CN114369545B - Taiwan copper-philic bacteria strain KY575 with heavy metal cadmium curing function and application thereof - Google Patents
Taiwan copper-philic bacteria strain KY575 with heavy metal cadmium curing function and application thereof Download PDFInfo
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- CN114369545B CN114369545B CN202111602254.7A CN202111602254A CN114369545B CN 114369545 B CN114369545 B CN 114369545B CN 202111602254 A CN202111602254 A CN 202111602254A CN 114369545 B CN114369545 B CN 114369545B
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Classifications
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N63/00—Biocides, pest repellants or attractants, or plant growth regulators containing microorganisms, viruses, microbial fungi, animals or substances produced by, or obtained from, microorganisms, viruses, microbial fungi or animals, e.g. enzymes or fermentates
- A01N63/20—Bacteria; Substances produced thereby or obtained therefrom
-
- 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
- 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
-
- 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
- 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
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- Engineering & Computer Science (AREA)
- Health & Medical Sciences (AREA)
- Microbiology (AREA)
- Chemical & Material Sciences (AREA)
- Biotechnology (AREA)
- General Health & Medical Sciences (AREA)
- Zoology (AREA)
- Organic Chemistry (AREA)
- Wood Science & Technology (AREA)
- Environmental & Geological Engineering (AREA)
- Virology (AREA)
- Biomedical Technology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Genetics & Genomics (AREA)
- Medicinal Chemistry (AREA)
- Biodiversity & Conservation Biology (AREA)
- Tropical Medicine & Parasitology (AREA)
- Molecular Biology (AREA)
- Mycology (AREA)
- Soil Sciences (AREA)
- Biochemistry (AREA)
- General Engineering & Computer Science (AREA)
- Hydrology & Water Resources (AREA)
- Water Supply & Treatment (AREA)
- Agronomy & Crop Science (AREA)
- Pest Control & Pesticides (AREA)
- Plant Pathology (AREA)
- Dentistry (AREA)
- Environmental Sciences (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention provides a Taiwan copper-philic bacteria (Cupriavidus taiwanensis) strain KY575 with a heavy metal cadmium curing function and application thereof, and relates to the technical field of microorganisms. The strain is preserved in China general microbiological culture Collection center (CGMCC) with a preservation number of CGMCC No.24095. According to the invention, a large number of soil samples are subjected to high-throughput screening to obtain the strain KY575 capable of tolerating the heavy metal cadmium (II) with the concentration of 500mg/L, and the strain can solidify cadmium in soil or water, reduce the harm of cadmium to plants and reduce the enrichment of cadmium in plants.
Description
Technical Field
The invention relates to the technical field of microorganisms, in particular to a Taiwan copper bacterium (Cupriavidus taiwanensis) strain KY575 with a heavy metal cadmium curing function and application thereof.
Background
Soil is a very important and life-rich limited resource, is an important component of the ecological environment, and is one of the main resources for human survival. In recent decades, with the development of modern industry and the rapid increase of urban household garbage and the massive use of pesticides and fertilizers, the soil on which human beings live is severely polluted by heavy metals. The main pollution sources of the heavy metals in the soil are pollution irrigation and solid waste application, mining and smelting, pesticides and fertilizers, and the heavy metals released into the environment each year mainly comprise lead, arsenic, cadmium and the like (Meng Xianghe and the like, 2000). Soil heavy metal pollution is an irreversible process, has serious hazard, directly affects the safety of agricultural products, and further threatens the health of animals and human beings.
