CN110283729B - Plant rhizosphere fungus F25 capable of adsorbing copper ions and application thereof - Google Patents
Plant rhizosphere fungus F25 capable of adsorbing copper ions and application thereof Download PDFInfo
- Publication number
- CN110283729B CN110283729B CN201910585045.2A CN201910585045A CN110283729B CN 110283729 B CN110283729 B CN 110283729B CN 201910585045 A CN201910585045 A CN 201910585045A CN 110283729 B CN110283729 B CN 110283729B
- Authority
- CN
- China
- Prior art keywords
- copper ions
- plant rhizosphere
- fungus
- sewage
- fungi
- 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.)
- Expired - Fee Related
Links
- 241000233866 Fungi Species 0.000 title claims abstract description 47
- JPVYNHNXODAKFH-UHFFFAOYSA-N Cu2+ Chemical compound [Cu+2] JPVYNHNXODAKFH-UHFFFAOYSA-N 0.000 title claims abstract description 35
- 229910001431 copper ion Inorganic materials 0.000 title claims abstract description 30
- 239000010865 sewage Substances 0.000 claims abstract description 30
- 239000002689 soil Substances 0.000 claims abstract description 24
- 238000012216 screening Methods 0.000 claims abstract description 17
- 238000004321 preservation Methods 0.000 claims abstract description 11
- 241000228129 Penicillium janthinellum Species 0.000 claims abstract description 5
- 241000228143 Penicillium Species 0.000 claims abstract description 3
- 239000002068 microbial inoculum Substances 0.000 claims abstract description 3
- 238000009629 microbiological culture Methods 0.000 claims abstract description 3
- 239000002351 wastewater Substances 0.000 claims description 15
- 238000001179 sorption measurement Methods 0.000 abstract description 14
- 229910001385 heavy metal Inorganic materials 0.000 abstract description 10
- 244000005700 microbiome Species 0.000 abstract description 5
- 230000000694 effects Effects 0.000 abstract description 4
- 238000010521 absorption reaction Methods 0.000 abstract 1
- 241000196324 Embryophyta Species 0.000 description 33
- 238000000034 method Methods 0.000 description 14
- 239000000725 suspension Substances 0.000 description 11
- 229920001817 Agar Polymers 0.000 description 10
- 239000008272 agar Substances 0.000 description 10
- 238000000746 purification Methods 0.000 description 10
- 239000010949 copper Substances 0.000 description 9
- 239000001963 growth medium Substances 0.000 description 9
- 230000001580 bacterial effect Effects 0.000 description 8
- 238000007865 diluting Methods 0.000 description 7
- 239000002609 medium Substances 0.000 description 7
- 229910021642 ultra pure water Inorganic materials 0.000 description 7
- 239000012498 ultrapure water Substances 0.000 description 7
- 108020004414 DNA Proteins 0.000 description 6
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 6
- 239000007788 liquid Substances 0.000 description 6
- 238000000926 separation method Methods 0.000 description 6
- 230000000813 microbial effect Effects 0.000 description 5
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 5
- 241000894006 Bacteria Species 0.000 description 4
- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 description 4
- 238000012408 PCR amplification Methods 0.000 description 4
- 241000228150 Penicillium chrysogenum Species 0.000 description 4
- 230000002538 fungal effect Effects 0.000 description 4
- 238000005070 sampling Methods 0.000 description 4
- 239000007787 solid Substances 0.000 description 4
- 238000004458 analytical method Methods 0.000 description 3
- 230000000877 morphologic effect Effects 0.000 description 3
- 239000002773 nucleotide Substances 0.000 description 3
- 125000003729 nucleotide group Chemical group 0.000 description 3
- 239000000243 solution Substances 0.000 description 3
- 238000012360 testing method Methods 0.000 description 3
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 2
- RYGMFSIKBFXOCR-UHFFFAOYSA-N Copper Chemical compound [Cu] RYGMFSIKBFXOCR-UHFFFAOYSA-N 0.000 description 2
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 2
- 239000007836 KH2PO4 Substances 0.000 description 2
- 239000001888 Peptone Substances 0.000 description 2
- 108010080698 Peptones Proteins 0.000 description 2
- 235000014676 Phragmites communis Nutrition 0.000 description 2
- 241001521648 Thalia dealbata Species 0.000 description 2
- 244000284012 Vetiveria zizanioides Species 0.000 description 2
- 235000007769 Vetiveria zizanioides Nutrition 0.000 description 2
- 230000003213 activating effect Effects 0.000 description 2
- 229940041514 candida albicans extract Drugs 0.000 description 2
