CN106591166A - In-situ bioremediation preparation and remediation method for chlorothalonil contaminated soil - Google Patents
In-situ bioremediation preparation and remediation method for chlorothalonil contaminated soil Download PDFInfo
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- CN106591166A CN106591166A CN201510679796.2A CN201510679796A CN106591166A CN 106591166 A CN106591166 A CN 106591166A CN 201510679796 A CN201510679796 A CN 201510679796A CN 106591166 A CN106591166 A CN 106591166A
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- 239000002689 soil Substances 0.000 title claims abstract description 105
- 239000005747 Chlorothalonil Substances 0.000 title claims abstract description 64
- CRQQGFGUEAVUIL-UHFFFAOYSA-N chlorothalonil Chemical compound ClC1=C(Cl)C(C#N)=C(Cl)C(C#N)=C1Cl CRQQGFGUEAVUIL-UHFFFAOYSA-N 0.000 title claims abstract description 64
- 238000002360 preparation method Methods 0.000 title claims abstract description 55
- 238000011065 in-situ storage Methods 0.000 title claims abstract description 37
- 238000005067 remediation Methods 0.000 title claims abstract description 32
- 238000000034 method Methods 0.000 title claims abstract description 21
- 241000894006 Bacteria Species 0.000 claims abstract description 47
- 241000233866 Fungi Species 0.000 claims abstract description 47
- 241000228245 Aspergillus niger Species 0.000 claims abstract description 28
- 241000589517 Pseudomonas aeruginosa Species 0.000 claims abstract description 28
- 241000122973 Stenotrophomonas maltophilia Species 0.000 claims abstract description 20
- 241000338144 Caballeronia zhejiangensis Species 0.000 claims abstract description 7
- 241000293018 Cunninghamella bertholletiae Species 0.000 claims abstract description 3
- 238000004321 preservation Methods 0.000 claims description 27
- 241001453380 Burkholderia Species 0.000 claims description 20
- 241000235402 Rhizomucor Species 0.000 claims description 19
- 239000003337 fertilizer Substances 0.000 claims description 18
- 241000235555 Cunninghamella Species 0.000 claims description 17
- 239000000203 mixture Substances 0.000 claims description 17
- 230000000813 microbial effect Effects 0.000 claims description 16
- 238000009472 formulation Methods 0.000 claims description 15
- 239000007952 growth promoter Substances 0.000 claims description 15
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 6
- 239000001301 oxygen Substances 0.000 claims description 5
- 229910052760 oxygen Inorganic materials 0.000 claims description 5
- 239000005696 Diammonium phosphate Substances 0.000 claims description 3
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 claims description 3
- 239000001888 Peptone Substances 0.000 claims description 3
- 108010080698 Peptones Proteins 0.000 claims description 3
- 240000004808 Saccharomyces cerevisiae Species 0.000 claims description 3
- 229920002472 Starch Polymers 0.000 claims description 3
- XSQUKJJJFZCRTK-UHFFFAOYSA-N Urea Chemical compound NC(N)=O XSQUKJJJFZCRTK-UHFFFAOYSA-N 0.000 claims description 3
- 229940041514 candida albicans extract Drugs 0.000 claims description 3
- 239000004202 carbamide Substances 0.000 claims description 3
- 239000003795 chemical substances by application Substances 0.000 claims description 3
- MNNHAPBLZZVQHP-UHFFFAOYSA-N diammonium hydrogen phosphate Chemical compound [NH4+].[NH4+].OP([O-])([O-])=O MNNHAPBLZZVQHP-UHFFFAOYSA-N 0.000 claims description 3
- 229910000388 diammonium phosphate Inorganic materials 0.000 claims description 3
- 235000019838 diammonium phosphate Nutrition 0.000 claims description 3
- 239000008103 glucose Substances 0.000 claims description 3
- 239000003895 organic fertilizer Substances 0.000 claims description 3
- 235000019319 peptone Nutrition 0.000 claims description 3
- OTYBMLCTZGSZBG-UHFFFAOYSA-L potassium sulfate Chemical compound [K+].[K+].[O-]S([O-])(=O)=O OTYBMLCTZGSZBG-UHFFFAOYSA-L 0.000 claims description 3
- 229910052939 potassium sulfate Inorganic materials 0.000 claims description 3
- 235000011151 potassium sulphates Nutrition 0.000 claims description 3
- 239000000843 powder Substances 0.000 claims description 3
- 230000001737 promoting effect Effects 0.000 claims description 3
- 239000011780 sodium chloride Substances 0.000 claims description 3
- 239000008107 starch Substances 0.000 claims description 3
- 235000019698 starch Nutrition 0.000 claims description 3
- 239000012138 yeast extract Substances 0.000 claims description 3
- 235000013877 carbamide Nutrition 0.000 claims description 2
- 230000000593 degrading effect Effects 0.000 claims description 2
- 241000588748 Klebsiella Species 0.000 claims 2
- 241000218378 Magnolia Species 0.000 claims 2
- 230000015556 catabolic process Effects 0.000 abstract description 10
- 238000006731 degradation reaction Methods 0.000 abstract description 9
- 241000293031 Rhizomucor variabilis Species 0.000 abstract description 6
- 238000005286 illumination Methods 0.000 abstract description 6
- 239000003153 chemical reaction reagent Substances 0.000 abstract description 5
- 238000009313 farming Methods 0.000 abstract description 5
- 235000013305 food Nutrition 0.000 abstract description 4
- 244000005700 microbiome Species 0.000 description 10