The pollution of harmful metals in the soil directly affects the quality of the soil, the water quality, the crop growth, the agricultural yield, the quality and the like, and is enriched into human bodies and animals through food chains, so that the health of the human bodies and the animals is endangered, and human cancers, other diseases and the like are caused. Heavy metal cadmium is a highly toxic heavy metal element, and cadmium is one of the heavy metal elements with the largest area and the largest hazard in the current heavy metal pollution, and is called as the head of five toxins. Toxicology studies of cadmium have shown (Xue Bin et al, 1999 and Liu Jie, 1998) that cadmium is difficult to eliminate in the environment by self-cleaning action of the environment, and difficult to exclude after entry into organisms, and that its biological half-life can be as long as 8-30 years, is one of the most readily known heavy metals to accumulate in the body (Jin T et al, 1999; hart B A et al, 2001; berglund M et al, 2000 and Antonio M T et al, 1999). The excessive intake of cadmium can inhibit the growth of human body, influence the activity of enzyme systems such as amino acid isocitrate, histidine enzyme, amylase, catalase and the like, interfere the metabolism of trace elements such as Cu, co, zn and the like, and cause a series of diseases; cadmium also binds strongly to phosphorus causing excessive excretion of calcium in bone calcium phosphate, leading to osteoporosis softening, deformity, fracture and pain (Liu Guosheng, 2004).
Therefore, the problem of treating the heavy metal pollution of the soil is not solved. The current treatment method for heavy metal pollution of soil mainly converts heavy metal into stable state through a series of reactions such as adsorption, precipitation, complexation and the like of physics, chemistry, biology and the like so as to reduce migration capacity and plant effectiveness, thereby achieving the purpose of repairing the heavy metal polluted soil. However, physical and chemical techniques have the problems of high restoration cost, easiness in causing secondary pollution, changing soil ecology and the like. Bioremediation (in particular microbial remediation) is becoming more and more important due to its full, simple, efficient and low cost characteristics.
Microbial remediation techniques include the use of the activity of living organisms, algae, bacteria, fungi to reduce or recover heavy metal contaminants into less harmful forms. Microorganisms cannot degrade and destroy heavy metals, but can affect migration and transformation of heavy metals by changing physicochemical properties of heavy metals. Research shows that the microorganism can remove heavy metal cadmium in the environment. The Pseudoalteromonas sp.scse709-6 can synchronously remove phosphorus and cadmium (Zhang Haiou, 2014) while resisting cadmium, the removal rate of the spherical rhodobacter sphaeroides reaches 97% when the cadmium concentration is 40mg/L (Bai H J et al, 2008), and the bacillus can remove 75.78% of cadmium when the cadmium concentration is 10mg/L (Guo H et al, 2010).
Because microorganisms have important advantages in heavy metal fixation and recovery and great application prospects, more types of microorganisms are necessary to be developed to solve the problem of pollution of heavy metals (particularly heavy metal cadmium) in soil or water.
In view of this, the present invention has been made.
Disclosure of Invention
The invention aims to provide a Taiwan copper bacterium (Cupriavidus taiwanensis) strain KY575 with a heavy metal cadmium curing function and application thereof. The strain obtained by screening can not only resist 500mg/L high-concentration heavy metal cadmium (II), but also solidify cadmium in soil or water, reduce the harm of cadmium to plants and reduce the enrichment of cadmium in plants.
The technical scheme provided by the invention is as follows:
in one aspect, the invention provides a taiwan copper bacterium (Cupriavidus taiwanensis) strain KY575 with a heavy metal cadmium curing function, wherein the strain is preserved in China general microbiological culture collection center (CGMCC) with a preservation number of CGMCC No.24095.
In the invention, the nucleotide sequence of the 16S rDNA of the strain is shown as SEQ ID No. 1.
According to the invention, through screening more than 170 collected soil samples, a strain KY575 capable of tolerating 500mg/L high-concentration heavy metal cadmium (II) is obtained, the strain is separated from soil in the Henan Zhoukou region, and the strain can solidify and remove the heavy metal cadmium in the soil and water.
In another aspect, the present invention provides a composite microbial inoculant comprising the aforementioned strain KY575 of taiwan copper (Cupriavidus taiwanensis); the composite microbial inoculum comprises a solid microbial inoculum and a liquid microbial inoculum.