- 238000006243 chemical reaction Methods 0.000 description 2
- 239000011248 coating agent Substances 0.000 description 2
- 238000000576 coating method Methods 0.000 description 2
- 229910052802 copper Inorganic materials 0.000 description 2
- 238000010790 dilution Methods 0.000 description 2
- 239000012895 dilution Substances 0.000 description 2
- 238000011049 filling Methods 0.000 description 2
- 239000008103 glucose Substances 0.000 description 2
- 229910052943 magnesium sulfate Inorganic materials 0.000 description 2
- 238000002156 mixing Methods 0.000 description 2
- 238000012544 monitoring process Methods 0.000 description 2
- 229910000402 monopotassium phosphate Inorganic materials 0.000 description 2
- 235000019319 peptone Nutrition 0.000 description 2
- 239000000843 powder Substances 0.000 description 2
- 230000008569 process Effects 0.000 description 2
- 239000000047 product Substances 0.000 description 2
- 238000012163 sequencing technique Methods 0.000 description 2
- 239000008223 sterile water Substances 0.000 description 2
- 238000005303 weighing Methods 0.000 description 2
- 239000012138 yeast extract Substances 0.000 description 2
- 239000002028 Biomass Substances 0.000 description 1
- 238000007400 DNA extraction Methods 0.000 description 1
- 108091023242 Internal transcribed spacer Proteins 0.000 description 1
- 238000012300 Sequence Analysis Methods 0.000 description 1
- 238000003723 Smelting Methods 0.000 description 1
- 208000005392 Spasm Diseases 0.000 description 1
- 239000003463 adsorbent Substances 0.000 description 1
- 238000000246 agarose gel electrophoresis Methods 0.000 description 1
- 208000007502 anemia Diseases 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 238000012512 characterization method Methods 0.000 description 1
- 238000009388 chemical precipitation Methods 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 238000012258 culturing Methods 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 238000004925 denaturation Methods 0.000 description 1
- 230000036425 denaturation Effects 0.000 description 1
- 238000001784 detoxification Methods 0.000 description 1
- 238000000502 dialysis Methods 0.000 description 1
- 238000004821 distillation Methods 0.000 description 1
- 238000009713 electroplating Methods 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 235000013305 food Nutrition 0.000 description 1
- 230000002496 gastric effect Effects 0.000 description 1
- 230000012010 growth Effects 0.000 description 1
- 238000011534 incubation Methods 0.000 description 1
- 230000006799 invasive growth in response to glucose limitation Effects 0.000 description 1
- 238000005342 ion exchange Methods 0.000 description 1
- 150000002500 ions Chemical class 0.000 description 1
- 238000002955 isolation Methods 0.000 description 1
- 210000003734 kidney Anatomy 0.000 description 1
- 238000009630 liquid culture Methods 0.000 description 1
- 210000004185 liver Anatomy 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 239000012528 membrane Substances 0.000 description 1
- 229910052751 metal Inorganic materials 0.000 description 1
- 239000002184 metal Substances 0.000 description 1
- 239000010814 metallic waste Substances 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 235000016709 nutrition Nutrition 0.000 description 1
- 230000035764 nutrition Effects 0.000 description 1
- 238000012257 pre-denaturation Methods 0.000 description 1
- 238000012545 processing Methods 0.000 description 1
- 238000011027 product recovery Methods 0.000 description 1
- 238000011084 recovery Methods 0.000 description 1
- 238000004153 renaturation Methods 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 238000012827 research and development Methods 0.000 description 1
- 239000011347 resin Substances 0.000 description 1
- 229920005989 resin Polymers 0.000 description 1
- 238000001223 reverse osmosis Methods 0.000 description 1
- 238000000638 solvent extraction Methods 0.000 description 1
- 239000006228 supernatant Substances 0.000 description 1
- 238000004381 surface treatment Methods 0.000 description 1
- 208000024891 symptom Diseases 0.000 description 1
- 230000001988 toxicity Effects 0.000 description 1
- 231100000419 toxicity Toxicity 0.000 description 1
- 239000011573 trace mineral Substances 0.000 description 1
- 235000013619 trace mineral Nutrition 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
- 238000004065 wastewater treatment Methods 0.000 description 1