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 description 6
- 238000009629 microbiological culture Methods 0.000 description 5
- 241000235525 Rhizomucor pusillus Species 0.000 description 4
- 239000000126 substance Substances 0.000 description 3
- 239000006228 supernatant Substances 0.000 description 3
- 244000174681 Michelia champaca Species 0.000 description 2
- 230000001580 bacterial effect Effects 0.000 description 2
- 239000013043 chemical agent Substances 0.000 description 2
- 238000011109 contamination Methods 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 230000035558 fertility Effects 0.000 description 2
- 230000002538 fungal effect Effects 0.000 description 2
- UHOVQNZJYSORNB-UHFFFAOYSA-N monobenzene Natural products C1=CC=CC=C1 UHOVQNZJYSORNB-UHFFFAOYSA-N 0.000 description 2
- 238000006303 photolysis reaction Methods 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 2
- HIXDQWDOVZUNNA-UHFFFAOYSA-N 2-(3,4-dimethoxyphenyl)-5-hydroxy-7-methoxychromen-4-one Chemical compound C=1C(OC)=CC(O)=C(C(C=2)=O)C=1OC=2C1=CC=C(OC)C(OC)=C1 HIXDQWDOVZUNNA-UHFFFAOYSA-N 0.000 description 1
- 241000134107 Burkholderia plantarii Species 0.000 description 1
- 240000007594 Oryza sativa Species 0.000 description 1
- 235000007164 Oryza sativa Nutrition 0.000 description 1
- 244000046052 Phaseolus vulgaris Species 0.000 description 1
- 235000010627 Phaseolus vulgaris Nutrition 0.000 description 1
- PMZURENOXWZQFD-UHFFFAOYSA-L Sodium Sulfate Chemical compound [Na+].[Na+].[O-]S([O-])(=O)=O PMZURENOXWZQFD-UHFFFAOYSA-L 0.000 description 1
- 241000209140 Triticum Species 0.000 description 1
- 235000021307 Triticum Nutrition 0.000 description 1
- 230000000844 anti-bacterial effect Effects 0.000 description 1
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 1
- 239000003899 bactericide agent Substances 0.000 description 1
- 150000001555 benzenes Chemical class 0.000 description 1
- 238000007664 blowing Methods 0.000 description 1
- 239000012159 carrier gas Substances 0.000 description 1
- 230000006652 catabolic pathway Effects 0.000 description 1
- 238000012258 culturing Methods 0.000 description 1
- 230000018044 dehydration Effects 0.000 description 1
- 238000006297 dehydration reaction Methods 0.000 description 1
- 239000008367 deionised water Substances 0.000 description 1
- 229910021641 deionized water Inorganic materials 0.000 description 1
- 229910003460 diamond Inorganic materials 0.000 description 1
- 239000010432 diamond Substances 0.000 description 1
- 238000007865 diluting Methods 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 235000013399 edible fruits Nutrition 0.000 description 1
- RTZKZFJDLAIYFH-UHFFFAOYSA-N ether Substances CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 description 1
- 238000001704 evaporation Methods 0.000 description 1
- 230000008020 evaporation Effects 0.000 description 1
- 238000004817 gas chromatography Methods 0.000 description 1
- 239000001963 growth medium Substances 0.000 description 1
- 238000003973 irrigation Methods 0.000 description 1
- 230000002262 irrigation Effects 0.000 description 1
- 231100000915 pathological change Toxicity 0.000 description 1
- 230000036285 pathological change Effects 0.000 description 1
- 239000000575 pesticide Substances 0.000 description 1
- 239000003208 petroleum Substances 0.000 description 1
- 230000015843 photosynthesis, light reaction Effects 0.000 description 1
- 229920006391 phthalonitrile polymer Polymers 0.000 description 1
- 239000000047 product Substances 0.000 description 1
- 230000001681 protective effect Effects 0.000 description 1
- 235000009566 rice Nutrition 0.000 description 1
- 239000002904 solvent Substances 0.000 description 1
- 238000005507 spraying Methods 0.000 description 1
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- 230000003442 weekly effect Effects 0.000 description 1
- 238000005303 weighing Methods 0.000 description 1
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- Agricultural Chemicals And Associated Chemicals (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
Relating to the soil remediation field, the invention discloses a preparation for in-situ remediation of chlorothalonil contaminated soil. The preparation contains fungi and/or bacteria with chlorothalonil degradation ability. The fungi can be at least one of Aspergillus niger, rhizomucor variabilis and Cunninghamella bertholletiae, and the bacteria can be at least one of Burkholderia zhejiangensis, pseudomonas aeruginosa and Stenotrophomonas maltophilia. According to the invention, the provided preparation is employed for in-situ bioremediation of chlorothalonil contaminated soil, not only overcomes the problems that photochemical degradation is easily limited by illumination conditions and can easily cause secondary chemical reagent pollution to arable land, at the same time makes full use of fungi and/or bacteria for catabolism of chlorothalonil residual in soil, thus solving the problem that high residual chlorothalonil in soil severely endangers food safety. At the same time, the micro-ecology of soil is improved, so that soil can recover farming ability more rapidly. Also, the method is low in cost, thus having broad application prospects.