In one embodiment, the composite microbial inoculant further comprises excipients for use therewith, such as one or more of turf, bran powder, wheat bran, kaolin, light calcium carbonate, diatomaceous earth, white carbon, talc, fine sand, and clay, for example; or a mixture of one or more of sucrose, glucose, peptone, soybean meal, sodium dodecylbenzenesulfonate, sodium lignin sulfonate, and sodium alkyl naphthalene sulfonate polycondensate.
In another aspect, the invention provides an application of the taiwan copper bacterium (Cupriavidus taiwanensis) strain KY575 or the composite microbial inoculum in removing or passivating heavy metal cadmium.
In one embodiment, the application is in the treatment of heavy metal cadmium pollution in water and soil. The strain or the microbial inoculum containing the strain can remove cadmium pollution in water and fix cadmium in soil.
In one embodiment, the application comprises applying the aforementioned strain KY575 of taiwan copper bacterium (Cupriavidus taiwanensis) or the aforementioned composite microbial agent to a heavy metal cadmium-contaminated water body or soil.
In one embodiment, the strain or a complex inoculant comprising the same or a culture or metabolite of the strain is capable of reducing the content of cadmium ions in a liquid medium.
In one embodiment, the invention provides the use of said taiwan copper bacterium (Cupriavidus taiwanensis) strain KY575 or said complex inoculant in crop production.
In a specific embodiment, the use is in passivating the hazard of heavy metal cadmium to plants and/or enhancing the plant's tolerance to heavy metal stress environments; the strain or the composite microbial inoculum containing the strain can reduce the absorption and enrichment of cadmium ions by plants.
In one embodiment, the use comprises applying the taiwan copper (Cupriavidus taiwanensis) strain KY575 or the composite microbial agent to the plant rhizosphere or planting after seed dressing of the plant.
In a specific embodiment, the plant comprises maize. The strain KY575 or the composite microbial inoculum can reduce the absorption of cadmium by the root and the stem and leaf of corn and the cadmium content of overground part and underground part of stored corn, and reduce the harm of heavy metal cadmium in soil to plants.
Strain preservation information: the strain Taiwan copper bacterium (Cupriavidus taiwanensis) strain KY575 of the invention is preserved in China general microbiological culture Collection center; deposit unit address: beijing, chaoyang area, north Chenxi Lu No.1, 3; accession number No.24095; the preservation time is as follows: 2021, 12 and 13. The viable strains were detected by the collection center and deposited.
The beneficial effects are that:
the Taiwan copper bacterium (Cupriavidus taiwanensis) strain KY575 provided by the invention has stronger tolerance to metal cadmium and can tolerate 500mg/L high-concentration heavy metal cadmium (II);
the invention provides Taiwan copper-philic alloyThe passivation rate of the strain KY575 of the bacterium (Cupriavidus taiwanensis) to heavy metal cadmium in liquid is high and can reach 84.21%; cd can be quickly dissolved in 6d 2+ The concentration was passivated from 152mg/L to 24mg/L.
The Taiwan copper bacterium (Cupriavidus taiwanensis) strain KY575 provided by the invention can solidify or remove heavy metal cadmium in the environment (such as soil and water body); can be used for reducing the content of cadmium in a liquid culture medium, passivating the harm of heavy metal cadmium to plants and reducing the absorption and enrichment of the plants to the cadmium.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings that are needed in the description of the embodiments or the prior art will be briefly described, and it is obvious that the drawings in the description below are some embodiments of the present invention, and other drawings can be obtained according to the drawings without inventive effort for a person skilled in the art.
FIG. 1 shows the growth of KY27 in a series of cadmium solutions of the present invention.
Fig. 2 shows the morphology of KY575 provided by the present invention at a 100-fold oil mirror.
Detailed Description
The technical solutions of the present invention will be clearly and completely described in connection with the embodiments, and it is apparent that the described embodiments are some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
Example 1 high throughput screening of heavy metal cadmium (Cd) tolerant microorganisms
1.1 Soil sample collection
170 or more soil samples including various soil samples such as black soil, clay and red soil are collected from various places of the whole country (particularly areas severely polluted by heavy metal cadmium) and are respectively sourced from forests, grasslands, wheat lands, paddy fields and the like. These soil samples are all designated as collection sites (province, city, county), collection time, collection sources (forest, grassland, wheat land, paddy field, etc.).
