CN118147126A - Preparation method of flora composite repairing agent for cooperatively treating PAEs and Cd - Google Patents
Preparation method of flora composite repairing agent for cooperatively treating PAEs and Cd Download PDFInfo
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- CN118147126A CN118147126A CN202410572600.9A CN202410572600A CN118147126A CN 118147126 A CN118147126 A CN 118147126A CN 202410572600 A CN202410572600 A CN 202410572600A CN 118147126 A CN118147126 A CN 118147126A
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- montmorillonite
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- 239000002131 composite material Substances 0.000 title claims abstract description 52
- 238000002360 preparation method Methods 0.000 title claims abstract description 16
- 239000003795 chemical substances by application Substances 0.000 claims abstract description 64
- 230000001580 bacterial effect Effects 0.000 claims abstract description 24
- 230000004048 modification Effects 0.000 claims abstract description 11
- 238000012986 modification Methods 0.000 claims abstract description 11
- GUJOJGAPFQRJSV-UHFFFAOYSA-N dialuminum;dioxosilane;oxygen(2-);hydrate Chemical compound O.[O-2].[O-2].[O-2].[Al+3].[Al+3].O=[Si]=O.O=[Si]=O.O=[Si]=O.O=[Si]=O GUJOJGAPFQRJSV-UHFFFAOYSA-N 0.000 claims description 95
- 229910052901 montmorillonite Inorganic materials 0.000 claims description 95
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 39
- 238000000034 method Methods 0.000 claims description 30
- 239000000243 solution Substances 0.000 claims description 30
- 239000011229 interlayer Substances 0.000 claims description 28
- 238000002156 mixing Methods 0.000 claims description 28
- 239000000725 suspension Substances 0.000 claims description 26
- 239000002245 particle Substances 0.000 claims description 22
- 238000005406 washing Methods 0.000 claims description 21
- 239000008367 deionised water Substances 0.000 claims description 20
- 229910021641 deionized water Inorganic materials 0.000 claims description 20
- 238000001035 drying Methods 0.000 claims description 20
- 238000003756 stirring Methods 0.000 claims description 19
- UUEWCQRISZBELL-UHFFFAOYSA-N 3-trimethoxysilylpropane-1-thiol Chemical compound CO[Si](OC)(OC)CCCS UUEWCQRISZBELL-UHFFFAOYSA-N 0.000 claims description 18
- IDGUHHHQCWSQLU-UHFFFAOYSA-N ethanol;hydrate Chemical compound O.CCO IDGUHHHQCWSQLU-UHFFFAOYSA-N 0.000 claims description 18
- 229920002554 vinyl polymer Polymers 0.000 claims description 18
- 229910052708 sodium Inorganic materials 0.000 claims description 17
- 239000011734 sodium Substances 0.000 claims description 17
- 229920000049 Carbon (fiber) Polymers 0.000 claims description 16
- DGAQECJNVWCQMB-PUAWFVPOSA-M Ilexoside XXIX Chemical compound C[C@@H]1CC[C@@]2(CC[C@@]3(C(=CC[C@H]4[C@]3(CC[C@@H]5[C@@]4(CC[C@@H](C5(C)C)OS(=O)(=O)[O-])C)C)[C@@H]2[C@]1(C)O)C)C(=O)O[C@H]6[C@@H]([C@H]([C@@H]([C@H](O6)CO)O)O)O.[Na+] DGAQECJNVWCQMB-PUAWFVPOSA-M 0.000 claims description 16
- 239000004917 carbon fiber Substances 0.000 claims description 16
- PXQLVRUNWNTZOS-UHFFFAOYSA-N sulfanyl Chemical class [SH] PXQLVRUNWNTZOS-UHFFFAOYSA-N 0.000 claims description 16
- 230000000593 degrading effect Effects 0.000 claims description 15
- VNWKTOKETHGBQD-UHFFFAOYSA-N methane Chemical compound C VNWKTOKETHGBQD-UHFFFAOYSA-N 0.000 claims description 15
- 230000010355 oscillation Effects 0.000 claims description 15
- 239000001963 growth medium Substances 0.000 claims description 13
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 claims description 12
- 239000000203 mixture Substances 0.000 claims description 12
- 238000007873 sieving Methods 0.000 claims description 12
- 150000001875 compounds Chemical class 0.000 claims description 11
- 230000002195 synergetic effect Effects 0.000 claims description 11
- 238000000227 grinding Methods 0.000 claims description 10
- 238000000197 pyrolysis Methods 0.000 claims description 10
- 241000196324 Embryophyta Species 0.000 claims description 9
- 239000000084 colloidal system Substances 0.000 claims description 9
- 239000004115 Sodium Silicate Substances 0.000 claims description 8
- 238000013329 compounding Methods 0.000 claims description 8
- 239000000835 fiber Substances 0.000 claims description 8
- 238000010438 heat treatment Methods 0.000 claims description 8
- 229910052911 sodium silicate Inorganic materials 0.000 claims description 8
- 241001337904 Gordonia <angiosperm> Species 0.000 claims description 7
- 230000000813 microbial effect Effects 0.000 claims description 7
- NTHWMYGWWRZVTN-UHFFFAOYSA-N sodium silicate Chemical compound [Na+].[Na+].[O-][Si]([O-])=O NTHWMYGWWRZVTN-UHFFFAOYSA-N 0.000 claims description 7
- 241000193830 Bacillus <bacterium> Species 0.000 claims description 6
- 241000316848 Rhodococcus <scale insect> Species 0.000 claims description 6
- 230000003213 activating effect Effects 0.000 claims description 6
- 238000001914 filtration Methods 0.000 claims description 6
- 230000001939 inductive effect Effects 0.000 claims description 6
- 239000010410 layer Substances 0.000 claims description 6
- 239000011259 mixed solution Substances 0.000 claims description 6
- 239000011148 porous material Substances 0.000 claims description 6
- 238000001338 self-assembly Methods 0.000 claims description 6
- -1 sulfhydryl montmorillonite Chemical compound 0.000 claims description 5
- 238000001816 cooling Methods 0.000 claims description 4
- 238000010304 firing Methods 0.000 claims description 4
- 238000007710 freezing Methods 0.000 claims description 4
- 230000008014 freezing Effects 0.000 claims description 4
- 238000007789 sealing Methods 0.000 claims description 4
- 239000012298 atmosphere Substances 0.000 claims description 2
- 239000000969 carrier Substances 0.000 claims description 2
- 238000005119 centrifugation Methods 0.000 claims description 2
- 238000007865 diluting Methods 0.000 claims description 2
- 230000015556 catabolic process Effects 0.000 abstract description 16
- 238000006731 degradation reaction Methods 0.000 abstract description 16
- 229910001385 heavy metal Inorganic materials 0.000 abstract description 16
- 239000002689 soil Substances 0.000 abstract description 16
- 230000000694 effects Effects 0.000 abstract description 8
- 238000002161 passivation Methods 0.000 abstract description 8
- 229910052793 cadmium Inorganic materials 0.000 abstract description 7
- BDOSMKKIYDKNTQ-UHFFFAOYSA-N cadmium atom Chemical compound [Cd] BDOSMKKIYDKNTQ-UHFFFAOYSA-N 0.000 abstract description 7
- 239000003802 soil pollutant Substances 0.000 abstract description 2
- 125000003396 thiol group Chemical group [H]S* 0.000 abstract description 2
- 239000000047 product Substances 0.000 description 15
- 230000000052 comparative effect Effects 0.000 description 13
- 238000012360 testing method Methods 0.000 description 12
- 239000002068 microbial inoculum Substances 0.000 description 7
- 238000001179 sorption measurement Methods 0.000 description 6
- 241000194110 Bacillus sp. (in: Bacteria) Species 0.000 description 4
- IRIAEXORFWYRCZ-UHFFFAOYSA-N Butylbenzyl phthalate Chemical compound CCCCOC(=O)C1=CC=CC=C1C(=O)OCC1=CC=CC=C1 IRIAEXORFWYRCZ-UHFFFAOYSA-N 0.000 description 4