Description
Technical Field
The invention relates to the field of soil remediation, in particular to an in-situ bioremediation preparation and a remediation method for chlorothalonil-contaminated soil.
Background
Chlorothalonil is a protective stem and leaf spray-substituted benzene bactericide developed by American Diamond alkali-making company, has the chemical name of 2,4,5, 6-tetrachloro-1, 3-phthalonitrile, and is widely used for preventing and treating diseases of various crops such as vegetables, fruit trees, beans, rice, wheat and the like. But is composed ofThe nonstandard application and excessive spraying of the chlorothalonil cause a great amount of residues on the surface of agricultural products and in the farming soil, which not only seriously endangers the food safety, but also can destroy the micro-ecological environment of the soil and bring hidden troubles for the loss of the fertility of the farming soil and the pathological changes of the crops. Currently, photochemical degradation is the major degradation pathway for pesticides. Penuela and other researches find that the photolysis half-life period of chlorothalonil in deionized water reaches more than one hundred hours, and Fe is added3+The half-life of the post-photolysis can be shortened to about 4h, however, the photochemical degradation method is not suitable for the large-scale repair of chlorothalonil residual agricultural soil due to the limitation of illumination conditions and the avoidance of secondary pollution of chemical reagents to cultivated lands.
Disclosure of Invention
The invention provides a preparation for in-situ remediation of chlorothalonil-contaminated soil and an in-situ bioremediation method of chlorothalonil-contaminated soil, aiming at solving the problems that a photochemical degradation method is easily limited by illumination conditions and easily causes secondary chemical reagent pollution to cultivated land in the remediation of chlorothalonil-contaminated soil.
The present inventors have unexpectedly found in their research that some fungi, such as Aspergillus niger, Rhizomucor varibilis, Cunninghamella grayani and bacteria, such as Burkholderia plantarii, Pseudomonas aeruginosa and Stenotrophomonas maltophilia, have the ability to degrade chlorothalonil and do not require light conditions, and the culture medium for culturing the above fungi and bacteria does not contain substances that cause harmful chemical contamination of the soil, thereby avoiding the problem of secondary contamination of cultivated lands by chemical agents in photochemical degradation. Furthermore, the inventors have also found that when a fungus and a bacterium are used in combination, or different fungi are used in combination, and different bacteria are used in combination, a better repairing effect is obtained as compared with when one kind of fungus or bacterium is used alone. Further, the present inventors have found that when a microbial growth promoter and a fertilizer are used in combination, chlorothalonil-contaminated soil can be more advantageously restored, thereby completing the present invention.
The invention provides a preparation for in-situ remediation of chlorothalonil contaminated soil, which contains fungi and/or bacteria with chlorothalonil degradation capability;
wherein the fungus is at least one of Aspergillus niger (Aspergillus niger) with the preservation number of CGMCC No.11113, Rhizomucor varilabilis (Rhizomucor varilabilis) with the preservation number of CGMCC No.11114 and Cunninghamella tabescens (Cunninghamella bertholletiae) with the preservation number of CGMCC No. 11115;
the bacteria is at least one of Burkholderia (Burkholderia zhejiangensis) with the preservation number of CGMCC No.10848, Pseudomonas aeruginosa (Pseudomonas aeruginosa) with the preservation number of CGMCC No.10842 and Stenotrophomonas maltophilia with the preservation number of CGMCC No. 10846.
The invention also provides an in-situ bioremediation method of the chlorothalonil contaminated soil, which comprises the step of carrying out in-situ remediation on the chlorothalonil contaminated soil by using the preparation for in-situ remediation of the chlorothalonil contaminated soil.
When the preparation for in-situ remediation of chlorothalonil contaminated soil is used for in-situ bioremediation of chlorothalonil contaminated soil, the adopted fungi and/or bacteria do not need illumination conditions and do not adopt chemical reagents polluting the environment, so that the problems that photochemical degradation is easily limited by illumination conditions and secondary chemical reagents polluting cultivated land are easily caused are solved, and meanwhile, the fungi and/or bacteria are sufficiently utilized to decompose and metabolize residual chlorothalonil in the soil by the aid of a microbial growth promoter and a fertilizer, so that the problem that the residual chlorothalonil in the soil seriously endangers food safety is solved, the microecology of the soil is improved, the soil can recover the farming capability more quickly, and the method is low in cost and has wide application prospects.
