Uranium-tolerant bacillus cereus and application thereof in biological combined remediation of uranium-contaminated soil

Abstract

The invention discloses uranium-tolerant bacillus cereus and application thereof in biological combined remediation of uranium-contaminated soil. According to the uranium-tolerant bacillus cereus B6 provided by the invention, the uranium-tolerant bacillus cereus B6 with a growth promoting function is obtained by screening and separating from soil in a decommissioned uranium mine area, and the strain can normally grow under the uranium concentration of 400mg. L <-1 > and can also grow under the uranium concentration of 400mg. L <-1 >; the strain also has various growth promoting characteristics of fixing nitrogen, dissolving potassium, dissolving inorganic phosphorus, producing cellulase, producing siderophores, producing organic acids, synthesizing protease and the like. Meanwhile, the bacillus cereus B6 has good growth promoting ability, can relieve toxicity of uranium to plant germination and growth, can significantly improve stress-resistant enzyme activity of plants, significantly increase biomass of plants and uranium enrichment amount, and is expected to become an excellent strain for microorganism-plant combined remediation of uranium contaminated soil.

Description

Technical Field

[0001] The invention relates to the technical field of microorganism-plant combined soil restoration, and in particular to the technical field of uranium-resistant Bacillus cereus and application thereof in biological combined restoration of uranium-contaminated soil. Background Art

[0002] The rapid development of the nuclear industry has increased the demand for nuclear fuel and accelerated uranium mining and smelting. However, in the process of uranium mining and smelting, a large amount of waste and tailings will be produced. The uranium content of these wastes and tailings is much higher than that of natural soil. The uranium elements in these wastes are spread into the soil, air or groundwater, which will have a significant impact on the ecological environment. Compared with uranium pollution in the atmosphere and water bodies, uranium pollution in the soil has the characteristics of concealment, accumulation, long repair cycle and strong toxicity, and has attracted more and more attention from scholars and the public.

[0003] Indigenous strains in polluted areas often have strong tolerance and environmental adaptability. Domestic and foreign studies have isolated heavy metal-resistant plant growth-promoting bacteria from polluted areas, mainly focusing on several genera such as Bacillus sp., Citrobacter sp. and Terrabacter sp., while there are very limited reports on the uranium resistance of Bacillus cereus, which mainly focuses on tolerance to other heavy metals such as Cd, Cr, Pb, and Ni.

[0004] The microbial-plant joint remediation technology combines microbial remediation and plant remediation together, giving full play to the advantages of the two remediation materials. It can improve the plant's remediation efficiency for radioactive nuclide contaminated soil by improving the plant rhizosphere microenvironment, cooperating with plants, promoting plant growth, etc. However, most of the current uranium contamination in soil uses a single microbial remediation or plant remediation alone for soil pollution, and these methods have limitations. Therefore, the use of microorganisms to enhance plant remediation is a mutually beneficial method that will not cause secondary pollution to the environment and is the direction of sustainable development in the future. Summary of the invention

[0005] In view of the increasingly serious uranium pollution caused by the current large-scale mining of uranium mines, and the technical problem that most of the existing methods for remediating uranium pollution in soil use single microbial remediation or plant remediation alone, which has limitations in the use of uranium pollution in soil, the present invention aims to provide a uranium-resistant Bacillus cereus and its application in the biological joint remediation of uranium-contaminated soil. The present invention screened and separated from the soil in the retired uranium mining area to obtain a uranium-resistant, plant growth-promoting bacterium Bacillus cereus B6, which not only has good tolerance to uranium and its associated heavy metals such as manganese and lead, but also has the ability to promote growth such as nitrogen fixation, phosphorus solubilization, siderophore production, and protease. At the same time, Bacillus cereus B6 has good growth-promoting ability, can alleviate the toxicity of uranium to plant growth, can improve the plant's stress-resistant enzyme activity and rhizosphere soil enzyme activity, significantly increase the plant's biomass and uranium enrichment, the uranium content of Shanghai green roots increased by 141%, the root's BCF for uranium exceeded 1, and the uranium content of the aboveground part also increased by 23%; the uranium content of Sudan grass roots increased by 12%, and the uranium content of the aboveground part increased by 274%, which has the potential to be used as a microorganism combined with plants to repair soil uranium pollution.

[0006] In order to achieve the above technical effects, the present invention is implemented through the following technical solutions.