- MQIUGAXCHLFZKX-UHFFFAOYSA-N Di-n-octyl phthalate Chemical compound CCCCCCCCOC(=O)C1=CC=CC=C1C(=O)OCCCCCCCC MQIUGAXCHLFZKX-UHFFFAOYSA-N 0.000 description 4
- NIQCNGHVCWTJSM-UHFFFAOYSA-N Dimethyl phthalate Chemical compound COC(=O)C1=CC=CC=C1C(=O)OC NIQCNGHVCWTJSM-UHFFFAOYSA-N 0.000 description 4
- 241001657434 Gordonia sp. Species 0.000 description 4
- 241000187562 Rhodococcus sp. Species 0.000 description 4
- 239000013078 crystal Substances 0.000 description 4
- DOIRQSBPFJWKBE-UHFFFAOYSA-N dibutyl phthalate Chemical compound CCCCOC(=O)C1=CC=CC=C1C(=O)OCCCC DOIRQSBPFJWKBE-UHFFFAOYSA-N 0.000 description 4
- FLKPEMZONWLCSK-UHFFFAOYSA-N diethyl phthalate Chemical compound CCOC(=O)C1=CC=CC=C1C(=O)OCC FLKPEMZONWLCSK-UHFFFAOYSA-N 0.000 description 4
- 239000002609 medium Substances 0.000 description 4
- 241000894006 Bacteria Species 0.000 description 3
- 239000004014 plasticizer Substances 0.000 description 3
- 229920006395 saturated elastomer Polymers 0.000 description 3
- BVKZGUZCCUSVTD-UHFFFAOYSA-L Carbonate Chemical compound [O-]C([O-])=O BVKZGUZCCUSVTD-UHFFFAOYSA-L 0.000 description 2
- 229910000616 Ferromanganese Inorganic materials 0.000 description 2
- 240000008042 Zea mays Species 0.000 description 2
- 235000005824 Zea mays ssp. parviglumis Nutrition 0.000 description 2
- 235000002017 Zea mays subsp mays Nutrition 0.000 description 2
- 238000004458 analytical method Methods 0.000 description 2
- 238000006243 chemical reaction Methods 0.000 description 2
- 235000005822 corn Nutrition 0.000 description 2
- 238000012258 culturing Methods 0.000 description 2
- FBSAITBEAPNWJG-UHFFFAOYSA-N dimethyl phthalate Natural products CC(=O)OC1=CC=CC=C1OC(C)=O FBSAITBEAPNWJG-UHFFFAOYSA-N 0.000 description 2
- 229960001826 dimethylphthalate Drugs 0.000 description 2
- 229910017053 inorganic salt Inorganic materials 0.000 description 2
- DALUDRGQOYMVLD-UHFFFAOYSA-N iron manganese Chemical compound [Mn].[Fe] DALUDRGQOYMVLD-UHFFFAOYSA-N 0.000 description 2
- 238000011068 loading method Methods 0.000 description 2
- 239000000463 material Substances 0.000 description 2
- 239000012299 nitrogen atmosphere Substances 0.000 description 2
- XNGIFLGASWRNHJ-UHFFFAOYSA-L phthalate(2-) Chemical compound [O-]C(=O)C1=CC=CC=C1C([O-])=O XNGIFLGASWRNHJ-UHFFFAOYSA-L 0.000 description 2
- JQCXWCOOWVGKMT-UHFFFAOYSA-N phthalic acid diheptyl ester Natural products CCCCCCCOC(=O)C1=CC=CC=C1C(=O)OCCCCCCC JQCXWCOOWVGKMT-UHFFFAOYSA-N 0.000 description 2
- 230000008569 process Effects 0.000 description 2
- 230000008439 repair process Effects 0.000 description 2
- 238000000926 separation method Methods 0.000 description 2
- 240000007594 Oryza sativa Species 0.000 description 1
- 235000007164 Oryza sativa Nutrition 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- BJQHLKABXJIVAM-UHFFFAOYSA-N bis(2-ethylhexyl) phthalate Chemical compound CCCCC(CC)COC(=O)C1=CC=CC=C1C(=O)OCC(CC)CCCC BJQHLKABXJIVAM-UHFFFAOYSA-N 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 230000007423 decrease Effects 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 238000004108 freeze drying Methods 0.000 description 1
- 230000006698 induction Effects 0.000 description 1
- 229910052500 inorganic mineral Inorganic materials 0.000 description 1
- 238000009342 intercropping Methods 0.000 description 1
- 238000005342 ion exchange Methods 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 239000007791 liquid phase Substances 0.000 description 1
- 230000007246 mechanism Effects 0.000 description 1
- 239000011707 mineral Substances 0.000 description 1
- 244000052769 pathogen Species 0.000 description 1
- 230000001717 pathogenic effect Effects 0.000 description 1
- 230000005501 phase interface Effects 0.000 description 1
- 239000008029 phthalate plasticizer Substances 0.000 description 1
- 230000018612 quorum sensing Effects 0.000 description 1
- 238000005067 remediation Methods 0.000 description 1
- 235000009566 rice Nutrition 0.000 description 1
- 239000004575 stone Substances 0.000 description 1
- 239000010902 straw Substances 0.000 description 1
- 239000006228 supernatant Substances 0.000 description 1
- 238000012876 topography Methods 0.000 description 1
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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
- C12N11/14—Enzymes or microbial cells immobilised on or in an inorganic carrier
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B09—DISPOSAL OF SOLID WASTE; RECLAMATION OF CONTAMINATED SOIL
- B09C—RECLAMATION OF CONTAMINATED SOIL
- B09C1/00—Reclamation of contaminated soil
- B09C1/10—Reclamation of contaminated soil microbiologically, biologically or by using enzymes
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- C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
- C09K—MATERIALS FOR MISCELLANEOUS APPLICATIONS, NOT PROVIDED FOR ELSEWHERE
- C09K17/00—Soil-conditioning materials or soil-stabilising materials
- C09K17/40—Soil-conditioning materials or soil-stabilising materials containing mixtures of inorganic and organic compounds
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- C—CHEMISTRY; METALLURGY
- C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
- C09K—MATERIALS FOR MISCELLANEOUS APPLICATIONS, NOT PROVIDED FOR ELSEWHERE
- C09K17/00—Soil-conditioning materials or soil-stabilising materials
- C09K17/40—Soil-conditioning materials or soil-stabilising materials containing mixtures of inorganic and organic compounds
- C09K17/42—Inorganic compounds mixed with organic active ingredients, e.g. accelerators
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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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
- C12N1/20—Bacteria; Culture media therefor
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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
- C12N1/36—Adaptation or attenuation of cells
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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
- C12N11/084—Polymers containing vinyl alcohol units
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- C12R2001/00—Microorganisms ; Processes using microorganisms
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- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
- C12R2001/07—Bacillus
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Abstract
The invention relates to the technical field of soil pollutant treatment, in particular to a preparation method of a flora composite repairing agent for cooperatively treating PAEs and Cd, which comprises the following steps: preparing functional flora; preparing a flora carrier; preparing a flora composite repairing agent; the composite repairing agent designed by the invention carries out cadmium-resistant domestication on functional bacterial groups, has broad-spectrum degradability on PAEs, and solves the problem that the degradation efficiency of a single functional bacterial strain is reduced under the stress of heavy metal Cd pollution in composite polluted soil; moreover, the composite repairing agent designed by the invention has excellent passivation effect on heavy metals such as cadmium and the like through sulfhydryl modification, and can realize the effect of synchronously removing PAEs and Cd in soil under the cooperation of functional flora.
