CN114058507A - Carbon-coupled compound microbial inoculum and preparation method and application thereof - Google Patents
Carbon-coupled compound microbial inoculum and preparation method and application thereof Download PDFInfo
- Publication number
- CN114058507A CN114058507A CN202111228892.7A CN202111228892A CN114058507A CN 114058507 A CN114058507 A CN 114058507A CN 202111228892 A CN202111228892 A CN 202111228892A CN 114058507 A CN114058507 A CN 114058507A
- Authority
- CN
- China
- Prior art keywords
- carbon
- soil
- microbial inoculum
- preparing
- coupled
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
- 239000002068 microbial inoculum Substances 0.000 title claims abstract description 57
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 title claims abstract description 35
- 229910052799 carbon Inorganic materials 0.000 title claims abstract description 35
- 238000002360 preparation method Methods 0.000 title claims abstract description 15
- 150000001875 compounds Chemical class 0.000 title claims description 10
- 239000002689 soil Substances 0.000 claims abstract description 76
- 239000002131 composite material Substances 0.000 claims abstract description 71
- 230000001580 bacterial effect Effects 0.000 claims abstract description 40
- 238000011065 in-situ storage Methods 0.000 claims abstract description 33
- 239000007788 liquid Substances 0.000 claims abstract description 31
- 238000000034 method Methods 0.000 claims abstract description 30
- 241000894006 Bacteria Species 0.000 claims abstract description 25
- 238000002156 mixing Methods 0.000 claims abstract description 25
- 229910001385 heavy metal Inorganic materials 0.000 claims abstract description 23
- 239000003575 carbonaceous material Substances 0.000 claims abstract description 21
- 239000003344 environmental pollutant Substances 0.000 claims abstract description 19
- 231100000719 pollutant Toxicity 0.000 claims abstract description 16
- 230000000593 degrading effect Effects 0.000 claims abstract description 12
- 238000012258 culturing Methods 0.000 claims abstract description 11
- 238000012136 culture method Methods 0.000 claims abstract description 10
- 239000005445 natural material Substances 0.000 claims abstract description 10
- 230000008014 freezing Effects 0.000 claims abstract description 4
- 238000007710 freezing Methods 0.000 claims abstract description 4
- 238000012545 processing Methods 0.000 claims abstract description 4
- 238000010168 coupling process Methods 0.000 claims description 40
- 230000008878 coupling Effects 0.000 claims description 38
- 238000005859 coupling reaction Methods 0.000 claims description 38
- 230000015556 catabolic process Effects 0.000 claims description 34
- 238000006731 degradation reaction Methods 0.000 claims description 34
- 239000001963 growth medium Substances 0.000 claims description 34
- 238000009792 diffusion process Methods 0.000 claims description 30
- 239000000725 suspension Substances 0.000 claims description 27
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 27
- 244000061458 Solanum melongena Species 0.000 claims description 16
- 235000002597 Solanum melongena Nutrition 0.000 claims description 16
- 239000008223 sterile water Substances 0.000 claims description 15
- 229920001661 Chitosan Polymers 0.000 claims description 13
- 239000000203 mixture Substances 0.000 claims description 13
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 claims description 12
- 239000003795 chemical substances by application Substances 0.000 claims description 12
- 125000005575 polycyclic aromatic hydrocarbon group Chemical group 0.000 claims description 12
- 244000005700 microbiome Species 0.000 claims description 11
- IXPNQXFRVYWDDI-UHFFFAOYSA-N 1-methyl-2,4-dioxo-1,3-diazinane-5-carboximidamide Chemical compound CN1CC(C(N)=N)C(=O)NC1=O IXPNQXFRVYWDDI-UHFFFAOYSA-N 0.000 claims description 10
- 238000001035 drying Methods 0.000 claims description 10
- 239000000463 material Substances 0.000 claims description 10
- 239000000661 sodium alginate Substances 0.000 claims description 10
- 235000010413 sodium alginate Nutrition 0.000 claims description 10
- 229940005550 sodium alginate Drugs 0.000 claims description 10
- 229920001817 Agar Polymers 0.000 claims description 9
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 claims description 9
- 239000008272 agar Substances 0.000 claims description 9
- 239000012528 membrane Substances 0.000 claims description 9
- 239000000843 powder Substances 0.000 claims description 9
- 241000282994 Cervidae Species 0.000 claims description 8
- 238000000576 coating method Methods 0.000 claims description 8
- 210000003608 fece Anatomy 0.000 claims description 8
- 239000004417 polycarbonate Substances 0.000 claims description 8
- 229920000515 polycarbonate Polymers 0.000 claims description 8
- 229910001220 stainless steel Inorganic materials 0.000 claims description 8
- 239000010935 stainless steel Substances 0.000 claims description 8
- 240000008042 Zea mays Species 0.000 claims description 7
- 235000005824 Zea mays ssp. parviglumis Nutrition 0.000 claims description 7
- 235000002017 Zea mays subsp mays Nutrition 0.000 claims description 7
- 235000005822 corn Nutrition 0.000 claims description 7
- 230000000813 microbial effect Effects 0.000 claims description 7
- 230000008569 process Effects 0.000 claims description 7
- 238000007873 sieving Methods 0.000 claims description 7
- 238000007711 solidification Methods 0.000 claims description 7
- 230000008023 solidification Effects 0.000 claims description 7
- 238000001179 sorption measurement Methods 0.000 claims description 7
- 238000005406 washing Methods 0.000 claims description 7
- 239000012752 auxiliary agent Substances 0.000 claims description 6
- 238000001816 cooling Methods 0.000 claims description 6
- 238000000227 grinding Methods 0.000 claims description 6
- 238000011534 incubation Methods 0.000 claims description 6
- 238000005067 remediation Methods 0.000 claims description 6
- 238000003756 stirring Methods 0.000 claims description 6
- 239000011248 coating agent Substances 0.000 claims description 5
- 239000002957 persistent organic pollutant Substances 0.000 claims description 5
- 239000000575 pesticide Substances 0.000 claims description 5
- 230000001681 protective effect Effects 0.000 claims description 5
- 238000005070 sampling Methods 0.000 claims description 5
- 238000009210 therapy by ultrasound Methods 0.000 claims description 5
- 239000012880 LB liquid culture medium Substances 0.000 claims description 4
- 238000005336 cracking Methods 0.000 claims description 4
- 239000003292 glue Substances 0.000 claims description 4
- 238000012216 screening Methods 0.000 claims description 4
- 239000000565 sealant Substances 0.000 claims description 4
- 235000017166 Bambusa arundinacea Nutrition 0.000 claims description 3
- 235000017491 Bambusa tulda Nutrition 0.000 claims description 3
- UXVMQQNJUSDDNG-UHFFFAOYSA-L Calcium chloride Chemical compound [Cl-].[Cl-].[Ca+2] UXVMQQNJUSDDNG-UHFFFAOYSA-L 0.000 claims description 3
- 244000082204 Phyllostachys viridis Species 0.000 claims description 3
- 235000015334 Phyllostachys viridis Nutrition 0.000 claims description 3
- 240000002044 Rhizophora apiculata Species 0.000 claims description 3
- 230000003213 activating effect Effects 0.000 claims description 3
- 239000011425 bamboo Substances 0.000 claims description 3
- 238000004140 cleaning Methods 0.000 claims description 3
- 239000012153 distilled water Substances 0.000 claims description 3
- 239000012535 impurity Substances 0.000 claims description 3
- 239000002054 inoculum Substances 0.000 claims description 3
- 238000004519 manufacturing process Methods 0.000 claims description 3
- 239000003209 petroleum derivative Substances 0.000 claims description 3
- 238000007789 sealing Methods 0.000 claims description 3
- 238000002791 soaking Methods 0.000 claims description 3
- 238000005527 soil sampling Methods 0.000 claims description 3
- 230000001954 sterilising effect Effects 0.000 claims description 3
- 238000003860 storage Methods 0.000 claims description 3
- 229910021642 ultra pure water Inorganic materials 0.000 claims description 3
- 239000012498 ultrapure water Substances 0.000 claims description 3
- 244000146553 Ceiba pentandra Species 0.000 claims description 2
- 235000003301 Ceiba pentandra Nutrition 0.000 claims description 2
- 239000000835 fiber Substances 0.000 claims description 2
- 238000001879 gelation Methods 0.000 claims description 2
- 239000011343 solid material Substances 0.000 claims description 2
- 239000006228 supernatant Substances 0.000 claims description 2
- 238000005303 weighing Methods 0.000 claims description 2
- 239000002028 Biomass Substances 0.000 abstract description 2
- 241001052560 Thallis Species 0.000 abstract 1
- 238000004321 preservation Methods 0.000 abstract 1
- BBEAQIROQSPTKN-UHFFFAOYSA-N pyrene Chemical compound C1=CC=C2C=CC3=CC=CC4=CC=C1C2=C43 BBEAQIROQSPTKN-UHFFFAOYSA-N 0.000 description 65
