CN110628678A

CN110628678A - Heavy metal-resistant cupronickel bacterium, preparation method and application of bacterium agent

Info

Publication number: CN110628678A
Application number: CN201910935883.8A
Authority: CN
Inventors: 王桔红; 陈文�
Original assignee: Hanshan Normal University
Current assignee: Hanshan Normal University
Priority date: 2019-09-29
Filing date: 2019-09-29
Publication date: 2019-12-31
Anticipated expiration: 2039-09-29
Also published as: CN110628678B

Abstract

The invention belongs to the technical field of new strain screening technology and environmental microbiology, and particularly relates to heavy metal-resistant cuprinophilus (cupriavidus sp.) HXC-8. The cupreous cuprinus (Cupriavdus sp.) HXC-8 is separated from the root of Mimosa pudica (Mimosa pudica) of leguminous plant, can resist heavy metal copper, and improves Cu tolerance²⁺Contaminated seed germination rate and promotion of Cu exposure²⁺The contaminated seedling grows, can effectively remove copper ions in the environment, and can be used as Cu²⁺Contaminated soilThe repairing preparation is used and has wide application prospect.

Description

Heavy metal-resistant cupronickel bacterium, preparation method and application of bacterium agent

Technical Field

The invention belongs to the technical fields of new strain screening technology and environmental microbiology, and particularly relates to a heavy metal-resistant cuprioides (cupriavidus sp.) HXC-8 strain, and a preparation method and application of a microbial inoculum thereof.

Background

In recent years, mining, chemical industry and life have increasingly serious heavy metal pollution to soil. Heavy metals cannot be automatically decomposed in soil, have long residual time and great harm, and are easily absorbed by crops to influence the health of human bodies. Copper pollution is common heavy metal pollution, excessive copper elements in soil are accumulated and stay at roots after being absorbed by plants, the roots are prevented from absorbing other components, particularly iron elements, so that the plants are poor in growth, and poisoning phenomena such as nausea, vomiting, epigastric pain, acute hemolysis, renal tubule deformation and the like can occur when excessive copper enters into a body. Bioremediation, particularly microbial remediation, is gradually gaining attention because of its advantages of in-situ remediation, low cost, environmental friendliness, and the like.

The invention discloses a patent CN201510885689.5 < a strain of Cupriavidus capable of converting heavy metals and application thereof > discloses that the Cupriavidus strain has strong oxidizing capability to ferrous iron and can oxidize the ferrous iron into ore in a nitrate environment, and can be used for improving soil; the strain has strong reduction effect on arsenic, has stronger reduction effect on arsenic under the condition of no nitrate and ferrous ions, and can be used for repairing arsenic-polluted soil.

The invention patent CN201810855138.8 application of cuprioides (Cupriavidus sp) in heavy metal contaminated soil remediation discloses that cuprioides (Cupriavidus sp) can tolerate heavy metal ions, adsorb heavy metal ions, increase the pH of a soil water phase microenvironment and generate struvite with nitrogen and phosphorus slow-controlled fertilizer release effects, and is used for heavy metal contaminated soil remediation, particularly cadmium contaminated soil remediation, not only can fix cadmium ions in soil through adsorption and deposition, reduce the bioavailability of cadmium in soil, but also can improve soil fertility and solve the technical problems of soil acidification and the like.

However, the prior art does not disclose the use for the remediation of Cu in soil²⁺Cuprinobacter greedy. In the field of biotechnology, Cu²⁺The pollution is still an unsolved problem, the invention discovers a new cuppriavdus sp HXC-8 strain in mimosa pudica, and the cuppriavdus sp HXC-8 strain can resist heavy metal copper and improve the Cu tolerance²⁺Contaminated seed germination rate and promotion of Cu exposure²⁺Contaminated seedlings were grown.

Disclosure of Invention

The invention aims to provide a heavy metal-resistant cuprioides (cupriavidus sp.) HXC-8, wherein the cuprioides is classified and named as cupriavidus sp, is preserved in Guangdong province collection center of microbial strains in 2019, 4 and 12 days, and has the preservation number as follows: 60632, accession number: guangzhou city, first furious Zhonglu No. 100 large yard No. 59 building No. 5.

A copper greedy bacterium agent containing the copper greedy bacterium (Cupriavdus sp.) HXC-8 is provided.

The cupreous cuprinus (cupriavidus sp.) HXC-8 can resist heavy metal Cu²⁺。

The preparation method of the cupprid polyporus umbellatus microbial inoculum comprises the following steps: taking a culture solution of the cuprioides (Cupriavdus sp.) HXC-8 in logarithmic phase, centrifuging at low temperature, removing supernatant, adding sterile water, centrifuging, removing supernatant, washing mixed strains on the wall of a centrifugal tube with sterile water, and preparing into a cupriodes (Cupriavdus sp.) HXC-8 microbial inoculum.