Additional features and advantages of the invention will be set forth in the detailed description which follows.
Biological preservation
The strains of the invention are as follows:
1. aspergillus niger (Aspergillus niger) was deposited at the general microbiological culture Collection center of China Committee for culture Collection of microorganisms (address: No. 3, West Lu 1, the institute of microbiology, national academy of sciences, Japan) (CGMCC No.11113, abbreviation of the unit of deposit) on 14 days 7 months 7 and 2015.
2. Rhizomucor mutabilis (Rhizomucor variabilis) is preserved in the ordinary microorganism center of China Committee for culture Collection of microorganisms (address: No. 3 of Xilu 1 of North Chen of the south-facing-Yang district, Beijing, institute of microbiology, China academy of sciences, postal code: 100101) within 7-month and 14-day 2015, wherein the preservation number is CGMCC No. 11114.
3. Cunninghamella grayish (Cunninghamella berthlletiae) is preserved in the China general microbiological culture Collection center (address: West Luo No.1 of Beijing Kogyo Beichen province, 3, institute of microbiology, China academy of sciences, postal code: 100101) within 7-month and 14-day of 2015 (the preservation unit is abbreviated as CGMCC), and the preservation number is CGMCC No. 11115.
4. Burkholderia (Burkholderia zhejiangensis) is preserved in the China general microbiological culture Collection center (address: Beijing, West Luo No.1, North Cheng, Xiu, No. 3, institute of microbiology, China academy of sciences, Japan) within 5 months and 22 days of 2015 for 5 months and 10 days (the preservation unit is abbreviated as CGMCC), and the preservation number is CGMCC No. 10848.
5. Pseudomonas aeruginosa (Pseudomonas aeruginosa) was deposited at the general microbiological culture Collection center of China Committee for culture Collection of microorganisms (address: No. 3, institute of microbiology, national academy of sciences, Ministry of China, West Luo No.1, Beijing, the south China, and the like, in 5-22.2015, the name "CGMCC"), and the number of the deposit was CGMCC No. 10842.
6. Stenotrophomonas maltophilia (Stenotrophoromonas maltophilia) was deposited at the general microbiological culture Collection center of China Committee for culture Collection of microorganisms (address: No. 3, institute of microbiology, national academy of sciences, Ministry of China, West Lu No.1, North Chen, Inward, Beijing) within 22 days 5-2015, and the deposit number is CGMCC No. 10846.
Detailed Description
The following describes in detail specific embodiments of the present invention. It should be understood that the detailed description and specific examples, while indicating the present invention, are given by way of illustration and explanation only, not limitation.
According to a first aspect of the present invention, there is provided a formulation for in situ remediation of chlorothalonil contaminated soil, the formulation comprising fungi and/or bacteria having chlorothalonil degrading ability;
wherein the fungi are: at least one of Aspergillus niger (Aspergillus niger) having a collection number of CGMCC No.11113, Rhizomucor variabilis (Rhizomucor variabilis) having a collection number of CGMCC No.11114, and Cunninghamella tabescens (Cunninghamella berthlletiae) having a collection number of CGMCC No. 11115;
the bacteria is at least one of Burkholderia (Burkholderia zhejiangensis) with the preservation number of CGMCC No.10848, Pseudomonas aeruginosa (Pseudomonas aeruginosa) with the preservation number of CGMCC No.10842 and Stenotrophomonas maltophilia with the preservation number of CGMCC No. 10846.
According to the preparation provided by the invention, bacteria can be not contained in the preparation, and the total content of fungi is 20-90mg dry weight/mL, preferably 50-80mg dry weight/mL.
When the preparation does not contain bacteria, the preparation can be any one of aspergillus niger, rhizomucor polytrichoides and kummer graying, or a combination of any two of the three.
In a preferred embodiment, the preparation contains aspergillus niger, rhizomucor polytropic and michelia grayii simultaneously, and the dry weight ratio of aspergillus niger to rhizomucor polytropic to michelia grayii is 1: (0.01-100): (0.01-100), preferably 1: (0.2-5): (0.2-5).
According to the preparation provided by the invention, the preparation can contain no fungi, and the total content of bacteria is 1013-1019one/mL, preferably 1015-1017one/mL.
When the preparation does not contain fungi, the preparation can be any one of burkholderia, pseudomonas aeruginosa and maltophilia oligomonas, any two of the three, or three of the three.
In a preferred embodiment, the formulation contains both Burkholderia, Pseudomonas aeruginosa and maltophilia oligoamonas in a ratio of 1: (10-6-106)：(10-6-106) Preferably 1: (10-2-102)：(10-2-102)。
According to the preparation provided by the invention, the preparation can contain fungi and bacteria at the same time, and the total content of the fungi is 10-80mg dry weight/mL, preferably 40-50mg dry weight/mL; the total content of bacteria is 1010-1018one/mL, preferably 1014-1015one/mL.