Description
Technical Field
The invention relates to the technical field of soil pollutant treatment, in particular to a preparation method of a flora composite repairing agent for cooperatively treating PAEs and Cd.
Background
The problem of the combined pollution of Phthalate (PAEs) plasticizers and heavy metal Cd exists in farmland soil in karst areas.
For solving the problems, the current economical and efficient mode is to use an immobilized microbial inoculum, and the immobilized microbial inoculum is changed into a compound formed by functional flora and a carrier thereof.
Regarding the problem of the treatment of the complex pollution of phthalate plasticizers and heavy metal Cd, the flora is required to be capable of not only tolerating the damage of Cd to cells, but also effectively removing PAEs. The inventor constructs a functional flora capable of efficiently degrading PAEs through a quorum sensing mechanism based on the degradation capability of the strain.
In preparing a vector based on the functional flora described above, the inventors have found that the following problems exist with the current technology:
for treating PAEs in farmlands, the existing microbial community or biochar carrier has good effect on adsorbing and removing organic matters, but the adsorption passivation capability of the microbial community or biochar carrier on heavy metals is insufficient, and the microbial community or biochar carrier is small in binding coefficient and easy to desorb.
In order to solve the problems, the inventor tries to introduce sulfhydryl modified montmorillonite to increase the passivation capability of the repairing agent to heavy metals, and designs a flora fixing composite repairing agent, so as to provide technical support for solving the problem of exceeding the standard of the phthalate plasticizer and heavy metal Cd composite pollution in farmland soil in karst areas.
Disclosure of Invention
In order to achieve the aim, the invention provides a preparation method of a flora composite repairing agent for cooperatively treating PAEs and Cd, and the composite repairing agent prepared by the method can not only efficiently degrade the PAEs in soil through functional flora, but also passivate the Cd in the soil through a sulfhydrylation carrier, so that the PAEs and Cd in the soil are synchronously removed.
The invention relates to a preparation method of a flora composite repairing agent for cooperatively treating PAEs and Cd, which comprises the following steps:
s1, preparing functional flora;
S1-1, domesticating Cd-resistant strains: under the premise of ensuring the normal growth of the strain and the function of degrading PAEs by the strain, inducing and domesticating the strains of the Gordonia, the rhodococcus and the bacillus in an LB culture medium with gradient concentration Cd 2+;
s1-2, activating the three strains treated by the S1-1 in LB culture medium for 24h, and adjusting OD 600 = 1.0;
s1-3, according to the volume ratio of 1:1:1, mixing the three strains treated by the S1-2 in proportion to prepare functional bacterial groups with functions of degrading PAEs and resisting Cd;
S2, preparing a flora carrier;
S2-1, preparing sulfhydryl montmorillonite;
s2-2, preparing a biochar carrier;
S2-3, compounding a carrier: firstly, putting the mercapto montmorillonite in the S2-1 and the biochar carrier in the S2-2 into pure water, uniformly mixing, and then obtaining a flora carrier after centrifugation, washing and drying;
s3, preparing a flora composite repairing agent;
s3-1, uniformly mixing the functional flora prepared in the S1 with the flora carrier prepared in the S2 to obtain a mixed system;
n is marked as a multiplying factor and n is R +, the adding amount of the flora carrier is 1n g, and the adding amount of the functional flora is 10n and 30n mL;
S3-2, firstly, carrying out shake culture on the mixed system obtained in the step S3-1 for 24 hours under the conditions of 150-200 r/min, constant temperature of 30 ℃ and light shielding; then filtering, washing, and drying at 25 ℃ under aseptic condition to obtain the flora compound repairing agent.
Further, the mercapto montmorillonite in S2-1 is sodium silicate-mercapto montmorillonite or polyvinyl alcohol-mercapto montmorillonite.
Description: the interlayer spacing of the montmorillonite structure is generally less than 1 nm when the montmorillonite structure is not modified, so that the montmorillonite and the biochar carrier are difficult to adsorb and fix, a stable organic-mineral combination structure is formed, and the risk of falling off exists in the use process; it is therefore necessary to expand the interlayer gap of montmorillonite.
Further, the method for preparing the sodium silicate-mercapto montmorillonite comprises the following steps:
S2-1-A, adding montmorillonite and sodium silicate into an ethanol-water solution of 3-mercaptopropyl trimethoxy silane, uniformly mixing, controlling the temperature to 25 ℃, controlling the pH to 9.5-10, and continuously stirring and reacting for 6-7 hours to obtain small-pore montmorillonite after mercapto modification, namely sodium silicate-mercaptomontmorillonite;
wherein, the mass ratio of montmorillonite, sodium silicate mercapto compound and 3-mercaptopropyl trimethoxy silane is as follows: 1:0.06:0.1;
the mass concentration of the ethanol-water solution is 95%;
Note that the mass sum of montmorillonite and sodium silicate is M 1, and the volume of 3-mercaptopropyl trimethoxysilane, ethanol-water solution is V 1, then M 1：V1 =1.06 g:20 And (3) mL.
Description: after sodium modification, original Ca 2+ between montmorillonite layers is replaced by Na +, montmorillonite particles are further reduced, stone plate-shaped characteristics are prominent, adsorption capacity is enhanced, and ion exchange capacity in a unit cell interlayer region is enhanced.
Further, the method for preparing the polyvinyl alcohol-mercapto montmorillonite comprises the following steps:
S2-1-B-1, and inserting organic matters between layers: firstly, dissolving montmorillonite into deionized water to prepare a suspension with the mass concentration of 2-4%, then adding PVA into the deionized water to prepare a mixed solution with the mass concentration of 2-3%, and then according to the volume ratio of 1: adding the solution into the suspension, and carrying out ultrasonic oscillation for 5-7 d to obtain a colloid suspension;
S2-1-B-2, self-assembly at low temperature: freezing 24h at-10deg.C after treating colloid suspension obtained in S2-1-B-1 to obtain large interlayer montmorillonite with expanded interlayer spacing;
S2-1-B-3, mercapto modification: adding the large interlayer montmorillonite in S2-1-B-2 into an ethanol-water solution of 3-mercaptopropyl trimethoxy silane, uniformly mixing, controlling the temperature to 25 ℃, controlling the pH to 9.5-10, and continuously stirring and reacting for 6-7 hours to obtain the mercapto-modified large interlayer montmorillonite, which is named as polyvinyl alcohol-mercapto montmorillonite;
Wherein, the mass ratio of the large interlayer montmorillonite to the 3-mercaptopropyl trimethoxy silane is 1:0.1;
the mass concentration of the ethanol-water solution is 95%;
Note that the mass of the interlayer montmorillonite is M 2, and the volume of the 3-mercaptopropyl trimethoxysilane and ethanol-water solution is V 2, then M 2：V2 =1 g:20 And (3) mL.
Description: the interlayer spacing of the original montmorillonite is not large, which is unfavorable for stable combination with the biochar material in the subsequent process, so that the interlayer spacing of each layer in the montmorillonite needs to be expanded: when preparing polyvinyl alcohol-mercapto montmorillonite, PVA is used to insert into the montmorillonite gaps, and the montmorillonite gaps are expanded in a low-temperature self-assembly mode, so that a foundation is provided for subsequent combination.