- GVEPBJHOBDJJJI-UHFFFAOYSA-N fluoranthrene Natural products C1=CC(C2=CC=CC=C22)=C3C2=CC=CC3=C1 GVEPBJHOBDJJJI-UHFFFAOYSA-N 0.000 description 32
- 230000000694 effects Effects 0.000 description 19
- 239000000243 solution Substances 0.000 description 17
- 238000010586 diagram Methods 0.000 description 11
- 239000010902 straw Substances 0.000 description 8
- 239000011148 porous material Substances 0.000 description 7
- 239000000969 carrier Substances 0.000 description 6
- 239000008188 pellet Substances 0.000 description 6
- 239000002671 adjuvant Substances 0.000 description 5
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 description 4
- 241000193830 Bacillus <bacterium> Species 0.000 description 4
- 239000007789 gas Substances 0.000 description 4
- 229910017053 inorganic salt Inorganic materials 0.000 description 4
- 239000000047 product Substances 0.000 description 4
- 239000007787 solid Substances 0.000 description 4
- 241000589516 Pseudomonas Species 0.000 description 3
- 230000009286 beneficial effect Effects 0.000 description 3
- XIEPJMXMMWZAAV-UHFFFAOYSA-N cadmium nitrate Inorganic materials [Cd+2].[O-][N+]([O-])=O.[O-][N+]([O-])=O XIEPJMXMMWZAAV-UHFFFAOYSA-N 0.000 description 3
- 238000011161 development Methods 0.000 description 3
- 230000018109 developmental process Effects 0.000 description 3
- 230000007613 environmental effect Effects 0.000 description 3
- 238000001914 filtration Methods 0.000 description 3
- 239000000499 gel Substances 0.000 description 3
- 239000010871 livestock manure Substances 0.000 description 3
- NMHMNPHRMNGLLB-UHFFFAOYSA-N phloretic acid Chemical compound OC(=O)CCC1=CC=C(O)C=C1 NMHMNPHRMNGLLB-UHFFFAOYSA-N 0.000 description 3
- 238000003900 soil pollution Methods 0.000 description 3
- XKRFYHLGVUSROY-UHFFFAOYSA-N Argon Chemical compound [Ar] XKRFYHLGVUSROY-UHFFFAOYSA-N 0.000 description 2
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 2
- 241000186359 Mycobacterium Species 0.000 description 2
- 238000007605 air drying Methods 0.000 description 2
- 239000003610 charcoal Substances 0.000 description 2
- 230000002860 competitive effect Effects 0.000 description 2
- 238000010276 construction Methods 0.000 description 2
- 238000007865 diluting Methods 0.000 description 2
- 238000010790 dilution Methods 0.000 description 2
- 239000012895 dilution Substances 0.000 description 2
- 235000013305 food Nutrition 0.000 description 2
- 238000011081 inoculation Methods 0.000 description 2
- 239000010410 layer Substances 0.000 description 2
- 238000005360 mashing Methods 0.000 description 2
- YNPNZTXNASCQKK-UHFFFAOYSA-N phenanthrene Chemical compound C1=CC=C2C3=CC=CC=C3C=CC2=C1 YNPNZTXNASCQKK-UHFFFAOYSA-N 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 238000012546 transfer Methods 0.000 description 2
- 239000002699 waste material Substances 0.000 description 2
- 230000002407 ATP formation Effects 0.000 description 1
- FMMWHPNWAFZXNH-UHFFFAOYSA-N Benz[a]pyrene Chemical compound C1=C2C3=CC=CC=C3C=C(C=C3)C2=C2C3=CC=CC2=C1 FMMWHPNWAFZXNH-UHFFFAOYSA-N 0.000 description 1
- 241000283690 Bos taurus Species 0.000 description 1
- 241000196324 Embryophyta Species 0.000 description 1
- 238000003915 air pollution Methods 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 229910052786 argon Inorganic materials 0.000 description 1
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 1
- 239000011324 bead Substances 0.000 description 1
- 230000008901 benefit Effects 0.000 description 1
- 229910052793 cadmium Inorganic materials 0.000 description 1
- BDOSMKKIYDKNTQ-UHFFFAOYSA-N cadmium atom Chemical compound [Cd] BDOSMKKIYDKNTQ-UHFFFAOYSA-N 0.000 description 1
- 230000000711 cancerogenic effect Effects 0.000 description 1
- 231100000315 carcinogenic Toxicity 0.000 description 1
- 239000012876 carrier material Substances 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 239000000356 contaminant Substances 0.000 description 1
- -1 crab shells Substances 0.000 description 1
- 230000002950 deficient Effects 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 238000009826 distribution Methods 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 230000009088 enzymatic function Effects 0.000 description 1
- 230000003203 everyday effect Effects 0.000 description 1
- 239000003517 fume Substances 0.000 description 1
- 230000006870 function Effects 0.000 description 1
- 150000004676 glycans Chemical class 0.000 description 1
- 230000012447 hatching Effects 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 238000010438 heat treatment Methods 0.000 description 1
- 239000001307 helium Substances 0.000 description 1
- 229910052734 helium Inorganic materials 0.000 description 1
- SWQJXJOGLNCZEY-UHFFFAOYSA-N helium atom Chemical compound [He] SWQJXJOGLNCZEY-UHFFFAOYSA-N 0.000 description 1
- 231100000086 high toxicity Toxicity 0.000 description 1
- 239000011261 inert gas Substances 0.000 description 1
- 238000009630 liquid culture Methods 0.000 description 1
- 230000007774 longterm Effects 0.000 description 1
- 238000001000 micrograph Methods 0.000 description 1
- 238000005065 mining Methods 0.000 description 1
- 239000010413 mother solution Substances 0.000 description 1
- 231100000219 mutagenic Toxicity 0.000 description 1
- 230000003505 mutagenic effect Effects 0.000 description 1
- 229910052757 nitrogen Inorganic materials 0.000 description 1
- 230000010355 oscillation Effects 0.000 description 1
- 239000001301 oxygen Substances 0.000 description 1
- 229910052760 oxygen Inorganic materials 0.000 description 1
- 230000002572 peristaltic effect Effects 0.000 description 1
- 231100001239 persistent pollutant Toxicity 0.000 description 1
- 239000004033 plastic Substances 0.000 description 1
- 229920001282 polysaccharide Polymers 0.000 description 1
- 239000005017 polysaccharide Substances 0.000 description 1
- 238000000197 pyrolysis Methods 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 230000008439 repair process Effects 0.000 description 1
- 238000001878 scanning electron micrograph Methods 0.000 description 1
- 239000002910 solid waste Substances 0.000 description 1
- 239000011550 stock solution Substances 0.000 description 1
- 239000010907 stover Substances 0.000 description 1
- 238000005092 sublimation method Methods 0.000 description 1
- 230000001502 supplementing effect Effects 0.000 description 1
- 239000002344 surface layer Substances 0.000 description 1
- 230000004083 survival effect Effects 0.000 description 1
- 230000002194 synthesizing effect Effects 0.000 description 1
- 239000008399 tap water Substances 0.000 description 1
- 235000020679 tap water Nutrition 0.000 description 1
- 231100000378 teratogenic Toxicity 0.000 description 1
- 230000003390 teratogenic effect Effects 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 239000002912 waste gas Substances 0.000 description 1
- 238000003911 water pollution Methods 0.000 description 1
Images
Classifications
-
- 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
- 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
- C12N1/02—Separating microorganisms from their culture media
-
- 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
-
- 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
- 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
-
- 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
- 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
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N11/00—Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof
- C12N11/02—Enzymes or microbial cells immobilised on or in an organic carrier
Abstract
The invention relates to the technical field of biomass resource utilization and environmental pollutant treatment, in particular to a preparation method of a carbon-coupled composite microbial inoculum; comprises the step S1 of processing the charrable natural material; s2, collecting a soil sample of soil at a typical pollution point, and carrying out isolated culture on a target strain by an in-situ culture method; s3, domesticating to obtain a strain capable of degrading the target pollutants under the stress of heavy metals; s4, separating and purifying to obtain a pure colony of the degrading bacteria; s5, repeating the step S4, collecting at least 3 thalli, carrying out enrichment culture until OD600= 1.0-1.2, collecting bacterial liquid, and carrying out freezing preservation; s6, mixing the multiple bacterial liquids to obtain a composite bacterial seed liquid; s7, mixing and culturing the composite bacteria seed liquid and the carbon-based material, and carrying out immobilization treatment to obtain a composite microbial inoculum; the method can obtain the carbon-coupled composite microbial inoculum which can degrade the content of target pollutants in the environment, fix heavy metals and improve the soil quality under the stress of heavy metals.
