CN110628678B - Heavy metal-resistant cupronickel bacterium, preparation method and application of bacterium agent - Google Patents

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⁺The preparation for repairing the polluted soil has wide application prospect.

Description

A strain of copper greedy bacteria resistant to heavy metals and preparation method and application of inoculum

technical field

The invention belongs to the field of new strain screening technology and environmental microorganism technology, and particularly relates to a heavy metal-resistant copper greedy bacteria (Cupriavidus sp.) HXC-8 and a preparation method and application thereof.

Background technique

In recent years, the heavy metal pollution of soil from mining, chemical industry and life has become increasingly serious. Heavy metals cannot be decomposed by themselves in the soil, the residual time is long, the harm is great, and they are easily absorbed by crops and affect human health. Copper pollution is a common heavy metal pollution. Too much copper in the soil accumulates and stays in the roots after being absorbed by plants, which hinders the absorption of other components, especially iron, by the root system, resulting in poor plant growth and excessive copper entering the body. Nausea, vomiting, epigastric pain, acute hemolysis and renal tubular deformation and other poisoning phenomena. Bioremediation, especially microbial remediation technology, has gradually attracted attention due to its advantages of in situ remediation, low cost and environmental friendliness.

It has become a current research trend to use copper greedy bacteria to remediate soil heavy metal pollution. Invention patent CN201510885689.5 "a strain of copper greedy bacteria that can transform heavy metals and its application" discloses that copper greedy bacteria strains can affect sub-substances in a nitrate environment. Iron has strong oxidizing ability and can oxidize ferrous ore to form minerals, which can be used for soil improvement; this strain also has strong reducing effect on arsenic. It has a stronger reducing effect and can be used to remediate arsenic-contaminated soil.

Invention patent CN201810855138.8 "A kind of copper greedy bacteria and copper greedy bacteria preparation and application of copper greedy bacteria preparation in the restoration of heavy metal contaminated soil" discloses that copper greedy bacteria (Cupriavidus sp.) can tolerate heavy metal ions, adsorb heavy metal ions, Increase the pH of the soil water microenvironment and produce struvite with slow and controlled release of nitrogen and phosphorus, which can be used for heavy metal contaminated soil remediation, especially cadmium contaminated soil remediation, not only can fix cadmium ions in soil by adsorption and deposition , reduce the bioavailability of soil cadmium, and can improve soil fertility and solve technical problems such as soil acidification.

However, the prior art does not disclose the copper-loving bacteria for remediation of Cu ²⁺ in soil. In the field of biotechnology, the contamination of Cu ²⁺ is still an unsolved problem, the present invention found a new strain of copper greedy bacteria (Cupriavidus sp.) HXC-8 in Mimosa, the copper greedy bacteria (Cupriavidus sp. ) HXC-8 is resistant to heavy metal copper, improves the germination rate of Cu ²⁺ -contaminated seeds and promotes the growth of Cu ²⁺ -contaminated seedlings.

SUMMARY OF THE INVENTION

The object of the present invention is to provide a heavy metal-resistant cupriavidus sp. HXC-8, the classification name of the cupriavidus sp. is Cupriavidus sp., which was preserved in Guangdong Province Microbial Bacteria on April 12, 2019 Species Collection Center, the deposit number is: GDMCC NO: 60632, and the deposit address is: 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou City.

A kind of copper greedy bacteria inoculation agent containing described copper greedy bacteria (Cupriavidus sp.) HXC-8.

The Cupriavidus sp. HXC-8 is resistant to heavy metal Cu ²⁺ .

Described copper greedy bacteria preparation method comprises the following steps: get the logarithmic phase copper greedy bacteria (Cupriavidus sp.) HXC-8 culture solution, centrifuge at low temperature, remove supernatant, add sterile water, centrifuge, remove supernatant, The mixed bacterial species on the wall of the centrifuge tube was rinsed with sterile water to prepare a Cupriavidus sp. HXC-8 bacterial preparation.

Application of Cupriavidus sp. HXC-8 inoculant in improving the germination rate of seeds contaminated with Cu ²⁺ .

Preferably, the seeds are Mimosa seeds.

Application of Cupriavidus sp. HXC-8 inoculant in promoting the growth of Cu ²⁺ contaminated seedlings.

Preferably, the seedling is a Mimosa seedling.