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F3/00—Biological treatment of water, waste water, or sewage
- C02F3/34—Biological treatment of water, waste water, or sewage characterised by the microorganisms used
- C02F3/347—Use of yeasts or fungi
-
- 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
-
- 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
- C12N1/14—Fungi; 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
- C12N1/14—Fungi; Culture media therefor
- C12N1/145—Fungal isolates
-
- C—CHEMISTRY; METALLURGY
- C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F2101/00—Nature of the contaminant
- C02F2101/10—Inorganic compounds
- C02F2101/20—Heavy metals or heavy metal compounds
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/645—Fungi ; Processes using fungi
- C12R2001/80—Penicillium
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Chemical & Material Sciences (AREA)
- Health & Medical Sciences (AREA)
- Organic Chemistry (AREA)
- Genetics & Genomics (AREA)
- Biotechnology (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Microbiology (AREA)
- Biomedical Technology (AREA)
- Biochemistry (AREA)
- Medicinal Chemistry (AREA)
- Tropical Medicine & Parasitology (AREA)
- Virology (AREA)
- General Health & Medical Sciences (AREA)
- General Engineering & Computer Science (AREA)
- Mycology (AREA)
- Botany (AREA)
- Environmental & Geological Engineering (AREA)
- Water Supply & Treatment (AREA)
- Hydrology & Water Resources (AREA)
- Biodiversity & Conservation Biology (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Purification Treatments By Anaerobic Or Anaerobic And Aerobic Bacteria Or Animals (AREA)
Abstract
The invention discloses a plant rhizosphere fungus F25 capable of adsorbing copper ions and application thereof, and belongs to the field of microorganisms. The plant rhizosphere fungi F25 of the invention is obtained by screening plant rhizosphere soil of constructed wetlands in sewage treatment plants through molecular biological identification and is classified and named as penicillium violaceum (A)Penicillium janthinellum) The microbial inoculum is preserved in the China general microbiological culture Collection center with the preservation number as follows: CGMCC NO. 17461; the preservation time is as follows: year 2019, 04 and 08. The plant rhizosphere fungi F25 has a high-efficiency absorption effect on copper ions, can be applied to biological adsorption of copper ions in sewage, and provides a basis for selection of high-efficiency strains in the urban comprehensive heavy metal sewage treatment system.
Description
Technical Field
The invention belongs to the field of microorganisms, and particularly relates to a plant rhizosphere fungus F25 capable of adsorbing copper ions and application thereof.
Background
Containing Cu2+The waste water is one of the most common heavy metal waste water, and is produced in the industries of metal surface treatment, electroplating, smelting, processing, petrochemical industry and the like. Although copper is a necessary trace element for human growth, the excessive and hard-to-degrade heavy metal copper is enriched in human body through food chain and has larger toxicity, and can cause the symptoms of human liver and kidney damage, gastrointestinal spasm, anemia and the like. At present, Cu is treated2+ The waste water mainly comprises the methods of chemical precipitation, membrane separation, dialysis, solvent extraction, reverse osmosis, distillation, resin ion exchange, activated carbon adsorption and the like, and the methods have good effects to a certain extent, but have the defects of secondary pollution, high cost and the like.The biological adsorption method is widely concerned with the advantages of high efficiency, low cost, wide applicable pH value and temperature range, good selectivity, good treatment effect on low-concentration wastewater and the like. The research hotspot is to use the resistance and the detoxification of microorganisms to heavy metals and use the live cells and biomass of the microorganisms as cheap and efficient adsorbents for purifying heavy metal sewage.
The plant rhizosphere fungus F25 capable of efficiently and biologically adsorbing copper ions is obtained by screening. The rhizosphere fungi can effectively adsorb copper ions in sewage, thereby providing a basis for selecting high-efficiency strains in a comprehensive heavy metal sewage treatment system in cities and also providing a reference for the research and development of microbial agents.
