CN109852567B - Microbial agent for accelerating heavy metal pollution remediation of soil and preparation method and application thereof - Google Patents
Microbial agent for accelerating heavy metal pollution remediation of soil and preparation method and application thereof Download PDFInfo
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- CN109852567B CN109852567B CN201910212083.3A CN201910212083A CN109852567B CN 109852567 B CN109852567 B CN 109852567B CN 201910212083 A CN201910212083 A CN 201910212083A CN 109852567 B CN109852567 B CN 109852567B
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Abstract
The invention relates to a microbial agent, which is characterized by comprising a complex microbial agent I, wherein the complex microbial agent I consists of a respiratory humus bacterium and a probiotic bacterium, wherein the respiratory humus bacterium consists of pseudomonas putreferans (Shewanella putrefacesiens), Bacillus metalloreductus (Geobacter metallardeus) and Shewanella alga (Shewanella alga), and the probiotic bacterium consists of anaerobic ammonia oxidation Brocadia (Brocadia anammoxidans) and Bacillus subtilis (Bacillus subtilis). The invention also relates to a preparation method and application of the microbial agent.
Description
Technical Field
The invention belongs to the field of environmental engineering, and particularly relates to a microbial agent for accelerating heavy metal pollution remediation of soil and a preparation method thereof.
Background
The quality of cultivated land in China generally falls, heavy metal pollution is the main problem of soil pollution at present, and the extracellular respiration microorganisms are used for enhancing the electron transfer and circulation capacity in the soil based on the existence of a large number of electron transfer mediators in the soil, so that the reductive degradation of various pollutants can be effectively promoted, and the environment effect is very obvious.
Disclosure of Invention
The present invention relates to one of the following aspects:
1. the microbial agent is characterized by comprising a complex microbial agent I, wherein the complex microbial agent I consists of humus respiratory bacteria and probiotics, wherein the said Humicola species consists of Pseudomonas putrefaciens (Shewanella putrefacesiens), Geobacter metalloreducens (Geobacter malrieducens) and Shewanella alga (Shewanella alga), the probiotics consist of anaerobic ammonia oxidation Brocadia (Brocadia anammoxans) and Bacillus subtilis (Bacillus subtilis), preferably, the mass ratio of the Pseudomonas putrefaciens (S.putrefacesiens), the metalloproteus (G.metallardeucens), the Shewanella alga (S.alga), the anaerobic ammonia oxidation Brocadia (B.anammoxans) and the Bacillus subtilis (B.subtilis) is A: B: C: D: E, wherein A is selected from the group consisting of numbers from 2 to 4, B is selected from the group consisting of numbers from 2 to 4, C is selected from the group consisting of numbers from 1 to 3, D is selected from the group consisting of numbers from 1 to 3, and E is selected from the group consisting of numbers from 3 to 5.
2. The microbial agent of the above 1, characterized by further comprising a bioactive substance, wherein the bioactive substance is composed of sugar (such as glucose, fructose, lactose, maltose, preferably glucose) and amino acid, the complex microbial agent I, sugar and amino acid are composed of complex microbial agent II, preferably the complex microbial agent I, and the mass ratio of sugar and amino acid is a: b: c, wherein a is selected from the number of 60-80, b is selected from the number of 15-40, and c is selected from the number of 10-30.
3. The microbial agent of any one of the above 1-2, characterized by further comprising biochar, preferably the biochar is from plants, more preferably from pine needles, more preferably the biochar is prepared by pyrolyzing pine needles at 400 ℃ for 4-6 hours and sieving through a 150-200 mesh sieve, and further preferably the composite bacteria II account for 6-12% by mass of the biochar.
4. The method for preparing the microbial agent of 3 is characterized by comprising the following steps:
(1) culturing humus respiratory bacteria and probiotics to logarithmic phase, and mixing uniformly to obtain the compound microbial inoculum I.
(2) Freeze-drying the compound bacterial agent I obtained in the step (1), and mixing with a bioactive substance to obtain a compound bacterial agent II.
(3) And (3) mixing and fixing the composite microbial inoculum II obtained in the step (2) with biochar.
5. The preparation method of the 4 is characterized in that the culture conditions of the humus respiratory bacteria and the anaerobic ammonia oxidation Brookadskya are 35-38 ℃ and anaerobic conditions are adopted; the culture condition of the bacillus subtilis is 35-38 ℃, and the aerobic condition is adopted.