1.2 Enrichment culture of cadmium-tolerant microorganisms
Five soil samples (0.2 g each) were mixed and placed in 50mL containing 250mg/LCd 2+ Is a liquid medium (0.5 g peptone, 0.5g yeast, 1g glucose, 0.5g tryptone, 0.3g sodium pyruvate, 0.05g MgSO) 4 ·7H 2 O, 987.5mL distilled water, and autoclaved at 121℃for 30min, followed by addition of 12.5mL 20g/L Cd 2+ Solution), shaking culture is carried out at 30 ℃ for 3 days at 200r/min, and changes of turbidity and the like of the liquid culture medium are observed and recorded.
1.3 Separation of heavy metal cadmium-resistant microorganisms
Taking a proper amount of the bacterial liquid in the step 1.2, and placing the bacterial liquid in a solid medium (0.5 g peptone, 0.5g yeast, 1g glucose, 0.5g tryptone, 0.3g sodium pyruvate, 0.05g MgSO) with cadmium of 250mg/L 4 ·7H 2 O, 987.5mL distilled water, 15g agar powder, and autoclaved at 121℃for 30min, 12.5mL 20g/L Cd were added 2+ Solution) is streaked on a flat plate, and the growth of microorganisms in the solid cadmium culture medium is observed and recorded.
1.4 Strain purification
Single colonies from step 1.3 were picked on R2A medium (0.5 g peptone, 0.5g yeast, 0.5g glucose, 0.5g tryptone, 0.5g soluble starch, 0.3g K) 2 HPO 4 0.3g sodium pyruvate, 0.05g MgSO 4 ·7H 2 O, 1000mL distilled water and 15g of agar powder) are streaked, cultured and purified to obtain pure bacterial colonies.
1.5 Repeated verification
Inoculating the microorganism obtained in step 1.4 into a microorganism containing different concentrations of Cd of 100mg/L, 250mg/L, 500mg/L and 1000mg/L 2+ And (3) streaking the solid culture medium to exclude false positive microorganisms, and finally obtaining cadmium-resistant microorganisms.
Experimental results of screening:
170 soil samples (nearly 15 ten thousand strains of microorganisms) are screened by a medium containing heavy metal cadmium, and 130 strains of cadmium-resistant microorganisms are obtained. The classifications of these functional microorganisms are shown in the following table:
TABLE 1 microbial statistics of heavy metal cadmium (Cd) resistance
Example 2 testing the cadmium-tolerant microorganisms' ability to inactivate heavy metals in liquids
2.1 Biological method for initially verifying cadmium passivation capability of cadmium-resistant microorganisms in liquid
The method can more intuitively and simply observe the cadmium passivating capability of cadmium-resistant microorganisms in the liquid.
2.1.1 Determination of biological method indicator Strain
1) Selecting microorganism strains of different species from microorganism strain resource pool of Kangshengyuan (Zhaoqing) biotechnology Co., ltd, inoculating to gradient Cd-containing strain of different concentration 2+ On a solid culture medium, observing the growth condition of a colony, and finally screening to obtain a strain KY27 (Enterobacter soli strainE) sensitive to the change of the cadmium concentration;
2) Gradient concentration of Cd 2+ Mixing the solution with KY27 bacterial liquid 1:1 subjected to gradient dilution, taking 10 microliters of mixed solution, placing the mixed solution on an R2A solid culture medium, culturing the mixed solution at 30 ℃ for 12 hours, and observing the growth condition of the strain;
results
As can be seen from FIG. 1, KY27 changes with the cadmium content of the medium. In the range of 12.5mg/L-200mg/L, the colony of KY27 gradually decreases as the cadmium content in the culture medium increases; conversely, as the cadmium content of the medium decreases, KY27 colonies gradually increase. In conclusion, the cadmium content in the sample can be primarily judged according to the KY27 growth condition. Therefore KY27 was chosen as indicator for this method.