Copper greedy bacterium (Cupriavdus sp.) HXC-8 microbial inoculum for improving Cu tolerance²⁺Use in the germination rate of contaminated seeds.

Preferably, the seeds are mimosa seeds.

Copper greedy bacterium (Cupriavdus sp.) HXC-8 microbial inoculum for promoting Cu-uptake²⁺Application in the growth of polluted seedlings.

Preferably, the seedling is a mimosa seedling.

Preparation of Cu by copper greedy (Cupriavdus sp.) HXC-8 microbial inoculum²⁺Application in contaminated soil remediation preparations.

The invention has the beneficial effects that: the invention provides a heavy metal-resistant copper greedy bacterium (cupriavidus sp.) HXC-8, wherein the copper greedy bacterium (cupriavidus sp.) HXC-8 is preserved in Guangdong province microorganism strain preservation center with the preservation number as follows: 60632, and preparing a corresponding cupreous bulimia agent, transfecting sensitive plant seeds with the cupreous bulimia agent and adding Cu with medium-low concentration²⁺The contaminated seeds are inoculated with copper greedy bacteria (Cupriavdus sp.) HXC-8, the germination rate is obviously higher than that of the non-inoculated copper greedy bacteria (Cupriavdus sp.) HXC-8, the inoculation can effectively slow down the pollution of heavy metal copper, and the germination rate of the sensitive plant seeds is improved. High concentration of Cu²⁺The contaminated seeds are inoculated with cuprinus (cupriavidus sp.) HXC-8, the germination rates of the inoculated group and the non-inoculated seeds are both greatly increased, the stress resistance is improved, and the growth promoting effect is realized. In the seedling growth process, the cupreous polyporus fungicide can effectively slow down Cu²⁺Stress to the growth of seedlings, promotion of substance accumulation and seedling growth; the cuprum bulimia microbial inoculum can obviously relieve copper pollution, increase tolerance to copper pollution, and promote the growth and biomass accumulation of seedlings of sensitive plants.

Drawings

FIG. 1 gram stain Pattern analysis

The long rod is Bacillus subtilis G +, the short rod is copper greedy bacterium G-

FIG. 2 Catalase Activity assay

FIG. 3 Congo Red experiment

FIG. 4 methyl Red reaction

FIG. 5 starch hydrolysis reaction

FIG. 6 gravy test

FIG. 7 BTB reaction

FIG. 8 influence of copper stress and inoculation of Cupriavidus mukuri inoculum on germination rate of Mimosa pudica seed

FIG. 9 influence of copper stress and inoculation of Cupriavidus mukuri inoculum on root length of Mimosa pudica seedlings

FIG. 10 effects of copper stress and inoculation of Cupriavidus inoculant on shoot growth of Mimosa pudica Linn

FIG. 11 copper stress and Effect of inoculation of Cupriavidus inoculant on Mimosa pudica Linn seedling Biomass

FIG. 12 shows the results of experiments on the minimum inhibitory concentration of copper greedy bacteria heavy metals

FIG. 13 copper ion removal Rate

Detailed Description

The present invention will be described below with reference to specific embodiments, but the scope of the present invention is not limited to the following examples. YMA medium (1L): 10g of mannitol, 4g of yeast powder and K₂HPO₃ 0.5g，MgSO₄·7H₂O 0.2g，NaCl 0.1g， CaCl₂0.05g, 4ml of RH trace element liquid, dH₂O996 ml and agar 15-16 g.

Rh trace element liquid (1L): h₃BO₃ 5.0g，Na₂MoO₄ 5.0g，dH₂0 1000ml

Example I isolation and culture of Rhizobium Mimosa

Collecting whole plant of Mimosa pudica Linn, cleaning with clear water, removing soil and stem, selecting large, full and fresh root nodule at root, washing off impurities, soaking in 95% ethanol for 5min, and adding 0.1% HgCl₂Surface sterilization for 5min, followed by 10 washes with sterile water under sterile conditions.

And (3) under the condition of aseptic operation, shaking up the sterile water flowing down from the root nodules washed for the last time, pouring a small amount of the sterile water into the YMA culture medium for uniform coating, and carrying out inverted culture at the temperature of 28 ℃ for 3-5 days. If no colony appears, the bacterial colony is proved to be cleaned before the inoculation of the copper greedy bacteria, otherwise, the reason of the existing colony needs to be separated out, and whether the colony appearing in the culture medium inoculated with the copper greedy bacteria is a rhizobium colony is verified.

Rhizobium characteristics: short rod-shaped gram-negative bacteria with large viscosity and fast growth speed grow into bacterial colony at 28 ℃ for 24-48h, and the bacterial colony is in a circular, milky white and semitransparent rhinorrhea-shaped bulge.