Application of Cupriavidus sp. HXC-8 inoculant in the preparation of Cu ²⁺ contaminated soil remediation preparations.

The beneficial effects of the present invention are as follows: the present invention provides a heavy metal-resistant copper bacterium (Cupriavidus sp.) HXC-8, which is preserved in the Guangdong Province Microorganism Culture Collection Center, deposit number is: GDMCC NO: 60632, and prepared corresponding copper greedy bacteria inoculum, utilizes described copper greedy bacteria inoculum to transfect Mimosa seeds, and the seeds polluted by medium and low concentration Cu ²⁺ are inoculated with copper greedy bacteria (Cupriavidus sp.) HXC-8, the germination rate was significantly higher than that without inoculation of copper greedy bacteria (Cupriavidus sp.) HXC-8, inoculation can effectively reduce the pollution of heavy metal copper and improve the germination rate of Mimosa seeds. Seeds contaminated with high concentrations of Cu ²⁺ were inoculated with Cupriavidus sp. HXC-8, and the germination rates of the inoculated and non-inoculated seeds increased significantly, improving their stress resistance and promoting growth. And in the process of seedling growth, the inoculum of copper greedy bacteria can effectively slow down the stress of Cu ²⁺ on the growth of seedlings, promote the accumulation of substances and the growth of seedlings; the inoculum of copper greedy bacteria can significantly relieve copper pollution and increase the tolerance to copper pollution. , to promote Mimosa seedling growth and biomass accumulation.

Description of drawings

Figure 1. Analysis of Gram staining pattern

The long rod is Bacillus subtilis G+, and the short rod is Copper greedy G-

Figure 2 Determination of catalase activity

Figure 3 Congo red experiment

Figure 4 Methyl red reaction

Fig. 5 Starch hydrolysis reaction

Figure 6 Gravy experiment

Figure 7BTB reaction

Fig. 8 Effects of copper stress and inoculation of copper fungus on the germination rate of Mimosa seeds

Fig. 9 Effects of copper stress and inoculation of copper fungus on root length of Mimosa mimosa seedlings

Fig. 10 Effects of copper stress and inoculation of copper fungus on the stem length of Mimosa seedlings

Fig. 11 The effects of copper stress and inoculation of copper fungus on the biomass of Mimosa mimosa seedlings

Fig. 12 The experimental results of the minimum inhibitory concentration of heavy metals in copper greedy bacteria

Figure 13 Copper ion removal rate

Detailed ways

The present invention will be described below with reference to specific embodiments, but the protection scope of the present invention is not limited to the following examples. YMA medium (1L): mannitol 10g, yeast powder 4g, K ₂ HPO ₃ 0.5g, MgSO ₄ 7H ₂ O 0.2g, NaCl 0.1g, CaCl ₂ 0.05g, RH trace element solution 4ml, dH ₂ O 996ml , Agar 15-16g.

Rh trace element solution (1L): H ₃ BO ₃ 5.0g, Na ₂ MoO ₄ 5.0g, dH ₂ 0 1000ml

Embodiment 1. Isolation and cultivation of Mimosa rhizobia

Collect the whole mimosa, wash the collected mimosa with clean water, remove the clods and stems, select a large, plump, fresh nodule at the root, wash away impurities, soak it in 95% ethanol for 5 minutes, then use 0.1 % HgCl ₂ surface sterilization for 5 min, then under aseptic conditions, rinse with sterile water 10 times.

In the case of aseptic operation, shake the sterile water flowing down from the last rinsing root nodule, pour a small amount into YMA medium for uniform coating, and invert at 28°C for 3-5 days. If no colonies appear, it proves that the copper greedy bacteria have been cleaned before inoculation, otherwise the reasons for the colonies should be analyzed, and it should be verified whether the colonies appearing in the medium inoculated with copper greedy bacteria are rhizobia colonies.

Rhizobium characteristics: short rod-shaped, gram-negative bacteria, large in size, sticky, fast growth, 24-48h at 28 ° C to grow into colonies, colonies are round, milky white, translucent snot-shaped bulges.

Under sterile conditions, cut a single nodule in half, hold the half nodule with sterile tweezers, place the incision facing the YMA medium, and place four halves of nodules evenly in each petri dish at 28°C Inverted and cultured for 3 to 5 days. After the colony appeared, a single colony that conformed to the rhizobia morphology was picked for purification and culture. After three times of purification and culture, a strain was obtained, which was named HXC-8.