[0007] In a first aspect, the present invention provides a strain of Bacillus cereus B6, which is deposited in Guangdong Microbiological Culture Collection Center with a deposit number of GDMCC No: 64588, and a 16Sr DNA gene sequence thereof is shown in SEQ ID NO: 1.

[0008] The Bacillus cereus numbered B6 provided by the present invention is obtained by screening and separation from the rhizosphere of plants in the contaminated soil around the uranium tailings. The strain is sequenced by 16Sr DNA sequence and whole genome sequencing. The length of the 16Sr DNA sequence is 1456bp. The obtained sequence is compared with the NCBI and EzBioCloud databases, and the phylogenetic tree analysis based on the 16S rDNA sequence shows that the Bacillus cereus numbered B6 belongs to the Bacillus cereus group of the genus Bacillus. The uranium-resistant Bacillus cereus numbered B6 is different from Bacillus arachidisSY8. TThe highest 16S rDNA sequence similarity was 99.5%, and the 16S rDNA sequence similarity with other species of the Bacillus cereus group was between 99.38% and 97.18%. However, comprehensive morphological, physiological and biochemical characteristics and evolutionary analysis showed that the uranium-resistant Bacillus cereus numbered B6 was different from closely related species in some physiological and biochemical characteristics. The strain numbered B6 belonged to a new species of the genus Bacillus and had the typical characteristics of a new species. It was tentatively named Bacillus cereus B6.

[0009] The Bacillus cereus B6 provided by the present invention is subjected to the above-mentioned well-known and recognized strain system molecular level identification in the art, combined with multiphase classification system identification analysis based on morphological identification, physiological and biochemical characteristics, etc. The present invention relates to uranium-resistant Bacillus cereus B6 and the standard model strain with the closest homology to the genus Bacillus. There are multiple differences, and it is confirmed that the strain numbered B6 belongs to a new strain of the genus Bacillus, and it is named Bacillus cereus B6. The strain has been deposited in Guangdong Provincial Microbiological Culture Collection Center, address: 5th Floor, Building 59, Compound 100 Xianlie Middle Road, Guangzhou, Institute of Microbiology, Guangdong Academy of Sciences, deposit date: May 7, 2024, and deposit number: GDMCCNo: 64588.

[0010] In the present invention, the culture conditions of Bacillus cereus B6 are growth temperature of 20° C.-50° C. and salinity (v / w) of 0%-8%.

[0011] In a second aspect, the present invention provides a method for biologically combining uranium-contaminated soil restoration using uranium-resistant Bacillus cereus B6, the specific steps of which are as follows:

[0012] Step 1: Preparation of bacterial solution, at 30°C, 150 rpm -1 The uranium-resistant Bacillus cereus B6 cultured at constant temperature and shaking for 24 h was centrifuged to collect the cells and resuspended in sterile water to OD 600 =1.0, i.e. bacterial solution is obtained;

[0013] Step 2: Seed disinfection: Select seeds with full grains, soak them in 1% sodium hypochlorite solution for 10 minutes, rinse them with sterile water 3 times, 1 minute each time, soak the seeds in sterile water for 7 hours, and finally soak them in bacterial solution for 2 hours, and then dry them in a sterile environment;

[0014] Step 3: Select planting soil. Take 5-30cm of soil without plants from the mining area 20km away. After natural air drying, sieve it through a 5mm sieve to remove impurities. Add uranyl nitrate to the homogenized soil. After fully stirring, let the soil stand in a cool place for 8 weeks and then pack it into 29cm×35cm flower pots, with 2.5kg of soil in each pot.

[0015] Step 4: The Shanghai green and Sudan grass seeds treated in step 2 are placed in a seedling tray for germination and seedling raising. When the seedlings grow the fourth true leaf, they are transplanted into the flower pots respectively placed in step 3. After one week of seedling acclimatization, 300 mL of bacterial solution is applied, and the bacterial count in the bacterial solution is 10 6 CFU mL -1 , the control group was given an equal amount of distilled water; thereafter, the bacteria were applied once every 7 days, for a total of 4 times, and the samples were collected after 35 days;

[0016] Step 5: After the plant samples are sterilized and dried, they are ground and mixed. Accurately weigh 300 mg of the sample into a 50 mL digestion container, add 6-8 mL of aqua regia and 1 mL of hydrogen peroxide, place it on a graphite heating plate at 120-200°C and digest for 20-60 min until the sample is completely digested, remove the acid, cool and filter, fix the volume, and use an ICP-MS instrument to determine the uranium content of above-ground and underground tissues.