When the preparation contains fungi and bacteria, the fungi in the preparation can be any one, any two or three of aspergillus niger, rhizomucor mutabilis and kummer grey; the bacteria in the preparation can be any one, any two or three of Burkholderia, Pseudomonas aeruginosa and maltophilia oligo-oxygen monad.
In a preferred embodiment, the preparation contains both the three fungi and the three bacteria, and the dry weight ratio of aspergillus niger, rhizomucor pustulosis and kummer grey is 1: (0.01-100): (0.01-100), preferably 1: (0.2-5): (0.2-5); the number ratio of Burkholderia, Pseudomonas aeruginosa and maltophilia oligo-oxygen monad is 1: (10-6-106)：(10-6-106) Preferably 1: (10-2-102)：(10-2-102)。
The preparation may also contain a microbial growth promoter and/or a fertilizer.
The microorganism growth promoter may be at least one of glucose, starch, yeast extract, yeast powder, peptone and sodium chloride. The microorganism growth promoter can promote the growth of microorganisms in the preparation for in-situ remediation of chlorothalonil-contaminated soil in the soil.
The content of the microbial growth promoter may be 10 to 80g/mL, preferably 30 to 50 g/mL.
The fertilizer can be at least one of organic fertilizer, urea, diammonium phosphate and potassium sulfate. The fertilizer may help the contaminated soil to restore fertility and to some extent promote the growth of the applied microorganisms.
The content of the fertilizer can be 1-10g/mL, preferably 4-6 g/mL.
According to a second aspect of the invention, the invention provides a method for the in situ bioremediation of chlorothalonil contaminated soil, which comprises the in situ remediation of chlorothalonil contaminated soil using the above preparation for the in situ remediation of chlorothalonil contaminated soil.
Wherein,
when the preparation does not contain bacteria, the fungus can be used in an amount such that the total content of fungus in the chlorothalonil-contaminated soil is 4-18g dry weight/m3Preferably 10 to 16g dry weight/m3。
When the preparation does not contain fungi, the bacteria can be used in such an amount that the total content of bacteria in the chlorothalonil-contaminated soil reaches 1015-1021Per m3Preferably 1017-1019Per m3。
When the preparation contains fungi and bacteria, the fungi can be used in such an amount that the total content of fungi in the chlorothalonil-contaminated soil reaches 2-16g dry weight/m3Preferably 8 to 10g dry weight/m3The amount of bacteria is such that the total content of bacteria in the chlorothalonil-contaminated soil reaches 1012-1020Per m3Preferably 1016-1017Per m3。
When the preparation also contains a microbial growth promoter and/or a fertilizer, the microbial growth promoter can be used in an amount which enables the content of the microbial growth promoter in the chlorothalonil-polluted soil to reach 20-160g/m3Preferably 60 to 100g/m3. The fertilizer is used in an amount that the content of the fertilizer in the chlorothalonil-polluted soil reaches 2-20g/m3Preferably 8 to 12g/m3。
According to the method for in-situ bioremediation of chlorothalonil contaminated soil, the in-situ remediation process can be operated for 5-30 days, preferably 7-14 days, at a soil moisture content of 5-30 wt% and a temperature of 15-40 ℃.
According to the method for in-situ bioremediation of chlorothalonil contaminated soil, irrigation and plowing operations can be adopted to ensure that the moisture content of the soil is 5-30 wt%. Wherein the period of the plowing operation can be 1 to 4 weeks and 1 time. The plowing operation can provide sufficient oxygen for the growth of microorganisms in the soil. The operation of covering the porous mulching film can be adopted to maintain the temperature in the soil at 15-40 ℃ and the moisture content at 5-30 wt% so as to provide the temperature and humidity required by the growth of fungi and/or bacteria, thereby providing a proper environment for the growth and propagation of microorganisms.
The present invention will be described in detail below by way of specific examples.
In the following examples, the content of chlorothalonil in the soil (in g/kg of soil) was determined as follows: weighing 5g of dried soil, adding 10mL of acetone and water in a volume ratio of 1:1, standing for 30 minutes, shaking for 5 minutes, centrifuging to obtain a supernatant, adding 5mL of petroleum ether, shaking for 5 minutes, repeating for three times, standing, taking 10mL of the supernatant, adding 0.4g of anhydrous sodium sulfate, shaking for dehydration for 1 minute, taking 4mL of the supernatant, rotating on a rotary evaporator at 40 ℃ for evaporation, dissolving with 2mL of chromatographically pure acetone, and diluting with 10 times of chromatographically pure acetone for later use.