Example 2. Gram staining identification of strain HXC-8

The purified strain HXC-8 was identified by Gram staining, and the identification result showed that it was a red short rod, so it was determined to be a Gram-negative Brevibacterium. The specific identification steps are as follows:

(1) Pick a small amount of bacteria in a circular translucent milky white snot-like single colony with an inoculation ring, and evenly spread it on a glass slide with sterile water; at the same time, pick a small amount of Bacillus subtilis and spread it on the same drop Sterile water, as a control; spread the bacteria evenly, after natural drying, overheat it on an alcohol lamp 2-3 times to fix it;

(2) Add crystal violet dye solution dropwise for 1 to 2 minutes, wash off the dye solution with a thin stream of water, and absorb the residual water with filter paper;

(3) mordant with iodine solution for 1min, wash with water;

(4) Absorb residual water with filter paper, tilt the glass slide, add 95% ethanol to decolorize with a dropper for 20-30s under a white background, until the ethanol that flows out is colorless, and wash with water immediately;

(5) dye with safranine dye solution for 1min, wash with water;

(6) Microscopic examination was carried out after drying. The microscopic examination results showed that the copper greedy bacteria were Brevibacterium and gram-negative bacteria (red), and Bacillus subtilis was long rod-shaped, gram-positive bacteria (purple), as shown in Figure 1 shown.

Example 3. 16S rDNA sequencing identification of strain HXC-8

1. Genomic DNA Extraction

1) Take the strains on the medium beside the flame of an alcohol lamp in a mortar and grind with liquid nitrogen.

2) Put the ground bacteria into a 1.5ml centrifuge tube, mark the name of the strain, add 0.6mL TE (pH 8.0), and pipette evenly with a pipette tip to fully suspend the bacteria.

3) Add 250 μL of 10% SDS, invert and mix.

4) Add 3 μL proteinase K (20 ng/μL), mix gently, and water bath at 37°C for 1 h.

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

6) Add 150 μL of 2% CTAB, mix gently, and water bath at 65°C for 20 minutes.

7) Centrifuge at 12000rpm at room temperature for 20min.

8) Carefully aspirate the supernatant into a new 1.5ml centrifuge tube, add an equal volume of isopropanol, mix well, place at room temperature for 30min, centrifuge at 12000rpm for 10min at 4°C.

9) Aspirate off the supernatant, dry the liquid on absorbent paper, add 750 μL of 70% ethanol, flick the tube wall to suspend the precipitate and invert several times, centrifuge at 12000 rpm and 4° C. for 2 min.

10) Add 30 μl of purified water to each tube to dissolve the precipitate (Rnase is added to the water, the final concentration is 10 ng/μl), flick the wall of the tube by hand, and dissolve at 4°C overnight.

2. DNA electrophoresis detection

3. PCR amplification

16s primer sequence:

Primer1(27f): AGAGTTTGATCMTGGCTCAG

Primer2(1492R): TACGGYTACCTTGTTACGACTT

reaction system:

H ₂ O 17.8 μL; Buffer 3 μL; dNTP 2 μL; Primer1 3 μL; Primer2 3 μL; DNA template 1 μL; Enzyme 0.2 μL; total volume 30 μL.

Reaction conditions:

1. 95℃ for 5min

2. 95℃ 30sec; 55℃ 30sec; 72℃ 1min; 35cycle

3. 72℃ for 10min

4. 12℃ forever

4. Identification of PCR products by electrophoresis

5. On-board sequencing, output peak map.

6. Results

(1) Picture of PCR products detected by agarose gel electrophoresis

Electrophoresis conditions: 3 μL sample + 1% agarose gel, Marker band composition: 100bp, 250bp, 500bp, 750bp, 1000bp, 2000bp, 3000bp, 5000bp. The concentration of the 750bp band was 60ng/3μL, which was shown as a highlighted band, and the concentration of the other bands was 30ng/3μL. The electrophoresis direction is from bottom to top.