Disclosure of Invention
The invention aims to provide a plant rhizosphere fungus F25 capable of adsorbing copper ions and application thereof, aiming at solving the problems in the existing heavy metal polluted wastewater treatment. The rhizosphere fungi can effectively adsorb copper ions in sewage, and provide a basis for selecting efficient strains in a comprehensive heavy metal sewage treatment system in a city.
In order to achieve the purpose, the invention adopts the following technical scheme:
a plant rhizosphere fungus F25 capable of adsorbing copper ions is obtained by screening plant rhizosphere soil of constructed wetlands in sewage treatment plants, identifying through molecular biology and classifying and naming as penicillium violaceum (A)Penicillium janthinellum) The microbial inoculum is preserved in the China general microbiological culture Collection center with the preservation number as follows: CGMCC NO. 17461; the preservation time is as follows: in 2019, 08.04.08, the preservation address is as follows: xilu No.1 Hospital No. 3, Beijing, Chaoyang, was detected as a viable strain and deposited.
The separation and purification method of the plant rhizosphere fungi F25 comprises the following steps:
collecting test soil: rhizosphere soil of artificial wetland plants of Thalia dealbata, vetiver grass and reed flowers in a domestic sewage treatment plant in Wuyeshan city is collected in 11 months in 2015. Collecting soil by 5-point sampling method with sampling depth of 0-20 cm, mixing root system and rhizosphere soil adhered thereon uniformly, taking 500 g of soil, filling into sterile bag, storing in refrigerator at 4 deg.C, wherein part of fresh soil sample is used for strain screening.
Fungus culture medium: the improved martin agar solid culture medium comprises the following components: 5.0g of peptone, 2.0 g of yeast extract powder, 20.0 g of glucose and KH2PO41.0 g，MgSO4•7H20.5 g of O, 15.0 g of agar and 1000 mL of ultrapure water, and the pH value is 6.2-6.6. The liquid medium was as above, but agar was not added.
Screening process of plant rhizosphere fungus F25:
(1) preparing rhizosphere soil suspension:
accurately weighing 5.0g of fresh soil sample in a sterile room, placing into a 250mL triangular flask containing 95mL sterile water, shaking on a shaking table for 20 min to disperse microbial cells, and standing for 20-30s to obtain 10-1Diluting the solution; then according to the method of gradually diluting by 10 times, continuously diluting to obtain 10-2、10-3、10-4Serially diluted bacteria liquid is used for separating microbes.
(2) Separation and purification of rhizosphere fungi and dominant strain screening
1) Separation: using a sterile pipette from 10-2、10-3、10-4And (3) sucking 100 mu L of each soil suspension from the diluted bacteria liquid and uniformly coating the soil suspension on the improved Martin agar solid culture medium. Each sample was set to 3 replicates. The flat plate is sealed and placed upside down in a constant temperature incubator at 29 ℃ for 3-5 d.
2) And (3) purification: single colonies were picked with an inoculating loop and streaked onto medium for purification, with 3 plates streaked per single colony. The flat plate is sealed and placed upside down in a constant temperature incubator at 29 ℃ for 3-5 d. And (4) repeatedly purifying for 2-3 times until pure culture is obtained, and performing slant culture preservation and glycerol culture preservation on the separated and purified single colony.
3) Efficient strain screening: the microbial numbers were recorded during the purification process and the strains of 5-10 colonies appearing on the highest dilution plates were taken as the dominant strains in the rhizosphere. And screening out high-efficiency strains from the dominant strains, wherein the specific method comprises the following steps: 0.5 mL of the bacterial suspension was inoculated into a flask containing 200 mL of medium concentration wastewater (sterilized) and added in equal amountsBacteria ultrapure water as blank control, 29 deg.C, 160r min-1After the constant temperature shaking culture for 48 hours, the wastewater Cd content is determined by referring to the Water and wastewater monitoring and analyzing method (fourth edition)2+Concentration, comparison of Cd pairs of strains2+The strain with high adsorption efficiency is screened out.