[0017] In a third aspect, the present invention provides an application of uranium-resistant Bacillus cereus in plant growth promotion.

[0018] In a fourth aspect, the present invention provides an application of uranium-resistant Bacillus cereus in biological combined remediation of uranium-contaminated soil.

[0019] Through the above technical scheme, the present invention obtains the following technical effects:

[0020] (1) The root lengths of Shanghai green seeds treated with the Bacillus cereus B6 bacterial solution provided by the present application were significantly increased by 21.4%, 26.1% and 442.3% (P<0.01) compared with the control group, and the root lengths of Sudan grass seeds were increased by 16.2%, 35.8% and 27.70% (P<0.01) compared with the control group, indicating that the strain Bacillus cereus B6 can promote seed germination and alleviate the stress of uranium on the seed germination stage.

[0021] (2) After the Shanghai green seeds were soaked in the uranium-resistant Bacillus cereus B6 bacterial solution provided by the present invention, the activities of stress-resistant enzymes POD, SOD, PPO, PAL and GSH / GSSG in the leaves were measured after the seedlings grew out, and the activities were increased by 7.5%, 48.81%, 50%, 5.52% and 80%, respectively. At the same time, the MDA content of the plants was reduced by 12.28%. The SOD, CAT, POD, PPO enzyme activities and GSH / GSSH of Sudan grass were increased by 15.76%, 24.79%, 17.88%, 11.01% and 51.7%, respectively, compared with the control group, and the MDA content was significantly reduced by 38.1%. It shows that the uranium-resistant Bacillus cereus B6 can alleviate the lipid peroxidation stress of U on plants, increase the GSH content, enhance the activity of plant antioxidant enzymes, have a positive regulatory effect on the activity of antioxidant enzymes in the growth process of plants, and can enhance the stress resistance of plants.

[0022] (3) The uranium-resistant Bacillus cereus B6 provided by the present invention can enhance the enrichment of uranium in the roots and improve the transport of uranium to the aboveground parts of the plants, thereby promoting the enrichment of uranium in the plants. After being treated with Bacillus cereus B6, the uranium content of the roots of Shanghai green grass increased by 141%, the BCF of the roots for uranium exceeded 1, and the uranium content of the aboveground parts also increased by 23%; the uranium content of the roots of Sudan grass increased by 12%, and the uranium content of the aboveground parts increased by 274%.

[0023] (4) The uranium-resistant Bacillus cereus B6 used in the present invention has been identified at the molecular level by a strain system well known in the art, and combined with multi-phase classification system identification analysis based on morphological identification, physiological and biochemical characteristics, etc., the uranium-resistant Bacillus cereus B6 provided by the present invention has multiple differences from the standard model strain of the genus Bacillus that is most homologous to the genus Bacillus, confirming that the strain numbered B6 belongs to a new strain of the genus Bacillus. The strain not only has good tolerance to uranium and its associated heavy metals such as manganese and lead, but also has the ability to promote growth such as nitrogen fixation, phosphate solubilization, siderophore production, and protease, and has potential and actual broad application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 Shown is the colony morphology characteristic of Bacillus cereus B6.

[0025] Figure 2Shown is a phylogenetic tree constructed from the Bacillus cereus B6 genome sequence using GBDP distances calculated from the genome sequence using FastME 2.1.6.1. The average branch support is 85.7%.

[0026] Figure 3 Shown is the growth curve of Bacillus cereus B6 under different uranium concentrations.

[0027] Figure 4 A shows the effect of Bacillus cereus B6 on phosphate solubilization; B shows the effect of Bacillus cereus B6 on cellulase production; C shows the effect of Bacillus cereus B6 on protease production; D shows the effect of Bacillus cereus B6 on siderophore production; E shows the effect of Bacillus cereus B6 on nitrogen fixation; F shows the effect of Bacillus cereus B6 on potassium solubilization; G shows the effect of Bacillus cereus B6 on organic acid production.