(2) Gene sequence

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

(3) Blast result

According to the blast results, the strain HXC-8 has the closest parental relationship with Cupriavidus sp. HBU52001. Therefore, the strain is determined to be a genus (Cupriavidus sp.) strain, whose full name is Cupriavidus sp. HXC-8, Copper greedy bacteria (Cupriavidus sp.) HXC-8 was deposited in the Guangdong Provincial Microbial Culture Collection Center, the deposit number is: GDMCC NO: 60632.

Embodiment 4. Physiological and biochemical characteristics of copper greedy bacteria HXC-8

1. H ₂ O ₂ enzyme activity assay

As shown in Figure 2, the described copper greedy bacteria HXC-8 strain was inoculated on YMA solid medium, cultivated at 28°C for 3 days, and 1ml of 3% H ₂ O ₂ was dropped on the bacterial lawn, and each treatment was repeated three times; If bubbles appear within 5 minutes, it is positive, otherwise it is negative. The results showed that the addition of H ₂ O ₂ quickly produced more bubbles, which indicated that the HXC-8H ₂ O ₂ had higher enzymatic activity.

2. Congo red experiment

The Congo red test is used to detect the presence of cellulose in cell surface components. Congo red can stain cellulose into a red complex, so the colony turns red, which is a positive reaction, indicating that there is cellulose on the cell surface; the colony color does not change, it is a negative reaction, and there is no or less cellulose on the cell surface. The described cupric bacteria HXC-8 was inoculated on Congo red medium, cultured at 28°C for 3 days, and each treatment was repeated three times. Congo red staining reaction showed that the color of the copper greedy bacteria HXC-8 colony changed from white to red, and absorbed the Congo red dye, indicating that the content of cellulose in the cell surface components was high, as shown in Figure 3.

3. MR experiment

MR experiments can detect whether the copper greedy bacteria HXC-8 produces organic acids. In the process of sugar metabolism, microorganisms decompose glucose to produce pyruvate, which is further decomposed into formic acid, acetic acid, lactic acid, etc., so that the medium becomes acidic, and its pH drops to 4.2 or lower, making the methyl group added to the medium. The red indicator changes from orange (pH 6.3) to red (pH 4.2) or does not change color (still yellow). The phenomenon of turning red is called a positive reaction of methyl red test, and if it is still yellow, it is a negative reaction of methyl red test. The discoloration of methyl red ranges from pH 4.2 (red) to pH 6.3 (yellow). Specific method: Inoculate copper greedy bacteria HXC-8 on methyl red medium, culture at 28°C for 3 days, and each treatment has three replicates. MR reaction showed that the colony of copper greedy bacteria HXC-8 was still yellow, indicating that in the process of sugar metabolism of copper greedy bacteria HXC-8, pyruvate was not further decomposed into formic acid, acetic acid, lactic acid, etc., as shown in Figure 4.

4. Hydrolyzed starch experiment

The starch hydrolysis experiment can detect the activity of amylase in copper greedy bacteria HXC-8. Amylase hydrolyzes starch into maltose and glucose, which will not change blue when encountering iodine solution, and will form a transparent circle on the medium. If there is a transparent circle, it is a positive reaction; if there is no transparent circle, it is a negative reaction. Specific method: inoculate copper greedy bacteria HXC-8 on starch medium, cultivate at 28℃ for 3 days, each treatment is repeated three times, drip an appropriate amount of iodine solution, and observe the results. The starch hydrolysis experiment showed that a white transparent circle appeared on the colony of copper greedy bacteria HXC-8, indicating that copper greedy bacteria HXC-8 contained amylase and had high activity, as shown in Figure 5.

5. Gravy Experiment

Detect the growth of the copper greedy bacteria HXC-8 on the gravy peptone. The described copper greedy bacteria HXC-8 was inoculated on the bouillon peptone growth medium, cultivated at 28°C for 3 days, and each treatment was repeated three times, and the results were observed. The gravy experiment showed that the copper greedy bacteria HXC-8 medium was very clear, and the colonies were few and almost no proliferation, indicating that the copper greedy bacteria HXC-8 was not suitable for growing on the gravy medium, as shown in FIG. 6 .