Gas chromatography conditions:
column: HT-pestides 1; a detector: an ECD detector; sample inlet temperature: 260 ℃; column temperature: 240 ℃; detector temperature: 280 ℃; the split ratio is as follows: 60, adding a solvent to the mixture; sample introduction amount: 1 mu L of the solution; blowing at the tail for 60 mL/min; column pressure: 60 kpa; carrier gas flow: 1.25 mL/min.
The soil clearance rate of the chlorothalonil is as follows: (content of chlorothalonil in the soil before treatment-content of chlorothalonil in the soil after treatment with the preparation for in-situ remediation of chlorothalonil-contaminated soil provided by the invention)/content of chlorothalonil in the soil before treatment x 100%.
In the following examples, the contents of the components in the preparation are based on each mL of the preparation for in-situ remediation of chlorothalonil contaminated soil.
Example 1
This example is intended to illustrate the preparation for in situ remediation of chlorothalonil contaminated soil provided by the present invention, and the method for in situ remediation of chlorothalonil contaminated soil using the preparation.
The preparation for in-situ remediation of chlorothalonil contaminated soil provided by the embodiment comprises the following components:
aspergillus niger (Aspergillus niger) with preservation number of CGMCC No.11113, the content of which is 13mg dry weight/mL;
rhizomucor mutabilis (Rhizomucor variabilis) with preservation number of CGMCC No.11114, the content of which is 13mg dry weight/mL;
cunninghamella bertholetiae (Cunninghamella berthletiae) with preservation number of CGMCC No.11115, the content is 14mg dry weight/mL;
burkholderia with preservation number CGMCC No.10848 (Burkholderia zhejiangensis) content of 1015Per mL;
pseudomonas aeruginosa (Pseudomonas aeruginosa) with preservation number of CGMCC No.10842, the content of the Pseudomonas aeruginosa is 1015Per mL;
the content of stenotrophomonas maltophilia (Stenotrophormonastrachalia) with preservation number of CGMCC No.10846 is 1015Per mL;
the content of glucose is 10g/mL, the content of starch is 5g/mL, the content of yeast extract is 5g/mL, the content of yeast powder is 5g/mL, the content of peptone is 10g/mL, and the content of sodium chloride is 10 g/mL;
the content of the organic fertilizer is 2g/mL, the content of the urea is 1g/mL, the content of the diammonium phosphate is 1g/mL, and the content of the potassium sulfate is 1 g/mL.
The preparation is uniformly applied to the soil polluted by the chlorothalonil, the content of the chlorothalonil is 10g/kg of soil, so that the content of Aspergillus niger in the soil reaches 2.6g dry weight/m3The content of Rhizomucor mutabilis (Rhizomucor variabilis) reaches 2.6g dry weight/m3The content of Cunninghamella grayish (Cunninghamella berthlletiae) amounts to 2.8g dry weight/m3The content of Burkholderia (Burkholderia zhejiangensis) reaches 1017Per m3The content of Pseudomonas aeruginosa reaches 1017Per m3The content of Stenotrophomonas maltophilia reaches 1017Per m3The content of the microbial growth promoter reaches 90g/m3The content of the fertilizer reaches 10g/m3. And covering a porous mulching film on the soil surface, so that the soil temperature is kept at 25 +/-2 ℃. The soil was irrigated and plowed weekly so that the moisture content in the soil was maintained at 20% to 30% by weight.
After 12 days, the content of the chlorothalonil in the soil is measured to be 0.039g/kg of soil, and the soil clearance rate of the chlorothalonil reaches 99.61%.
Examples 2 to 16
This example is intended to illustrate the preparation for in situ remediation of chlorothalonil contaminated soil provided by the present invention, and the method for in situ remediation of chlorothalonil contaminated soil using the preparation.
Examples 2-16 the formulations for in situ remediation of chlorothalonil contaminated soil were provided in the same compositions as in example 1, except for the contents of the components in tables 1-1 and 1-2, and the contents of the remaining components (microbial growth promoting agent and fertilizer).
The formulations for in situ remediation of chlorothalonil contaminated soil provided in examples 2 to 16 were uniformly applied to chlorothalonil contaminated soil having a chlorothalonil content of 10g/kg soil, so that the contents of the other components (microbial growth promoting agent and fertilizer) in the soil were the same as in example 1 except that the contents of fungi and bacteria were reached to the contents shown in tables 2-1 and 2-2.
In examples 2 to 16, the treatment of the chlorothalonil contaminated soil with the formulations for in situ remediation of chlorothalonil contaminated soil provided in examples 2 to 16 was carried out in the same manner as in example 1.
After 12 days, the contents of chlorothalonil in the soil and the soil clearance of chlorothalonil were measured as shown in table 3.