[0028] Figure 5 Schematic diagram showing the growth-promoting effect of Bacillus cereus B6 on Shanghai green and Sudan grass seeds at different uranium concentrations. A represents the uranium concentration of 0 mg·L -1 The effect diagram when B represents the uranium concentration of 50 mg·L -1 The effect diagram when C represents the uranium concentration of 100 mg·L -1 The effect diagram when .

[0029] Figure 6 Schematic diagram showing the effect of Bacillus cereus B6 treatment on plant growth in soils without uranium and with uranium. A represents Shanghai Qing planted in soil without uranium; B represents Shanghai Qing planted in soil containing uranium; C represents Sudan grass planted in soil without uranium; and D represents Sudan grass planted in soil without uranium. The left side of the figure shows CK, and the right side shows the inoculation treatment with Bacillus cereus B6.

[0030] Figure 7 Shown is a comparison of the activity of scutellaria baicalensis by different combinations of bacterial agents under uranium stress.

[0031] Figure 8 The figure shows the comparison of enzyme activity of Sudan grass under uranium stress by different combinations of bacterial agents. DETAILED DESCRIPTION

[0032] The following will be combined with the contents in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention, but the present invention is not limited to the following embodiments. All the raw and auxiliary materials selected in the present invention, as well as the selected bacterial culture method are conventional reagents and equipment in the technical field.

[0033] The main culture medium and reagents used in the present invention are:

[0034] The culture medium used in the present invention includes: nutrient broth medium (NB), R2A medium, LB medium, Ashby medium, organophosphate-dissolving bacteria medium, PKO bacteria medium, silicate bacteria medium, CAS detection medium, skim milk powder medium, sodium carboxymethyl cellulose medium, and bromocresol purple color development medium.

[0035] Uranium (U)-containing culture medium: 3.0 g·L beef extract in NA basic culture medium -1 , peptone 5.0 g·L -1 , glucose 2.5 g·L -1 , agar 18.0 g·L -1 , pH 7.0 ± 0.1, add 5.0 g·L of filter-sterilized -1 UO2(NO3)2 mother liquor to the corresponding concentration.

[0036] Example 1: Screening and Isolation of Bacillus cereus B6

[0037] Microorganisms were separated and purified by gradient dilution method. 5.0 g of air-dried soil sample was weighed and added into a triangular flask containing 45 mL of sterile water. The mixture was incubated at 30 °C and 150 r min. -1 The soil suspension was prepared into 10 -3 , 10 -4 , 10 -5 Gradient dilutions were prepared by taking 100 μL of soil suspensions of different dilutions with a pipette and adding them to a solution with a concentration of 200 mg L -1 The uranium-containing medium was evenly coated with a spreader and incubated at 30°C for 7 days. Under aseptic operation conditions, single colonies of different morphologies were picked up with sterilized and dried bamboo sticks and streaked on the new uranium-containing solid medium for separation and purification.

[0038] After preliminary screening, Bacillus cereus B6 was isolated from the uranium contaminated soil sample. Its colony morphology is shown in the attached Figure 1As shown, the colonies on NA medium are off-white, round, with irregular edges, rough, raised and opaque surfaces; microscopic observation shows that the bacteria are long rod-shaped, 2.4-3.2×1.1-1.4μm in size, spore-forming, and Gram-positive. The growth temperature range of Bacillus cereus B6 is 20℃~50℃, and the salinity (v / w) is 0%~8%. The strain is positive for cytochrome oxidase, catalase, gelatin liquefaction, nitrate reduction, pyruvate, arginine dihydratase, inositol, sorbitol, rhamnose and sucrose utilization, and negative for hydrogen sulfide production, citrate utilization, β-galactosidase, lysine decarboxylase, ornithine decarboxylase, urease, tryptophan deaminase, glucose, mannitol, melastose, amygdalin and arabinose utilization.

[0039] Example 2: Identification of Bacillus cereus B6

[0040] The length of the 16S rDNA sequence of Bacillus cereus B6 is 1456bp (GenBank accession number ON820114). After comparative analysis of NCBI and EzBioCloud databases, Bacillus cereus B6 belongs to the Bacillus cereus group of the genus Bacillus, and its similarity with members of the Bacillus cereus group is 99.5%-97.18%. It is difficult to effectively distinguish between species based on the comparison of 16S rRNA sequence similarity. Then the strain genome was sequenced, see Appendix Figure 2 The phylogenetic tree constructed based on the whole genome sequence of Bacillus cereus B6 strain showed that B6 strain and Bacillus cereus ATCC 14579 strain were in the same branch and had the closest relationship. The ANI of the two strains was >97% and the dDDH was >70%, so B6 could be considered as a Bacillus cereus species. However, after comprehensive morphological, physiological and biochemical characteristics and evolutionary analysis, it was found that Bacillus cereus B6 was different from closely related species in some physiological and biochemical characteristics, as shown in Table 1.

