably play a role in a complex soil environment, so that the porous hydrogel material microbial agent prepared by agarose and polyacrylamide is coated, so that the microbial agent is effectively protected, and meanwhile, heavy metal elements can be adsorbed and fixed to assist the microorganisms to play a role. Meanwhile, the material has good water retention effect, can regulate the soil environment, increase soil aggregate and restore the soil ecology.
In conclusion, the soil heavy metal pollution repairing agent disclosed by the invention can effectively realize the adsorption and removal of main heavy metal ions in soil by utilizing a means of combining chemical repairing and biological repairing, effectively reduce the effective state content of heavy metal elements, and has the effect of regulating the soil structure, so that the social benefit and the economic benefit are remarkable.
Detailed Description
The technical scheme of the present invention is further described below with reference to specific examples, but is not limited thereto.
Example 1
The soil heavy metal pollution repairing agent is prepared from the following raw materials in parts by weight: 5 parts of microbial passivating agent, 15 parts of sepiolite, 6 parts of biochar and 3 parts of chitosan.
The sepiolite is pretreated sepiolite, and the pretreatment method comprises the following steps: sintering sepiolite at 300 ℃ for 30min, cooling, ball milling, mixing with hydrochloric acid with the mass fraction of 20%, stirring, mixing, filtering, washing and drying.
The biochar is corn stalk biochar.
The preparation method of the microbial deactivation agent comprises the following steps:
(1) Preparing a microbial bacterial liquid: activating Pseudomonas stutzeri and bacillus megatherium respectively, inoculating to LB liquid culture medium, shake culturing at 30deg.C until the bacterial concentration is O.D600 apprxeq 2.0, and mixing according to the volume ratio of 1:1;
(2) Mixing 0.5g agarose and 0.5g polyacrylamide into 100mL water, fully stirring for 0.5h under the assistance of ultrasound, then placing the mixed solution at 95 ℃ for continuous stirring for 0.5h, and cooling the obtained solution at room temperature for 24h to obtain a mixed solution; and mixing the microbial liquid into the mixed solution under continuous stirring, fully drying and crushing to obtain the microbial deactivation agent.
The microorganism strains can be purchased through the purchasing process by the China general microbiological culture collection center, and the biological collection is not needed.
The pseudomonas stutzeri (Pseudomonas stutzeri) is purchased from China General Microbiological Collection Center (CGMCC) 1.15316, and the original preservation time is 2015, 6, 15 days.
The bacillus megatherium (Bacillus megaterium) is purchased from China general microbiological collection center, the preservation number is CGMCC1.10466, and the original preservation time is 2010, 3 months and 18 days.
The volume ratio of the microbial liquid to the mixed solution in the step (2) is 1:10.
The preparation method of the soil heavy metal pollution repairing agent comprises the following preparation steps:
(1) Pretreatment of sepiolite: sintering sepiolite at 300 ℃ for 30min, cooling, ball milling, mixing with hydrochloric acid with the mass fraction of 20% after ball milling, stirring, mixing, filtering, washing and drying;
(2) Preparing a microbial deactivation agent: activating Pseudomonas stutzeri and bacillus megatherium respectively, inoculating the activated Pseudomonas stutzeri and bacillus megatherium into an LB liquid culture medium, performing shake culture at 30 ℃ until the bacterial concentration is O.D600 apprxeq 2.0, and then mixing according to the volume ratio of 1:1 to obtain microbial bacterial liquid; mixing 0.5g agarose and 0.5g polyacrylamide into 100mL water, fully stirring for 0.5h under the assistance of ultrasound, then placing the mixed solution at 95 ℃ for continuous stirring for 0.5h, and cooling the obtained solution at room temperature for 24h to obtain a mixed solution; mixing the microbial liquid into the mixed solution under continuous stirring, fully drying and crushing to obtain the microbial passivating agent;
(3) Preparing the raw materials according to the parts by weight, crushing, uniformly mixing, and granulating.
Example 2
The soil heavy metal pollution repairing agent is prepared from the following raw materials in parts by weight: 6 parts of microbial passivating agent, 18 parts of sepiolite, 3 parts of biochar and 2 parts of chitosan.