6. The method according to any one of the above 4 to 5, wherein the logarithmic growth phase is an optical density value (OD) of the bacterial liquid at a wavelength of 600nm600) The range is 0.5 to 0.9.
7. The method for preparing any one of the above 4 to 5, characterized in that the method for preparing the biochar is as follows: pyrolyzing the pine needles at 400 ℃ for 4-6 h, and sieving the pine needles with a sieve of 150-200 meshes.
8. The production method according to any one of the above 4 to 5, characterized in that the fixing method is:
adding biochar (preferably 1-1.2 g) into 100mL of immobilized culture medium, sterilizing, and adding 6-12% of composite microbial inoculum II by mass at 35-38 ℃ for 16-20 h.
9. The microbial agent of any one of the above 1-3 for promoting remediation of heavy metal pollution of soil (preferably reducing the content of heavy metal in soil).
10. The use of 9 above, wherein the heavy metal is selected from the group consisting of Cd, Cr, U, Cu, Pb, Hg, Zn, and combinations thereof.
In a particular embodiment of the invention, the composition of the immobilization medium is: 10g of cane sugar, 6g of beef extract, 1.5g of yeast extract and 1000mL of distilled water, and adjusting the pH value to 7.0-7.5.
In the present invention, the bioactive substance is used to provide nutrition to the microorganism, wherein the sugar used is a sugar conventionally used in the art for culturing microorganisms, including, but not limited to, glucose, fructose, lactose, maltose. The amino acids used are also those conventionally used in the art for the cultivation of microorganisms.
The biochar is a substance with large specific surface area, looseness, porosity, rich carbon, high aromaticity and strong decomposition resistance, contains active functional groups such as hydroxyl, carboxyl, carbonyl and the like, has a good adsorption effect on various pollutants, can improve the pH value of soil, reduce the loss of nitrogen and improve the microbial characteristics of the soil, and is widely applied to soil improvement. The microbial technology restoration is a process of regulating or controlling biochemical reactions in the environment and degrading organic pollutants by utilizing the life activities of microorganisms, and has great application potential due to low cost and quick response.
In the development process of the invention, the inventors found that in the natural redox system of soil, humus respiring bacteria using humus as an electron acceptor can gradually degrade macromolecular pollutants into non-toxic or low-toxic micromolecular substances through an electron transfer process in the presence of humus, thereby removing the pollutants. In addition, the biochar can be used as a source of highly aromatic structural components in soil humus, so that the maturity of compost can be improved, and the organic matter content in soil can be increased. Through a large number of experimental researches and repeated practical verifications, the inventor finally finds that the microbial agent has excellent and ideal effects on the aspects of accelerating the heavy metal conversion, improving the soil structure and the like, thereby completing the invention.
On the basis of the common knowledge in the field, the above preferred conditions can be combined randomly to obtain the preferred embodiments of the invention. The reagents and starting materials used in the present invention are commercially available.
Compared with the prior art, the invention has the positive improvement effects that:
the method has the advantages that solid wastes such as pine needles, straws and the like which possibly cause environmental pollution are economically and efficiently treated, so that the solid wastes are changed into valuables, the solid wastes are used as beneficial factors to realize reutilization in environmental protection, and the problem of secondary pollution is avoided. In addition, the prepared microbial agent has both adsorption capacity and reduction degradation capacity, and has very excellent effects on degrading pollutants and increasing the content of organic matters in soil.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is further described in detail with reference to the following embodiments. It will be understood by those of ordinary skill in the art that the following examples are for illustrative purposes only and that the present invention is to be protected as set forth in the appended claims.
The following examples are presented to further illustrate the practice and technical effects of the present invention. In the following examples, the percentages are by mass unless otherwise specified.
The strains used in the following examples are publicly available, for example, from the China general microbiological culture Collection center, China network for querying microbial cultures, North Nami organism, China agricultural microbial culture Collection center, or published in the scientific journal, and other reagents including amino acids are commonly available from companies.