TABLE 1-1 content of fungal and bacterial components in the formulations
| Examples | 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 |
| Aspergillus niger | 13 | 20 | -- | -- | 35 | -- | -- | 10 |
| Rhizomucor pusillus polytropic | 13 | 20 | 25 | -- | 30 | -- | 75 | - |
| Gray Cunninghamella | 14 | -- | 25 | -- | -- | 70 | -- | - |
| Burkholderia sp | 1015 | 1014 | -- | 1016 | -- | -- | 1010 | 1018 |
| Pseudomonas aeruginosa | 1015 | 1014 | 1012 | -- | -- | 1017 | 1010 | - |
| Stenotrophomonas maltophilia | 1015 | -- | 1012 | 1016 | -- | 1017 | -- | - |
TABLE 1-2 content of fungal and bacterial Components in the formulations
| Examples | 9 | 10 | 11 | 12 | 13 | 14 | 15 | 16 |
| Aspergillus niger | -- | -- | 60 | -- | -- | -- | -- | -- |
| Rhizomucor pusillus polytropic | 45 | -- | -- | 65 | -- | -- | -- | -- |
| Gray Cunninghamella | -- | 80 | -- | -- | 75 | -- | -- | -- |
| Burkholderia sp | -- | -- | -- | -- | -- | 1015 | -- | -- |
| Pseudomonas aeruginosa | 1011 | -- | -- | -- | -- | -- | 1016 | -- |
| Stenotrophomonas maltophilia | -- | 1015 | -- | -- | -- | -- | -- | 1017 |
In tables 1-1 and 1-2, the content units of Aspergillus niger, Rhizomucor pustulosis variabilis and Cunninghamella grayanii are "mg dry weight/mL"; the content of Burkholderia, Pseudomonas aeruginosa and maltophilia oligo-monas was expressed in "one/mL".
TABLE 2-1 content of fungi and bacteria in soil
| Examples | 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 |
| Aspergillus niger | 2.6 | 4 | -- | -- | 7 | -- | -- | 2 |
| Rhizomucor pusillus polytropic | 2.6 | 4 | 5 | -- | 6 | -- | 15 | - |
| Gray Cunninghamella | 2.8 | -- | 5 | -- | -- | 14 | -- | - |
| Burkholderia sp | 1017 | 1016 | -- | 1018 | -- | -- | 1012 | 1020 |
| Pseudomonas aeruginosa | 1017 | 1016 | 1014 | -- | -- | 1019 | 1012 | - |
| Stenotrophomonas maltophilia | 1017 | -- | 1014 | 1018 | -- | 1019 | -- | - |
TABLE 2-2 content of fungi and bacteria in soil
| Examples | 9 | 10 | 11 | 12 | 13 | 14 | 15 | 16 |
| Aspergillus niger | -- | -- | 12 | -- | -- | -- | -- | -- |
| Rhizomucor pusillus polytropic | 9 | -- | -- | 13 | -- | -- | -- | -- |
| Gray Cunninghamella | -- | 16 | -- | -- | 15 | -- | -- | -- |
| Burkholderia sp | -- | -- | -- | -- | -- | 1017 | -- | -- |
| Pseudomonas aeruginosa | 1013 | -- | -- | -- | -- | -- | 1018 | -- |
| Stenotrophomonas maltophilia | -- | 1017 | -- | -- | -- | -- | -- | 1019 |
In tables 2-1 and 2-2, the contents of Aspergillus niger, Rhizomucor pustulatus and Cunninghamella grayanii are expressed in units of "g dry weight/m3"; the content unit of Burkholderia, Pseudomonas aeruginosa and maltophilia oligo-oxygen monad is' per m3”。
TABLE 3 content of chlorothalonil in the treated soil and the soil clearance of chlorothalonil
| The content of chlorothalonil in the treated soil (g/kg soil) | Chlorothalonil soil clearance (%) | |
| Example 1 | 0.039 | 99.61 |
| Example 2 | 0.103 | 98.97 |
| Example 3 | 0.105 | 98.95 |
| Example 4 | 0.294 | 97.06 |
| Example 5 | 0.290 | 97.10 |
| Example 6 | 0.124 | 98.76 |
| Example 7 | 0.127 | 98.73 |
| Example 8 | 0.285 | 97.15 |
| Example 9 | 0.274 | 97.26 |
| Example 10 | 0.259 | 97.41 |
| Example 11 | 0.438 | 95.62 |
| Example 12 | 0.416 | 95.84 |
| Example 13 | 0.385 | 96.15 |
| Example 14 | 0.394 | 96.06 |
| Example 15 | 0.377 | 96.23 |
| Example 16 | 0.348 | 96.52 |
As can be seen from examples 1 to 16 and Table 3, the preparation for in-situ remediation of chlorothalonil contaminated soil provided by the invention can be used for in-situ remediation of chlorothalonil contaminated soil, can efficiently remove chlorothalonil in soil, not only solves the problem that high residual chlorothalonil in soil seriously endangers food safety, improves the micro-ecology of soil and enables the soil to recover farming ability more quickly, but also overcomes the problems that a photochemical degradation method is easily limited by illumination conditions and is easily polluted by secondary chemical agents on cultivated lands. Meanwhile, when a fungus and a bacterium are used in combination, or different fungi are used in combination, and different bacteria are used in combination, a better repairing effect is obtained as compared with the case where one kind of fungus or bacterium is used alone.