Description
Technical Field
The invention relates to the technical field of biomass resource utilization and environmental pollutant treatment, in particular to a carbon-coupled composite microbial inoculum and a preparation method and application thereof.
Background
The national soil environment is not optimistic, and the water body and the soil are seriously damaged while the productivity development is increased. Among them, soil problems are most prominent in southern areas, and the main reason is due to the continuous development of industrial industries. According to the estimation, the soil area polluted by pesticides, heavy metals and the like in China reaches hundreds of thousands of km2Wherein the polluted soil in the mining area reaches 2 km2About 5 km of petroleum-polluted soil2About 500km of solid waste stacked polluted soil2Has already seriously developed the ecological environment quality, food safety and social economy of ChinaAnd (4) serious threats. Human activities are main factors causing soil pollution, pesticide pollution sources and part of heavy metal pollutants have high toxicity and strong mobility. In coastal areas with developed economy, the condition of soil pollution is very complex, and the soil pollution has the characteristics of diversity and complexity, and is represented by the diversity of pollution ways (air pollution type, water pollution type, solid pollution type and the like) and the diversity of pollution sources (industrial pollution sources, agricultural pollution sources, living pollution sources, traffic pollution sources, geological effects and the like). The development from the research of single pollutants to the research of composite pollutants is a typical trend of the current environmental problems and is a stage of the mature repair means.
Polycyclic aromatic hydrocarbons and heavy metals are trace persistent pollutants in the environment and can be detected in the environment at the same time. Polycyclic aromatic hydrocarbons are considered carcinogenic, teratogenic, and mutagenic contaminants, with phenanthrene (Phe), pyrene (Pyr), and benzo [ a ] pyrene (BAP) being widely discussed for their universality and distribution. The removal of polycyclic aromatic hydrocarbons from soil is imperative because they pose a serious threat to human health through the food chain. But remediation of polycyclic aromatic hydrocarbons is challenging in the presence of heavy metals, which can affect ATP production, C-mineralization, colony transfer, and enzyme function, among others, and thereby impair microbial activity.
Disclosure of Invention
The invention aims to provide a preparation method of a carbon-coupled composite microbial inoculum capable of degrading organic pollutants under the stress of heavy metals.
The technical scheme provided by the invention is as follows:
a preparation method of a carbon-coupled composite microbial inoculum comprises the following steps:
s1, processing the natural material capable of being carbonized to obtain a carbon-based material serving as one of the subsequent coupling materials;
s2, collecting a soil sample of the soil at a typical pollution point to prepare a soil suspension, and performing isolated culture on microorganisms in the soil by using a diffusion chamber in-situ culture method to obtain a soil microorganism enrichment solution containing a target strain;
s3, carrying out multiple periods of strain screening and domestication on the enrichment solution to obtain strains capable of degrading the polycyclic aromatic hydrocarbon under heavy metal stress;
s4, separating and purifying the strains with the degradation capability by using a plate coating method to obtain pure colonies of the degradation bacteria;
s5, repeating the step S4, respectively collecting the thallus after at least 3 strains are purified, carrying out enrichment culture by using an LB liquid culture medium until OD600= 1.0-1.2, and collecting bacterial liquid for freezing storage;
s6, mixing the activated multiple bacterial liquids in a certain proportion to obtain a composite bacterial seed liquid serving as one of the coupling materials;
s7, mixing and culturing the composite bacteria seed liquid and the carbon-based material in proportion for a period of time, and then carrying out immobilization treatment to obtain the composite bacteria agent.
Preferably, the carbonizable natural material described in step S1 includes one or more of corn stover, crab shells, eggplant stalks, bamboo leaves, kapok fibers, and deer feces.
Preferably, the carbonizable natural material is treated in step S1 by:
s11, cleaning the charrable natural material, removing impurities, drying and crushing into powder;
s12, slowly cracking for 1-3 hours at 300-700 ℃ in an oxygen-isolated smoldering high temperature;
and S13, cooling to room temperature under the protection of protective gas and condensed circulating water, sequentially grinding and sieving, soaking and activating with hydrochloric acid, washing with distilled water and drying to obtain the carbon-based material.
Preferably, the typical contaminated site soil refers to pesticide contaminated farmland, petroleum hydrocarbon contaminated soil, mangrove root system soil, soil near manufacturing plants or dry mud bed soil.
Preferably, the soil suspension is prepared by the following method:
weighing 10g of soil sample, adding 90ml of sterilized ultrapure water, uniformly mixing, performing ultrasonic treatment, and standing for 30-60 min to obtain a supernatant containing microorganisms;
the ultrasonic treatment was carried out 2 times under ultrasonic amplitude of 40 μm and ultrasonic time of 10s at 5s intervals.
Preferably, in step S2, the procedure of the diffusion chamber in situ culture method is as follows:
s100, preparing an in-situ culture medium: taking 1L of water near a soil sampling point, adding 1% (wt/vol) agar powder into the water at a natural pH value, uniformly mixing, sterilizing at a high temperature, and cooling to 45 ℃ to obtain warm agar;
s200, inoculating and assembling a diffusion chamber: adhering the sterilized polycarbonate membrane with the aperture of 0.03 mu m to the bottom surface of the sterilized stainless steel gasket by using a sealant, introducing a mixture of a soil suspension and warm agar into the hollow inner ring, and sealing the polycarbonate membrane with the aperture of 0.03 mu m and the top surface of the stainless steel gasket by using glue to form a sealed chamber;
s300, burying the diffusion chamber at an original sampling position or in a simulated natural environment for incubation, wherein the depth is 5-10 cm; after 1 week of incubation, the in situ culture device is taken back and cleaned, the in situ culture medium is transferred to a sterile 1.5ml EP tube, sterile water is added, the in situ culture medium is mashed and fully mixed with the sterile water, and the mixture is centrifuged to obtain a bacterial suspension.
Preferably, in step S3, the strain acclimation process is as follows:
and (4) absorbing the bacterial suspension obtained in the step (S2) and coating the bacterial suspension into an acclimation culture medium of a new diffusion chamber, culturing in situ for four acclimation periods by using the diffusion chamber, transferring the bacterial suspension into the newly constructed diffusion chamber in each period, setting four acclimation culture media with different concentration gradients, and adding organic pollutants and heavy metals with certain concentrations into the in situ culture media to screen and acclimate strains capable of degrading target pollutants under stress.
Preferably, in step S7, the composite bacteria seed liquid and the carbon-based material are mixed and cultured according to a ratio of 1: 10-30 (w/v).
Preferably, in step S7, the immobilization process includes mixing the composite bacterial liquid and the carbon-based material at a ratio of 1: 10-30 (w/v), culturing for 12-24 h, performing adsorption coupling, sieving in a sterile environment, and drying the solid material.
Preferably, in step S7, the immobilization process includes mixing and culturing the composite bacteria seed solution and the carbon-based material in proportion for a period of time, adding an equal volume of coupling adjuvant, stirring, dropping into the solidified solution drop by drop, repeatedly washing with sterile water after solidification or gelation, and naturally drying.
Preferably, the grinding and sieving are carried out by a sieve of 20-100 meshes; the concentration of the hydrochloric acid solution is 1-3 mol/L.
Preferably, 3 bacterial liquids are collected and mixed in a ratio of 1:1:1, 1:1:2, 1:2:1 or 2:1: 1.
Preferably, the coupling auxiliary agent and the corresponding curing liquid with the same volume are 3-5% of sodium alginate and 2% of calcium chloride solution as curing liquid or 4-6% of chitosan and 1mol/L of sodium hydroxide as curing liquid.
The invention also provides the carbon-coupled composite microbial inoculum prepared by the preparation method of the carbon-coupled composite microbial inoculum.