[0041] Table 1: Physiological and biochemical characteristics of Bacillus cereus B6 strain and related bacteria

[0042]

[0043] Example 3: Growth curve and adsorption characteristics of Bacillus cereus B6 at different uranium concentrations

[0044] The selected Bacillus cereus B6 colonies were inoculated on NA plates containing different concentrations of heavy metals such as cadmium, chromium, lead, manganese and zinc, and cultured at 30°C for 14 days to observe the growth of the strains. At the same time, by observing the growth of the colonies, the lowest heavy metal concentration tolerated by each strain, namely the minimum inhibitory concentration (MIC), was obtained.

[0045] Preparation of OD 600 The seed culture solution of Bacillus cereus B6 with a concentration of 1.0 was inoculated into a uranium-containing medium at a 2% (V / V) inoculation rate, and the uranium concentration was 0-800 mg L -1 The culture medium was uranium-containing, and each treatment was repeated 3 times. The culture was kept at 30°C for 48 hours, and the absorbance of the bacterial solution at 600 nm was measured every 2 hours. The time was used as the horizontal axis, and the absorbance OD was used as the horizontal axis. 600 The value is the vertical axis, and the strain growth curve is drawn.

[0046] The results are shown in the attached Figure 3 Bacillus cereus B6 grew on NA plates containing heavy metals, and the MIC concentrations of heavy metals such as cadmium, chromium, lead, manganese, and zinc were 200 mg kg -1 , 300mg·kg -1 , 500mg·kg -1 , 200mg·kg -1 and 500 mg kg -1 When the uranium concentration was lower than 400 mg·L -1 The growth curve of Bacillus cereus B6 was S-shaped. When the uranium concentration of the culture medium was 500 mg·L -1 When the uranium concentration in the culture medium reached 700 mg·L -1 When the concentration of iodine was above 2.0, the growth of strain Bacillus cereus B6 was completely inhibited.

[0047] The prepared Bacillus cereus B6 bacterial solution was heated at 10000 r min -1 Centrifuge for 5 minutes, take 300 μL of supernatant, refer to the method of drawing the uranium standard curve, add 0.5 mL of concentrated hydrochloric acid, 1.0 mL of ETA, and 2.0 mL of arsenazo in sequence, dilute to 10.0 mL with distilled water, mix well, and measure OD 652 , to determine the remaining uranium concentration in the bacterial solution. The adsorption rate is calculated using formula (1):

[0048]

[0049] Example 4: Determination of the Growth-Promoting Properties of Bacillus cereus B6

[0050] Select Bacillus cereus B6 strain and inoculate it into 50 mL of uranium (100 mg·L -1 )NB medium was shaken at 30℃ for 24h as seed solution, and 5μL of bacterial solution was taken with a pipette and spotted on Ashby medium, organophosphate-solubilizing bacteria medium, PKO bacteria medium, silicate bacteria medium, CAS detection medium, skim milk powder medium, sodium carboxymethyl cellulose medium and bromocresol purple color development medium respectively, and cultured in a 30℃ incubator for 7d to observe whether a transparent zone was formed around the colony to determine whether it had the characteristics of nitrogen fixation, phosphate and potassium solubilization, iron carrier synthesis, protease production and cellulase production.

[0051] The results are shown in the attached Figure 4 As shown, Bacillus cereus B6 can produce transparent hydrolysis circles in inorganic phosphorus culture medium, sodium carboxymethyl cellulose culture medium, skimmed milk powder culture medium and CAS detection culture medium respectively, and has the ability to degrade inorganic phosphorus, produce cellulase, protease and synthesize iron carrier; it forms smooth and transparent oil droplets on silicate culture medium, indicating that it has the ability to degrade potassium; it grows well on Ashby culture medium, indicating that it has the ability to fix nitrogen; and it produces a yellow transparent circle in bromocresol purple color development culture medium, indicating that it has the ability to produce organic acids.