Example 1
1. The preparation method of the microbial agent comprises the following steps:
(1) culturing Pseudomonas putrefaciens (S.putrefacesiens, such as Chinese microbial species query net, number bio-097659), Bacillus Metallurgicus (G.metallardeucens, such as Chinese microbial species query net, number bio-091064), Shewanella alga (S.alga, such as Chinese microbial species query net, number bio-74287), and Brevibacterium anammox (B.amamoxidans, such as metabolic pathway of anammox bacteria and research progress of key enzymes thereof, Bridgman, Wangshume, Zhang Mzhu, Chenghua, journal of ecology, 2012,31 (3); 738 and 744) in liquid LB culture medium at 37 ℃ in anaerobic environment to OD6000.6, culturing Bacillus subtilis in liquid LB culture medium at 37 deg.C in aerobic environment to OD600Is 0.5.
(2) Collecting thalli of pseudomonas putrefaction (S.putrefacesiens), pseudomonas metalloreducens (G.metalrieducens), Shewanella alga (S.alga), anaerobic ammonia oxidation Brookada (B.anammoxidans) and bacillus subtilis (B.subtilis), and mixing the thalli according to the mass ratio of 2: 1: 3 after freeze-drying to obtain the compound microbial agent I.
(3) And mixing the compound microbial inoculum with a bioactive substance to obtain a compound microbial inoculum II. The concrete proportion is as follows: a compound microbial inoculum I: glucose: the mass ratio of the amino acid is 60: 20.
(4) Fixing the compound microbial inoculum II on biochar (prepared by pyrolyzing pine needles at 400 ℃ for 6h and sieving through a 150-mesh sieve). Adding 1.2g of biochar into 100mL of immobilized culture medium (10 g of cane sugar, 6g of beef extract, 1.5g of yeast extract and 1000mL of distilled water, adjusting pH to 7.0-7.5), sterilizing at 121 ℃ for 20min, cooling, adding a composite microbial inoculum II with the mass ratio of 12%, and adjusting the rotating speed to 170r.min-1And (3) fixing for 16h at the temperature of 38 ℃, centrifuging, and cleaning the precipitate for 2 times by using sterile normal saline to obtain the microbial agent.
2. The prepared microbial agent is applied to a remediation test of Cd-polluted farmland soil (52.69mg/kg) near a certain mining area in Hunan Tan City to detect the reduction capability of the microbial agent on Cd. The result shows that the Cd-polluted soil added with 15% of the microbial agent disclosed by the invention in percentage by mass is treated for 10 days, and the removal rate of Cd is improved by 32% compared with that of a sample not added, so that the microbial agent has an obvious effect on promoting heavy metal pollution remediation.
Example 2
1. The preparation method of the microbial agent comprises the following steps:
(1) culturing Pseudomonas putrefaciens (S.putrefacesiens), Bacillus Metallus (G.metalrieducens), Shewanella alga (S.alga) and anaerobic ammonium oxidation Brookada (B.anammoxdans) in liquid LB medium at 35 deg.C under anaerobic condition to OD6000.8, culturing Bacillus subtilis in liquid LB culture medium at 35 deg.C in aerobic environment to OD600Is 0.6.
(2) Collecting thalli of pseudomonas putrefaction (S.putrefacesiens), pseudomonas metalloreducens (G.metalrieducens), Shewanella alga (S.alga), anaerobic ammonia oxidation Brucella (B.anammoxidans) and bacillus subtilis (B.subtilis), freeze-drying, and mixing according to the mass ratio of 4: 3: 5 to obtain the compound microbial inoculum I.
(3) And mixing the compound microbial inoculum with a bioactive substance to obtain a compound microbial inoculum II. The concrete proportion is as follows: a compound microbial inoculum I: glucose: the mass ratio of the amino acid is 65: 20: 15.
(4) Fixing the compound microbial inoculum II on organismsCharcoal (prepared by pyrolyzing pine needles at 400 ℃ for 5h and sieving with a 180-mesh sieve). Adding 1.2g of biochar into 100mL of immobilized culture medium (10 g of cane sugar, 6g of beef extract, 1.5g of yeast extract and 1000mL of distilled water, adjusting pH to 7.0-7.5), sterilizing at 121 ℃ for 20min, cooling, adding 10% of composite microbial inoculum II, and adjusting the rotating speed to 160r.min-1And (3) fixing for 20 hours at the temperature of 35 ℃, centrifuging, and cleaning the precipitate for 3 times by using sterile normal saline to obtain the microbial agent.