Properties of the plant rhizosphere fungus F25
Morphological characteristics of plant rhizosphere fungi F25:
plant rhizosphere fungi F25 culture characteristics: high nutrition, high temperature (29 deg.C), high humidity
Molecular biological identification of plant rhizosphere fungus F25: after activating the plant rhizosphere fungus F25 strain by using a modified Martin agar culture medium, extracting the total DNA of the strain. Using the fungal ITS sequence universal primer ITS 1: 5 '-TCCGTAGGTGAACCTGCGG-3' and ITS 4: 5 '-TCCTCCGCTTATTGATATGC-3' was PCR amplified with ITS sequences. And recovering and sequencing the PCR amplification product to obtain the ITS nucleotide sequence of the plant rhizosphere fungi F25 strain, wherein the length of the ITS nucleotide sequence is 563 bp. Submitting the obtained sequence to a GenBank database for BLAST analysis and alignment, finding that the strains with 99 percent of homology with F25 are all the penicillium chrysogenum, combining morphological characteristics and culture characteristics, and finally identifying the strain F25 as the penicillium chrysogenum (a) (B)Penicillium janthinellum）。
The plant rhizosphere fungus F25 is applied to biological adsorption of copper ions in sewage.
The plant rhizosphere fungi F25 is applied to biological adsorption of copper ions in sewage, and the concentration of the copper ions in the sewage is 1-10 mg.L-1。
Preferably, the concentration of copper ions in the wastewater is 1 mg.L-1。
The invention has the advantages that: the plant rhizosphere fungus F25 capable of adsorbing copper ions is obtained by screening plant rhizosphere soil of artificial wetlands in a sewage treatment plant. The plant rhizosphere fungi F25 can effectively adsorb copper ions and can be applied to biological adsorption of copper ions in sewage. The concentration of copper ions in the sewage is 1-10 mg.L-1When the microorganism is used, the rhizosphere fungi F25 has high-efficiency biological adsorption effect on copper ions. Especially for copper ion concentration of 1 mg.L-1The adsorption rate of the low-concentration sewage inoculated with rhizosphere fungi F2548 h to copper ions reaches 59.00%. The plant rhizosphere fungi F25 provide a basis for selecting high-efficiency strains in the urban comprehensive heavy metal sewage treatment system.
Description of the drawings:
FIG. 1 is a colony morphology of plant rhizosphere fungus F25.
Detailed Description
The present invention will be further illustrated with reference to the following examples, but the present invention is not limited thereto.
Example 1 isolation and purification of plant rhizosphere fungus F25
Collecting test soil: rhizosphere soil of artificial wetland plants of Thalia dealbata, vetiver grass and reed flowers in a domestic sewage treatment plant in Wuyeshan city is collected in 11 months in 2015. Collecting soil by 5-point sampling method with sampling depth of 0-20 cm, mixing root system and rhizosphere soil adhered thereon uniformly, taking 500 g of soil, filling into sterile bag, storing in refrigerator at 4 deg.C, wherein part of fresh soil sample is used for strain screening.
Fungus culture medium: improving a martin agar solid culture medium: 5.0g of peptone, 2.0 g of yeast extract powder, 20.0 g of glucose and KH2PO41.0 g，MgSO4•7H20.5 g of O, 15.0 g of agar and 1000 mL of ultrapure water, and the pH value is 6.2-6.6. The liquid medium was as above, but agar was not added.
Screening process of plant rhizosphere fungus F25:
(1) preparing rhizosphere soil suspension:
accurately weighing 5.0g of fresh soil sample in a sterile room, placing into a 250mL triangular flask containing 95mL sterile water, shaking on a shaking table for 20 min to disperse microbial cells, and standing for 20-30s to obtain 10-1Diluting the solution; then according to the method of gradually diluting by 10 times, continuously diluting to obtain 10-2、10-3、10-4Serially diluted bacteria liquid is used for separating microbes.
(2) Separation and purification of rhizosphere fungi and dominant strain screening
1) Separation: using a sterile pipette from 10-2、10-3、10-4And (3) sucking 100 mu L of each soil suspension from the diluted bacteria liquid and uniformly coating the soil suspension on the improved Martin agar solid culture medium. Each sample was set to 3 replicates. The flat plate is sealed and placed upside down in a constant temperature incubator at 29 ℃ for 3-5 d.
2) And (3) purification: single colonies were picked with an inoculating loop and streaked onto medium for purification, with 3 plates streaked per single colony. The flat plate is sealed and placed upside down in a constant temperature incubator at 29 ℃ for 3-5 d. And (4) repeatedly purifying for 2-3 times until pure culture is obtained, and performing slant culture preservation and glycerol culture preservation on the separated and purified single colony.