The sepiolite is pretreated sepiolite, and the pretreatment method comprises the following steps: sintering sepiolite at 400 ℃ for 40min, cooling, ball milling, mixing with 25% hydrochloric acid by mass fraction, stirring, mixing, filtering, washing and drying.
The biochar is corn stalk biochar.
The preparation method of the microbial deactivation agent comprises the following steps:
(1) Preparing a microbial bacterial liquid: activating Pseudomonas stutzeri and bacillus megatherium respectively, inoculating to LB liquid culture medium, shake culturing at 30deg.C until the bacterial concentration is O.D600 apprxeq 2.0, and mixing according to the volume ratio of 1:1;
(2) Mixing 0.5g agarose and 0.5g polyacrylamide into 100mL water, fully stirring for 0.5h under the assistance of ultrasound, then placing the mixed solution at 95 ℃ for continuous stirring for 0.5h, and cooling the obtained solution at room temperature for 24h to obtain a mixed solution; and mixing the microbial liquid into the mixed solution under continuous stirring, fully drying and crushing to obtain the microbial deactivation agent.
The microorganism strains can be purchased through the purchasing process by the China general microbiological culture collection center, and the biological collection is not needed.
The pseudomonas stutzeri (Pseudomonas stutzeri) is purchased from China General Microbiological Collection Center (CGMCC) 1.15316, and the original preservation time is 2015, 6, 15 days.
The bacillus megatherium (Bacillus megaterium) is purchased from China general microbiological collection center, the preservation number is CGMCC1.10466, and the original preservation time is 2010, 3 months and 18 days.
The volume ratio of the microbial liquid to the mixed solution in the step (2) is 1:10.
The preparation method of the soil heavy metal pollution repairing agent comprises the following preparation steps:
(1) Pretreatment of sepiolite: sintering sepiolite at 400 ℃ for 40min, cooling, ball milling, mixing with 25% hydrochloric acid by mass fraction, stirring, mixing, filtering, washing and drying;
(2) Preparing a microbial deactivation agent: activating Pseudomonas stutzeri and bacillus megatherium respectively, inoculating the activated Pseudomonas stutzeri and bacillus megatherium into an LB liquid culture medium, performing shake culture at 30 ℃ until the bacterial concentration is O.D600 apprxeq 2.0, and then mixing according to the volume ratio of 1:1 to obtain microbial bacterial liquid; mixing 0.5g agarose and 0.5g polyacrylamide into 100mL water, fully stirring for 0.5h under the assistance of ultrasound, then placing the mixed solution at 95 ℃ for continuous stirring for 0.5h, and cooling the obtained solution at room temperature for 24h to obtain a mixed solution; mixing the microbial liquid into the mixed solution under continuous stirring, fully drying and crushing to obtain the microbial passivating agent;
(3) Preparing the raw materials according to the parts by weight, crushing, uniformly mixing, and granulating.
Example 3
The soil heavy metal pollution repairing agent is prepared from the following raw materials in parts by weight: 5 parts of microbial passivating agent, 15 parts of sepiolite, 4 parts of biochar and 1 part of chitosan.
The sepiolite is pretreated sepiolite, and the pretreatment method comprises the following steps: sintering sepiolite at 300 ℃ for 30min, cooling, ball milling, mixing with hydrochloric acid with the mass fraction of 20%, stirring, mixing, filtering, washing and drying.
The biochar is corn stalk biochar.
The preparation method of the microbial deactivation agent comprises the following steps:
(1) Preparing a microbial bacterial liquid: activating Pseudomonas stutzeri and bacillus megatherium respectively, inoculating to LB liquid culture medium, shake culturing at 30deg.C until the bacterial concentration is O.D600 apprxeq 2.0, and mixing according to the volume ratio of 1:1;
(2) Mixing 0.5g agarose and 0.5g polyacrylamide into 100mL water, fully stirring for 1h under the assistance of ultrasound, then placing the mixed solution at 95 ℃ for continuous stirring for 0.5h, and cooling the obtained solution at room temperature for 24h to obtain a mixed solution; and mixing the microbial liquid into the mixed solution under continuous stirring, fully drying and crushing to obtain the microbial deactivation agent.