2. The prepared microbial agent is applied to a remediation test of Cd and Cr contaminated farmland soil (the Cd content is 48.31mg/kg, and the Cr content is 29.23mg/kg) near a certain mining area in Hunan Tan City to detect the reduction capability of the microbial agent on Cd and Cr, and the result shows that 15% of the contaminated soil added with the microbial agent is treated for 7 days, the removal rate of Cd and Cr is respectively improved by 18% and 15% compared with that of a sample which is not added, and the removal rate of Cd and Cr is respectively improved by 42% and 37% compared with that of the sample which is not added after 15 days of treatment, so that the microbial agent has an obvious effect on promoting the remediation of heavy metal composite pollution.
Example 3
1. The preparation method of the microbial agent comprises the following steps:
(1) culturing Pseudomonas putrefaciens (S.putrefacesiens), Bacillus Metallus (G.metalrieducens), Shewanella alga (S.alga) and anaerobic ammonium oxidation Brookada (B.anammoxdans) in liquid LB medium at 37 deg.C under anaerobic condition to OD6000.9, culturing Bacillus subtilis in liquid LB culture medium at 35 deg.C in aerobic environment to OD600Is 0.7.
(2) Collecting thalli of pseudomonas putrefaction (S.putrefacesiens), pseudomonas metalloreducens (G.metalrieducens), Shewanella alga (S.alga), anaerobic ammonia oxidation Brookada (B.anammoxidans) and bacillus subtilis (B.subtilis), and mixing the thalli according to the mass ratio of 3: 2: 4 after freeze-drying to obtain the compound microbial agent I.
(3) And mixing the compound microbial inoculum with a bioactive substance to obtain a compound microbial inoculum II. The concrete proportion is as follows: a compound microbial inoculum I: glucose: the mass ratio of the amino acid is 70: 15.
(4) Fixing the compound microbial inoculum II on biochar (prepared by pyrolyzing pine needles at 400 ℃ for 4h and sieving by a 200-mesh sieve). Adding 1g of biochar into 100mL of immobilized culture medium (10 g of cane sugar, 6g of beef extract, 1.5g of yeast extract and 1000mL of distilled water, adjusting pH to 7.0-7.5), sterilizing at 121 ℃ for 20min, cooling, adding 6% of composite microbial inoculum II, and adjusting the rotating speed to 170r.min-1And (3) fixing for 19h at the temperature of 37 ℃, centrifuging, and cleaning the precipitate for 3 times by using sterile normal saline to obtain the microbial agent.
2. The prepared microbial agent is applied to a U-polluted farmland soil (the U content is 10.87mg/kg) repair test near a uranium mine in Jiangxi to detect the reduction capability of the microbial agent to U, and the result shows that the contaminated soil added with 20% of the microbial agent in percentage by mass is treated for 7 days, the U removal rate is improved by 31% compared with that of an unadditized sample, and the U removal rate is respectively improved by 46% compared with that of the unadditized sample after 15 days of treatment, which indicates that the microbial agent has remarkable effect in promoting the repair of heavy metal pollution.
Example 4
The experiment of example 1 was repeated except that the heavy metals in the soil were replaced with Cu, Pb, Hg, Zn and a combination to examine the ability of the microbial inoculum to promote the degradation of heavy metals. The results show that 20 percent of the microbial inoculum is added according to the mass percentage and the soil is repaired for 20 days, and the data are shown in the following table
The implementation and results of the embodiments show that the biochar immobilized microbial agent is used for repairing the heavy metal contaminated soil, a synergistic effect can be generated, the heavy metal pollution removal efficiency in the soil is improved, the repairing time is shortened, the secondary pollution problem is avoided, the biochar immobilized microbial agent can be used for large-area heavy metal composite contaminated sites, the market popularization value is high, and the application prospect is excellent.
The above-mentioned embodiments are intended to illustrate the objects, technical solutions and advantages of the present invention in further detail, and it should be understood that the above-mentioned embodiments are only exemplary embodiments of the present invention and are not intended to limit the present invention, and any modifications, equivalents, improvements and the like made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (19)
1. Microbial agent, characterized by comprising a complex microbial agent I consisting of a humus respiring bacterium consisting of pseudomonas putrefying (Shewanella putrefacesiens), pseudomonas metalloreductans (Geobacter metalriensis) and Shewanella alga (Shewanella alga) and a probiotic bacterium consisting of anaerobic ammonium-oxidizing burcadres (brocdia and amoxidans) and Bacillus subtilis (Bacillus subtilis).