The principle of low-temperature self-assembly of PVA modified montmorillonite is as follows: at low temperature, water is frozen to form ice crystals, and at the solid-liquid phase interface, the solute is separated from the ice crystals; the ice crystals further force the montmorillonite particles to be enriched in the gaps of the ice crystals and slowly assemble under the effect of supermolecules; finally, the large interlayer montmorillonite material distributed by regular pores is obtained after freeze drying.
Further, the method for inserting the organic matters between the S2-1-B-1 and the middle layer comprises the following steps:
S2-1-B-1-1, firstly dissolving montmorillonite in deionized water to prepare a suspension with the mass concentration of 2-4%, stirring for 3-4 hours at the temperature of 45-50 ℃, and then adding PVA into the deionized water to prepare a mixed solution with the mass concentration of 2-3%;
S2-1-B-1-2 according to the volume ratio of 1:4 adding the solution to the suspension and inserting PVA into the montmorillonite interstices according to the following parameters:
firstly, carrying out ultrasonic oscillation for 3-5 min, and standing for 1 h; then ultrasonic oscillation is carried out for 4-5 min, and standing is carried out for 2 h; then ultrasonic oscillation is carried out for 5 min, and standing is carried out for 10 h; stirring at 45-50 ℃ for 3 h; finally, sealing and carrying out ultrasonic oscillation at room temperature for 5-7 d to obtain a colloid suspension;
S2-1-B-1-3, centrifugally separating the colloidal suspension isolate in S2-1-B-1-2, repeatedly washing the isolate with deionized water, and finally diluting the isolate with deionized water for later use.
Further, the biochar carrier paired with sodium silicate-mercapto montmorillonite is biochar particles, and the biochar carrier paired with polyvinyl alcohol-mercapto montmorillonite is reticular carbon fiber.
Description: the biochar particles have smaller particle size and larger specific surface area, so the biochar particles are easier to combine with montmorillonite, but at the same time, the combination surface with the flora is reduced because the combination surface of the biochar particles and the montmorillonite is beaten, so the loading amount of the flora is reduced; the reticular carbon fiber has a larger absolute surface area though the specific surface area is smaller than that of the biochar particles, and a large amount of bacteria are still loaded on the rest while one end is combined with the montmorillonite, so that the combination strength with the montmorillonite is reduced.
Further, the preparation method of the biochar particles comprises the following steps: crushing and grinding plant fibers, pyrolyzing under inert atmosphere, grinding the product after pyrolysis, and sieving with a 200-mesh sieve to obtain biochar particles;
The parameters of pyrolysis are: and (3) raising the furnace temperature from room temperature to 600 ℃ at a speed of 4-5 ℃/min, and keeping the furnace temperature at 2 h ℃.
Description: the plant fiber can be selected from corn, rice straw and other plant fibers, and is not limited; the biochar particles need to ensure small and uniform particle size as much as possible, so that the bonding strength during loading is improved.
Further, the preparation method of the netlike carbon fiber comprises the following steps:
Firstly, immersing lignocellulose with the length of 1-2 mm into a NaOH solution with the length of 0.1 mol/L, and stirring for 20-25 h at the speed of 500-550 r/min; then taking out lignocellulose, drying at 80 ℃, finally firing at a gradient temperature, and washing and drying the fired product to obtain the netlike carbon fiber;
the gradient temperature is as follows: firstly, heating from 20 ℃ to 250 ℃ at a speed of 4-5 ℃/min, and preserving heat for 5-6 min; then heating from 250 ℃ to 500 ℃ at a speed of 20-22 ℃/min, and preserving heat for 1-2 h; finally cooling to 20 ℃ along with the furnace.
Description: the lignocellulose can still keep the plant morphology after being fired at high temperature, and a netlike fiber structure with a plant fiber framework is formed.
Further, the method for compounding the carrier in S2-3 comprises the following steps:
S2-3-1, uniformly mixing the mercapto montmorillonite obtained in the S2-1 and the biochar carrier in the S2-2 in pure water to obtain a mixture; n is recorded as a multiplying factor and n is R +, the addition of the mercapto montmorillonite is 1n g, the addition of the biochar carrier is [3n,4n ] g, and the addition of the pure water is [100n,110n ] mL;
S2-3-2, separating the mixture obtained in the step S2-3-1, washing the separated product, drying at 100 ℃ for 5h, and finally sieving with a 200-mesh sieve to obtain the composite carrier.
Description: taking the reticular carbon fiber as an example, carbonized lignocellulose can retain the original plant morphology, has a reticular framework, is mixed with the polyvinyl alcohol-mercapto montmorillonite, is combined with the polyvinyl alcohol-mercapto montmorillonite and entangled on the surface of the reticular carbon fiber, and the surface of the reticular carbon fiber and the gaps of the reticular carbon fiber can provide a large number of attachment sites to provide structural support for the attachment of subsequent strains.
Compared with the existing microbial inoculum for degrading PAEs, the invention has the beneficial effects that:
(1) The composite repairing agent designed by the invention has cadmium-resistant domestication of functional bacterial groups, has broad-spectrum degradability to PAEs, and can form a intercropping relationship by group induction of various degrading bacterial strains in the bacterial groups, thereby solving the problems of degradation efficiency decline and weak competitiveness of single functional bacterial strains under the stress of heavy metal Cd pollution in composite polluted soil;
(2) The composite repairing agent designed by the invention has excellent passivation effect on heavy metals such as cadmium and the like through sulfhydryl modification, and can realize the effect of synchronously removing PAEs and Cd in soil under the cooperation of functional bacteria.
Drawings
FIG. 1 is a graph showing the growth of the cadmium-tolerant domesticated strain of example 1 for 0 to 48 hours; wherein Gordonia sp.is Gordonia sp.rhodococcus sp.is Rhodococcus sp.and Bacillus sp.is Bacillus sp;
FIG. 2 is a graph of OD 600 values of 24h of the cadmium-tolerant acclimated strain of example 1 grown in LB medium with different Cd 2+ concentrations;
FIG. 3 is a graph of OD 600 values of 24 h of the cadmium-tolerant acclimated strain of example 1 grown in LB medium at different pH values;
FIG. 4 is a graph of degradation rates of the cadmium tolerant domesticated strain of example 1 for 6 PAEs;
FIG. 5 is a graph showing the saturated adsorption amount of Cd 2+ by the microbial group carriers in example 4, comparative example 2, and comparative example 3; the error bars a and b are letter marks in an SPSS (specific pathogen free) significant difference analysis method, and represent significant differences of microbial inoculum on PAEs degradation rates;
FIG. 6 is a graph showing degradation efficiency of the composite restorative after culture of 5 d in examples 4-10, comparative examples 1-3; wherein a2 is example 4, a3 is example 5, a4 is example 6, a5 is example 7, a6 is example 8, a7 is example 9, a8 is example 10, b1 is comparative example 1, b2 is comparative example 2, and b3 is comparative example 3; the error bars a, b, d, f, ef, e, cd, bc are all letter marks in the SPSS significant difference analysis method, which indicate the significant difference of the microbial inoculum on the PAEs degradation rate;
FIG. 7 is an SEM topography of the composite repair agent of example 4;
FIG. 8 is a graph of the degradation efficiency of the composite remediation agent of example 4 on PAEs in soil;
FIG. 9 is a graph showing passivation efficacy of the composite repair agent of example 4 against heavy metal Cd.