2.1.2 Testing cadmium-tolerant microorganisms for their ability to inactivate heavy metals in liquids
1) Configuration 10 6 、10 5 、10 4 、10 3 KY27 bacterial liquid of (2) for standby;
2) The cadmium-resistant microorganism is inoculated into 200mg/L cadmium liquid culture medium and cultured for 6 days at the temperature of 200rpm and 30 ℃. Taking out and centrifuging, filtering with a 0.45 μm filter membrane, and taking the supernatant (namely the required sample).
3) Diluting the sample 8-fold (with medium without cadmium);
4) Take 10 6 、10 5 、10 4 、10 3 Mixing the KTY27 bacterial solution and the diluted sample 1:1 to obtain a solution A, B, C, D, and taking 10 microliters of the solution to be spotted on R2A;
5) After the solution is dried on a culture medium, placing the solution in a 30 ℃ incubator for 12 hours, observing the growth condition of microorganisms, and primarily judging the cadmium content of the sample according to the growth condition of the microorganisms.
Results:
the results of preliminary screening of the ability of 130 strains of cadmium-resistant microorganisms to passivate heavy metal cadmium by using a biological method primary screen are shown in Table 2. As can be seen from the table, through preliminary verification, the 130 strains of cadmium-resistant microorganisms find that 64 strains of cadmium-resistant microorganisms have better heavy metal cadmium passivating capability, 34 strains of cadmium-resistant microorganisms have inferior heavy metal cadmium passivating capability, and 32 strains of cadmium-resistant microorganisms have no heavy metal cadmium passivating capability.
TABLE 2 cadmium tolerant microbial passivation of heavy metals
Note that: when the passivation ability is absent, KY27 growth is inhibited, only at 10 5 And 10 6 A few colonies are grown, which means that the microorganism strain does not have the ability of passivating heavy metal cadmium; KY27 at 10 when the passivation capability is + 4 、10 5 And 10 6 The strain can grow out of colony, which means that the strain has the ability to passivate heavy metal cadmium, but the ability to passivate heavy metal cadmium is general; KY27 at 10 when the passivation capability is ++ 3 、10 4 、10 5 And 10 6 The microbial strain can grow more colonies without growth inhibition, and has stronger capability of passivating heavy metal cadmium.
2.2 Accurate determination of ability of cadmium-resistant microorganisms to passivate heavy metal cadmium in liquid
And precisely measuring the cadmium content of the solution treated by the cadmium-resistant microorganism by adopting a flame atomic absorption method, and determining the removal rate of the strain to heavy metals.
2.2.1 Preparing a bacterial suspension: placing the microorganism strain which grows on the R2A solid culture medium and has been verified to have heavy metal cadmium passivation capability in sterilized water to prepare bacterial suspension, and measuring OD 600 A value;
2.2.2 Inoculating: preparation of 50mL of 200mg/L cadmium liquid culture in 250mL Erlenmeyer flask, inoculation of the bacterial suspension in step 2.2.1 and OD 600 Approximately 0.05, and a blank is added with the sterilized water with the same volume;
2.2.3 Culturing: placing the conical flask in a shaking table, and culturing at 200rpm and 30 ℃ for 6 days;
2.2.4 Sample treatment: centrifuging the fermented culture solution, filtering the supernatant with a 0.45 μm filter membrane, and obtaining filtrate as a sample to be detected;
2.2.5 Sample measurement: and (3) taking cadmium standard solution (GSB G62040-90 of national iron and steel materials testing center) according to a gradient in a 100mL volumetric flask, adding 4mL of concentrated hydrochloric acid, adding distilled water to dilute to a scale, namely a standard curve, taking a proper amount of samples in the step 2.2.4 in the 100mL volumetric flask, adding 4mL of concentrated hydrochloric acid, adding distilled water to dilute to the scale, namely to-be-measured solution, and measuring the cadmium content by adopting a flame atomic absorption method.