In the case of aseptic manipulation, a single nodule was cut in half, the half nodule was held with sterile forceps, the cut was placed facing the YMA medium, four half-cut nodules were placed evenly in each dish, and inverted for 3-5 days at 28 ℃. After the occurrence of the strains, selecting a single colony conforming to the shape of the rhizobium to carry out purification culture, and carrying out purification culture for three times to obtain a strain, wherein the strain is named as HXC-8.

EXAMPLE II gram stain identification of Strain HXC-8

And (3) identifying the purified strain HXC-8 by a gram staining method, wherein the identification result shows that the strain is in a red short rod shape, so that the strain is judged to be a gram-negative short bacillus. The specific identification steps are as follows:

(1) picking a few bacteria in a round semitransparent milky rhinorrhea single bacterial colony by using an inoculating loop, and uniformly coating the bacteria on a glass slide dropped with sterile water; meanwhile, a small amount of bacillus subtilis is picked and coated in the same drop of sterile water as a reference; uniformly coating the fungi, naturally drying, and then burning for 2-3 times on an alcohol burner to fix;

(2) dripping crystal violet dye liquor for dyeing for 1-2 min, washing the dye liquor by using fine water flow, and absorbing residual water by using filter paper;

(3) mordant dyeing with iodine solution for 1min, and washing with water;

(4) absorbing residual water by using filter paper, inclining the glass slide, adding 95% ethanol in a dropping tube to decolor for 20-30 s under a white background until the flowing ethanol is colorless, and immediately washing with water;

(5) dyeing with safranine dye liquor for 1min, and washing with water;

(6) microscopic examination is carried out after drying, and the microscopic examination result shows that the cupriosis is short bacillus and gram-negative bacteria (red), and the bacillus subtilis is long rod-shaped and gram-positive bacteria (purple), as shown in figure 1.

EXAMPLE III 16S rDNA sequencing identification of Strain HXC-8

1. Genomic DNA extraction

1) The strain on the medium was placed in a mortar beside the alcohol burner flame and ground with liquid nitrogen.

2) The ground bacteria are put into a 1.5mL centrifuge tube, the name of the strain is marked, 0.6mL of TE (pH 8.0) is added, and the mixture is uniformly sucked and beaten by a gun head to fully suspend the bacteria.

3) Add 250. mu.L of 10% SDS, mix by gentle inversion.

4) Add 3. mu.L proteinase K (20 ng/. mu.L), mix gently, and water bath at 37 ℃ for 1 h.

5) Add 150. mu.L of 5mol/L NaCl and mix gently.

6) Add 150. mu.L of 2% CTAB, mix gently, and water bath at 65 ℃ for 20 min.

7) Centrifuging at 12000rpm for 20 min.

8) Carefully sucking the supernatant into a new 1.5ml centrifuge tube, adding equal volume of isopropanol, mixing well, standing at room temperature for 30min, 12000rpm, and centrifuging at 4 deg.C for 10 min.

9) The supernatant was aspirated off, the liquid drained on absorbent paper, 750. mu.L 70% ethanol was added, the tube wall was flicked, the precipitate was suspended and inverted several times, centrifuged at 12000rpm at 4 ℃ for 2 min.

10) Add 30. mu.l of purified water to each tube to dissolve the precipitate (RNase in water at a final concentration of 10 ng/. mu.l), flick the tube wall with hand and dissolve overnight at 4 ℃.

DNA electrophoresis detection

PCR amplification

16s primer sequence:

Primer1(27f)：AGAGTTTGATCMTGGCTCAG

Primer2(1492R)：TACGGYTACCTTGTTACGACTT

reaction system:

H₂o17.8 μ L; buffer 3. mu.L; dNTP 2. mu.L; primer 13 μ L; primer 23 μ L; 1 mu L of DNA template; enzyme 0.2 μ L; the total volume was 30. mu.L.

Reaction conditions are as follows:

1.95℃5min

2.95℃30sec；55℃30sec；72℃1min；35cycle

3.72℃10min

4.12 ℃ forever 4. identification of electrophoresis PCR product

5. And (4) performing computer sequencing and outputting a peak diagram.

6. Results

(1) Picture for detecting PCR product by agarose gel electrophoresis

Electrophoresis conditions: 3 μ L of sample + 1% agarose gel, Marker band composition: 100bp, 250bp, 500bp, 750bp, 1000bp, 2000bp, 3000bp and 5000 bp. The 750bp band concentration was 60 ng/3. mu.L, which is indicated as a highlight band, and the remaining band concentrations were 30 ng/3. mu.L. The electrophoresis direction is from bottom to top.

(2) Gene sequences

The sequence of 16S rDNA of the strain HXC-8 is shown in SEQ ID NO. 1.