6. BTB experiment

Check whether the bacteria produce acidic substances. BTB is a monobasic organic weak acid, which can be expressed as HIn. In acidic solution, molecular HIn is the main form of its existence, making the solution yellow; in alkaline solution, ion ^In- is the main form of its existence, so the solution is blue color. Specific method: inoculate the copper greedy bacteria HXC-8 on YMA medium containing 0.5% bromothymol blue, and place it in a constant temperature incubator at 28°C for 3 days, with three replicates for each treatment; observe result. The BTB experiment showed that the medium of the copper greedy bacteria HXC-8 turned yellow, indicating that acidic substances were produced during the growth process (Fig. 7).

To sum up, it can be seen that the HXC-8 H ₂ O ₂ enzyme activity of the copper greedy bacteria is high; the Congo red staining reaction is positive (the content of cellulose on the cell surface is more); the MR test is negative, that is, its pyruvate is not further metabolized. It is decomposed into formic acid, acetic acid, lactic acid, etc.; the cell contains amylase with high activity; copper greedy bacteria HXC-8 is not suitable for growth on gravy medium; certain acidic substances are produced during the growth process.

Embodiment 5. The growth-promoting effect of copper fungus inoculum on the seed germination and seedling growth of Mimosa mimosa polluted by Cu ²⁺

1. Preparation of copper fungus inoculum

The copper greedy bacteria HXC-8 strain was inoculated in the YMA liquid medium under aseptic conditions, and together with the sterile YMA liquid medium (blank control) without the inoculated strain, placed under the conditions of 175r/min and 28°C Shaker culture was performed, and after 6h, the OD value (wavelength 600) was measured every 2h, three replicate values were measured each time, the average value was taken, and the absorbance change curve was drawn.

The logarithmic phase of the growth of the copper greedy bacteria HXC-8 was measured to be 16-22h. Take 100ml of the logarithmic phase culture medium of Copper greedy bacteria HXC-8, carry out low temperature ultracentrifugation at 10000r/min, 4℃ for 3min, remove the supernatant, add a small amount of sterile water, and again at 10000r/min, 4℃ Centrifuge for 8 minutes, remove the supernatant, rinse the mixed bacterial species on the wall of the centrifuge tube with sterile water, and prepare 100 ml of copper greedy bacteria inoculum.

2. The collection and infection process of Mimosa seeds

Collect plump, mature, and similar-sized wild mimosa seeds in autumn, soak them in 95% alcohol for 7 minutes, disinfect them with 0.1% HgCl ₂ solution for 5 minutes, and then soak them in 0.1% HgCl ₂ solution for 5 minutes; under sterile conditions, use sterile Rinse with water for 5 times, blot up the residual water with sterilized absorbent paper, and break the dormancy (cut (or tie) a small opening at the outer seed coat of the Mimosa seeds near the hilum).

The mimosa seeds were soaked in the inoculum of copper greedy bacteria for 6 hours, and the blank control group was soaked in sterile water for 6 hours. During the soaking process, the bacterial suspension should just cover the seeds. After soaking, the seeds were naturally dried in the shade for 1 hour and used for later use.

3. Grouping of heavy metal copper solution concentration

Prepare CuCl ₂ solutions with concentrations of 0mg/L, 100mg/L, 200mg/L, 300mg/L, 400mg/L, 500mg/L, 600mg/L and 800mg/L respectively, and mix them with the plant nutrient solution at a ratio of 1:1. The ratio was thoroughly mixed to obtain a mixed culture solution of heavy metal Cu ²⁺ with final concentrations of 0mg/L, 50mg/L, 100mg/L, 150mg/L, 200mg/L, 250mg/L, 300mg/L and 400mg/L. to simulate soil contaminated with heavy metal Cu ²⁺ . Set up 4 replicates of 30 mimosa seeds each.

4. Sowing of Mimosa Seeds

Mimosa seeds and seedling culture environment made of absorbent paper and petri dish, soaked with Cu ²⁺ mixed culture solution and just without filter paper, to simulate heavy metal polluted soil environment, to cultivate Mimosa seeds, replace the water absorption every two days paper and medium. The culture dish was placed in an RXZ type (multi-stage programming) intelligent artificial climate box for cultivation, and the cultivation conditions were 10h/d of light, 28°C, 50% relative humidity, and good ventilation.