Results: atomic absorption verification is carried out on 64 strains of microorganisms, and results show that the passivation rate of heavy metal cadmium is higher for 30 strains of microorganisms (the passivation rate of heavy metal cadmium refers to the percentage of the cadmium content or concentration in the liquid after being treated by the microorganisms, which is reduced in the liquid before being treated). As shown in table 3.
TABLE 3 passivation Rate of different microorganism strains on heavy cadmium in liquid culture for 6d
Among these strains, KY575 can rapidly cleave Cd within 6d 2+ The concentration is passivated from 152mg/L to 24mg/L, and the passivation rate is as high as 84.21%.
Example 3 identification of Strain KY575
Extraction of bacterial DNA by CTAB method
1) Inoculating a single colony in 5mLR A, and culturing overnight at 30 ℃;
2) 1mL of seed culture solution is taken and inoculated into 100mLR A2A liquid, and the culture is carried out for 16 hours at 37 ℃ and 220 r/min;
3) Centrifuge at 5000r/min for 10 min, discard supernatant. Adding 10mLTE, centrifugally washing, dissolving thalli with 10mLTE, uniformly mixing, and preserving at-20 ℃ for later use;
4) Taking 3.5mL of bacterial suspension, adding 184 mu L of 10% SDS, uniformly mixing, adding 37 mu L of 10mg/mL proteinase K, uniformly mixing, and incubating for 1 hour at 37 ℃;
5) 740. Mu.L of 5mol/LNaCl and 512. Mu.LCTAB/NaCl were added, mixed well and incubated at 65℃for 10 minutes;
6) Adding equal volume of chloroform/isoamyl alcohol, mixing, centrifuging at 10000r/min for 5 min, and retaining supernatant;
7) Adding an equal volume of phenol to the supernatant: chloroform: isoamyl alcohol (25:24:1), evenly mixed, centrifuged at 10000r/min for 5 minutes, and the supernatant is reserved
8) Adding 0.6 times of isopropanol, mixing, centrifuging at 10000r/min for 5 min, collecting DNA precipitate, and centrifuging with 70% ethanol to wash the DNA precipitate;
9) The DNA was dissolved in 1mL of TE, and RNaseA was added at a final concentration of 20. Mu.g/mL and stored at 4 ℃.
Amplification and sequencing
PCR amplification of 16S rDNA was performed using the 16S rDNA universal primer 27f (5'-AGAGTTTGATCCTGGCTCAG-3' (SEQ ID No. 2)) and 1492r (5'-GGTTACCTTGTTACGACTT-3' (SEQ ID No. 3)).
PCR reaction conditions: pre-denaturation at 94 ℃ for 30s; denaturation at 94℃for 30s, annealing at 52℃for 30s, elongation at 72℃for 60s,35 cycles.
The PCR products were subjected to 1.5% agarose gel electrophoresis. Sequencing (Shanghai Biotechnology Co., ltd.) the PCR products after passing the test, searching homologous sequences in GenBank by Blast according to the obtained 16S rDNA sequences, and simultaneously, comparing the sequences with the approved 16sRNA database to determine the classification status (Yoon, S.H., ha, S.M., kwon, S.Lim., J., kim, Y, seo, H.and Chun, J. (2017) Introducing EzBioCloud: A taxonomically united database of 16S rRNA and whole genome assemblies.Int J Syst Evol Microbiol.67:1613-1617) of the target microorganism.