(3) Blast results

According to the blast result, the strain HXC-8 has a closest affinity relationship with the Pythium (Cupriavdus sp.) HBU52001, so the strain is judged to be a genus (Cupriavdus sp.) strain which is totally called as copper greedy (Cupriavdus sp.) HXC-8, and the copper greedy (Cupriavdus sp.) HXC-8 is preserved in the Guangdong province collection center of microorganism strains with the preservation number: 60632.

EXAMPLE four physiological and Biochemical Properties of cupreous cuprinus HXC-8

1.H₂O₂Enzyme Activity assay

As shown in FIG. 2, the cupronickel strain HXC-8 was inoculated on YMA solid medium, cultured at 28 ℃ for 3 days, and 1ml of 3% H was taken₂O₂Dripping on lawn, each treatment is repeated three times; if the bubble appears within 5min, the bubble is positive, and the bubble is negative. The results show that H is added₂O₂More bubbles are rapidly generated, which indicates that the cupreous polyporus HXC-8H₂O₂The enzyme activity is higher.

2. Congo Red test

Congo Red assay was used to determine whether the cell surface components contained cellulose. Congo red can dye cellulose into red compound, so bacterial colony turns red, which is a positive reaction and shows that cellulose exists on the surface of cells; if the colony color is not changed, the reaction is negative, and the cell surface has no or little cellulose. Inoculating the copper greedy bacterium HXC-8 on Congo red culture medium, and culturing at 28 ℃ for 3d, wherein each treatment is repeated three times. Congo red staining reaction shows that the colony color of the cupreous greedy bacterium HXC-8 changes from white to red, and absorbs Congo red dye, which indicates that the content of cellulose in the cell surface components is high, as shown in FIG. 3.

MR experiment

The MR experiment can detect whether the cupreous greedy bacterium HXC-8 generates organic acid. During the sugar metabolism, the microorganism decomposes glucose to produce pyruvic acid, which is further decomposed into formate, acetate, lactate, etc., to make the medium acidic, and its pH is lowered to 4.2 or less, so that the methyl red indicator added to the medium changes from orange (pH6.3) to red (pH4.2) or does not change color (still yellow). The phenomenon of red color change is called methyl red test positive reaction, and if it is still yellow, it is called methyl red test negative reaction. The color change range of methyl red is from pH4.2 (red) to pH6.3 (yellow). The specific method comprises the following steps: the cupreous greedy strain HXC-8 was inoculated on methyl red medium and cultured at 28 ℃ for 3 days, 3 replicates for each treatment. The MR reaction shows that the colony of the cuprioides HXC-8 is still yellow, which indicates that pyruvic acid is not further decomposed into formic acid, acetic acid, lactic acid and the like in the sugar metabolism process of the cuprioides HXC-8, and is shown in figure 4.

4. Experiment of hydrolyzed starch

The activity of amylase in cupronickel bacteria HXC-8 can be detected by a starch hydrolysis experiment. Amylase hydrolyzes starch into maltose and glucose, which do not turn blue in iodine solution and form transparent circles on the culture medium. If a transparent ring appears, the reaction is positive; no transparent circle is negative reaction. The specific method comprises the following steps: inoculating copper greedy bacterium HXC-8 on a starch culture medium, culturing at 28 ℃ for 3d, repeating each treatment for three times, dripping a proper amount of iodine solution, and observing the result. The starch hydrolysis experiment shows that white transparent circles appear on the colonies of the cupreous greedy bacterium HXC-8, which indicates that the cupreous greedy bacterium HXC-8 contains amylase and has higher activity, and is shown in FIG. 5.

5. Gravy test

And detecting the growth condition of the cupreous polyprioides HXC-8 on the meat juice peptone. Inoculating the cupreous polyprioides HXC-8 on a meat extract peptone growth medium, culturing at 28 ℃ for 3d, repeating three times for each treatment, and observing the result. The gravy experiment shows that the culture medium of the cupreous polyprioides HXC-8 is very clear, and the bacterial colony is rarely and almost not proliferated, which indicates that the cupreous polyprioides HXC-8 is not suitable for growing on the gravy culture medium, as shown in FIG. 6.

BTB experiment

Detecting whether the bacteria produce acidic substances. BTB is a weak monobasic organic acid, which can be represented as HIn, in acidic solution, molecule HIn is the predominant form it exists, giving the solution a yellow color; in alkaline solution, ion In^-Is the predominant form in which it is present, the solution is blue in color. The specific method comprises the following steps: inoculating the copper greedy bacterium HXC-8 on YMA culture medium containing 0.5% of bromothymol blue, and culturing in a constant-temperature incubator at 28 ℃ for 3d, wherein each treatment is repeated three times; and (6) observing the result. The BTB experiment showed that the culture medium of Cupriavium HXC-8 turned yellow, indicating the production of acidic substances during growth (FIG. 7).