Detailed Description
In order to further illustrate the manner in which the invention is made and the effects obtained, a clear and complete description of the technical solution of the invention will be provided in connection with experiments.
In the following examples, PAEs include 6 EPA-controlled plasticizers: dimethyl phthalate (DMP), diethyl phthalate (DEP), dibutyl phthalate (DBP), butyl Benzyl Phthalate (BBP), di (2-ethylhexyl) phthalate (DEHP), and di-n-octyl phthalate (DOP); the reagents were all analytically pure and purchased from Shanghai Meilin Biochemical technologies Co.
Example 1: the description of the embodiment is a preparation method of a flora composite repairing agent for cooperatively treating PAEs and Cd, wherein a flora carrier is formed by combining polyvinyl alcohol-mercapto montmorillonite and reticular carbon fibers, and the preparation method comprises the following steps of:
s1, preparing functional flora;
S1-1, domesticating Cd-resistant strains: under the premise of ensuring the normal growth of the strain and the function of degrading PAEs by the strain, inducing and domesticating the strains of the Gordonia, the Rhodococcus and the Bacillus in an LB culture medium containing Cd 2+;
s1-2, activating the three strains treated by the S1-1 in LB culture medium for 24h, and adjusting OD 600 = 1.0;
s1-3, according to the volume ratio of 1:1:1, mixing the three strains treated by the S1-2 in proportion to prepare functional bacterial groups with functions of degrading PAEs and resisting Cd;
The domestication results are shown in fig. 1-4, wherein fig. 1 is a growth curve diagram of a cadmium-resistant domesticated strain for 0-48 h; wherein Gordonia sp.is Gordonia sp.rhodococcus sp.is Rhodococcus sp.and Bacillus sp.is Bacillus sp; FIG. 2 is a graph of OD 600 values of a cadmium tolerant acclimated strain grown 24h in LB medium with different Cd 2+ concentrations; FIG. 3 is a graph of OD 600 values of 24h of cadmium-tolerant acclimated strains grown in LB medium at different pH; FIG. 4 is a graph of degradation rates of cadmium-tolerant acclimatized strains for 6 PAEs;
S2, preparing a flora carrier;
S2-1-B-1-1, firstly dissolving montmorillonite in deionized water to prepare suspension with the mass concentration of 4%, stirring at 50 ℃ for 4h, and then adding PVA into the deionized water to prepare mixed solution with the mass concentration of 3%;
S2-1-B-1-2 according to the volume ratio of 1:4 adding the solution to the suspension and inserting PVA into the montmorillonite interstices according to the following parameters:
Firstly, ultrasonically oscillating 5min, and standing 1: 1 h; then carrying out ultrasonic oscillation for 5min and standing for 2: 2 h; then ultrasonic oscillation is carried out for 5min, and standing is carried out for 10 h; stirring at 50deg.C for 3 h; finally, sealing and carrying out ultrasonic oscillation at room temperature for 7 d to obtain a colloid suspension;
S2-1-B-1-3, centrifugally separating the separated colloidal suspension in S2-1-B-1-2, repeatedly washing the separated matter with deionized water, and finally obtaining the product according to the following formula 1 g:50 The separation product is diluted and stored with deionized water for standby;
S2-1-B-2, self-assembly at low temperature: freezing 24h at-10deg.C after treating colloid suspension obtained in S2-1-B-1 to obtain large interlayer montmorillonite with expanded interlayer spacing;
S2-1-B-3, mercapto modification: adding the large interlayer montmorillonite in S2-1-B-2 into an ethanol-water solution of 3-mercaptopropyl trimethoxy silane, uniformly mixing, controlling the temperature to 25 ℃, controlling the pH to 10, and continuously stirring for reacting 7 h to obtain the mercapto-modified large interlayer montmorillonite, namely polyvinyl alcohol-mercapto montmorillonite;
the addition amount of the large interlayer montmorillonite is 1g, and the addition amount of the ethanol-water solution of the 3-mercaptopropyl trimethoxysilane is 25 mL;
s2-2, preparing a reticular carbon fiber:
Lignocellulose with the length of 2mm is immersed into a NaOH solution with the concentration of 0.1 mol/L, and stirred for 25 h at the speed of 550 r/min; then taking out lignocellulose, drying at 80 ℃, finally firing at a gradient temperature, and washing and drying the fired product to obtain a netlike carbon fiber, namely a biochar carrier;
The gradient temperature is as follows: firstly, heating from 20 ℃ to 250 ℃ at a speed of 5 ℃/min, and preserving heat for 6 min ℃; then heating from 250 ℃ to 500 ℃ at a speed of 22 ℃/min, and preserving heat for 2 h; finally cooling to 20 ℃ along with the furnace;
S2-3, compounding a carrier:
s2-3-1, uniformly mixing the polyvinyl alcohol-mercapto montmorillonite in S2-1-B-3 and the biochar carrier in S2-2 in pure water to obtain a mixture;
The addition amount of the polyvinyl alcohol-mercapto montmorillonite is 1 g, the addition amount of the biochar carrier is 4 g, and the addition amount of the pure water is 110 mL;
S2-3-2, separating the mixture in S2-3-1, washing the separated product, drying at 100 ℃ for 5h, and finally sieving with a 200-mesh sieve to obtain a composite carrier, namely a flora carrier;
S3, preparing a fixed composite repairing agent;
s3-1, uniformly mixing the functional flora prepared in the S1 with the flora carrier prepared in the S2 to obtain a mixed system;
the addition amount of the flora carrier is 5 g, and the addition amount of the functional flora is 300 mL;
S3-2, firstly, carrying out shake culture on the mixed system obtained in the step S3-1 at 200 r/min under the conditions of constant temperature and 30 ℃ and light shielding for 24 h; then filtering, washing, and drying at 25 ℃ under aseptic condition to obtain the flora compound repairing agent.