[0052] Example 5: Quantitative determination of iron carriers in Bacillus cereus B6

[0053] Bacillus cereus B6 was inoculated into NB liquid medium and incubated at 28°C and 150 r·min -1 After 48 h of incubation, 5 mL of bacterial solution was placed in a centrifuge tube and centrifuged at 10,000 r / min. -1 Centrifuge for 10 minutes, take 1mL of the supernatant and mix it with an equal amount of CAS detection solution, let it stand for 1 hour, then dilute it twice with deionized water, measure the absorbance (As) at a wavelength of 630nm, and take deionized water as a control to adjust to zero. Use the same method to measure the absorbance of the uninoculated culture solution as a reference value (Ar) and calculate the SU value. The larger the SU value, the stronger the ability of Bacillus cereus B6 to produce siderophores. Bacteria with higher siderophore production ability generally have an As / Ar value lower than 0.5, that is, the SU value is greater than 50%, and the calculation formula is as follows:

[0054] SU=[(Ar-As) / Ar]×100%

[0055] The results showed that the SU value of siderophore produced by Bacillus cereus B6 was 42.20%.

[0056] Example 5: Effect of Bacillus cereus B6 on seed germination under uranium stress

[0057] 30℃, 150r min -1 The bacterial solution of Bacillus cereus B6 cultured at constant temperature and shaking for 24 hours was centrifuged, the cells were collected, and the bacterial solution was resuspended and mixed with sterile water to OD = 1.0, which was used as the bacterial solution for later use. Seeds with full grains were selected, soaked and disinfected in 1% (m / V) sodium hypochlorite solution for 10 minutes, rinsed with sterile water 3 times, 1 minute each time, and then soaked in sterile water for 7 hours. After being removed and drained, they were soaked in bacterial solution for 2 hours, dried in a sterile environment, and set aside. Three layers of filter paper were laid in the culture dish and soaked with 10 mL of sterile water; in the germination test under uranium stress, 0, 50 and 100 mg L were used in different treatments. -1 Soak the filter paper with uranium solution, place the seeds treated with bacterial solution on it, place 15 seeds on each plate, set 3 replicates for each treatment group, and culture them in a light incubator at 20℃, 12h / 12h light / dark alternation, and 80% humidity. Seeds not treated with bacterial solution were used as controls. After 7 days, the germination rate was counted, the root length, shoot length and fresh weight were measured, and the germination potential, germination index and vitality index were calculated as shown in (2) to (5).

[0058]

[0059]

[0060]

[0061] In the formula: Gt refers to the number of seeds germinated on day t, and Dt is the corresponding number of germination days for seeds.

[0062] Vitality index (VI) = GI × Lr (5)

[0063] Where: Lr is the average root length.

[0064] The results are shown in the attached Figure 5 , at 0, 50 and 100 mg L -1The seed germination experiment of uranium concentration treatment showed that the root length of both plants decreased significantly with the increase of uranium concentration, indicating that the roots of seedlings were more sensitive to uranium and were most affected by uranium; compared with Shanghai green, the germination rate of Sudan grass seeds decreased significantly, and was more obviously affected by uranium. According to the data in Table 2, with the increase of uranium concentration, the germination rate, germination index, vitality index, root length and sprout length of plants treated with strain Bacillus cereus B6 were all improved compared with CK with the same uranium concentration. The root length of Shanghai green seeds was significantly increased by 21.4%, 26.1% and 442.3% (P<0.01) compared with the control group, and the root length of Sudan grass seeds was increased by 16.2%, 35.8% and 27.70% (P<0.01) compared with the control group, indicating that strain Bacillus cereus B6 can promote seed germination and alleviate the stress of uranium on the seed germination stage.

[0065] Table 2: Effects of Bacillus cereus B6 on germination of Shanghai green leaves

[0066]

[0067] Note: Different letters indicate significant longitudinal differences (P<0.05).

[0068] Example 6: Effects of Bacillus cereus B6 on plant growth and uranium enrichment under uranium stress

[0069] Take the soil from the 5-30cm surface layer without plants 20km away from the mining area, dry it naturally, and pass it through a 5mm sieve to remove large stones, plant residues and other impurities. Add uranyl nitrate to the homogenized soil to simulate 25mg / kg contaminated soil. After fully stirring, let the soil stand in a cool place for 8 weeks and pack it into 29cm×35cm flower pots, with 2.5kg soil in each pot, for later use.