3) Efficient strain screening: the microbial numbers were recorded during the purification process and the strains of 5-10 colonies appearing on the highest dilution plates were taken as the dominant strains in the rhizosphere. And screening out high-efficiency strains from the dominant strains, wherein the specific method comprises the following steps: inoculating 0.5 mL of the bacterial suspension into a triangular flask containing 200 mL of medium-concentration sewage (sterilized), and adding equal amount of sterile ultrapure water as a blank control at 29 ℃ for 160r min-1After the incubation for 48 hours with constant temperature shaking, the Cu content in the wastewater was measured by reference to "Water and wastewater monitoring and analyzing method" (fourth edition)2+Concentration, comparison of Cu for each strain2+The strain with high adsorption efficiency is screened out. Screening to obtain plant rhizosphere fungi F25 to Cu2+The adsorption efficiency of (2) is highest.
Example 2 molecular biological characterization of plant rhizosphere fungi F25
(1) Extraction of total DNA of bacterial strain
Activating plant rhizosphere fungus F25 strain with improved Martin agar culture medium, and extracting total DNA of strain with OMEGA genome DNA extraction kit (D3485-01) and DP 302-02).
(2) PCR amplification of fungal ITS sequences
Using the fungal ITS sequence universal primer ITS 1: 5 '-TCCGTAGGTGAACCTGCGG-3' and ITS 4: 5 '-TCCTCCGCTTATTGATATGC-3' is a positive primer and a negative primer for amplifying the ITS sequence of the plant rhizosphere fungi F25.
25 mu L PCR amplification reaction system
And (3) PCR reaction conditions: pre-denaturation at 93 ℃ for 3 min; denaturation at 93 ℃ for 45s, renaturation at 55 ℃ for 45s, extension at 72 ℃ for 1.5min, 35 cycles; terminal extension at 72 ℃ for 10 min.
(3) PCR product recovery
Detecting the PCR amplification product by 1% agarose gel electrophoresis, cutting a target band, purifying and recovering by using a Tiangen recovery kit (DP 214-03), and finally sequencing.
The nucleotide sequence of the ITS sequence of the plant rhizosphere fungi F25 is as follows:
5’-CTTCCGTAAGGGGACCTGCGGAAGGATCATTACCGAGTGCTGGGTCCTTCGGGGCCCAACCTCCCACCCGTGCTTACCGTACCCTGTTGCTTCGGCGGGCCCGCCTTCGGGCGGCCCGGGGCCTGCCCCCGGGACCGCGCCCGCCGGAGACCCCAATGGAACACTGTCTGAAAGCGTGCAGTCTGAGTCGATTGATACCAATCAGTCAAAACTTTCAACAATGGATCTCTTGGTTCCGGCATCGATGAAGAACGCAGCGAAATGCGATAACTAATGTGAATTGCAGAATTCAGTGAATCATCGAGTCTTTGAACGCACATTGCGCCCCCTGGTATTCCGGGGGGCATGCCTGTCCGAGCGTCATTTCTCCCCTCCAGCCCCGCTGGTTGTTGGGCCGCGCCCCCCCGGGGGCGGGCCTCGAGAGAAACGGCGGCACCGTCCGGTCCTCGAGCGTATGGGGCTCTGTCACCCGCTCTATGGGCCCGGCCGGGGCTTGCCTCGACCCCCAATCTTCTCAGATTGACCTCGGATCAGGTAGGGATACCCGCTGAACTTAAGCATAT-3’
(4) fungal ITS sequence analysis
And performing BLAST analysis comparison analysis on the obtained ITS sequence in a GenBank database, selecting a sequence with homology of more than 99 percent with the GenBank database, and preliminarily determining the genus of the strain. The strains with 99% homology to F25 were all Penicillium chrysogenum, and combined with morphological and cultural characteristics, strain F25 was finally identified as Penicillium chrysogenum (F25: (Penicillium janthinellum）。
Example 3 biological adsorption of copper ions in wastewater by plant rhizosphere fungi F25
The rhizosphere fungus F25 obtained by screening and identification is inoculated into 50mL of modified martin liquid culture medium and is 160 r/mL at 29 DEG CShake culturing for 36 hr at min, centrifuging the bacterial solution at 10000 r/min and 4 deg.C for 5min, discarding supernatant, washing thallus with sterile ultrapure water for 3 times, diluting thallus with sterile ultrapure water, and making into 5 × 10 thallus by hemacytometer6CFU/mL of bacterial suspension. The concentration gradients of 3 sewage with high concentration, medium concentration and low concentration are set in the test, and the specific index concentrations of the sewage with different concentrations are shown in the table 1. 0.5 mL of the prepared bacterial suspension is added into 200 mL of sterilized sewage with different concentrations, and the same amount of sterile ultrapure water is added into a control group, and the mixture is subjected to shake culture in a constant-temperature shake incubator at 29 ℃ and 114 r/min. Each treatment group was set with 3 replicates. The concentration of heavy metal ions in the sewage is measured once every 12 hours, and the continuous measurement is carried out for 48 hours.