Claims (10)
1. A preparation for in-situ remediation of chlorothalonil contaminated soil, wherein the preparation comprises fungi and/or bacteria having chlorothalonil degrading ability;
wherein the fungus is at least one of Aspergillus niger (Aspergillus niger) with the preservation number of CGMCC No.11113, Rhizomucor varilabilis (Rhizomucor varilabilis) with the preservation number of CGMCC No.11114 and Cunninghamella tabescens (Cunninghamella bertholletiae) with the preservation number of CGMCC No. 11115;
the bacteria is at least one of Burkholderia (Burkholderia zhejiangensis) with the preservation number of CGMCC No.10848, Pseudomonas aeruginosa (Pseudomonas aeruginosa) with the preservation number of CGMCC No.10842 and Stenotrophomonas maltophilia with the preservation number of CGMCC No. 10846.
2. The formulation according to claim 1, wherein the formulation is free of bacteria and the total content of fungi is 20-90mg dry weight/mL, preferably 50-80mg dry weight/mL;
preferably, the preparation simultaneously contains aspergillus niger, rhizomucor polytropic and michelia grey, and the dry weight ratio of the aspergillus niger to the rhizomucor polytropic to the michelia grey is 1: (0.01-100): (0.01-100), preferably 1: (0.2-5): (0.2-5).
3. The formulation of claim 1, wherein the formulation is free of fungi and has a bacteria content of 1013-1019one/mL, preferably 1015-1017Per mL;
preferably, the preparation simultaneously contains Burkholderia, Pseudomonas aeruginosa and maltophilia oligooxydans, and the number ratio of the Burkholderia, the Pseudomonas aeruginosa and the maltophilia oligooxydans is 1: (10-6-106)：(10-6-106) Preferably 1: (10-2-102)：(10-2-102)。
4. The preparation of claim 1, wherein the preparation contains both fungi and bacteria, and the fungi content is 10-80mg dry weight/mL, preferably 40-50mg dry weight/mL; the content of bacteria is 1010-1018one/mL, preferably 1014-1015Per mL;
preferably, the preparation simultaneously contains aspergillus niger, rhizomucor polytropic, klebsiella grayanii, burkholderia, pseudomonas aeruginosa and maltophilia oligomonas oryzae, and the dry weight ratio of the aspergillus niger to the rhizomucor polytropic to the klebsiella grayanii is 1: (0.01-100): (0.01-100),preferably 1: (0.2-5): (0.2-5), the number ratio of the Burkholderia, the pseudomonas aeruginosa and the maltophilia oligo-oxygen monad is 1: (10-6-106)：(10-6-106) Preferably 1: (10-2-102)：(10-2-102)。
5. The preparation according to any one of claims 1 to 4, wherein the preparation further comprises a microbial growth promoter and/or a fertilizer.
6. The formulation of claim 5, wherein the microbial growth promoter is at least one of glucose, starch, yeast extract, yeast powder, peptone and sodium chloride;
the fertilizer is at least one of organic fertilizer, urea, diammonium phosphate and potassium sulfate.
7. The formulation according to claim 6, wherein the content of the microbial growth promoting agent is 10-80g/mL, preferably 30-50 g/mL;
the content of the fertilizer is 1-10g/mL, preferably 4-6 g/mL.
8. A method for the in situ bioremediation of chlorothalonil contaminated soil, the method comprising the in situ remediation of chlorothalonil contaminated soil using a formulation as claimed in any one of claims 1 to 7.
9. The method of claim 8, wherein,
when the preparation does not contain bacteria, the usage amount of the fungi is that the content of the fungi in the chlorothalonil contaminated soil reaches 4-18g dry weight/m3Preferably 10 to 16g dry weight/m3；
When the preparation does not contain fungi, the bacteria are used in an amount which enables the content of the bacteria in the chlorothalonil-polluted soil to reach 1015-1021Per m3Preferably 1017-1019Per m3；
When the preparation contains fungi and bacteria, the fungi are used in such an amount that the content of the fungi in the chlorothalonil-contaminated soil reaches 2-16g dry weight/m3Preferably 8 to 10g dry weight/m3The amount of bacteria is such that the total content of bacteria in the chlorothalonil-contaminated soil reaches 1012-1020Per m3Preferably 1016-1017Per m3；
When the preparation also contains a microbial growth promoter and/or a fertilizer, the microbial growth promoter is used in an amount which enables the content of the microbial growth promoter in the chlorothalonil-polluted soil to reach 20-160g/m3Preferably 60 to 100g/m3. The fertilizer is used in an amount that the content of the fertilizer in the chlorothalonil-polluted soil reaches 2-20g/m3Preferably 8 to 12g/m3。
10. The method according to claim 8 or 9, wherein the in situ remediation process is carried out at a soil moisture content of 5-30 wt% and a temperature of 15-40 ℃ for 5-30 days, preferably 7-14 days.
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