[0070] Take the sterilized and bacterial solution-soaked Shanghai green and Sudan grass seeds, place them in a seedling tray for germination, and transplant them into round flower pots filled with 2.5 kg of soil after the seedlings grow the fourth true leaf. After one week of seedling acclimatization, apply 300 mL of the prepared 100-fold diluted bacterial solution (the bacterial count is about 10 6 CFU mL -1 ), and the control group was given an equal amount of distilled water. After that, the bacteria were applied once every 7 days, for a total of 4 times, and samples were collected after 35 days. Ten plants were taken from each treatment group to measure plant height, root length, stem diameter, number of leaves, leaf width, leaf length, aboveground fresh and dry weight, and underground fresh and dry weight.

[0071] The malondialdehyde (MDA), chlorophyll and soluble protein contents of leaves were measured. The activities of catalase (CAT), phenylalanine ammonia lyase (PAL), peroxidase (POD), polyphenol oxidase (PPO) and superoxide dismutase (SOD) were measured. The activities of reduced glutathione (GSH) and oxidized glutathione (GSSG) were also measured.

[0072] After the plant samples are inactivated and dried, they are ground and mixed. 300 mg of the sample is accurately weighed into a 50 mL digestion container, 6-8 mL of aqua regia and 1 mL of hydrogen peroxide are added. The sample is placed on a graphite heating plate at 120-200 ° C for 20-60 min until the sample is completely digested. The acid is removed, and the sample is filtered after cooling and the volume is fixed. The uranium content of the above-ground and underground tissues is determined by an ICP-MS instrument.

[0073] Example 7: Effects of Bacillus cereus B6 on plant growth and uranium enrichment under uranium stress

[0074] The results are shown in the attached Figure 6 As shown in Table 3, by comparing the plants in the control group without inoculation in the uranium-free soil and the uranium-containing soil, it was found that the plant height, root length, and dry and fresh weight of the plants under uranium stress were significantly lower than those in the uranium-free soil. As shown in Table 3, the average plant height and root length of Shanghai Qing decreased by 35.4% and 58.9% respectively, and the dry and fresh weight decreased by more than 60%; the plant height and root length of Sudan grass decreased by 30.8% and 36.3% respectively, and the biomass decreased significantly; the growth of both plants was significantly inhibited, indicating that the uranium in the soil caused stress and toxicity to the growth of plants.

[0075] When Bacillus cereus B6 strain was inoculated into uranium-containing soil, it was found that the average plant height and biomass of Shanghai green grass increased significantly; the plant height, root length and dry weight of Sudan grass increased significantly; it can be seen that Bacillus cereus B6 strain can alleviate the inhibition of uranium stress on plant growth and promote the growth of plants under uranium stress.

[0076] After being treated with Bacillus cereus B6, the uranium content in the aboveground and roots of the two plants increased significantly; the uranium content in the roots of Shanghai green grass increased by 141%, the BCF of uranium in the roots exceeded 1, and the uranium content in the aboveground parts also increased by 23%; the uranium content in the roots of Sudan grass increased by 12%, and the uranium content in the aboveground parts increased by 274%. It can be seen that the roots are the main part of the two plants that accumulate uranium. Bacillus cereus B6 can enhance the enrichment of uranium in the roots and increase the transport of uranium to the aboveground parts of the plants, promoting the enrichment of uranium in the plants.

[0077] Table 3: Effects of Bacillus cereus B6 treatment on plant growth

[0078]

[0079] Note: Different letters indicate significant longitudinal differences (P<0.05).

[0080] Example 8: Effect of Bacillus cereus B6 on plant oxidase activity under uranium stress

[0081] By the attached Figure 7 It can be seen that the activities of antioxidant enzymes such as peroxidase (POD), superoxide dismutase (SOD), polyphenol oxidase (PPO), phenylalanine ammonia lyase (PAL) and glutathione / oxidized glutathione (GSH / GSSG) in Shanghai Qing treated with Bacillus cereus B6 were increased by 7.5%, 48.81%, 50%, 5.52% and 80% respectively compared with the control group, and the malondialdehyde (MDA) content was reduced by 12.2% compared with the control group.