The invention also provides application of the carbon-coupled composite microbial inoculum in pollutant treatment and soil remediation.
The invention has the beneficial effects that:
1. the preparation method is simple in preparation process and low in economic cost, waste biomasses such as corn straws, crab shells, bamboo leaves, eggplant stalks, deer dung and the like are used as carrier materials, polluted soil is used for automatically extracting and synthesizing target flora, the price is low, the raw materials are sufficient and easily available, the combination and matching of the materials are flexible, the materials can be completed in a laboratory, the waste resources can be secondarily utilized, and the environmental protection is facilitated;
2. according to the invention, the eggplant stalk, the deer feces and the crab shells are firstly made into the carbon-based material to be used as a coupling carrier, and the chitosan is used as a coupling auxiliary agent to prepare the charcoal gel balls, so that more choices are provided for preparing immobilized bacteria agents with specific functions;
3. the invention uses the diffusion chamber in situ culture method to separate and screen the target strains, the diffusion chamber aims at creating a hatching strategy which is very close to the natural habitat, and fundamentally 'lures' cells to think that the cells grow in the natural environment; compared with the traditional laboratory culture method, the in-situ culture method can obtain traditional 'unculturable' microorganisms and also can enable the strains to keep the symbiotic relationship between the natural environment and the microorganisms, and the most important point is that the obtained target strains can be controlled not to deviate from the survival conditions of the treatment environment to generate variation, so that the subsequently prepared composite microbial inoculum can better keep the activity of the pollutants when the pollutants are treated in situ, and the performance of the composite microbial inoculum can be better exerted.
4. The invention provides a method for coupling three strains belonging to pseudomonas respectively by using carbon-based materialsPseudomonas) Bacillus (B) and (C)Bacillus) And Mycobacterium (A), (B), (C)Cytobacillus) Compared with the prior art using a single strain, the composite flora composed of the strains has stronger stability, higher possibility of establishing tolerance and acquiring competitive advantage in a complex environment and larger degradable components, is more beneficial to improving the richness and diversity of soil communities and has obvious influence on the composition of the microbial communities and the effect of root system microorganisms;
5. in the microbial inoculum prepared by the invention, the biochar has the characteristics of higher pore structure, specific surface area and the like, pollutants can be quickly adsorbed into the pore diameter or around the pore diameter to form aggregates, then the pollutants are adsorbed and metabolically degraded by carried heavy metal-resistant degradation flora, and heavy metals can be fixed, so that the threat of soil is reduced in a short time, and an excellent degradation effect is achieved in a composite polluted environment;
6. the carbon-coupled composite microbial inoculum prepared by the invention has good mechanical strength and mass transfer performance and strong environmental durability and stability, when the microbial inoculum is used for degrading a polycyclic aromatic hydrocarbon polluted liquid culture medium, 2% (w/v) of the composite microbial inoculum is added when the initial concentration of pyrene is 50mg/L, and after 7d culture tests, the degradation rates of different components are all over 70%, and the degradation rate can reach 95.77% at most, so that the environmental pollutants can be safely and effectively treated, and the carbon-coupled composite microbial inoculum has a wide application prospect in pollutant treatment and soil remediation.
Drawings
FIG. 1 is a schematic diagram of a preparation process of a carbon-coupled composite microbial inoculum;
FIG. 2 is a schematic diagram showing the yields of 9 biochar-coupled supports;
FIG. 3 is a scanning electron micrograph of 9 biochar-coupled carriers;
FIG. 4 is an exploded schematic view of the diffusion chamber;
FIG. 5 is a schematic diagram showing the pyrene-degrading ability of a single strain and a complex flora;
FIG. 6 is a schematic diagram showing the pyrene degradation capability of a complex microbial inoculum prepared by sodium alginate embedding;
FIG. 7 is a schematic diagram showing the pyrene degradation capability of a complex microbial inoculum prepared by chitosan embedding;
FIG. 8 is a schematic diagram showing the pyrene degradation capability of a complex microbial inoculum prepared by adsorption coupling;
FIG. 9 is a schematic diagram showing the comparison of pyrene degradation capability of a complex microbial inoculum prepared from different carbon-based materials by three coupling methods.
Detailed Description
The invention will be further elucidated with reference to the accompanying figures 1 to 9:
the first embodiment is as follows:
the preparation method of the carbon-coupled composite microbial inoculum shown in figure 1 comprises the following steps:
s1, processing the natural material capable of being carbonized to obtain a carbon-based material serving as one of the subsequent coupling materials;
preparation of carbon-based material as one of the coupling materials:
removing impurities from the collected eggplant straws, washing the eggplant straws clean by using tap water, naturally drying the eggplant straws in an air drying oven at 105 ℃ for 12 hours, crushing the eggplant straws into powder by using a crusher, putting the powder into an atmosphere box furnace, sealing the atmosphere box furnace, vacuumizing the atmosphere box furnace, introducing protective gas nitrogen, rapidly heating the powder to a set temperature of 300-700 ℃ at a speed of 5-10 ℃/min, and continuously carrying out oxygen-insulated cracking for 1-3 hours. In the embodiment, the temperature is rapidly increased to 500 ℃ at the speed of 5 ℃/min, the oxygen-insulated cracking is continuously carried out for 2 hours, the generated waste gas is pumped by an exhaust system to prevent the environment pollution, and the biological tar obtained by condensing part of the steam generated in the pyrolysis process is recycled. The protective gas introduced into the atmosphere furnace can also be inert gas such as helium or argon, and the inner cavity of the atmosphere furnace is in an oxygen-deficient or oxygen-insulated state by vacuumizing and introducing the protective gas.
Cooling to room temperature, taking out the product, grinding, sieving with a 80-mesh sieve, soaking and activating with 1mol/L hydrochloric acid, washing with distilled water, and drying to obtain eggplant stalk biochar (marked as 500 QZ), wherein the yield of 500QZ is 35.21%, and the pH is 10.38.
In other embodiments, the deer manure or the corn straw is adopted to replace eggplant stalks to prepare the biochar coupling carrier, the obtained deer manure biochar and the obtained corn straw biochar are respectively marked as 500LF and 500YM, and other operating conditions are the same as those of the eggplant stalks to prepare the eggplant stalk biochar.
The temperature rise of the biochar coupling carrier prepared from the eggplant stalks, the deer dung and the corn straws is set to be 300 ℃ or 700 ℃, the obtained corresponding biochar is respectively and correspondingly marked as 300QZ, 300LF, 300YM, 700QZ, 700LF and 700YM, and the operation conditions are the same as the conditions for preparing the eggplant stalk biochar from the eggplant stalks.
Fig. 2 shows the yields of the 9 biochar coupled carriers, and it can be seen that the yields of the three materials, deer manure, eggplant stalk and corn stalk, are the largest at the preparation temperature of 300 ℃, and the yield of the biochar prepared at 700 ℃ is the lowest. Fig. 3 shows a scanning electron microscope image of 9 biochar coupled carriers, which shows that biochar prepared at 300 ℃ has a smooth surface and almost no pore structure, while biochar prepared at 500 ℃ and 700 ℃ has a rough surface and a plurality of pore structures with different sizes on the surface layer and inside, and the structure is beneficial to the attachment and growth of microorganisms according to the analysis of results.
S2, collecting a soil sample of soil at a typical pollution point, and enriching and culturing target strains with degradation effects by using a diffusion chamber in-situ culture method; the typical contaminated site soil refers to soil near farmlands polluted by pesticides, soil polluted by petroleum hydrocarbon, soil polluted by heavy metal, soil near manufacturing plants such as electronic factories, mangrove root system soil, dry mud beds and the like, and long-term contaminated or moderately heavily contaminated soil.
With reference to FIG. 4, the procedure of the diffusion chamber in situ culture method is described as follows:
preparing an in-situ culture medium: taking 1L of water near a soil sampling point, adding 1% (wt/vol) agar powder into the water at a natural pH value, uniformly mixing, sterilizing at high temperature, and cooling to 45 ℃ for later use (warm agar). The sterilized 0.03 μm pore size polycarbonate membrane 1 was attached to the bottom surface of the sterilized stainless steel gasket 2 with a sealant, 3ml of inoculum 3 (soil suspension mixed with warm agar, soil suspension added at 1%) was introduced into the hollow inner ring to a position where there was a thin air layer from the top, and then the 0.03 μm pore size polycarbonate membrane 1 and the stainless steel gasket 2 were sealed with glue on the top surface to form a sealed chamber (the membrane allowed mass exchange between the chamber and the environment but restricted movement of the cells).