2. The microbial agent of claim 1, wherein the mass ratio of pseudomonas putrefactive (s.putrefacesens), pseudomonas metalloreducens (g.metalreducens), shewanella alga (s.alga), anaerobic ammonia oxidizing burkholderia (b.amammoxidans) and bacillus subtilis (b.subtilis) is a: b: c: d: e, wherein A is selected from the group consisting of numbers from 2 to 4, B is selected from the group consisting of numbers from 2 to 4, C is selected from the group consisting of numbers from 1 to 3, D is selected from the group consisting of numbers from 1 to 3, and E is selected from the group consisting of numbers from 3 to 5.
3. The microbial agent according to claim 1, characterized by further comprising a biologically active substance consisting of a sugar and an amino acid, wherein said complex microbial agent I, the sugar and the amino acid constitute a complex microbial agent II.
4. The microbial agent according to claim 3, wherein the mass ratio of the complex microbial agent I, the sugar and the amino acid is a: b: c, wherein a is selected from the group consisting of numbers from 60 to 80, b is selected from the group consisting of numbers from 15 to 40, and c is selected from the group consisting of numbers from 10 to 30.
5. The microbial inoculant of claim 3 or 4, wherein the sugar is glucose, fructose, lactose or maltose.
6. The microbial inoculant according to any one of claims 3 to 4, further comprising biochar.
7. The microbial inoculant of claim 6, wherein the biochar is from a plant.
8. The microbial inoculant according to claim 6, wherein the plant is pine needles.
9. The microbial inoculant according to claim 6, wherein the biochar is prepared by pyrolyzing pine needles at 400 ℃ for 4-6 h and sieving the pine needles with a 150-200-mesh sieve.
10. The microbial agent of claim 6, wherein the mass percent of the composite bacteria II in the biochar is 6-12%.
11. A method for preparing the microbial inoculant of claim 6, comprising the steps of:
(1) culturing humus respiratory bacteria and probiotics to logarithmic phase, mixing uniformly to obtain a complex microbial inoculum I,
(2) freeze-drying the compound bacterial agent I obtained in the step (1), mixing the freeze-dried compound bacterial agent I with a bioactive substance to obtain a compound bacterial agent II,
(3) and (3) mixing and fixing the compound microbial inoculum II obtained in the step (2) with the biochar.
12. The preparation method of claim 11, wherein the culture conditions of the respiratory humus bacteria and the anaerobic ammonium oxidation Brookadskyi bacteria are 35-38 ℃ and anaerobic conditions; the culture condition of the bacillus subtilis is 35-38 ℃, and the aerobic condition is adopted.
13. The method according to any one of claims 11 to 12, wherein the logarithmic growth phase is an optical density value of the bacterial liquid at a wavelength of 600nm in a range of 0.5 to 0.9.
14. The method according to any one of claims 11 to 12, wherein the biochar is produced by: pyrolyzing the pine needles at 400 ℃ for 4-6 h, and sieving the pine needles with a sieve of 150-200 meshes.
15. The method of any one of claims 11 to 12, wherein the immobilization method is:
adding biochar into 100mL of immobilized culture medium, sterilizing, adding a composite microbial inoculum II with the mass ratio of 6-12%, and fixing for 16-20 h at the temperature of 35-38 ℃.
16. The method of claim 15, wherein the amount of biochar is 1-1.2 g.
17. The use of the microbial inoculant of any one of claims 1-6 for promoting remediation of heavy metal contamination of soil.
18. The use of claim 17, wherein the promotion of remediation of heavy metal contamination of soil is a reduction in the heavy metal content of soil.
19. The use of claim 17 or 18, wherein the heavy metal is selected from Cd, Cr, U, Cu, Pb, Hg, Zn and combinations.
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| CN113861997B (en) * | 2021-11-02 | 2022-07-01 | 创想未来生物工程(北京)有限公司 | Heavy metal passivation and soil remediation type microbial agent and preparation method thereof |
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| WO2012060417A1 (en) * | 2010-11-02 | 2012-05-10 | 森下仁丹株式会社 | Capsule for non-ferrous metal collection and method for collecting non-ferrous metal |
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