KY575 16s DNA sequence (SEQ ID No. 1):
GATGTGGCGGGCTGTCTTACCATGCAAGTCGAACGGCAGCGCGGGCTTCGGCCTGGCGGCGAGTGGCGAACGGGTGAGTAATACATCGGAACGTGCCCTGTCGTGGGGGATAACTAGTCGAAAGATTAGCTAATACCGCATACGACCTGAGGGTGAAAGCGGGGGACCGCAAGGCCTCTCGCGATATGAGCGGCCGATGTCTGATTATCTATTTGGTGGGGTAAAGGCCCACCAAGGCGACAATCTCTAGCTGGGCTGAGAGGACGATCTCCCACACTGGGACTGAGACACGGCCCCCACTCCTACGGGAGGCAGCAGTGGGGAATTTTGGACAATGGGGGCAACCCTGATCCCCCAATGCCGCGTGTGTGAAGAAGGCCTTCTGGTTGTAAAGCACTTTTGTCCGGAAAGAAATGGGCCTGGGTAATACCCGGGGTCGATGACAGTACCGGAAAAATAAGCACCGGCTAACTACATGCCACCAGCCGCGGTAATACGTAGGGTGCGAGCGTTAATCGGAATACTGGGCGTAAAGCGTGCGCAGGCGGTTGATAAGACAGGCGTGAAATCCCCGGGCTCAACCTGGGAATGGCGCTTGTGACTGTCAGGCTAGAGTGCGTCAGAGGGGGGTAGAATTCCACGTGTAGCAGTGAAATGCGTAGAGATGTGGAGGAATACCGATGGCGAAGGCAGCCCCCTGGGACGTGACTGACGCTCATGCACGAAAGCGTGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCACGCCCTAAACGATGTCAACTAGTTGTTGGGGATTCATTTCTTCAGTAACGTAGCTAACGCGTGAAGTTGACCGCCTGGGGAGTACGGTCGCAAGATTAAAACTCAAAGGAATTGACGGGGACCCGCACAAGCGGTGGATGATGTGGATTAATTCGATGCAACGCGAAAAACCTTACCTACCCTTGACATGCCACTAACGAAGCAGAGATGCATTAGGTGCCCGAAAGGGAAAGTGGACACAGGTGCTGCATGGCTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTGTCTCTAGTTGCTACGCAAGAGCACTCTAGAGAGACTGCCGGTGACAAACCGGAGGAAGGTGGGGATGACGTCAAGTCCTCATGGCCCTTATGGGTAGGGCTTCACACGTCATACAATGGTGCGTACAGAGGGTTGCCAACCCGCGAGGGGGAGCTAATCCCAGAAAACGCATCGTAGTCCGGATCGTAGTCTGCAACTCGACTACGTGAAGCTGGAATCGCTAGTAATCGCGGATCAGCATGCCGCGGTGAATACGTTCCCGGGTCTTGTACACACCGCCCGTCACACCATGGGAGTGGGTTTTGCCAGAAGTAGTTAGCCTAACCGCAAGGAGGGCGATACCACGTCAGCTAAAA。
by analysis of 16S rDNA, KY575 was shown to be highly homologous to Cupriavidus taiwanensis and the strain was identified as Cupriavidus taiwanensis.
The strain KY575 is gram negative bacteria, short rod-shaped, and cultured on R2A culture medium at 30 ℃ for 48 hours, and the colony is round, neat in edge, light yellow, transparent, about 1 mm in diameter, dark in middle color and light in edge color. In addition, the morphology of KY575 at 100 times the scope is shown in fig. 2.
3. Potting experiment
The experiment designs three treatments of heavy metal cadmium (negative control), heavy metal cadmium+humic acid (positive control) and heavy metal cadmium+bacterial liquid (experimental group) in sequence, namely treatment 1, treatment 2 and treatment 3.
Each pot was filled with 450mL of soil (available from Ind. Chemicals Inc. of Guangzhou, inc.), 79mL of 25mg/L Cd was poured before planting 2+ Selecting uniformly-sized Beijing-sticky first corn seeds, planting 1 seed in each pot, setting 10 repeated seeds, randomly arranging and planting, treating 1-pouring 35mL of water, treating 2-pouring 35mL of 500mg/L of humic acid, and treating 3-pouring 35mL of bacterial liquid with OD=0.05 when the seeds are just planted; during one week of growth, treatment 1 poured 30mL of water, treatment 2 poured 30mL of 500mg/L humic acid, and treatment 3 poured 30mL of bacterial liquid with od=0.05. And the rest time is timely, proper and balanced irrigation according to the growth requirement of corn. And harvesting corn seedlings about 20 days.