TABLE 1 concentration of various indexes (mg. L) of sewage with different concentrations-1）
TABLE 2 plant rhizosphere fungi F25 treatment of Cu at different concentrations2+Dynamic change of
Note: the lower case letters in the same column indicate that the treatment differed significantly at 0.05 level for the same treatment time, and the upper case letters in the same row indicate that the treatment differed significantly at 0.05 level for the same strain.
The results in Table 2 show that in 3 concentrations of wastewater, the strain F25 treated Cu in the wastewater with the passage of treatment time2+The concentration showed a clear decrease and Cu was added at different treatment times under the treatment of strain F252+The adsorption rates are all obviously higher than that of the CK in the control group. After inoculating the bacterial suspension for 48 hours, the bacterial strain F25 is used for treating high, medium and low concentration sewage Cu2+The adsorption rates were respectively: 55.73%,57.73%, 59.00%, both significantly higher than the blank CK. Shows that the strain F25 can obviously reduce Cu2+The concentration range is 1-10 mg.L-1Cu in sewage2+Concentration, capable of highly efficient bioadsorption of Cu2+。
The above description is only a preferred embodiment of the present invention, and all equivalent changes and modifications made in accordance with the claims of the present invention should be covered by the present invention.
SEQUENCE LISTING
<110> Fujian agriculture and forestry university
<120> plant rhizosphere fungus F25 capable of adsorbing copper ions and application thereof
<130> 3
<160> 3
<170> PatentIn version 3.3
<210> 1
<211> 19
<212> DNA
<213> ITS1
<400> 1
tccgtaggtg aacctgcgg 19
<210> 2
<211> 20
<212> DNA
<213> ITS4
<400> 2
tcctccgctt attgatatgc 20
<210> 3
<211> 563
<212> DNA
<213> F25 ITS
<400> 3
cttccgtaag gggacctgcg gaaggatcat taccgagtgc tgggtccttc ggggcccaac 60
ctcccacccg tgcttaccgt accctgttgc ttcggcgggc ccgccttcgg gcggcccggg 120
gcctgccccc gggaccgcgc ccgccggaga ccccaatgga acactgtctg aaagcgtgca 180
gtctgagtcg attgatacca atcagtcaaa actttcaaca atggatctct tggttccggc 240
atcgatgaag aacgcagcga aatgcgataa ctaatgtgaa ttgcagaatt cagtgaatca 300
tcgagtcttt gaacgcacat tgcgccccct ggtattccgg ggggcatgcc tgtccgagcg 360
tcatttctcc cctccagccc cgctggttgt tgggccgcgc ccccccgggg gcgggcctcg 420
agagaaacgg cggcaccgtc cggtcctcga gcgtatgggg ctctgtcacc cgctctatgg 480
gcccggccgg ggcttgcctc gacccccaat cttctcagat tgacctcgga tcaggtaggg 540
atacccgctg aacttaagca tat 563
Claims (4)
1. A plant rhizosphere fungus F25 capable of adsorbing copper ions is characterized in that: the plant rhizosphere fungi F25 is obtained by screening plant rhizosphere soil of constructed wetlands in sewage treatment plants and identifying through molecular biology, and is classified and named as penicillium violaceum (A)Penicillium janthinellum) The microbial inoculum is preserved in the China general microbiological culture Collection center with the preservation number as follows: CGMCC NO: 17461, preservation time is: year 2019, 04 and 08.