As can be seen from the above, the cupreous bacterium HXC-8H₂O₂The enzyme activity is higher; congo red staining reaction is positive (the cellulose content on the cell surface is high); the MR experiment is negative, namely the pyruvic acid is not further decomposed into formic acid, acetic acid, lactic acid and the like in the metabolism; the thallus contains amylase and has higher activity; cupreous greedy HXC-8 is not suitable for growth on gravy medium(ii) a During the growth process, certain acidic substances are generated.

Example five bacterial agent pair of cupreous griseus²⁺Growth promoting effect of contaminated mimosa seed germination and seedling growth

1. Preparation of cuprum bulimia agent

Inoculating cuppridinium bulimia HXC-8 strain into YMA liquid culture medium under aseptic condition, performing shake culture at 28 deg.C at 175r/min together with the aseptic YMA liquid culture medium (blank control) without strain, measuring OD value (wavelength 600) every 2h after 6h, measuring three repeated values every time, taking the average value, and drawing the absorbance change curve.

The growth log phase of the cupreous greedy strain HXC-8 is measured to be 16-22 h. Taking 100ml of culture solution of copper greedy bacteria HXC-8 in logarithmic phase, carrying out low-temperature ultracentrifugation for 3min at 10000r/min and 4 ℃, removing supernatant, adding a small amount of sterile water, centrifuging for 8min at 10000r/min and 4 ℃ again, removing supernatant, washing mixed strains on the wall of a centrifugal tube with sterile water, and preparing into 100ml of copper greedy bacteria agent.

2. Mimosa pudica seed collecting and bacteria staining process

Collecting full, mature and similar wild Mimosa pudica seed in autumn, soaking in 95% ethanol for 7min, and soaking in 0.1% HgCl₂Sterilizing the solution for 5min, and adding 0.1% HgCl₂Soaking in the solution for 5 min; washing with sterile water under sterile condition for 5 times, and removing dormancy (cutting (or tying) a small opening at the position near hilum of seed coat of Mimosa pudica Linn) with sterilized absorbent paper.

Soaking Mimosa pudica seed in copper-philic bacteria for 6h, soaking blank control group in sterile water for 6h, wherein the bacteria suspension is just over the seed during soaking, and naturally drying in shade for 1h after soaking.

3. Grouping of heavy metal copper solution concentrations

CuCl with the preparation concentrations of 0mg/L, 100mg/L, 200mg/L, 300mg/L, 400mg/L, 500mg/L, 600mg/L and 800mg/L respectively₂Mixing the solution with plant nutrient solution at a ratio of 1:1 to obtain final concentrations of 0mg/L, 50mg/L, 100mg/L, and 15 mg/LHeavy metal Cu of 0mg/L, 200mg/L, 250mg/L, 300mg/L, 400mg/L²⁺To simulate the heavy metal Cu exposure²⁺Contaminated soil. 4 replicates were set, 30 mimosa seeds per replicate.

4. Sowing of mimosa seeds

Mimosa pudica seed and seedling culture environment made of absorbent paper and culture dish, and Cu²⁺Soaking the mixed culture solution just without filter paper, simulating the heavy metal polluted soil environment to culture the mimosa seeds, and replacing the absorbent paper and the culture solution every two days. The culture dish is placed in an RXZ type (multi-stage programming) intelligent artificial climate box for culture under the conditions of illumination for 10h/d, temperature of 28 ℃, relative humidity of 50 percent and good ventilation.

5. Determination of sensitive plant seed germination and seedling growth conditions

(1) And observing and recording the germination condition of the mimosa seeds every 24h until no seed continues to germinate (germination standard: germ breaks through seed coat). Germination rate is the number of sprouts/number of seeds

(2) After the sensitive plant seeds basically germinate, continuously culturing for more than 7 days, separating roots and stems of the seedlings, measuring the root length and the stem length (excluding cotyledon length) of the seedlings by using a measuring ruler, weighing the fresh root weight and the fresh stem weight (including cotyledon) of the seedlings by using an electronic balance to obtain the biomass of the seedlings, and calculating the average value to obtain the growth indexes (the root length, the stem length and the biomass) of the sensitive plant seedlings.

(3) Tolerance index is often used to comprehensively assess plant response to heavy metal treatment.

Plant traits of heavy metal-free treated group/plant traits of heavy metal-free treated group

When the tolerance index is more than 0.5, the plant has stronger tolerance to heavy metals and better growth, and the higher the tolerance index is, the stronger the tolerance is; when the tolerance index is less than 0.5, the toxic effect of the heavy metal on the plant is obvious, and the heavy metal of the plant at the concentration is difficult or incapable of growing.

6. Results of the experiment

(1) Cuprum greedy bacterium agentEffectively improve the Cu receiving capacity²⁺Germination rate of contaminated seeds.