[0082] By the attached Figure 8 It can be seen that after inoculation with Bacillus cereus B6 under uranium stress, the SOD, CAT, POD, PPO enzyme activities and GSH / GSSH of Sudan grass increased by 15.76%, 24.79%, 17.88%, 11.01% and 51.7% respectively compared with the control group, and the MDA content was significantly reduced by 38.1%. The results show that inoculation with B6 can alleviate the lipid peroxidation stress of U on plants, increase the GSH content, enhance the activity of plant antioxidant enzymes, have a positive regulatory effect on the activity of antioxidant enzymes during plant growth, and can enhance the stress resistance of plants.

[0083] The present invention provides a uranium-resistant Bacillus cereus and its application in the biological combined remediation of uranium-contaminated soil. With Shanghai green and Sudan grass as plant materials, it is found that although uranium stress has a great influence on the germination of Sudan grass seeds, the germination rate of the two plant seeds is acceptable under 100 mg / L uranium stress, and the tolerance is good. In the pot experiment, the plants can still grow in 100 mg / kg uranium-contaminated soil, but are strongly inhibited and poisoned by the high concentration of uranium, and the plants become short. Inoculation of Bacillus cereus B6 has a significant promoting effect on seed germination and plant growth, especially root growth, improves the tolerance of plants to uranium, and strengthens the enrichment and transport of uranium by plants.

[0084] The embodiments described above are only some embodiments of the present invention, not all embodiments. The detailed description of the embodiments of the present invention is not intended to limit the scope of the invention claimed for protection, but only represents selected embodiments of the present invention. All other embodiments obtained without creative work and related deductions and substitutions made by ordinary technicians in the field under the conditions of the concept of the present invention belong to the scope of protection of the present invention.

Claims

1. A uranium-resistant Bacillus cereus B6, characterized in that: The strain is deposited in Guangdong Microbiological Culture Collection Center with a deposit number of GDMCC No: 64588, and its 16S rDNA gene sequence is shown in SEQ ID NO:

1.

2. The strain of Bacillus cereus B6 according to claim 1, characterized in that: The culture conditions of the B6 strain are a growth temperature of 20° C.-50° C. and a salinity of 0%-8%.

3. A method for biologically combining uranium-contaminated soil restoration using uranium-resistant Bacillus cereus B6, characterized in that: The specific steps are as follows: Step 1: Preparation of bacterial solution, at 30°C, 150 rpm -1 The uranium-resistant Bacillus cereus B6 cultured at constant temperature and shaking for 24 hours was centrifuged to collect the bacterial cells, and the bacterial solution was resuspended and mixed with sterile water until OD = 1.0, thereby obtaining a bacterial solution; Step 2: Seed disinfection: Select seeds with full grains, soak them in 1% sodium hypochlorite solution for 10 minutes, rinse them with sterile water 3 times, 1 minute each time, soak the seeds in sterile water for 7 hours, and finally soak them in bacterial solution for 2 hours, and then dry them in a sterile environment; Step 3: Select planting soil. Take 5-30cm of soil without plants from the mining area 20km away. After natural air drying, sieve it through a 5mm sieve to remove impurities. Add uranyl nitrate to the homogenized soil. After fully stirring, let the soil stand in a cool place for 8 weeks and then pack it into 29cm×35cm flower pots, with 2.5kg of soil in each pot. Step 4: The Shanghai green and Sudan grass seeds treated in step 2 are placed in a seedling tray for germination and seedling raising. When the seedlings grow the fourth true leaf, they are transplanted into the flower pots respectively placed in step 3; after one week of seedling acclimatization, 300 mL of bacterial solution is applied, and the bacterial count in the bacterial solution is 10 6 CFU mL -1 , the control group was given an equal amount of distilled water; thereafter, the bacteria were applied once every 7 days, for a total of 4 times, and the samples were collected after 35 days; Step 5: After the plant samples are sterilized and dried, they are ground and mixed. Accurately weigh 300 mg of the sample into a 50 mL digestion container, add 6-8 mL of aqua regia and 1 mL of hydrogen peroxide, place it on a graphite heating plate at 120-200°C and digest for 20-60 min until the sample is completely digested, remove the acid, cool and filter, fix the volume, and use an ICP-MS instrument to determine the uranium content of above-ground and underground tissues.

4. Use of the uranium-resistant Bacillus cereus B6 as claimed in claim 1 in plant growth promotion.

5. Application of a uranium-resistant Bacillus cereus B6 in biological combined remediation of uranium-contaminated soil.

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