5. Determination of Mimosa Seed Germination and Seedling Growth Status

(1) Germination rate Observe and record the germination of Mimosa seeds every 24h until no seeds continue to germinate (germination standard: the germ breaks through the seed coat). Germination rate = number of germinations / number of seeds

(2) Seedling growth After the Mimosa seeds are basically germinated and continue to be cultivated for more than 7 days, the roots and stems of the seedlings are separated, the root length and stem length of the seedlings (not counting the cotyledon lengths) are measured with a measuring ruler, and the roots of the seedlings are weighed with an electronic balance. Fresh weight and stem fresh weight (including cotyledons) were used to obtain seedling biomass, and the average value was calculated to obtain the growth index (root length, stem length, biomass) of Mimosa seedlings.

(3) Tolerance index The tolerance index is often used to comprehensively evaluate the response of plants to heavy metal treatment.

Tolerance index = plant traits in heavy metal treatment group/plant traits in no heavy metal treatment

When the tolerance index is greater than 0.5, it indicates that the plant has strong tolerance to heavy metals and grows better. The higher the tolerance index, the stronger the tolerance; when the tolerance index is less than 0.5, it indicates that the heavy metal is toxic to the plant. The effect is obvious, and it is difficult or impossible for plants to grow at this concentration of heavy metals.

6. Experimental results

(1) The inoculum of copper greedy bacteria can effectively improve the germination rate of seeds contaminated by Cu ²⁺ .

As shown in Figure 8, when the concentration of ^{Cu 2+} was ^0-200 mg·L ^-1 , the ^effect of inoculation on the germination rate was not obvious. The seed germination rate of the fungus was significantly higher than that of the non-inoculated copper greedy fungus. Specific index: when the seeds were not contaminated with Cu ²⁺ , the germination rate was 96-100%; when the Cu ²⁺ contamination was 250 mg·L ^-1 and 300 mg·L ^-1 , the germination rate of the seeds without inoculation was reduced to 52.5% (SD ± 11) and 51.6% (SD ± 7.6); after seeds were transfected with the copper fungus inoculum, the germination rate increased to 84.2% (SD ± 11) and 84.0% (SD ± 14), respectively. 1.60 times and 1.63 times; the tolerance index of seed germination to Cu ²⁺ pollution increased from 0.62 (250 mg·L ^-1 ) and 0.51 (300 mg·L ^-1 ) before inoculation to 0.88 and 0.87, respectively. Therefore, inoculation can effectively reduce the impact of heavy metal copper pollution on seeds and improve the germination rate of Mimosa seeds. When Cu ²⁺ was 400 mg·L ^-1 , the seed germination rate of the inoculated group and the non-inoculated group increased significantly. It may be that Mimosa has resistance to heavy metal copper at this concentration, which has a growth-promoting effect.

(2) Copper greedy fungus agent can effectively promote the growth of Mimosa seedlings

As shown in Fig. 9, the root length of the seedlings without inoculation was 11 mm when they were not polluted by copper, and the root length decreased to 3.5 mm (250 mg·L ^-1 Cu ²⁺ pollution) with the increase of Cu ²⁺ pollution concentration. ). Transfection of copper greedy bacteria agent can promote the growth of roots, but the effect is small, under 100mg·L ^-1 , 150mg·L ^-1 , 250mg·L ^-1 copper contamination, copper greedy bacteria agent makes the roots grow respectively 1.23, 1.19, 1.31 times.

However, the inoculum of copper greedy bacteria can increase the tolerance of stem growth to Cu ²⁺ and effectively promote the growth of seedlings. As shown in Figure 10, when the seedlings without inoculation were not contaminated by Cu ²⁺ , the stem length was 7.9 mm, and when the Cu ²⁺ pollution increased to 200 mg·L ^-1 , the stem length decreased to 4.3 mm; Cu When the ²⁺ pollution concentration increased to 250-300mg·L ^-1 , the stems stopped growing (0mm); after transfection with copper fungus inoculum, the stem lengths increased significantly to 1.45mm (250mg·L ^-1 ), 2.84 mm (300 mg·L ^-1 ), 8.0 mm (400 mg·L ^-1 ). The growth of the seedling stems of the inoculants showed tolerance to Cu ²⁺ , and the tolerance index increased from 0.56 to 1.03 (150 mg·L ^-1 ), from 0 to 0.17 and 0.33 (250-300 mg·L ^-1 ), It increased from 0.36 to 0.94 (400 mg·L ^-1 ). It was shown that the copper-loving bacteria can effectively reduce the stress of Cu ²⁺ pollution on the growth of seedlings, enhance the tolerance of Mimosa to copper when the copper concentration is 150-400 mg·L ^-1 , and promote the growth of plants.