As shown in FIG. 8, Cu²⁺The concentration is 0-200 mg.L^-1In time, the influence of the inoculation on the germination rate is not obvious; in Cu²⁺The concentration is 250 mg.L^-1And 300 mg. L^-1In time, the seed germination rate of the inoculated copper greedy bacterium is obviously higher than that of the non-inoculated copper greedy bacterium. Specific indexes are as follows: seed not receiving Cu²⁺When in pollution, the germination rate is 96-100%; cu²⁺Contamination was 250 mg.L^-1And 300 mg. L^-1When the method is used, the germination rates of seeds which are not inoculated with the microbial inoculum are respectively reduced to 52.5 percent (SD +/-11) and 51.6 percent (SD +/-7.6); after the cuprum bulimia microbial inoculum is transfected by seeds, the germination rates are respectively improved to 84.2% (SD +/-11) and 84.0% (SD +/-14), and are improved by 1.60 times and 1.63 times; seed germination vs. Cu²⁺The tolerance index to contamination was determined from 0.62(250 mg. multidot.L) before inoculation^-1) And 0.51(300 mg. L)^-1) To 0.88 and 0.87, respectively. Therefore, the inoculation can effectively slow down the influence of heavy metal copper pollution on the seeds and improve the germination rate of the sensitive plant seeds. In Cu²⁺Is 400 mg.L^-1In the process, the germination rates of the inoculated group and the non-inoculated seed are greatly increased, and probably the sensitive plant generates stress resistance to the heavy metal copper at the concentration and has the growth promoting effect.

(2) Cuprum donavani microbial inoculum for effectively promoting growth of Mimosa pudica seedlings

As shown in FIG. 9, the root length of the seedlings not inoculated with the microbial inoculum was 11mm when they were not contaminated with Cu²⁺The pollution concentration is increased, and the root length is reduced to 3.5mm (250 mg. L)^-1Cu²⁺Contamination). The copper-transfecting bacterium agent can promote the growth of roots, but has a small effect of 100 mg.L^-1、150mg·L^-1、250mg·L^-1Under the copper pollution, the cupreous bacteria agent respectively increases the roots by 1.23 times, 1.19 times and 1.31 times.

However, cupreous trivialis agents can increase stem growth vs. Cu²⁺The tolerance of the compound fertilizer can effectively promote the growth of seedlings. As shown in FIG. 10, seedlings that were not inoculated with the inoculum were not Cu-inoculated²⁺When contaminated, the stem length was 7.9mm, when Cu²⁺The pollution is increased to 200 mg.L^-1When the stem length is reduced to 4.3 mm; cu²⁺The pollution concentration is increased to 250-300mg·L^-1When this happens, the stem stops growing (0 mm); when the cuprum bulimia agent is transfected, the stem length is remarkably increased to 1.45mm (250 mg.L)^-1)、2.84mm(300mg·L^-1)、8.0mm(400mg·L^-1). Growth of seedling stem of microbial inoculum to Cu²⁺Tolerance appeared, and the tolerance index increased from 0.56 to 1.03(150 mg. L)^-1) Increases from 0 to 0.17 and 0.33(250-300 mg. L)^-1) Increased from 0.36 to 0.94(400 mg. L)^-1). Shows that the cupriosis can effectively slow down Cu²⁺The stress of pollution on the growth of seedlings is enhanced, and the copper concentration of the sensitive plant is 150-^-1The tolerance to copper can promote the growth of plants.

(3) Cuprum donavani microbial inoculum for effectively promoting increase of biomass of Mimosa pudica seedlings

As shown in FIG. 11, seedlings that were not inoculated with the inoculum were not Cu-inoculated²⁺When contaminated, the biomass was 18.5mg, Cu²⁺The concentration is 250 mg.L^-1、300mg·L^-1、400mg·L^-1When the biomass of the seedlings is reduced to 1.48mg, 2.15mg and 5.22 mg; after the copper greedy bacterium is transfected, the biomass of seedlings is respectively increased to 4.36mg, 6.16mg and 16.32mg, namely the biomass of the seedlings of the sensitive plants can be increased by about 3 times by the infection of the bacteria, and the Cu is slowed down²⁺Stress, which is beneficial to the accumulation of substances in the growth of seedlings and promotes the growth of the seedlings of the sensitive plants. After transfection with a cuprum bulimia agent, seedling biomass accumulation is performed on Cu²⁺The tolerance index of (2) is increased from 0.34 to 0.95(150 mg. L)^-1) From 0.08 to 0.23(250 mg. L)^-1) Increased from 0.11 to 0.32(300 mg. L)^-1) From 0.28 to 0.87(400 mg. L)^-1). Shows that the cupreous polyporus fungicide can effectively slow down Cu in the growth process of seedlings²⁺Stress to the growth of seedlings, promotion of substance accumulation and seedling growth.