Example 2: the description of this example is based on example 1, and mainly illustrates a preparation method under another parameter, including the following steps:
s1, preparing functional flora;
S1-1, domesticating Cd-resistant strains: under the premise of ensuring the normal growth of the strain and the function of degrading PAEs by the strain, inducing and domesticating the strains of the Gordonia, the Rhodococcus and the Bacillus in an LB culture medium containing Cd 2+;
s1-2, activating the three strains treated by the S1-1 in LB culture medium for 24h, and adjusting OD 600 = 1.0;
s1-3, according to the volume ratio of 1:1:1, mixing the three strains treated by the S1-2 in proportion to prepare functional bacterial groups with functions of degrading PAEs and resisting Cd;
S2, preparing a flora carrier;
S2-1-B-1-1, firstly dissolving montmorillonite in deionized water to prepare a suspension with the mass concentration of 2%, stirring at 50 ℃ for 4h, and then adding PVA into the deionized water to prepare a mixed solution with the mass concentration of 2%;
S2-1-B-1-2 according to the volume ratio of 1:4 adding the solution to the suspension and inserting PVA into the montmorillonite interstices according to the following parameters:
Firstly, ultrasonically oscillating 3 min, and standing 1:1 h; then ultrasonic oscillating 4 min, standing 2 h; then ultrasonic oscillation is carried out for 5min, and standing is carried out for 10 h; stirring at 45 ℃ for 3 h; finally, sealing and carrying out ultrasonic oscillation at room temperature for 5 d to obtain a colloid suspension;
S2-1-B-1-3, centrifugally separating the separated colloidal suspension in S2-1-B-1-2, repeatedly washing the separated matter with deionized water, and finally obtaining the product according to the following formula 1 g:50 The separation product is diluted and stored with deionized water for standby;
S2-1-B-2, self-assembly at low temperature: freezing 24h at-10deg.C after treating colloid suspension obtained in S2-1-B-1 to obtain large interlayer montmorillonite with expanded interlayer spacing;
S2-1-B-3, mercapto modification: adding the large interlayer montmorillonite in S2-1-B-2 into an ethanol-water solution of 3-mercaptopropyl trimethoxy silane, uniformly mixing, controlling the temperature to 25 ℃, controlling the pH to 9, and continuously stirring for reaction to 6h to obtain the mercapto-modified large interlayer montmorillonite, namely polyvinyl alcohol-mercapto montmorillonite;
The addition amount of the large interlayer montmorillonite is 1 g, and the addition amount of the ethanol-water solution of the 3-mercaptopropyl trimethoxysilane is 20 mL;
s2-2, preparing a reticular carbon fiber:
Firstly, immersing lignocellulose with the length of 1mm into a NaOH solution with the length of 0.1 mol/L, and stirring for 20 h at the speed of 500 r/min; then, the lignocellulose after treatment is separated and dried at 80 ℃, and finally, the lignocellulose is fired at a gradient temperature, and after the firing is finished, the product is washed and dried to obtain the netty carbon fiber, namely the biochar carrier;
the gradient temperature is as follows: firstly, heating from 20 ℃ to 250 ℃ at a speed of 4 ℃/min, and preserving heat for 5 min; then heating from 250 ℃ to 500 ℃ at a speed of 20 ℃/min, and preserving heat for 1h; finally cooling to 20 ℃ along with the furnace;
S2-3, compounding a carrier:
s2-3-1, uniformly mixing the polyvinyl alcohol-mercapto montmorillonite in S2-1-B-3 and the biochar carrier in S2-2 in pure water to obtain a mixture;
The addition amount of the polyvinyl alcohol-mercapto montmorillonite is 1g, the addition amount of the biochar carrier is 3g, and the addition amount of the pure water is 100 mL;
S2-3-2, separating the mixture in S2-3-1, washing the separated product, drying at 100 ℃ for 5h, and finally sieving with a 200-mesh sieve to obtain a composite carrier, namely a flora carrier;
s3, preparing a flora composite repairing agent;
S3-1, uniformly mixing the functional flora prepared in the S1 with the flora carrier prepared in the S2 to obtain a mixed system;
The addition amount of the flora carrier is 5 g, and the addition amount of the functional flora is 100 mL;
S3-2, firstly, carrying out shake culture on the mixed system in the S3-1 at 150 r/min under the conditions of constant temperature of 30 ℃ and light shielding for 24 h; then filtering, washing, and drying at 25 ℃ under aseptic condition to obtain the flora compound repairing agent.
Example 3: the description of the embodiment is a preparation method of a flora composite repairing agent for cooperatively treating PAEs and Cd, wherein a flora carrier is formed by combining sodium silicate-mercapto montmorillonite and biochar particles, and the preparation method comprises the following steps of:
s1, preparing functional flora;
S1-1, domesticating Cd-resistant strains: under the premise of ensuring the normal growth of the strain and the function of degrading PAEs by the strain, inducing and domesticating the strains of the Gordonia, the Rhodococcus and the Bacillus in an LB culture medium containing Cd 2+;
s1-2, activating the three strains treated by the S1-1 in LB culture medium for 24h, and adjusting OD 600 = 1.0;
s1-3, according to the volume ratio of 1:1:1, mixing the three strains treated by the S1-2 in proportion to prepare functional bacterial groups with functions of degrading PAEs and resisting Cd;
S2, preparing a flora carrier;
S2-1-A, adding 10g montmorillonite and 0.6 g sodium silicate into 200 mL ethanol-water solution containing 1 g of 3-mercaptopropyl trimethoxy silane with the mass concentration of 95%, uniformly mixing, controlling the temperature to 25 ℃, controlling the pH to 10, and continuously stirring for reacting for 7 h to obtain small-pore montmorillonite after mercapto modification, which is denoted as sodium silicate-mercapto montmorillonite;
S2-2, preparing biochar particles: crushing and grinding plant fibers, pyrolyzing under nitrogen atmosphere, grinding the product after pyrolysis, and sieving with a 200-mesh sieve to obtain biochar particles, namely a biochar carrier;
the parameters of pyrolysis are: raising the furnace temperature to 600 ℃ at a speed of 5 ℃/min, keeping the temperature at 2h ℃, and grinding the biochar after pyrolysis is finished and sieving the biochar with a 200-mesh sieve to prepare biochar particles;
S2-3, compounding a carrier:
s2-3-1, uniformly mixing sodium silicate-mercapto montmorillonite in S2-1-A and biochar carrier in S2-2 in pure water to obtain a mixture;
The addition amount of sodium silicate-mercapto montmorillonite is 1g, the addition amount of biochar carrier is 4 g, and the addition amount of pure water is 110 mL;
S2-3-2, separating the mixture in S2-3-1, washing the separated product, drying at 100 ℃ for 5h, and finally sieving with a 200-mesh sieve to obtain a composite carrier, namely a flora carrier;
s3, preparing a flora composite repairing agent;
S3-1, uniformly mixing the functional flora prepared in the S1 with the flora carrier prepared in the S2 to obtain a mixed system;
The addition amount of the flora carrier is 5 g, and the addition amount of the functional flora is 100 mL;
S3-2, firstly, carrying out shake culture on the mixed system in the S3-1 at 150 r/min under the conditions of constant temperature of 30 ℃ and light shielding for 24 h; then filtering, washing, and drying at 25 ℃ under aseptic condition to obtain the flora compound repairing agent.
Example 4: the description of this example is based on example 3, and mainly illustrates the preparation method under another parameter, including the following steps:
s1, preparing functional flora;
S1-1, domesticating Cd-resistant strains: under the premise of ensuring the normal growth of the strain and the function of degrading PAEs by the strain, inducing and domesticating the strains of the Gordonia, the Rhodococcus and the Bacillus in an LB culture medium containing Cd 2+;
s1-2, activating the three strains treated by the S1-1 in LB culture medium for 24h, and adjusting OD 600 = 1.0;
s1-3, according to the volume ratio of 1:1:1, mixing the three strains treated by the S1-2 in proportion to prepare functional bacterial groups with functions of degrading PAEs and resisting Cd;
S2, preparing a flora carrier;
S2-1-A, adding 10 g montmorillonite and 0.6g sodium silicate into 200 mL ethanol-water solution with the mass concentration of 1 g 3-mercaptopropyl trimethoxy silane of 95%, uniformly mixing, controlling the temperature to 25 ℃, controlling the pH to 9.5, and continuously stirring for reacting 6 h to obtain small-pore montmorillonite after mercapto modification, which is named as sodium silicate-mercapto montmorillonite;
S2-2, preparing biochar particles: crushing and grinding plant fibers, pyrolyzing under nitrogen atmosphere, grinding the product after pyrolysis, and sieving with a 200-mesh sieve to obtain biochar particles, namely a biochar carrier;
The parameters of pyrolysis are: raising the furnace temperature to 600 ℃ at a speed of 4 ℃/min, keeping the temperature at 2h, and grinding the biochar after pyrolysis is finished and sieving the biochar with a 200-mesh sieve to prepare biochar particles;
S2-3, compounding a carrier:
s2-3-1, uniformly mixing sodium silicate-mercapto montmorillonite in S2-1-A and biochar carrier in S2-2 in pure water to obtain a mixture;
The addition amount of sodium silicate-mercapto montmorillonite is 1g, the addition amount of biochar carrier is 3 g, and the addition amount of pure water is 100 mL;
S2-3-2, separating the mixture in S2-3-1, washing the separated product, drying at 100 ℃ for 5h, and finally sieving with a 200-mesh sieve to obtain a composite carrier, namely a flora carrier;
s3, preparing a flora composite repairing agent;
S3-1, uniformly mixing the functional flora prepared in the S1 with the flora carrier prepared in the S2 to obtain a mixed system;
The addition amount of the flora carrier is 5 g, and the addition amount of the functional flora is 100 mL;
S3-2, firstly, carrying out shake culture on the mixed system obtained in the step S3-1 at 150 r/min under the conditions of constant temperature and 30 ℃ and light shielding for 24h; then filtering, washing, and drying at 25 ℃ under aseptic condition to obtain the flora compound repairing agent.
Example 5: the description of this example is a method for preparing a complex bacterial colony repairing agent for synergistic treatment of PAEs and cds, and the conditions of this example are changed based on example 4, except the following contents are all the same:
S3-1: the addition amount of the flora carrier is 5g, and the addition amount of the functional flora is 50mL.
Example 6: the description of this example is a method for preparing a complex bacterial colony repairing agent for synergistic treatment of PAEs and cds, and the conditions of this example are changed based on example 4, except the following contents are all the same:
s3-1: the addition amount of the flora carrier is 5g, and the addition amount of the functional flora is 150 mL.
Example 7: the description of this example is a method for preparing a complex bacterial colony repairing agent for synergistic treatment of PAEs and cds, and the conditions of this example are changed based on example 4, except the following contents are all the same:
s3-2: constant temperature 25 ℃.
Example 8: the description of this example is a method for preparing a complex bacterial colony repairing agent for synergistic treatment of PAEs and cds, and the conditions of this example are changed based on example 4, except the following contents are all the same:
S3-2: constant temperature is 35 ℃.
Example 9: the description of this example is a method for preparing a complex bacterial colony repairing agent for synergistic treatment of PAEs and cds, and the conditions of this example are changed based on example 4, except the following contents are all the same:
S3-2: shake culturing under light-proof condition 48 h.
Example 10: the description of this example is a method for preparing a complex bacterial colony repairing agent for synergistic treatment of PAEs and cds, and the conditions of this example are changed based on example 4, except the following contents are all the same:
S3-2: shake-culturing under light-shielding condition for 72 h.
Comparative example 1: the description of this example is a method for preparing a complex bacterial colony repairing agent for synergistic treatment of PAEs and cds, and the conditions of this example are changed based on example 4, except the following contents are all the same:
The functional flora is replaced by the genus gordonia.
Comparative example 2: the description of this example is a method for preparing a complex bacterial colony repairing agent for synergistic treatment of PAEs and cds, and the conditions of this example are changed based on example 4, except the following contents are all the same:
the corn stalk biochar carbonized at 600 ℃ is used for replacing the flora carrier.
Comparative example 3: the description of this example is a method for preparing a complex bacterial colony repairing agent for synergistic treatment of PAEs and cds, and the conditions of this example are changed based on example 4, except the following contents are all the same:
the flora carrier is not subjected to sulfhydrylation treatment.
Test example 1: the test example is used for measuring the saturated adsorption amount of the flora carrier in the embodiment 4 and the comparative examples 2 and 3 to Cd 2+, and comprises the following specific steps: adding 0.1 g flora carrier into 20. 20 ml Cd 2+ solution containing 600 mg/L KNO 3 of 0.1. 0.1 mol/L, maintaining solid-to-liquid ratio of 1g to 200 ml, adjusting pH to 6.0, oscillating at 25deg.C for 1.1 h, standing for 16 h, centrifuging for 30min, collecting supernatant, and measuring Cd 2+ concentration, and the test result is shown in FIG. 5.
The results showed that: the saturated adsorption capacity of the flora carrier of the embodiment 4 on Cd 2+ is obviously larger than that of the comparative examples 2 and 3 (P < 0.05), which shows that compared with the composite repairing agent based on the biochar carrier and the composite repairing agent without sulfhydrylation treatment, the adsorption capacity of the complex repairing agent on heavy metal Cd 2+ is obviously improved, and the passivation efficiency is obvious.
Test example 2: the test example is used for measuring the degradation efficiency of the composite repairing agent in the examples 4-10 and the comparative examples 1 and 2 on PAEs in an inorganic salt system, and comprises the following specific steps: under the aseptic condition, 0.2 g compound repairing agent is weighed and added into a 20 ml inorganic salt culture medium containing 6 PAEs with the total concentration of 30 mg/L, after being cultured for 5d at 150 rpm and 30 ℃ in a dark place, the degradation rate of the sigma PAEs is measured and calculated, and the test result is shown in figure 6.
The results showed that: the total degradation rate of the composite repairing agent in example 4 for 6 PAEs is highest and can reach 86.70%, and the sigma PAEs degradation rate of the composite repairing agent in examples 5-10 can be known: the optimal preparation condition of the immobilized microbial inoculum is that the microbial inoculum ratio is 1 g to 20 ml, the temperature is 30 ℃, and the shake culture is carried out for 24 hours in a dark place; as can be seen from comparative example 1, the complex restorative agent prepared from the functional flora significantly increases the degradation capability to Σpaes compared with the functional single bacteria.
Test example 3: the composite repairing agent in the example 4 is observed by using a scanning electron microscope in the test, and the test result is shown in fig. 7.
The results showed that: SEM bioelectricity microscope of the composite repairing agent in example 4 shows that a large amount of functional strains are attached to the surface and pore structure of the composite repairing agent, and the strains are attached to the composite material in an agglomerated form or a dispersed state, which indicates that the prepared composite repairing agent can effectively enrich and fix functional bacterial groups.
Test example 4: the test example is used for measuring the degradation efficiency of the composite restoration agent in the example 4 on PAEs in the polluted soil and the passivation efficiency of heavy metal Cd.
The compound repairing agent in the example 4 is applied to PAEs and heavy metal Cd compound contaminated soil in an adding amount of 1%, after the PAEs and heavy metal Cd compound contaminated soil are subjected to dark culture for 50 d, the PAEs content and heavy metal occurrence form are measured, and test results are shown in fig. 8 and 9.
Conclusion: as can be seen from fig. 8, in the embodiment 4, the composite restoration agent is applied to the composite contaminated soil, the content of Σpae is reduced from 2.56 mg/kg to 1.17 mg/kg, the degradation rate is 54.14%, and the efficiency of degrading pae is higher; as can be seen from fig. 9, the exchangeable Cd content in the treated soil is reduced by 37.06%, and the carbonate bound state, the ferro-manganese oxide bound state and the residual Cd are respectively increased by 19.26%, 13.00% and 12.55%, which indicates that the application of the composite repairing agent promotes the conversion of the soil Cd from the exchangeable state with higher activity to the stable carbonate bound state, the ferro-manganese oxide bound state and the residual state, and has higher heavy metal passivation efficiency.