(3) Copper greedy fungus agent can effectively promote the increase of biomass of Mimosa seedlings

As shown in Figure 11, the biomass of the seedlings without inoculation without Cu ²⁺ contamination was 18.5 mg, and the Cu ²⁺ concentrations were 250 mg·L ^-1 , 300 mg·L ^-1 , and 400 mg·L ^-1 . , the seedling biomass decreased to 1.48mg, 2.15mg, 5.22mg; after transfection with copper greedy bacteria, the seedling biomass increased to 4.36mg, 6.16mg, 16.32mg respectively, that is, the infection of bacteria can increase the biomass of Mimosa seedlings About 3 times, slow down the Cu ²⁺ stress, facilitate the material accumulation in the growth of seedlings, and promote the growth of Mimosa seedlings. After transfection with copper greedy bacteria agent, the tolerance index of seedling biomass accumulation to Cu ²⁺ increased from 0.34 to 0.95 (150mg·L ^-1 ) and from 0.08 to 0.23 (250mg·L ^-1 ) , increased from 0.11 to 0.32 (300 mg·L ^-1 ), and increased from 0.28 to 0.87 (400 mg·L ^-1 ). It showed that during the growth of seedlings, the inoculum of copper greedy bacteria could effectively alleviate the stress of Cu ²⁺ on the growth of seedlings, and promote the accumulation of substances and the growth of seedlings.

Embodiment 6. The adsorption and removal of copper greedy bacteria to heavy metal Cu ²⁺

1 Test strain

Cupriavidus sp. HXC-8

2 Research methods

(1) Activation of copper greedy bacteria:

First, the strains of copper greedy bacteria were streaked on solid YMA medium for inoculation, and then cultured in an incubator with a temperature condition of 28 °C for 3 days until the bacterial lawn was full.

(2) Preparation of copper greedy bacteria suspension:

In the cultured plump bacterial lawn, a ring of colonies was selected with an inoculation loop and placed in a liquid medium of 80 mL YMA (250 mL conical flask), and was fully shaken for 15 h with a shaker (rotation speed 120 r/min, temperature 28 °C) to obtain bacteria. suspension.

(3) Determination of the highest tolerance concentration of heavy metal Cu ²⁺

The YMA solid medium with heavy metal Cu ²⁺ concentration (mmol/L): 0, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6 was prepared for use.

1 mL of the bacterial suspension prepared in step (2) was added dropwise to the YMA solid medium containing the above heavy metal concentration for coating culture, and the colony growth was observed every 4 hours. Use this to verify the maximum MIC value.

(4) Determination of removal rate of heavy metal ions

①Preparation of copper greedy bacteria suspension under copper stress: add the bacterial suspension to 100 mL of heavy metal-containing liquid medium (concentration of heavy metals: 0, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6 mmol/L) , Shaker culture (speed of 120r/min, temperature of 28 ℃), cultured for 24h.

②Preparation of copper standard mother solution (100μg/ml): Accurately weigh 0.390g copper sulfate pentahydrate, dilute to a 1000ml volumetric flask with 1% nitric acid, and mix well. That is, the mother liquor of 100ug/ml is prepared.

Preparation of copper standard stock solution: According to the measurable range of the instrument (0-2.5μg/mL), the mother solution was diluted 250 times, 200 times, 125 times, 100 times, 50 times, and 40 times with 1% nitric acid, respectively. Obtain 0.4 μg/mL, 0.5 μg/mL, 0.8 μg/mL, 1.0 μg/mL, 2.0 μg/mL, 2.5 μg/mL copper standard stock solution, and take out 7 mL of standard stock solution in centrifuge tubes respectively. (The centrifuge tube should be soaked in 10% nitric acid for 12-24 hours before use, rinsed with clean water, and then rinsed with deionized water for use.)

③ Dilution of sample solution: Take out the culture solution of heavy metal (copper) bacteria with different concentrations for 24h (stable period), and filter with 0.45μm microfiltration membrane. Since the range of copper measured by flame atomic absorption spectrometer is 0-2.5 μg/mL, and the concentration of Cu ²⁺ in the bacterial solution is 0-0.6 mmol/L, the bacterial solution is reduced by 16 times to prepare a sample solution.