Example six copper greedy bacteria to heavy metal Cu²⁺Adsorption and removal of

1 test strains

Cupriavdus sp HXC-8

2 research methods

(1) Activation of cupreous polyprioides:

firstly, streaking the copper greedy strain on a solid YMA culture medium for inoculation, and then culturing in an incubator at the temperature of 28 ℃ for 3 days until the lawn is full.

(2) Preparing a cuprum bulimia bacterial suspension:

in the cultivation of the full lawn, a ring colony is selected by an inoculating loop in 80mL YMA liquid medium (250mL conical flask), and the mixture is fully shaken for 15h by a shaking table (the rotating speed is 120r/min, and the temperature is 28 ℃) to obtain bacterial suspension.

(3) Heavy metal Cu²⁺Determination of the highest tolerated concentration

Preparation of heavy Metal Cu²⁺Concentration (mmol/L): 0. 0.1, 0.2, 0.3, 0.4, 0.5, 0.6 YMA solid medium for use.

And (3) respectively dripping 1mL of the bacterial suspension prepared in the step (2) into YMA solid culture medium containing the heavy metal concentration for coating culture, and observing the growth condition of the bacterial colony every 4 h. Thereby verifying the maximum MIC value.

(4) Determination of removal rate of heavy metal ions

Preparing a copper-greedy bacterium suspension under copper stress: the bacterial suspensions were added to 100mL of liquid medium containing heavy metals (heavy metal concentration: 0, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6mmol/L), cultured in a shaker (rotation speed 120r/min, temperature 28 ℃) for 24 h.

② preparation of copper standard mother liquor (100 mug/ml): accurately weighing 0.390g of blue vitriol, fixing the volume in a 1000ml volumetric flask by 1% nitric acid, and mixing evenly. Thus preparing 100ug/ml mother liquor.

Preparation of copper standard stock solution: according to the measurable range of the instrument (0-2.5. mu.g/mL), the mother liquor is diluted 250 times, 200 times, 125 times, 100 times, 50 times and 40 times with 1% nitric acid to obtain 0.4. mu.g/mL, 0.5. mu.g/mL, 0.8. mu.g/mL, 1.0. mu.g/mL, 2.0. mu.g/mL and 2.5. mu.g/mL copper standard stock solutions, and 7mL of the standard stock solutions are taken out of the centrifuge tube. (the centrifuge tube is soaked in 10% nitric acid for 12-24 hours before use, washed with clean water and then rinsed with deionized water for later use).

③ diluting the sample liquid: the heavy metal (copper) bacteria culture solution with different concentrations after 24h (stationary phase) culture is taken out and filtered by a microfiltration membrane with the diameter of 0.45 mu m. Due to the flameThe range of copper measured by an atomic absorption spectrometer is 0-2.5 mu g/mL, and Cu in the bacterial liquid²⁺The concentration is 0-0.6mmol/L, so the bacterial liquid is reduced by 16 times to prepare a sample liquid.

Determination of copper content: and (3) putting the standard solution into a flame atomic absorption spectrometer to measure the copper content and drawing a standard curve. And (4) measuring the OD value of the prepared sample liquid in a flame atomic absorption spectrometer.

Obtaining a standard curve formula according to the standard curve obtained by measurement as follows: y is 0.0121x +0.0019, and the Cu after 24h is calculated according to the standard curve formula and the measured absorbance²⁺The concentration is calculated by the following formula²⁺The removal rate of (2):

3 results

From experiments, Cu²⁺When the concentration is 0.2mmol/L, the copper greedy (cupriavidus sp.) HXC-8 strain grows better, and the colony amount reaches more than 90% after 20 hours. Cu²⁺When the concentration is 0.4 and 0.6mmol/L, the bacterium is cultured for 20-24h, the growth of the bacterium is slow, and the colony amount is only 10%; culturing for 28-32h, wherein bacterial colonies are gradually increased, and the bacterial colony amount is 15%; the number of colonies increased at 40h to 50%. When Cu²⁺At concentrations of 0.8, 1.0, 1.2, 1.4, 1.6mmol/L, the Cupriavdus sp HXC-8 strain did not grow. Thus, the Cupriavdus sp HXC-8 strain is responsible for Cu²⁺The highest tolerated concentration of (2) was 0.6mmol/L (FIG. 12).

From the calculation results, it was found that the Cupriavdus sp HXC-8 strain is present in Cu²⁺The concentration is in the range of 0.1-0.6mmol/L, with Cu²⁺The removal rate gradually decreased with increasing concentration, and the maximum removal rate reached 97.94% (fig. 13). The prior art discloses removal rates greater than 30% as effective removal rates. Therefore, the copper-greedy bacterium (cupriavidus sp.) HXC-8 can effectively remove copper ions in the environment, and the maximum removal rate is 97.94%.