After inoculation and assembly, the diffusion chamber is buried in an original sampling position or is incubated in a simulated natural environment, and the depth is 5-10 cm. After 1 week of incubation, a large number of colonies with different forms can be observed, the diffusion chamber is taken back and cleaned, the in-situ culture medium is transferred to a sterile 1.5mL EP tube, sterile water is added, the in-situ culture medium is mashed and fully mixed with the sterile water, and the bacteria suspension is obtained by centrifugation.
S3, performing strain domestication on the target strain for multiple periods to obtain a strain capable of degrading the polycyclic aromatic hydrocarbon under the stress of heavy metal; the strain domestication process is as follows:
and (4) sucking the bacterial suspension subjected to in-situ enrichment culture in the step S2, coating the bacterial suspension in an acclimation culture medium of a new diffusion chamber, carrying out in-situ culture for a plurality of acclimation periods by using the diffusion chamber, and transferring the bacterial suspension to the newly constructed diffusion chamber after each acclimation period is finished. The acclimatization culture medium is obtained by adding organic pollutants and heavy metals with certain concentration into the in-situ culture medium. The concentration of organic pollutants in the domestication culture medium in a plurality of domestication periods is sequentially increased, and strains which can efficiently degrade target pollutants under stress are screened and domesticated through the plurality of domestication periods.
S4, separating and purifying the strains with the degradation capability by using a plate coating method to obtain pure colonies of degradation bacteria;
s5, repeating the step S4, respectively collecting the thallus after at least 3 strains are purified, carrying out enrichment culture by using an LB liquid culture medium until OD600= 1.0-1.2, and collecting bacterial liquid for freezing storage;
s6, mixing the activated multiple bacterial liquids in a certain proportion to obtain a composite bacterial seed liquid serving as one of the coupling materials;
s7, mixing and culturing the composite bacteria seed liquid and the carbon-based material in proportion for a period of time, and then carrying out immobilization treatment to obtain the composite bacteria agent.
In this embodiment, the steps S2 to S6 are described in detail by taking the construction of the heavy metal-resistant polycyclic aromatic hydrocarbon efficient degradation flora as an example.
The construction method of the heavy metal-resistant polycyclic aromatic hydrocarbon high-efficiency degradation flora comprises the following steps:
1) contaminated farmland soil polluted by PAHs and heavy metals near an industrial garden of Wang cattle mound towns in Dongguan city is taken as a bacteria source for extracting strains, 10g of contaminated soil is weighed, 90ml of sterile ultrapure water is added, ultrasonic treatment is carried out twice at an interval of 5s every time by using ultrasonic amplitude of 40 micrometers for 10s once, so that microorganisms on the soil are removed, and the soil suspension is obtained after standing for 30 min. The sterilized polycarbonate membrane 1 with the aperture of 0.03 mu m is stuck to the bottom surface of the sterilized stainless steel gasket 2 by using a sealant, the inoculum 3 is introduced into the hollow inner ring to the position away from the top end and provided with a thin air layer, and then the polycarbonate membrane 1 with the aperture of 0.03 mu m and the upper end of the stainless steel gasket 2 are sealed by using the glue to form a sealed chamber. After inoculation and assembly, the diffusion chamber is buried in an original sampling position or a simulated natural environment, the depth is 5-10 cm, and incubation is carried out.
And taking back the diffusion chamber for cleaning, transferring the in-situ culture medium into a sterile 1.5ml EP tube, adding sterile water, mashing the in-situ culture medium, fully mixing the in-situ culture medium with the sterile water, and centrifuging to obtain a bacterial suspension. 5ml of 10g/L pyrene mother solution passing through a 0.22 mu m filter head is added into a container, 1L of in-situ culture medium is added after complete volatilization to prepare the acclimatization culture medium, the final concentration of pyrene is 50mg/L, and artificially prepared cadmium nitrate solution is added, and the addition amount is 50 mg/L. And (3) sucking the bacterial suspension, coating the bacterial suspension in an acclimation culture medium of a new diffusion chamber, and carrying out in-situ culture by using the diffusion chamber, wherein an acclimation period is 7 days.
And 7 days later, transferring the domestication culture medium in the diffusion chamber into a sterile 1.5ml EP tube, adding sterile water, mashing the domestication culture medium, fully and uniformly mixing the domestication culture medium with the sterile water, centrifuging to obtain a bacterial suspension, transferring the prepared bacterial suspension into a newly constructed diffusion chamber, entering the next domestication period, and circulating the steps. Totally performing four domestication periods, wherein the concentrations of pyrene in the four domestication periods are respectively 50mg/L, 100 mg/L, 150 mg/L and 200 mg/L, adding a manually prepared cadmium nitrate solution, and completing the process of screening, domesticating and culturing PAHs degrading bacteria after 4 periods.
2) Diluting the finally obtained bacterial suspension according to gradient with the dilution concentration of 10-3、10-5、10-7And then respectively coating the strain on an inorganic salt solid plate, forming a film by using a sublimation method for pyrene, covering the film on the inorganic salt solid plate coated with the dilution enrichment liquid, placing the plate in a constant temperature incubator (37 ℃) for dark culture, observing a plate culture medium, and enabling a transparent aperture to appear around the strain to be the strain with the capability of degrading pyrene.
3) Firstly, selecting a strain with pyrene degradation capability, gently and carefully selecting a one-third ring colony on an LB solid culture medium by using a sterilized inoculating ring, then scribing a new LB culture medium for three times, carrying out flat plate scribing, separating and purifying to obtain a pure colony of the degradation bacteria, namely the degradation bacteria obtained by screening and separating, and finally obtaining three pure colonies with high pyrene degradation capability. The three pure strains which have high efficiency degradation effect on pyrene and strong tolerance to heavy metal and are screened out by detection belong to bacillus (Bacillus) respectivelyBacillus) Mycobacterium genus (A), (B), (C), (B), (C), (B), (C), (B), (C), (B), (C), (B), (C), (B), (C), (B), (C)Cytobacillus) And Pseudomonas (Pseudomonas) Respectively designated as P1, P2 and P3.
4) Streaking the three strains on LB plate to activate, inoculating the obtained single colony to LB liquid culture medium, and culturing in a shaking table under dark condition (37 deg.C, 180 r min)-1) To obtain a strain seed solution with OD600 of about 1.0.
5) The seed liquids of the three strains are mixed according to the proportion of 1:1:1 to construct a compound flora (recorded as HH 1) with the capability of degrading pyrene under the stress of heavy metal, and the removal rate of 50mg/L pyrene at 7d is 81.08 percent, which is higher than the removal effect of any single strain.
In other examples, the seed solutions of the strains P1, P2, and P3 were mixed at a ratio of 1:1:2, 1:2:1, and 2:1:1, respectively, and the mixed complex bacterial colonies were designated as HH2, HH3, and HH4, respectively.
The effect of single strains P1, P2 and P3 and composite strains HH1, HH2, HH3 and HH4 on the removal of 50mg/L pyrene at 7d is shown in FIG. 5. As can be seen from FIG. 5, the degradation effect of the composite strain is improved compared with that of the single strain, wherein the degradation effect of HH1 is the best, which indicates that the optimal mixing ratio of the seed liquid of the three strains is 1:1: 1.
In this embodiment, sodium alginate is used as a coupling adjuvant to prepare a complex microbial inoculum, and the method comprises the following steps:
mixing carbon-based coupling carrier 500LF and composite flora HH1 at a ratio of 1:30 (w/v), placing in a shaking table at 37 deg.C and 180 r min-1After oscillating for 2 hours under the condition, adding 4% sodium alginate solution with the same volume, stirring evenly and standing for 30min, dropwise adding the mixture into 2% calcium chloride solution by using a peristaltic pump, wrapping and filtering the mixture by using sterile gauze after curing for 12 hours, and repeatedly washing the mixture by using sterile water to obtain black hard pellets, namely the composite microbial inoculum (recorded as 500 LFPH), wherein the removal rate of 50mg/L pyrene in 7 days is 93.01%, and the degradation rate is improved by 12% compared with HH 1.