The microorganism strains can be purchased through the purchasing process by the China general microbiological culture collection center, and the biological collection is not needed.
The pseudomonas stutzeri (Pseudomonas stutzeri) is purchased from China General Microbiological Collection Center (CGMCC) 1.15316, and the original preservation time is 2015, 6, 15 days.
The bacillus megatherium (Bacillus megaterium) is purchased from China general microbiological collection center, the preservation number is CGMCC1.10466, and the original preservation time is 2010, 3 months and 18 days.
The volume ratio of the microbial liquid to the mixed solution in the step (2) is 1:10.
The preparation method of the soil heavy metal pollution repairing agent comprises the following preparation steps:
(1) Pretreatment of sepiolite: sintering sepiolite at 300 ℃ for 30min, cooling, ball milling, mixing with 20-30% hydrochloric acid by mass fraction, stirring, mixing, filtering, washing and drying;
(2) Preparing a microbial deactivation agent: activating Pseudomonas stutzeri and bacillus megatherium respectively, inoculating the activated Pseudomonas stutzeri and bacillus megatherium into an LB liquid culture medium, performing shake culture at 30 ℃ until the bacterial concentration is O.D600 apprxeq 2.0, and then mixing according to the volume ratio of 1:1 to obtain microbial bacterial liquid; mixing 0.5g agarose and 0.5g polyacrylamide into 100mL water, fully stirring for 1h under the assistance of ultrasound, then placing the mixed solution at 95 ℃ for continuous stirring for 0.5h, and cooling the obtained solution at room temperature for 24h to obtain a mixed solution; mixing the microbial liquid into the mixed solution under continuous stirring, fully drying and crushing to obtain the microbial passivating agent;
(3) Preparing the raw materials according to the parts by weight, crushing, uniformly mixing, and granulating.
Example 4
The soil heavy metal pollution repairing agent is prepared from the following raw materials in parts by weight: 8 parts of microbial passivating agent, 18 parts of sepiolite, 6 parts of biochar and 3 parts of chitosan.
The sepiolite is pretreated sepiolite, and the pretreatment method comprises the following steps: sintering sepiolite at 500 ℃ for 60min, cooling, ball milling, mixing with 30% hydrochloric acid by mass fraction, stirring, mixing, filtering, washing and drying.
The biochar is corn stalk biochar.
The preparation method of the microbial deactivation agent comprises the following steps:
(1) Preparing a microbial bacterial liquid: activating Pseudomonas stutzeri and bacillus megatherium respectively, inoculating to LB liquid culture medium, shake culturing at 30deg.C until the bacterial concentration is O.D600 apprxeq 2.0, and mixing according to the volume ratio of 1:1;
(2) Mixing 0.5g agarose and 0.5g polyacrylamide into 100mL water, fully stirring for 1h under the assistance of ultrasound, then placing the mixed solution at 95 ℃ for continuous stirring for 0.5h, and cooling the obtained solution at room temperature for 24h to obtain a mixed solution; and mixing the microbial liquid into the mixed solution under continuous stirring, fully drying and crushing to obtain the microbial deactivation agent.
The microorganism strains can be purchased through the purchasing process by the China general microbiological culture collection center, and the biological collection is not needed.
The pseudomonas stutzeri (Pseudomonas stutzeri) is purchased from China General Microbiological Collection Center (CGMCC) 1.15316, and the original preservation time is 2015, 6, 15 days.
The bacillus megatherium (Bacillus megaterium) is purchased from China general microbiological collection center, the preservation number is CGMCC1.10466, and the original preservation time is 2010, 3 months and 18 days.
The volume ratio of the microbial liquid to the mixed solution in the step (2) is 1:10.