Claims (9)
1. The preparation method of the flora composite repairing agent for cooperatively treating PAEs and Cd is characterized by comprising the following steps of:
s1, preparing functional flora;
S1-1, domesticating Cd-resistant strains: inducing and domesticating the strains of Gordonia, rhodococcus and Bacillus in LB culture medium with gradient concentration Cd 2+;
s1-2, activating the three strains treated by the S1-1 in LB culture medium for 24h, and adjusting OD 600 = 1.0;
s1-3, according to the volume ratio of 1:1:1, mixing the three strains treated by the S1-2 in proportion to prepare functional bacterial groups with functions of degrading PAEs and resisting Cd;
S2, preparing a flora carrier;
S2-1, preparing sulfhydryl montmorillonite;
s2-2, preparing a biochar carrier;
S2-3, compounding a carrier: firstly, putting the mercapto montmorillonite in the S2-1 and the biochar carrier in the S2-2 into pure water, uniformly mixing, and then obtaining a flora carrier after centrifugation, washing and drying;
s3, preparing a flora composite repairing agent;
s3-1, uniformly mixing the functional flora prepared in the S1 with the flora carrier prepared in the S2 to obtain a mixed system;
N is marked as a multiplying factor and n is R +, the adding amount of the flora carrier is 1n g, and the adding amount of the functional flora is [10n,30n ] mL;
S3-2, firstly, carrying out shake culture on the mixed system obtained in the S3-1 at 150-200 r/min under the conditions of constant temperature and 30 ℃ and light shielding for 24h; then filtering, washing, and drying at 25 ℃ under aseptic condition to obtain the flora compound repairing agent.
2. The method for preparing a complex bacterial colony repairing agent for synergistic treatment of PAEs and Cd as claimed in claim 1, wherein the mercapto montmorillonite in S2-1 is sodium silicate-mercapto montmorillonite or polyvinyl alcohol-mercapto montmorillonite.
3. The method for preparing the flora composite repairing agent for cooperatively treating PAEs and Cd according to claim 2, which is characterized in that the method for preparing the sodium silicate-mercapto montmorillonite comprises the following steps:
S2-1-A, adding montmorillonite and sodium silicate into an ethanol-water solution of 3-mercaptopropyl trimethoxy silane, uniformly mixing, controlling the temperature to 25 ℃, controlling the pH to 9.5-10, and continuously stirring and reacting for 6-7 hours to obtain small-pore montmorillonite after mercapto modification, namely sodium silicate-mercaptomontmorillonite;
Wherein, the mass ratio of montmorillonite, sodium silicate and 3-mercaptopropyl trimethoxy silane is 1:0.06:0.1;
the mass concentration of the ethanol-water solution is 95%;
Note that the mass sum of montmorillonite and sodium silicate is M 1, and the volume of 3-mercaptopropyl trimethoxysilane, ethanol-water solution is V 1, then M 1：V1 =1.06 g:20 And (3) mL.
4. The method for preparing the flora composite repairing agent for cooperatively treating PAEs and Cd according to claim 2, wherein the method for preparing the polyvinyl alcohol-mercapto montmorillonite is as follows:
S2-1-B-1, and inserting organic matters between layers: firstly, dissolving montmorillonite into deionized water to prepare a suspension with the mass concentration of 2-4%, then adding PVA into the deionized water to prepare a mixed solution with the mass concentration of 2-3%, and then according to the volume ratio of 1: adding the solution into suspension, and carrying out ultrasonic oscillation for 5-7 d to obtain colloid suspension;
S2-1-B-2, self-assembly at low temperature: freezing the colloidal suspension obtained in S2-1-B-1 at-10deg.C for 24 h to obtain large interlayer montmorillonite with expanded interlayer spacing;
S2-1-B-3, mercapto modification: adding the large interlayer montmorillonite in S2-1-B-2 into an ethanol-water solution of 3-mercaptopropyl trimethoxy silane, uniformly mixing, controlling the temperature to 25 ℃, controlling the pH to 9.5-10, and continuously stirring and reacting for 6-7 hours to obtain the mercapto-modified large interlayer montmorillonite, which is named as polyvinyl alcohol-mercapto montmorillonite;
Wherein, the mass ratio of the large interlayer montmorillonite to the 3-mercaptopropyl trimethoxy silane is 1:0.1;
the mass concentration of the ethanol-water solution is 95%;
Note that the mass of the interlayer montmorillonite is M 2, and the volume of the 3-mercaptopropyl trimethoxysilane and ethanol-water solution is V 2, then M 2：V2 =1 g:20 And (3) mL.
5. The method for preparing the complex microbial community repairing agent for cooperatively treating PAEs and Cd according to claim 4, wherein the method for inserting organic matters between the S2-1-B-1 and the middle layer is as follows:
S2-1-B-1-1, firstly dissolving montmorillonite in deionized water to prepare a suspension with the mass concentration of 2-4%, stirring for 3-4 hours at the temperature of 45-50 ℃, and then adding PVA into the deionized water to prepare a mixed solution with the mass concentration of 2-3%;
S2-1-B-1-2 according to the volume ratio of 1:4 adding the solution to the suspension and inserting PVA into the montmorillonite interstices according to the following parameters:
firstly, carrying out ultrasonic oscillation for 3-5 min, and standing for 1 h; then ultrasonic oscillation is carried out for 4-5 min, and standing is carried out for 2 h; then ultrasonic oscillation is carried out for 5 min, and standing is carried out for 10 h; stirring at 45-50 ℃ for 3 h; finally, sealing and carrying out ultrasonic oscillation at room temperature for 5-7 d to obtain a colloid suspension;
S2-1-B-1-3, centrifugally separating the colloidal suspension isolate in S2-1-B-1-2, repeatedly washing the isolate with deionized water, and finally diluting the isolate with deionized water for later use.
6. The method for preparing a complex microbial community repairing agent for synergistic treatment of PAEs and cds according to claim 2, wherein the biochar carrier paired with the sodium silicate-mercapto montmorillonite is biochar particles, and the biochar carrier paired with the polyvinyl alcohol-mercapto montmorillonite is a reticular carbon fiber.
7. The method for preparing the complex microbial community restoration agent for cooperatively treating PAEs and cds according to claim 6, wherein the method for preparing the biochar particles is as follows: crushing and grinding plant fibers, pyrolyzing under inert atmosphere, grinding the product after pyrolysis, and sieving with a 200-mesh sieve to obtain biochar particles;
The parameters of the pyrolysis are as follows: and (3) raising the furnace temperature from room temperature to 600 ℃ at a speed of 4-5 ℃/min, and keeping the furnace temperature at 2 h ℃.
8. The method for preparing the flora composite repairing agent for cooperatively treating PAEs and Cd according to claim 6, wherein the method for preparing the reticular carbon fiber is as follows:
Firstly, immersing lignocellulose with the length of 1-2 mm into a NaOH solution with the length of 0.1 mol/L, and stirring for 20-25 h at the speed of 500-550 r/min; then taking out lignocellulose, drying at 80 ℃, finally firing at a gradient temperature, and washing and drying the fired product to obtain the netlike carbon fiber;
The gradient temperature is as follows: firstly, heating from 20 ℃ to 250 ℃ at a speed of 4-5 ℃/min, and preserving heat for 5-6 min; then heating from 250 ℃ to 500 ℃ at a speed of 20-22 ℃/min, and preserving heat for 1-2 h; finally cooling to 20 ℃ along with the furnace.
9. The method for preparing the complex bacterial colony repairing agent for cooperatively treating PAEs and Cd according to claim 1, wherein the method for compounding the carriers in S2-3 is as follows:
S2-3-1, uniformly mixing the mercapto montmorillonite in S2-1 and the biochar carrier in S2-2 in pure water to obtain a mixture; n is recorded as a multiplying factor and n is R +, the addition of the mercapto montmorillonite is 1n g, the addition of the biochar carrier is [3n,4n ] g, and the addition of the pure water is [100n,110n ] mL;
S2-3-2, separating the mixture obtained in the step S2-3-1, washing the separated product, drying at 100 ℃ for 5h, and finally sieving with a 200-mesh sieve to obtain the composite carrier.
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