In conclusion, the microbial inoculum prepared from the cupriopsis cupriadus sp HXC-8 claimed by the invention can tolerate copper pollution with a certain concentration, can obviously relieve the copper pollution, relieves part of sensitive plant seeds polluted by copper, promotes the seed germination, and improves the germination rate; promoting the growth of Mimosa pudica Linn seedlings and biomass accumulation. Meanwhile, the cuprinus greedy (cupriavidus sp.) HXC-8 strain claimed by the invention can effectively remove copper ions in the environment.

Sequence listing

<110> Han mountain teaching college

<120> heavy metal-resistant cupronickel bacterium, preparation method and application of bacterium agent

<160> 1

<170> SIPOSequenceListing 1.0

<210> 1

<211> 1433

<212> DNA

<213> cupronickel bacterium (cupriavidus sp.)

<400> 1

ggttggcggc tgcttaacat gcagtcggac ggcagcgcgg gcttcggcct ggcggcgagt 60

ggcgaacggg tgagtaatac atcggaacgt gtcctggagt gggggatagc ccggcgaaag 120

ccggattaat accgcatacg ctctgaggag gaaagcgggg gatcttcgga cctcgcgctc 180

aaggggcggc cgatggcaga ttagctagtt ggtggggtaa aggcctacca aggcgacgat 240

ctgtagctgg tctgagagga cgaccagcca cactgggact gagacacggc ccagactcct 300

acgggaggca gcagtgggga attttggaca atgggggcaa ccctgatcca gcaatgccgc 360

gtgtgtgaag aaggccttcg ggttgtaaag cacttttgtc cggaaagaaa acttcgccgc 420

caataccggt ggaggatgac ggtaccggaa gaataagcac cggctaacta cgtgccagca 480

gccgcggtaa tacgtagggt gcgagcgtta atcggaatta ctgggcgtaa agcgtgcgca 540

ggcggttcgc taagaccgat gtgaaatccc cgggcttaac ctgggaactg cattggtgac 600

tggcgggcta gagtatggca gaggggggta gaattccacg tgtagcagtg aaatgcgtag 660

agatgtggag gaataccgat ggcgaaggca gccccctggg ccaatactga cgctcatgca 720

cgaaagcgtg gggagcaaac aggattagat accctggtag tccacgccct aaacgatgtc 780

aactagttgt cgggtcttca ttgacttggt aacgtagcta acgcgtgaag ttgaccgcct 840

ggggagtacg gtcgcaagat taaaactcaa aggaattgac ggggacccgc acaagcggtg 900

gatgatgtgg attaattcga tgcaacgcga aaaaccttac ctacccttga catgtacgga 960

accttgccga gaggtgaggg tgcccgaaag ggagccgtaa cacaggtgct gcatggctgt 1020

cgtcagctcg tgtcgtgaga tgttgggtta agtcccgcaa cgagcgcaac ccttgtccct 1080

agttgctacg caagagcact ctagggagac tgccggtgac aaaccggagg aaggtgggga 1140

tgacgtcaag tcctcatggc ccttatgggt agggcttcac acgtcataca atggtcggaa 1200

cagagggttg ccaagccgcg aggtggagcc aatcccagaa aaccgatcgt agtccggatc 1260

gcagtctgca actcgactgc gtgaagctgg aatcgctagt aatcgcggat cagcatgccg 1320

cggtgaatac gttcccgggt cttgtacaca ccgcccgtca caccatggga gtgggtttta 1380

ccagaagtgg ctagtctaac cgcaaggagg acggtcacca cggtagatcg tgc 1433

Claims

1. A heavy metal-resistant cuppriavdus sp HXC-8 strain is characterized in that the cuppriavdus sp HXC-8 strain is preserved in Guangdong culture collection of microorganisms with the preservation number as follows: 60632.

2. A cupreous bulimia agent, wherein the cupreous bulimia agent comprises cupreous bulimia (cupruvsp.) HXC-8 of claim 1.

3. A method of preparing the cuppridinium bulimia agent of claim 2, wherein the method comprises the steps of: taking culture solution of cupriosis cuprioides (Cupriavdus sp.) HXC-8 in logarithmic phase, centrifuging at low temperature, removing supernatant, adding sterile water, centrifuging, removing supernatant, washing mixed strains on the wall of a centrifugal tube with sterile water, and preparing into cupriosis cuprioides microbial inoculum.

4. The cupreous bulimia agent as claimed in claim 2 for increasing Cu tolerance²⁺Use in the germination rate of contaminated seeds.

5. The use of claim 4, wherein the seed is Mimosa pudica seed of Leguminosae.

6. The cupronickel bacterium agent of claim 2 for promoting Cu tolerance²⁺Application in the growth of polluted seedlings.

7. Use of cuprinusgreedy (cupriavidus sp.) HXC-8 as claimed in claim 1 for the preparation of Cu²⁺Application in contaminated soil remediation preparations.