2. Use of the plant rhizosphere fungus F25 of claim 1 for biosorpting copper ions in wastewater.
3. The use of the plant rhizosphere fungus F25 in biosorption of copper ions in wastewater according to claim 2, wherein: the concentration of copper ions in the sewage is 1-10 mg.L-1。
4. The use of the plant rhizosphere fungus F25 in biosorption of copper ions in wastewater according to claim 3, wherein: the concentration of copper ions in the sewage is 1 mg.L-1。
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910585045.2A CN110283729B (en) | 2019-07-01 | 2019-07-01 | Plant rhizosphere fungus F25 capable of adsorbing copper ions and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910585045.2A CN110283729B (en) | 2019-07-01 | 2019-07-01 | Plant rhizosphere fungus F25 capable of adsorbing copper ions and application thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN110283729A CN110283729A (en) | 2019-09-27 |
| CN110283729B true CN110283729B (en) | 2020-12-29 |
Family
ID=68021419
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201910585045.2A Expired - Fee Related CN110283729B (en) | 2019-07-01 | 2019-07-01 | Plant rhizosphere fungus F25 capable of adsorbing copper ions and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN110283729B (en) |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1316008C (en) * | 2004-06-02 | 2007-05-16 | 广西大学 | High metal-salt-resistant micro penicillium purpurogerum |
-
2019
- 2019-07-01 CN CN201910585045.2A patent/CN110283729B/en not_active Expired - Fee Related
Also Published As
| Publication number | Publication date |
|---|---|
| CN110283729A (en) | 2019-09-27 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN108587915B (en) | Chlorella W5 capable of removing heavy metals in water with high heavy metal content and application thereof | |
| CN114703095B (en) | Pseudomonas adulthood and application thereof in field of sewage and wastewater purification | |
| CN110791454A (en) | Efficient ammonia nitrogen degrading strain and application thereof | |
| CN103833144B (en) | A kind of method utilizing flcos producing bacteria fermented liquid to remove heavy metal ion in water | |
| CN109337832A (en) | A kind of anthropi of resistance to high ammonia nitrogen heterotrophic nitrification-aerobic denitrification and its application | |
| CN110656066A (en) | Acinetobacter strain for shortcut nitrification and denitrification and application thereof | |
| CN111394260B (en) | Separation and application of microorganisms for treating wastewater | |
| CN105567575A (en) | New Purpureocillium sp strain F1 and application thereof | |
| CN112961790A (en) | Heterotrophic nitrifying bacteria resistant to high-salt environment and application thereof | |
| CN110643553B (en) | Bacterial strain capable of efficiently removing organic matters in water body and application thereof | |
| CN110343620B (en) | Plant rhizosphere fungus F9 capable of adsorbing cadmium ions and application thereof | |
| CN110283729B (en) | Plant rhizosphere fungus F25 capable of adsorbing copper ions and application thereof | |
| CN114292798B (en) | Anaerobic denitrifying strain and application thereof in riverway water body remediation | |
| CN110241031B (en) | Plant rhizosphere fungus F30 capable of adsorbing zinc ions and application thereof | |
| CN113151090B (en) | Copper pollution treating agent and application thereof | |
| CN110643554B (en) | Strain capable of efficiently removing inorganic phosphorus in water body and application thereof | |
| CN115851450A (en) | Cladosporium tenuissima NXY8, mycelial ball and application thereof in high-salt wastewater treatment | |
| CN111484939B (en) | Separation and application of spectinomyceliophthora | |
| CN113862163A (en) | Penicillium and microbial inoculum with heavy metal ion removal effect and application thereof | |
| CN113881582A (en) | Rhodotorula MF4 for removing heavy metal ions, microbial inoculum and application thereof | |
| CN112920958B (en) | High-concentration wastewater biological pretreatment functional bacteria and application thereof | |
| CN117383709B (en) | Treatment method of high-salt and high-concentration organic wastewater | |
| CN107022505A (en) | A kind of citric acid bacillus and its application for heavy metal copper ion remaval | |
| CN114836355B (en) | Pseudomonas DT04 and application thereof in sewage denitrification | |
| CN113201475B (en) | Actinomycetes capable of enriching heavy metals 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 | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20201229 |