In other embodiments, other carbon-based coupling carriers can be used for generating corresponding composite microbial agents with HH2 and the like in other composite bacteria. For example, we used carbon-based materials 300QZ, 300LF, 300YM, 500QZ, 500YM, 700QZ, 700LF, 700YM instead of 500LF to mix with Complex flora HH1 to obtain the corresponding complex bacterial agents 300QZPH, 300LFPH, 300YMPH, 500QZPH, 500YMPH, 700QZPH, 700LFPH, 700YMPH under the same conditions.
FIG. 6 shows a schematic diagram of the degradation effect of the composite microbial inoculum pellets with sodium alginate as the adjuvant on pyrene at a concentration of 50mg/L in 7d, and it can be seen from the diagram that the degradation rate of the composite microbial inoculum 7d prepared with sodium alginate as the adjuvant on pyrene at a concentration of 50mg/L exceeds 70%, wherein the composite microbial inoculum prepared by coupling the biochar at 500 ℃ with HH1 has the best effect, and can reach more than 90%.
Example two:
in this example, chitosan was used as a coupling adjuvant to prepare a complex microbial inoculum, which was prepared as follows:
mixing carbon-based coupling carrier 700QZ and composite bacteria HH1 at a ratio of 1:30 (w/v), placing in a shaking table, and standing at 37 deg.C for 180 r min-1After 2h of oscillation under the conditions of (1), an equal volume of 6% shell was addedThe polysaccharide solution (2% hydrochloric acid solution is used for dissolving chitosan powder to prepare 6% chitosan), the chitosan solution is uniformly mixed to be sticky, the sticky chitosan solution is dropwise added into 1mol/L sodium hydroxide solution, sterile gauze is used for wrapping and filtering after 12 hours of solidification, sterile water is used for repeatedly washing, the obtained gel beads are composite microbial inoculum which is recorded as 700QZPQ, the removal rate of 50mg/L pyrene at 7 days is 76.77%, and the removal rate reaches 95.6% after 15 days.
In other embodiments, other carbon-based coupling carriers can be used for generating corresponding composite microbial agents with HH2 and the like in other composite bacteria. For example, carbon-based materials 300QZ, 300LF, 300YM, 500LF, 500QZ, 500YM, 700LF and 700YM were mixed with complex flora HH1 in place of 700QZ, and the corresponding complex bacterial agents 300QZPQ, 300LFPQ, 300YMPQ, 500LFPQ, 500QZPQ, 500YMPQ, 700LFPQ and 700YMPQ were obtained under the same conditions.
FIG. 7 shows a schematic diagram of the degradation effect of the composite microbial inoculum pellets using chitosan as an auxiliary agent on pyrene at 7d at 50mg/L, and it can be seen from the diagram that 9 composite microbial inocula prepared using chitosan as an auxiliary agent have the degradation rate of more than 70% on pyrene at 7d at 50mg/L and the best effect on the composite microbial inoculum prepared using 500 ℃ biochar as a coupling carrier, wherein the degradation rate of 500LFPQ is 86.74% at most.
Example three:
in this embodiment, the method for preparing the composite microbial inoculum pellet in the adsorption coupling manner is as follows:
mixing 9 carbon-based coupling carriers 300QZ, 300LF, 300YM, 500QZ, 500LF, 500YM, 500QZ, 700LF and 700YM with composite bacteria HH1 at a ratio of 1:30 (w/v), placing in a shaking table, and standing at 37 deg.C for 180 r min-1After shaking for 24h under the condition of (1), filtering by using a 75 mu m sterile cell sieve, and drying to obtain corresponding complex microbial inoculum pellets 300QZP, 300LFP, 300YMP, 500QZP, 500LFP, 500YMP, 500QZP, 700LFP and 700 YMP.
FIG. 8 shows the effect of the compound bacterial agent 7d prepared by the above 9 adsorption coupling methods on removing 50mg/L pyrene, and it can be seen that the degradation rates of the 9 compound bacterial agents are all greater than 70%, wherein the compound bacterial agent prepared by using biochar prepared at 500 ℃ and 700 ℃ as a coupling carrier has relatively good effect, and the degradation rate can reach up to 88.34% and is 500 LFP.
The following table shows the property comparison results of the charcoal-coupled complex microbial agents (respectively named as product 2, product 3 and product 1) prepared by sodium alginate embedding coupling, chitosan embedding coupling and adsorption coupling in the first to third embodiments:
and (3) measuring the degradation performance of the composite microbial inoculum obtained by different biochar in different coupling modes:
preparing 60ml of pyrene-containing inorganic salt culture medium with the initial concentration of 50mg/L by using a sterile inorganic salt culture medium, and then adding a certain amount of composite microbial inoculum, wherein 0.5g of the composite microbial inoculum prepared by adsorption coupling is added; adding 1g of the compound microbial inoculum pellets prepared by coupling sodium alginate; 1g of chitosan is added into the gel balls of the composite microbial inoculum prepared by coupling. All treatment groups were placed in a shaker at 37 ℃ and 180 r.min-1The sample was taken after shaking for 7 days under the conditions of (1), the residual concentration of pyrene in the sample was determined, five replicates were performed for each treatment group, and the final removal effect is shown in fig. 9, in which the removal rate of 500YMPH by sodium alginate embedding coupling was 95.77% at the highest, and the removal rate of all components was higher than 70%.
Example four:
the compound microbial inoculum prepared by the preparation method of the first to third embodiments is applied to the polycyclic aromatic hydrocarbon soil with heavy metal stress for remediation.
This embodiment mainly takes a 500LFPH complex microbial inoculum as an example for explanation.
Collecting soil in Dongguan college of science and technology, collecting 0-20 cm of surface soil without pollution history, removing plant residues and sundries from the collected soil sample, naturally drying, grinding and sieving by a 2mm sieve for later use. Slowly dripping acetone stock solution dissolved with pyrene onto soil to be tested, stirring and mixing uniformly while dripping, placing in a fume hood, continuously diluting with the rest soil after acetone is completely volatilized, stirring and mixing uniformly to obtain 100 mg.kg-1Pyrene contaminated soil. Then 1/2 pyrene-contaminated soil was taken and droppedContinuously stirring and uniformly mixing a certain amount of cadmium nitrate solution, and air-drying to obtain pyrene and cadmium (Cd) with the concentration of 100 mg-kg-1The polluted soil sample is stored in dark and is aged for 2 months for later use.
The pre-prepared polluted soil is filled into a small plastic basin according to 200 g/basin, and the treatment group comprises: 1) the compound contaminated soil is not treated at all to serve as a blank; 2) adding 20ml of free flora HH1 seed solution of 10% (v/w) into the composite contaminated soil; 3) 5% (w/w) of 500LFPH 10g is added into the composite soil. And (3) supplementing sterile water to all treatment groups every day to control the soil moisture to be about 35%, placing the treatment groups in an incubator at 25 ℃ in the dark for 45 days, and sampling to determine the residual quantity of the polycyclic aromatic hydrocarbon pyrene in the soil.
After 45d of culture, the removal rate of pyrene in the soil sample of the treatment group added with the immobilized flora is obviously higher than that of other two treatment groups. The removal rate of pyrene in the soil sample of the added free flora is 19.87%, which is slightly higher than that of a blank Control (CK), and the free flora is in weak competitive power in soil, so that the stability is poor. And after the 500LFPH is repaired for 45 days, the removal rate of the pollutants can reach 85.31%, which shows that the 500LFPH has better stability, stronger capability of removing pyrene and good degradation effect.
Claims (15)
1. A preparation method of a carbon-coupled compound microbial inoculum is characterized by comprising the following steps: the method comprises the following steps:
s1, processing the natural material capable of being carbonized to obtain a carbon-based material serving as one of the subsequent coupling materials;
s2, collecting a soil sample of the soil at a typical pollution point to prepare a soil suspension, and performing isolated culture on microorganisms in the soil by using a diffusion chamber in-situ culture method to obtain a soil microorganism enrichment solution containing a target strain;
s3, carrying out multiple periods of strain screening and domestication on the enrichment solution to obtain strains capable of degrading the polycyclic aromatic hydrocarbon under heavy metal stress;
s4, separating and purifying the strains with the degradation capability by using a plate coating method to obtain pure colonies of the degradation bacteria;
s5, repeating the step S4, respectively collecting the thallus after at least 3 strains are purified, carrying out enrichment culture by using an LB liquid culture medium until OD600= 1.0-1.2, and collecting bacterial liquid for freezing storage;
s6, mixing the activated multiple bacterial liquids in a certain proportion to obtain a composite bacterial seed liquid serving as one of the coupling materials;
s7, mixing and culturing the composite bacteria seed liquid and the carbon-based material in proportion for a period of time, and then carrying out immobilization treatment to obtain the composite bacteria agent.