The morphological observation of corn shows that the corn plants treated by heavy metal cadmium (negative control) are short, the leaves are narrow, and the root system is short; corn plants treated with biological humic acid with heavy metal cadmium passivation capability (positive control) and corn irrigated with KY575 bacteria liquid (experimental group) are higher than those of the negative control, the leaves are wider, and the root system is developed and leaf extraction is normal.
And (5) respectively drying the overground part and the underground part of the corn seedlings, and measuring the cadmium content of the dried samples according to GB 5009.15-2014. The results are shown in the following table:
TABLE 3 results of corn potting experiments
Therefore, the strain can passivate heavy metal cadmium in soil, obviously lighten the harm of the heavy metal cadmium to plants and obviously reduce the absorption of the heavy metal cadmium by roots, stems and leaves of plant corns.
In view of the above experimental results, the strain Cupriavidus taiwanensis KY575 of the present invention is capable of tolerating Cd at a concentration of 500mg/L 2+ Meanwhile, the strain can solidify heavy metal cadmium in water and soil and reduce the cadmium content in plants, and can be applied to the treatment of heavy metal cadmium polluted soil.
Finally, it should be noted that: the above embodiments are only for illustrating the technical solution of the present invention, and not for limiting the same; although the invention has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: the technical scheme described in the foregoing embodiments can be modified or some or all of the technical features thereof can be replaced by equivalents; such modifications and substitutions do not depart from the spirit of the invention.
SEQUENCE LISTING
<110> Hebei lovely water-soluble fertilizer shares limited; kangshengyuan (Zhaoqing) biotechnology Co.Ltd
<120> a Taiwan copper-philic bacteria strain KY575 with heavy metal cadmium curing function and application thereof
<130> PA21028444
<160> 3
<170> PatentIn version 3.3
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<213> Taiwan copper (Cupriavidus taiwanensis) Strain KY575 16S rDNA
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actgtcaggc tagagtgcgt cagagggggg tagaattcca cgtgtagcag tgaaatgcgt 660
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cacgaaagcg tggggagcaa acaggattag ataccctggt agtccacgcc ctaaacgatg 780
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Claims (8)
1. A Taiwan copper bacterium (Cupriavidus taiwanensis) strain KY575 with a heavy metal cadmium curing function is characterized in that the strain is preserved in China general microbiological culture collection center (CGMCC) with a preservation number of 24095.
2. A composite microbial agent comprising the strain KY575 of taiwan copper bacterium (Cupriavidus taiwanensis) of claim 1; the composite microbial inoculum comprises a solid microbial inoculum and a liquid microbial inoculum.
3. Use of taiwan copper bacterium (Cupriavidus taiwanensis) strain KY575 as claimed in claim 1 or a composite microbial agent as claimed in claim 2 for removing or passivating heavy metal cadmium.
4. The use according to claim 3, characterized in that it is the use in the treatment of heavy metal cadmium pollution of water bodies and soils.
5. The use according to claim 4, comprising applying the strain KY575 of taiwan copper bacterium (Cupriavidus taiwanensis) of claim 1 or the composite microbial agent of claim 2 to a heavy metal cadmium contaminated water or soil.
6. Use of a strain KY575 of taiwan copper (Cupriavidus taiwanensis) as claimed in claim 1 or a composite microbial agent as claimed in claim 2 in crop production;
the application is in the aspects of reducing the harm of heavy metal cadmium to plants and/or enhancing the environmental tolerance of the plants to heavy metal cadmium stress.
7. The use according to claim 6, characterized in that it comprises applying the taiwan copper bacterium (Cupriavidus taiwanensis) strain KY575 or the complex inoculant to the rhizosphere of plants or planting after seed dressing of the plants.
8. The use according to claim 6 or claim 7, wherein the plant comprises maize.
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| 耐镉菌株对土壤镉形态及土壤微生物群落结构的影响;王京文;李丹;柳俊;侯昌萍;张奇春;;农业环境科学学报(第09期);第1693-1699页 * |
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