The preparation method of the soil heavy metal pollution repairing agent comprises the following preparation steps:
(1) Pretreatment of sepiolite: sintering sepiolite at 500 ℃ for 60min, cooling, ball milling, mixing with 30% hydrochloric acid by mass fraction, stirring, mixing, filtering, washing and drying;
(2) Preparing a microbial deactivation agent: activating Pseudomonas stutzeri and bacillus megatherium respectively, inoculating the activated Pseudomonas stutzeri and bacillus megatherium into an LB liquid culture medium, performing shake culture at 30 ℃ until the bacterial concentration is O.D600 apprxeq 2.0, and then mixing according to the volume ratio of 1:1 to obtain microbial bacterial liquid; mixing 0.5g agarose and 0.5g polyacrylamide into 100mL water, fully stirring for 1h under the assistance of ultrasound, then placing the mixed solution at 95 ℃ for continuous stirring for 0.5h, and cooling the obtained solution at room temperature for 24h to obtain a mixed solution; mixing the microbial liquid into the mixed solution under continuous stirring, fully drying and crushing to obtain the microbial passivating agent;
(3) Preparing the raw materials according to the parts by weight, crushing, uniformly mixing, and granulating.
Example 5
The soil heavy metal pollution repairing agent is prepared from the following raw materials in parts by weight: 10 parts of microbial deactivation agent, 25 parts of sepiolite, 5 parts of biochar and 1 part of chitosan.
The sepiolite is pretreated sepiolite, and the pretreatment method comprises the following steps: sintering sepiolite at 500 ℃ for 60min, cooling, ball milling, mixing with 30% hydrochloric acid by mass fraction, stirring, mixing, filtering, washing and drying.
The biochar is corn stalk biochar.
The preparation method of the microbial deactivation agent comprises the following steps:
(1) Preparing a microbial bacterial liquid: activating Pseudomonas stutzeri and bacillus megatherium respectively, inoculating to LB liquid culture medium, shake culturing at 30deg.C until the bacterial concentration is O.D600 apprxeq 2.0, and mixing according to the volume ratio of 1:1;
(2) Mixing 0.5g agarose and 0.5g polyacrylamide into 100mL water, fully stirring for 1h under the assistance of ultrasound, then placing the mixed solution at 95 ℃ for continuous stirring for 0.5h, and cooling the obtained solution at room temperature for 24h to obtain a mixed solution; and mixing the microbial liquid into the mixed solution under continuous stirring, fully drying and crushing to obtain the microbial deactivation agent.
The microorganism strains can be purchased through the purchasing process by the China general microbiological culture collection center, and the biological collection is not needed.
The pseudomonas stutzeri (Pseudomonas stutzeri) is purchased from China General Microbiological Collection Center (CGMCC) 1.15316, and the original preservation time is 2015, 6, 15 days.
The bacillus megatherium (Bacillus megaterium) is purchased from China general microbiological collection center, the preservation number is CGMCC1.10466, and the original preservation time is 2010, 3 months and 18 days.
The volume ratio of the microbial liquid to the mixed solution in the step (2) is 1:10.
The preparation method of the soil heavy metal pollution repairing agent comprises the following preparation steps:
(1) Pretreatment of sepiolite: sintering sepiolite at 500 ℃ for 60min, cooling, ball milling, mixing with 20-30% hydrochloric acid by mass fraction, stirring, mixing, filtering, washing, and drying;
(2) Preparing a microbial deactivation agent: activating Pseudomonas stutzeri and bacillus megatherium respectively, inoculating the activated Pseudomonas stutzeri and bacillus megatherium into an LB liquid culture medium, performing shake culture at 30 ℃ until the bacterial concentration is O.D600 apprxeq 2.0, and then mixing according to the volume ratio of 1:1 to obtain microbial bacterial liquid; mixing 0.5g agarose and 0.5g polyacrylamide into 100mL water, fully stirring for 1h under the assistance of ultrasound, then placing the mixed solution at 95 ℃ for continuous stirring for 0.5h, and cooling the obtained solution at room temperature for 24h to obtain a mixed solution; mixing the microbial liquid into the mixed solution under continuous stirring, fully drying and crushing to obtain the microbial passivating agent;
(3) Preparing the raw materials according to the parts by weight, crushing, uniformly mixing, and granulating.
Comparative example 1
The soil heavy metal pollution repairing agent is prepared from the following raw materials in parts by weight: 10 parts of microbial deactivation agent, 25 parts of sepiolite, 5 parts of biochar and 1 part of chitosan.
The sepiolite is pretreated sepiolite, and the pretreatment method comprises the following steps: sintering sepiolite at 500 ℃ for 60min, cooling, ball milling, mixing with 30% hydrochloric acid by mass fraction, stirring, mixing, filtering, washing and drying.
The biochar is corn stalk biochar.
The preparation method of the microbial deactivation agent comprises the following steps:
(1) Preparing a microbial bacterial liquid: activating Pseudomonas stutzeri and Bacillus megaterium respectively, inoculating into LB liquid culture medium, shake culturing at 30deg.C until the bacterial concentration is O.D600 ≡2.0, mixing according to the volume ratio of 1:1, drying thoroughly, and pulverizing to obtain microbial passivator.
The microorganism strains can be purchased through the purchasing process by the China general microbiological culture collection center, and the biological collection is not needed.
The pseudomonas stutzeri (Pseudomonas stutzeri) is purchased from China General Microbiological Collection Center (CGMCC) 1.15316, and the original preservation time is 2015, 6, 15 days.
The bacillus megatherium (Bacillus megaterium) is purchased from China general microbiological collection center, the preservation number is CGMCC1.10466, and the original preservation time is 2010, 3 months and 18 days.
The preparation method of the soil heavy metal pollution repairing agent comprises the following preparation steps:
(1) Pretreatment of sepiolite: sintering sepiolite at 500 ℃ for 60min, cooling, ball milling, mixing with 20-30% hydrochloric acid by mass fraction, stirring, mixing, filtering, washing, and drying;
(2) Preparing a microbial deactivation agent: activating Pseudomonas stutzeri and bacillus megatherium respectively, inoculating the activated pseudomonas stutzeri and bacillus megatherium into an LB liquid culture medium, carrying out shake culture at 30 ℃ until the concentration of bacteria is O.D600 apprxeq 2.0, mixing according to the volume ratio of 1:1, fully drying and crushing to obtain a microbial passivating agent;
(3) Preparing the raw materials according to the parts by weight, crushing, uniformly mixing, and granulating.
In this comparative example, the raw materials and the preparation method were the same as in example 5, except that the microorganism was not coated, that is, the microorganism bacterium liquid was directly used.
Comparative example 2
The composition and proportion of the microbial liquid in the preparation of the microbial deactivation agent are changed, and the other raw materials and the preparation method are the same as those in example 5.
TABLE 1 composition of the strains (volume ratio)
| Pseudomonas stutzeri | Bacillus megaterium | |
| Comparative example 2 | 1 | 2 |
| Comparative example 3 | 2 | 1 |
| Comparative example 4 | 0 | 1 |
| Comparative example 5 | 1 | 0 |
Experimental test
Test soil: collecting soil on the surface layer of farmland polluted by the periphery of Shandong Jining mining area, removing impurities, airing, grinding, and storing by a nylon sieve with the diameter of 2mm for later use.
Test plants: little cabbage (Shanghai green)
The test method comprises the following steps: in the test, 11 groups of treatments are arranged, wherein the blank group treatment is used for directly filling the tested soil into a flowerpot with the length of 15cm multiplied by 20 cm; the other experimental groups respectively add the soil restoration agents obtained in examples 1-5 and comparative examples 1-5 according to 5 kg/mu (about 1.4% of soil weight), uniformly mix soil and passivating agent, then fill the soil into flowerpots, each pot contains 1kg of soil, each group of experiments is provided with 3 parallel samples, and the results are averaged. Deionized water is added by a weighing method to maintain the water content of the soil to about 60 percent. After one week of maintenance, sowing about 10 seeds of the Chinese cabbage in each pot, fixing 3 seedlings, harvesting when the plants grow and mature, and performing index analysis and test. Collecting edible parts of the cabbages, washing, deactivating enzymes in a baking oven at 105 ℃ for 1h, drying at 60 ℃, weighing, crushing, and preserving to be detected; collecting a root soil sample of the Chinese cabbage, removing impurities, naturally air-drying, grinding, and respectively sieving with 10 mesh and 100 mesh sieve for storage.