2. The method for preparing the carbon-coupled composite microbial inoculum according to claim 1, which is characterized by comprising the following steps: the charrable natural material in the step S1 includes one or more of corn stalk, crab shell, eggplant stalk, bamboo leaf, kapok fiber and deer feces.
3. The method for preparing the carbon-coupled composite microbial inoculum according to claim 1, which is characterized by comprising the following steps: in step S1, the carbonizable natural material is treated by:
s11, cleaning the charrable natural material, removing impurities, drying and crushing into powder;
s12, slowly cracking for 1-3 hours at 300-700 ℃ in an oxygen-isolated smoldering high temperature;
and S13, cooling to room temperature under the protection of protective gas and condensed circulating water, sequentially grinding and sieving, soaking and activating with hydrochloric acid, washing with distilled water and drying to obtain the carbon-based material.
4. The method for preparing the carbon-coupled composite microbial inoculum according to claim 1, which is characterized by comprising the following steps: the typical polluted point soil refers to farmland polluted by pesticides, soil polluted by petroleum hydrocarbon, mangrove root system soil, soil near manufacturing plants or dry mud bed soil.
5. The method for preparing the carbon-coupled composite microbial inoculum according to claim 1, which is characterized by comprising the following steps: the soil suspension is prepared by the following method:
weighing 10g of soil sample, adding 90ml of sterilized ultrapure water, uniformly mixing, performing ultrasonic treatment, and standing for 30-60 min to obtain a supernatant containing microorganisms;
the ultrasonic treatment was carried out 2 times under ultrasonic amplitude of 40 μm and ultrasonic time of 10s at 5s intervals.
6. The method for preparing the carbon-coupled composite microbial inoculum according to claim 1, which is characterized by comprising the following steps: in step S2, the procedure of the diffusion chamber in situ culture method is as follows:
s100, preparing an in-situ culture medium: taking 1L of water near a soil sampling point, adding 1% (wt/vol) agar powder into the water at a natural pH value, uniformly mixing, sterilizing at a high temperature, and cooling to 45 ℃ to obtain warm agar;
s200, inoculating and assembling a diffusion chamber: adhering the sterilized polycarbonate membrane with the aperture of 0.03 mu m to the bottom surface of the sterilized stainless steel gasket by using a sealant, introducing a mixture of a soil suspension and warm agar into the hollow inner ring, and sealing the polycarbonate membrane with the aperture of 0.03 mu m and the top surface of the stainless steel gasket by using glue to form a sealed chamber;
s300, burying the diffusion chamber at an original sampling position or in a simulated natural environment for incubation, wherein the depth is 5-10 cm; after 1 week of incubation, the in situ culture device is taken back and cleaned, the in situ culture medium is transferred to a sterile 1.5ml EP tube, sterile water is added, the in situ culture medium is mashed and fully mixed with the sterile water, and the mixture is centrifuged to obtain a bacterial suspension.
7. The method for preparing the carbon-coupled composite microbial inoculum according to claim 1, which is characterized by comprising the following steps: in step S3, the strain acclimation process is as follows:
and (4) absorbing the bacterial suspension obtained in the step (S2) and coating the bacterial suspension into an acclimation culture medium of a new diffusion chamber, culturing in situ for four acclimation periods by using the diffusion chamber, transferring the bacterial suspension into the newly constructed diffusion chamber in each period, setting four acclimation culture media with different concentration gradients, and adding organic pollutants and heavy metals with certain concentrations into the in situ culture media to screen and acclimate strains capable of degrading target pollutants under stress.
8. The method for preparing the carbon-coupled composite microbial inoculum according to claim 1, which is characterized by comprising the following steps: in step S7, the composite bacteria seed liquid and the carbon-based material are mixed and cultured according to the proportion of 1: 10-30 (w/v).
9. The method for preparing the carbon-coupled composite microbial inoculum according to claim 1, which is characterized by comprising the following steps: in the step S7, the immobilization treatment is to mix and culture the composite bacterial liquid and the carbon-based material according to the ratio of 1: 10-30 (w/v) for 12-24 h, and after adsorption coupling is completed, the mixture is screened in a sterile environment, and solid materials are reserved and dried.
10. The method for preparing the carbon-coupled composite microbial inoculum according to claim 1, which is characterized by comprising the following steps: in step S7, the immobilization treatment is to mix and culture the composite bacteria seed liquid and the carbon-based material in proportion for a period of time, add coupling auxiliary agent with the same volume, stir the mixture evenly, drop the mixture into the solidification liquid drop by drop, repeatedly wash the mixture with sterile water after solidification or gelation, and naturally dry the mixture.
11. The method for preparing the carbon-coupled composite microbial inoculum according to claim 2, which is characterized in that: the grinding and sieving are carried out by a sieve of 20-100 meshes; the concentration of the hydrochloric acid solution is 1-3 mol/L.
12. The method for preparing the carbon-coupled composite microbial inoculum according to claim 1, which is characterized by comprising the following steps: collecting 3 bacterial liquids, and mixing the 3 bacterial liquids according to the proportion of 1:1:1, 1:1:2, 1:2:1 or 2:1: 1.
13. The method for preparing a carbon-coupled complex microbial inoculum of claim 10, which is characterized in that: the coupling auxiliary agent and the corresponding solidification solution with the same volume are 3-5% of sodium alginate and 2% of calcium chloride solution as solidification solution or 4-6% of chitosan and 1mol/L of sodium hydroxide as solidification solution.
14. The carbon-coupled complex microbial inoculum prepared by the method for preparing the carbon-coupled complex microbial inoculum according to any one of claims 1 to 13.
15. The use of the carbon-coupled complex microbial inoculant of claim 14 in pollutant remediation and soil remediation.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202111228892.7A CN114058507A (en) | 2021-10-21 | 2021-10-21 | Carbon-coupled compound microbial inoculum and preparation method and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202111228892.7A CN114058507A (en) | 2021-10-21 | 2021-10-21 | Carbon-coupled compound microbial inoculum and preparation method and application thereof |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN114058507A true CN114058507A (en) | 2022-02-18 |
Family
ID=80235134
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202111228892.7A Pending CN114058507A (en) | 2021-10-21 | 2021-10-21 | Carbon-coupled compound microbial inoculum and preparation method and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN114058507A (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114602967A (en) * | 2022-03-04 | 2022-06-10 | 南京农业大学 | Method for removing USEPA PAHs in soil by using functional indigenous flora solid microbial inoculum |
| CN115446108A (en) * | 2022-10-12 | 2022-12-09 | 西安交通大学 | Method for preparing biochar immobilized fungus-bacterium combined microbial inoculum for degrading oily sludge petroleum hydrocarbon from oily sludge |
Citations (15)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101735996A (en) * | 2009-12-10 | 2010-06-16 | 中国科学院地理科学与资源研究所 | Mixed bactericide for restoring places polluted by polynuclear aromatic hydrocarbons, preparation method and application method |
| US20100209988A1 (en) * | 2005-11-08 | 2010-08-19 | The University of Surrey The Forrestry Commission | Bioremediation Materials |
| CN104475444A (en) * | 2014-11-04 | 2015-04-01 | 上海交通大学 | Preparation, application and usage method for biocarbon-immobilized composite pollution-degrading bacterium particle |
| CN104946620A (en) * | 2015-04-24 | 2015-09-30 | 南开大学 | Immobilized microbial inoculant for restoring petroleum-hydrocarbon-polluted alkaline-saline soil and preparation method thereof |