④ Determination of copper content: Put the standard solution into the flame atomic absorption spectrometer to measure the copper content and draw a standard curve. Measure the OD value of the prepared sample solution in a flame atomic absorption spectrometer.

According to the standard curve obtained by the measurement, the formula of the standard curve is: y=0.0121x+0.0019. According to the formula of the standard curve and the measured absorbance, the concentration of Cu ²⁺ after 24 hours is calculated, and the following formula is used to calculate the effect of HXC-8 on Cu ^{2 +} removal rate:

3 results

It can be seen from the experiment that when the concentration of Cu ²⁺ is 0.2mmol/L, the strain of Cupriavidus sp. HXC-8 grows better, and the colony amount reaches more than 90% after 20h. When the Cu ²⁺ concentration was 0.4 and 0.6 mmol/L, the bacteria grew slowly and the colony amount was only 10% after culturing for 20-24 hours; after culturing for 28-32 hours, the colonies gradually increased, and the colony amount was 15%; after 40 hours, the number of colonies increased , is 50%. When the Cu ²⁺ concentration was 0.8, 1.0, 1.2, 1.4, 1.6 mmol/L, the strain of Cupriavidus sp. HXC-8 did not grow. Therefore, the highest tolerance concentration of Cu ²⁺ by Cupriavidus sp. HXC-8 strain was 0.6 mmol/L ( FIG. 12 ).

It can be seen from the calculation results that the removal rate of Cupriavidus sp. HXC-8 strain is in the range of Cu ²⁺ concentration of 0.1-0.6 mmol/L. With the increase of Cu ²⁺ concentration, the removal rate gradually decreases, and the maximum removal rate is higher. The rate can reach 97.94% (Figure 13). The prior art discloses that a removal rate greater than 30% is an effective removal rate. Therefore, Cupriavidus sp. HXC-8 can effectively remove copper ions in the environment, and the maximum removal rate is 97.94%.

To sum up, the inoculum prepared by the copper-loving bacteria (Cupriavidus sp.) HXC-8 claimed in the present invention can withstand a certain concentration of copper pollution, can significantly remove the copper pollution, and relieve some of the mimosa polluted by copper. Seeds, promote seed germination, increase the germination rate; promote the growth and biomass accumulation of Mimosa seedlings. Meanwhile, the Cupriavidus sp. HXC-8 strain claimed in the present invention can effectively remove copper ions in the environment.

sequence listing

<110> Hanshan Teachers College

<120> A strain of copper-loving bacteria resistant to heavy metals and its preparation method and application

<160> 1

<170> SIPOSequenceListing 1.0

<210> 1

<211> 1433

<212> DNA

<213> Cupriavdus 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 (6)

1. a heavy metal-resistant copper bacterium ( Cupriavidus sp. ) HXC-8 is characterized in that, the described copper greedy bacterium ( Cupriavidus sp. ) HXC-8 is preserved in the Guangdong Microorganism Culture Collection Center, and the deposit number is: GDMCC NO: 60632. 2. a kind of cupriavidus inoculum, it is characterized in that, described cupriavidus inoculum contains as claimed in claim 1 copper inoculum ( Cupriavidus sp. ) HXC-8. 3. a preparation method of cupriavidus inoculum as claimed in claim 2, is characterized in that, described preparation method may further comprise the steps: get the cupriavidus ( Cupriavidus sp. ) HXC-8 culture of logarithmic phase liquid, centrifuge at low temperature, remove the supernatant, add sterile water, centrifuge, remove the supernatant, rinse the mixed bacterial species on the wall of the centrifuge tube with sterile water, and configure it as a copper greedy bacteria agent. 4. the application of the copper fungus inoculum as claimed in claim 2 in improving the germination rate of seeds polluted by Cu ²⁺ , wherein the seeds are legume Mimosa seeds. 5. the application of the copper fungus inoculum as claimed in claim 2 in promoting the growth of seedlings polluted by Cu ²⁺ , wherein the seedlings are legume Mimosa seedlings. 6. the application of copper greedy bacteria ( Cupriavidus sp. ) HXC-8 as claimed in claim 1 in the preparation of Cu ²⁺ polluted soil remediation preparation.

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