Analytical measurement method
The pH of the soil is measured by a glass electrode method, and the form of heavy metals in the soil is measured by a BCR extraction method. Soil samples are digested by adopting a tetra-acid digestion method, and plant samples are digested by adopting HNO 3 -HClO 4 And carrying out wet digestion, wherein the content of heavy metals in soil and plants is measured by adopting an inductively coupled plasma mass spectrometer (ICP-MS), and the measurement error of the content of each heavy metal is less than +/-10%.
Enrichment factor (BCF): heavy metal content in plant/heavy metal content in soil.
Table 2 experimental results
TABLE 3 enrichment factor of edible parts of Brassica oleracea for heavy metals
It should be noted that the above-mentioned embodiments are merely some, but not all embodiments of the preferred mode of carrying out the invention. It is evident that all other embodiments obtained by a person skilled in the art without making any inventive effort, based on the above-described embodiments of the invention, shall fall within the scope of protection of the invention.
Claims (7)
1. The soil heavy metal pollution repairing agent is characterized by comprising the following raw materials in parts by weight: 5-10 parts of microbial passivating agent, 15-25 parts of sepiolite, 3-6 parts of biochar and 1-3 parts of chitosan.
2. The soil heavy metal pollution remediation agent of claim 1, wherein the sepiolite is pretreated sepiolite by the following steps: sintering sepiolite at 300-500 ℃ for 30-60min, cooling, ball milling, mixing with 20-30% hydrochloric acid by mass fraction, stirring, mixing, filtering, washing, and drying.
3. The soil heavy metal pollution remediation agent of claim 1, wherein the preparation method of the microbial deactivation agent comprises the following steps:
(1) Preparing a microbial bacterial liquid: activating Pseudomonas stutzeri and bacillus megatherium respectively, inoculating to LB liquid culture medium, shake culturing at 30deg.C until the bacterial concentration is O.D600 apprxeq 2.0, and mixing according to the volume ratio of 1:1;
(2) Mixing 0.5g agarose and 0.5g polyacrylamide into 100mL water, fully stirring for 0.5-1h under the assistance of ultrasound, then placing the mixed solution at 95 ℃ for continuous stirring for 0.5h, and cooling the obtained solution at room temperature for 24h to obtain a mixed solution; and mixing the microbial liquid into the mixed solution under continuous stirring, fully drying and crushing to obtain the microbial deactivation agent.
4. The soil heavy metal pollution remediation agent of claim 3 wherein pseudomonas stutzeri (Pseudomonas stutzeri) is purchased from the China general microbiological collection center with a collection number of CGMCC1.15316.
5. The soil heavy metal pollution remediation agent of claim 3 wherein bacillus megaterium (CGMCC 1.10466) is purchased from the chinese common microbiological collection center.
6. The soil heavy metal pollution remediation agent of claim 3, wherein the volume ratio of the microbial liquid to the mixed solution in the step (2) is 1:10.
7. A method for preparing a soil heavy metal pollution remediation agent as claimed in any one of claims 1 to 6, comprising the steps of:
(1) Pretreatment of sepiolite: sintering sepiolite at 300-500 ℃ for 30-60min, cooling, ball milling, mixing with 20-30% hydrochloric acid by mass fraction, stirring, mixing, filtering, washing, and drying;
(2) Preparing a microbial deactivation agent: activating Pseudomonas stutzeri and bacillus megatherium respectively, inoculating the activated Pseudomonas stutzeri and bacillus megatherium into an LB liquid culture medium, performing shake culture at 30 ℃ until the bacterial concentration is O.D600 apprxeq 2.0, and then mixing according to the volume ratio of 1:1 to obtain microbial bacterial liquid; mixing 0.5g agarose and 0.5g polyacrylamide into 100mL water, stirring thoroughly under the assistance of ultrasound for 0.5-1h,
then placing the mixed solution at 95 ℃ for continuous stirring for 0.5h, and cooling the obtained solution at room temperature for 24h to obtain a mixed solution;
mixing the microbial liquid into the mixed solution under continuous stirring, fully drying and crushing to obtain the microbial passivating agent;
(3) Preparing the raw materials according to the parts by weight, crushing, uniformly mixing, and granulating.
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