| CN106111687A (en) * | 2016-06-17 | 2016-11-16 | 广东省农业科学院农业资源与环境研究所 | The application in heavy-metal contaminated soil is improved of a kind of charcoal based composition and use thereof in packaging |
| CN106118664A (en) * | 2016-06-17 | 2016-11-16 | 广东省农业科学院农业资源与环境研究所 | A kind of preparation method and applications of charcoal based composition and use thereof in packaging |
| CN108192631A (en) * | 2017-12-26 | 2018-06-22 | 湖南恒凯环保科技投资有限公司 | A kind of charcoal base flora composite soil conditioner and its preparation and application |
| CN110396488A (en) * | 2019-07-18 | 2019-11-01 | 中国科学院城市环境研究所 | A kind of mixed culture and application method of degrading polycyclic aromatic hydrocarbons pollutant |
| CN110964535A (en) * | 2019-11-28 | 2020-04-07 | 湖北同惠生物工程有限公司 | Soil remediation agent and preparation method and application thereof |
| CN111088246A (en) * | 2019-12-30 | 2020-05-01 | 东华大学 | Biochar immobilized naphthalene degrading microbial inoculum and preparation method and application thereof |
| CN111088187A (en) * | 2019-12-31 | 2020-05-01 | 华东理工大学 | Charcoal-based immobilized polycyclic aromatic hydrocarbon efficient degradation microbial inoculum and preparation method and application thereof |
| CN111187766A (en) * | 2020-02-21 | 2020-05-22 | 西安交通大学 | Preparation and application of Polycyclic Aromatic Hydrocarbon (PAHs) degrading microbial inoculum |
| CN111906141A (en) * | 2020-07-14 | 2020-11-10 | 西南石油大学 | Soil remediation method based on biochar immobilized degrading bacteria |
| CN113293157A (en) * | 2021-06-18 | 2021-08-24 | 华东理工大学 | Ferroferric oxide modified charcoal immobilized polycyclic aromatic hydrocarbon degrading microbial inoculum and preparation and application thereof |
| CN113462395A (en) * | 2021-06-28 | 2021-10-01 | 锦州市科学技术研究院 | Method for improving soil by combined action of mixed microbial inoculum and chemical fertilizer |
-
2021
- 2021-10-21 CN CN202111228892.7A patent/CN114058507A/en active Pending
Patent Citations (15)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20100209988A1 (en) * | 2005-11-08 | 2010-08-19 | The University of Surrey The Forrestry Commission | Bioremediation Materials |
| CN101735996A (en) * | 2009-12-10 | 2010-06-16 | 中国科学院地理科学与资源研究所 | Mixed bactericide for restoring places polluted by polynuclear aromatic hydrocarbons, preparation method and application method |
| CN104475444A (en) * | 2014-11-04 | 2015-04-01 | 上海交通大学 | Preparation, application and usage method for biocarbon-immobilized composite pollution-degrading bacterium particle |
| CN104946620A (en) * | 2015-04-24 | 2015-09-30 | 南开大学 | Immobilized microbial inoculant for restoring petroleum-hydrocarbon-polluted alkaline-saline soil and preparation method thereof |
| CN106111687A (en) * | 2016-06-17 | 2016-11-16 | 广东省农业科学院农业资源与环境研究所 | The application in heavy-metal contaminated soil is improved of a kind of charcoal based composition and use thereof in packaging |
| CN106118664A (en) * | 2016-06-17 | 2016-11-16 | 广东省农业科学院农业资源与环境研究所 | A kind of preparation method and applications of charcoal based composition and use thereof in packaging |
| CN108192631A (en) * | 2017-12-26 | 2018-06-22 | 湖南恒凯环保科技投资有限公司 | A kind of charcoal base flora composite soil conditioner and its preparation and application |
| CN110396488A (en) * | 2019-07-18 | 2019-11-01 | 中国科学院城市环境研究所 | A kind of mixed culture and application method of degrading polycyclic aromatic hydrocarbons pollutant |
| CN110964535A (en) * | 2019-11-28 | 2020-04-07 | 湖北同惠生物工程有限公司 | Soil remediation agent and preparation method and application thereof |
| CN111088246A (en) * | 2019-12-30 | 2020-05-01 | 东华大学 | Biochar immobilized naphthalene degrading microbial inoculum and preparation method and application thereof |
| CN111088187A (en) * | 2019-12-31 | 2020-05-01 | 华东理工大学 | Charcoal-based immobilized polycyclic aromatic hydrocarbon efficient degradation microbial inoculum and preparation method and application thereof |
| CN111187766A (en) * | 2020-02-21 | 2020-05-22 | 西安交通大学 | Preparation and application of Polycyclic Aromatic Hydrocarbon (PAHs) degrading microbial inoculum |
| CN111906141A (en) * | 2020-07-14 | 2020-11-10 | 西南石油大学 | Soil remediation method based on biochar immobilized degrading bacteria |
| CN113293157A (en) * | 2021-06-18 | 2021-08-24 | 华东理工大学 | Ferroferric oxide modified charcoal immobilized polycyclic aromatic hydrocarbon degrading microbial inoculum and preparation and application thereof |
| CN113462395A (en) * | 2021-06-28 | 2021-10-01 | 锦州市科学技术研究院 | Method for improving soil by combined action of mixed microbial inoculum and chemical fertilizer |
Non-Patent Citations (2)
| Title |
|---|
| 于红艳等: "生物炭菌剂对多环芳烃的吸附及降解作用研究", 《环境科学与技术》, vol. 43, no. 12, pages 120 - 124 * |
| 任静等: "生物炭固定化多环芳烃高效降解菌剂的制备及稳定性", 《环境科学学报》, vol. 40, no. 12, pages 4517 - 4523 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114602967A (en) * | 2022-03-04 | 2022-06-10 | 南京农业大学 | Method for removing USEPA PAHs in soil by using functional indigenous flora solid microbial inoculum |
| CN114602967B (en) * | 2022-03-04 | 2022-12-23 | 南京农业大学 | Method for removing USEPA PAHs in soil by using functional indigenous flora solid microbial inoculum |
| CN115446108A (en) * | 2022-10-12 | 2022-12-09 | 西安交通大学 | Method for preparing biochar immobilized fungus-bacterium combined microbial inoculum for degrading oily sludge petroleum hydrocarbon from oily sludge |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN106148318B (en) | A kind of preparation and application of charcoal immobilized microbial inoculum | |
| CN104017757B (en) | The one plant height effect end, changed bacillus and the composite base made changes microbial inoculum and application | |
| CN109536173B (en) | Composite material for simultaneously repairing heavy metal and glyphosate and preparation method thereof | |
| CN109534513B (en) | Method for purifying aquaculture wastewater in situ by biochar-enhanced bacteria-algae immobilized preparation | |
| CN104450597B (en) | Preparation method of petroleum degrading bacteria solid microbial inoculum and method for repairing petroleum-polluted soil by using solid microbial inoculum prepared by preparation method | |
| CN104475444A (en) | Preparation, application and usage method for biocarbon-immobilized composite pollution-degrading bacterium particle | |
| CN114058507A (en) | Carbon-coupled compound microbial inoculum and preparation method and application thereof | |
| CN108689781A (en) | Biomass carbon base soil-repairing agent and preparation method thereof | |
| CN104651342B (en) | A kind of cold-resistant ammonia oxidizing bacteria process for fixation of salt tolerant and application | |
| CN106834269A (en) | A kind of immobilized microspheres of PAEs degradation bacterias and its preparation method and application | |
| CN101073311A (en) | Method for treating heavy metals in living garbage by using crop stalks and lawn plants | |
| CN105039211A (en) | Polycyclic aromatic hydrocarbon removal composite material, and preparation method and use thereof | |
| CN107056409B (en) | Functional biochar-based nutrient medium for preventing and treating root rot and preparation method thereof | |
| CN111117993B (en) | Biomass immobilized fungus-bacterium mixed particle for degrading PAHs in soil as well as preparation method and application thereof | |
| CN114107092B (en) | Endophyte Gordonia L191 for degrading phthalate and application thereof | |
| CN109554305B (en) | Screening method of probiotics simulating micro-ecology, repairing agent and application of repairing agent | |
| CN107217017B (en) | Acinetobacter and application thereof in petroleum degradation | |
| CN108823195A (en) | A kind of preparation method and application method of mushroom bran immobilized microbial inoculum | |
| CN113234714A (en) | Plant growth promoting microbial inoculum for tailing restoration and preparation method and application thereof | |
| CN109797120B (en) | Preparation method and application of microecological preparation for removing nitrate in soil | |
| CN111117909B (en) | Strain capable of resisting multiple heavy metals and promoting plant growth and application thereof | |
| CN114602967B (en) | Method for removing USEPA PAHs in soil by using functional indigenous flora solid microbial inoculum | |
| CN114107095B (en) | Rhodococcus ruber and application of preparation thereof in pyrethroid pesticide pollution remediation | |
| CN115537366A (en) | Microbial agent for biochar coupling synergistic degradation of TCC (TCC) and preparation method thereof | |
| CN112080447B (en) | Bacterium ZG2 capable of repairing cadmium-nickel moderately polluted soil and application thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination |