Molybdenum-tolerant potassium-dissolving bacteria with molybdenum reduction function, isolation and screening method and application

Abstract

The present application relates to a kind of molybdenum-tolerant potassium-dissolving bacteria with molybdenum reduction, which is classified and named as Bacillus amyloliquefaciens MoTB 79, and was preserved in China Center for Type Culture Collection (CCTCC) on April 7, 2024, with the accession number of CCTCC No.M2024639.A kind of molybdenum-tolerant potassium-dissolving bacteria with molybdenum reduction is applied in soil or wastewater containing molybdenum ion pollution.A kind of molybdenum-tolerant potassium-dissolving bacteria with molybdenum reduction is applied in soil potassium-dissolving.The beneficial effect is that Bacillus amyloliquefaciens MoTB 79 can reduce self-adsorbed Mo (Ⅵ), and the adsorbed Mo (Ⅵ) is reduced to Mo (V) and Mo (IV) with relatively low toxicity, to reduce the toxicity and ecological harm of Mo (Ⅵ) in soil or water body, and ultimately realize the effective management of Mo (Ⅵ) pollution, with relatively high Mo (Ⅵ) reduction efficiency and certain potassium-dissolving capacity.

Description

Technical Field

[0001] This invention relates to the field of environmental microbiology, specifically to a molybdenum-resistant potassium-solubilizing bacterium with molybdenum reduction activity, its isolation and screening method, and its application. Background Technology

[0002] Molybdenum is a multivalent element with various oxidation states from 0 to VI. Mo(IV) and Mo(VI) are the most stable. Compared with Mo(IV), Mo(VI) is more easily absorbed and more toxic. Long-term exposure to high concentrations of molybdenum can lead to gout, increase the risk of cardiovascular disease, and in severe cases, cause cancer. It has been reported that the concentration of molybdenum in mine soil can reach 268.13 mg / kg, which is 134 times the background value of Chinese soil (2.0 mg / kg). In recent years, the problem of soil molybdenum pollution caused by molybdenum mining and tailings dam leakage has received increasing attention. Therefore, it is very necessary to remediate molybdenum pollution risk areas.

[0003] Molybdenum blue (Mo-Blue) is a product of molybdenum-reducing strains fixing soluble molybdenum in the environment into a less toxic, insoluble form. It plays a certain role in the remediation of molybdenum-contaminated soil. Currently, microbial molybdenum reduction is a sustainable method for remediating molybdenum pollution and has been recognized globally. The key to using microbial technology to control molybdenum pollution is to obtain strains with high molybdenum tolerance and high molybdenum reduction capacity. However, there are few methods for screening and identifying molybdenum-reducing strains with molybdenum tolerance, and related technologies are still weak. Summary of the Invention

[0004] The technical problem to be solved by this invention is to overcome the defects of existing soil molybdenum pollution remediation technologies, and to provide a molybdenum-resistant potassium-solubilizing bacterium with molybdenum reduction capabilities, along with its isolation, screening, and application methods. This bacterium can tolerate sodium molybdate with a Mo(VI) concentration of up to 100,000 mg / L. Within 48 hours, it can remove 21.66% of Mo(VI) from BPM medium containing 39.55 mg / L Mo(VI), and within 2 days, it can reduce the available molybdenum content in the soil by 11.14%. Furthermore, in potassium-solubilizing liquid medium, this strain can dissolve insoluble potassium into soluble potassium at a concentration of 0.14 ± 0.02 mg / L. Preliminary studies indicate that the strain of this invention can provide bacterial resources for the bioremediation of molybdenum-polluted areas with low soil potassium utilization.

[0005] The technical solution of the present invention to solve the above-mentioned technical problems is as follows:

[0006] A molybdenum-resistant potassium-solubilizing bacterium with molybdenum-reducing activity, classified as Bacillus amyloliquefaciens MoTB 79, was deposited on April 7, 2024, at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC No. M2024639.

[0007] Based on the above technical solution, the present invention can be further improved as follows.

[0008] Furthermore, the bacterium grows well in an environment with a Mo(VI) content of 3000 mg / L.

[0009] Furthermore, the maximum Mo(VI) tolerance concentration of this bacterium is 100,000 mg / L.

[0010] Furthermore, the bacterium reduces Mo(VI) to generate Mo-Blue, and the absorbance of the product Mo-Blue at 865 nm can reach 4.0997.

[0011] Based on the above technical solution, the present invention also provides a method for isolating and screening molybdenum-resistant potassium-solubilizing bacteria with molybdenum reduction activity, comprising the following steps:

[0012] S1. Take rhizosphere soil from pioneer plants in the molybdenum tailings area, prepare it into a soil suspension, then dilute it serially and streak it on beef extract peptone solid medium containing high concentration of Mo(VI) for screening and isolation of molybdenum resistant bacteria, so as to isolate surviving strains that are resistant to Mo(VI).

[0013] S2. After the colonies grow, the colonies are streaked on the plate surface and transferred multiple times to purify the strain and obtain molybdenum-resistant strains.

[0014] S3. The above molybdenum-resistant bacteria were cultured in a low-phosphorus molybdate solid medium. The formation of blue colonies after 24 hours of culture was used as the initial screening criterion, and strains with molybdenum reduction ability were screened out.

[0015] S4. Quantitative analysis of strains with molybdenum reducing ability was performed using LPM liquid medium to screen out strains with strong molybdenum reducing ability.

[0016] S5. Characterize the potassium-solubilizing effect of the obtained molybdenum-resistant strains to obtain molybdenum-resistant potassium-solubilizing bacteria with molybdenum-reducing activity.

[0017] Furthermore, the Mo(VI) source added to the beef extract peptone solid medium in S1 was sodium molybdate, with a Mo(VI) concentration of 3000 mg / L.

[0018] Based on the above technical solution, the present invention also provides an application of a molybdenum-resistant potassium-solubilizing bacterium with molybdenum reduction activity in soil or wastewater contaminated with molybdenum ions.

[0019] Based on the above technical solution, the present invention also provides the application of molybdenum-resistant potassium-solubilizing bacteria with molybdenum reduction function in soil potassium solubilization.

[0020] Furthermore, the molybdenum-resistant potassium-solubilizing bacteria with molybdenum-reducing properties dissolved insoluble potassium to a soluble potassium content of 0.14 ± 0.02 mg / L.

[0021] Furthermore, the insoluble potassium element is potassium feldspar.

[0022] Compared with existing technologies, this invention has the following beneficial technical effects: Bacillus amyloliquefaciens MoTB 79 was identified through 16S rDNA sequencing, and its molybdenum reducing ability and potassium solubility were measured. In the study of Mo(VI) adsorption and conversion in the environment, Bacillus amyloliquefaciens MoTB 79 was able to reduce its own adsorbed Mo(VI), with a reduction rate of 62.54%–65.75%. The adsorbed Mo(VI) was reduced to the relatively less toxic Mo(V) and Mo(IV). Bacillus amyloliquefaciens MoTB 79 can survive in solutions or soils with high Mo(VI) content, effectively controlling the migration and transformation of Mo(VI), Mo(IV), and Mo(V) in soil or water, reducing the toxicity and ecological harm of Mo(VI) in soil or water, and ultimately achieving effective treatment of Mo(VI) pollution. While having a high Mo(VI) reduction efficiency, it also has a certain potassium solubilization ability. Bacillus amyloliquefaciens MoTB 79 can dissolve insoluble potassium feldspar, improve the utilization rate of potassium in soil, and is environmentally friendly. It is a strain with good application prospects and is of great significance to the development of bioremediation technology. Attached Figure Description

[0023] Figure 1 This is a photograph of the screening results of Bacillus amyloliquefaciens MoTB 79 plates in this invention;

[0024] Figure 2 This is the phylogenetic tree of Bacillus amyloliquefaciens MoTB 79 in this invention;

[0025] Figure 3 The growth curves of Bacillus amyloliquefaciens MoTB 79 in this invention are shown below, with different initial concentrations of Mo(VI).

[0026] Figure 4 This is a spectral scan of Mo-Bluue, the product of the reduction of Mo(VI) by Bacillus amyloliquefaciens MoTB 79 in LPM liquid medium in this invention.

[0027] Figure 5 The XPS results are as follows: Bacillus amyloliquefaciens MoTB 79 adsorbs Mo(VI) into BPM and LPM culture media.

[0028] Figure 6 This is a characteristic diagram of the potassium-solubilizing effect of Bacillus amyloliquefaciens MoTB 79 in this invention. Detailed Implementation

[0029] The principles and features of the present invention are described below with reference to the accompanying drawings. The examples given are only for explaining the present invention and are not intended to limit the scope of the present invention.

[0030] A molybdenum-resistant potassium-solubilizing bacterium with molybdenum-reducing activity, classified as *Bacillus amyloliquefaciens* MoTB 79, was deposited on April 7, 2024, at the China Center for Type Culture Collection (CCTCC) No. M2024639, at Wuhan University, Wuhan, China.

[0031] This bacterium grows well in an environment with a Mo(VI) concentration of 3000 mg / L. The test environment is solid or liquid culture medium. Specifically, the growth rate is fast in liquid BPM medium with a Mo(VI) concentration of 3000 mg / L, and the growth rate decreases significantly in liquid BPM medium with a Mo(VI) concentration exceeding 60000 mg / L. The maximum tolerated Mo(VI) concentration is 100000 mg / L, and the test environment is liquid culture medium containing Mo(VI).

[0032] Bacillus amyloliquefaciens MoTB 79 can reduce its own adsorbed Mo(VI), and the proportion of reduced Mo(VI) can reach 62.54% to 65.75%.

[0033] This bacterium reduces Mo(VI) to produce Mo-Blue, and the absorbance of the product Mo-Blue at 865 nm can reach 4.0997. The test environment is LPM liquid medium containing Mo(VI) source.

[0034] like Figure 1 , Figure 2 As shown, the strains were screened:

[0035] Take rhizosphere soil samples from pioneer plants in molybdenum tailings areas. For example, take 5.0g of the soil sample to be tested from the rhizosphere soil samples of pioneer plants in Luanchuan molybdenum tailings area, Henan Province. Add the soil sample to a triangular flask containing 45ml of sterile water and incubate it in a shaker at 30℃ and 150r / min for 30min to obtain a soil suspension.

[0036] After serial dilution, the strains were streaked on beef extract peptone solid medium containing a high concentration of Mo(VI) (Mo(VI) source: sodium molybdate dihydrate, concentration of 3000 mg Mo / L) for screening. Strains that could survive under high concentrations of Mo(VI) and were tolerant to several pairs of Mo(VI) were isolated. After the colonies grew, the colonies were streaked on the plates and subcultured more than 3 times to purify the strains, and a total of 142 strains were obtained.

[0037] Screening for molybdenum-resistant bacteria with molybdenum-reducing ability:

[0038] The 142 molybdenum-resistant bacteria were inoculated into low phosphomolybdate (LPM) solid medium. The formation of blue colonies (phosphomolybdate blue reaction) after 24 hours of incubation was used as the initial screening criterion, identifying 44 strains with molybdenum reducing ability. Then, these molybdenum-reducing strains were inoculated at a 1% inoculum rate into 250 ml Erlenmeyer flasks containing 100 ml of liquid LPM medium. Three replicate shake flasks were prepared for each strain, with sterile LPM liquid medium serving as a control. The cultures were incubated at 30°C and 150 rpm for 24 hours. The supernatant was scanned using a UV-Vis spectrophotometer to determine its molybdenum blue production ability by measuring the absorbance at 865 nm. One strain with the strongest molybdenum reducing ability was selected. Its full-spectrum scan (molybdenum blue reduction product) and the reading at 865 nm (Mo(VI) reduction effect) are shown below. Figure 4 As shown;

[0039] Molybdenum-resistant growth curve test of molybdenum-reducing potassium-solubilizing bacteria (Bacillus amyloliquefaciens MoTB 79)

[0040] The isolated pure culture strain was inoculated into fresh BPM liquid medium and cultured for about 12 hours. Then, a certain amount of bacterial suspension (1% inoculum) was taken and inoculated into BPM liquid medium with different Mo(VI) concentrations (0, 3000, 6000, 9000, 15000, 30000, 60000, 70000, 80000, 90000, 100000 mg / L). The culture was carried out at 30℃ and 150 rpm. The uninoculated liquid medium was used as a control. The OD value was measured at a wavelength of 600 nm using a spectrophotometer. The growth curve was plotted to analyze the growth of the strain in liquid medium with different Mo(VI) concentrations.

[0041] Figure 3To investigate the growth curves of the molybdenum-reducing potassium-solubilizing bacterium (Bacillus amyloliquefaciens MoTB79) obtained by inoculating it into culture media with initial Mo(VI) concentrations of 0, 3000, 6000, 9000, 15000, 30000, 60000, 70000, 80000, 90000, and 100000 mg / L, respectively, after 30 hours of cultivation, the following figures are shown: [Figure showing growth curves of the molybdenum-reducing potassium-solubilizing bacterium (Bacillus amyloliquefaciens MoTB79) with initial Mo(VI) concentrations of 0, 3000, 6000, 9000, 15000, 30000, 60000, 70000, 80000, 90000, and 100000 mg / L]. At concentrations of 0 mg / L and 6000 mg / L, Mo(VI) had no significant inhibitory effect on the growth of the strain. However, when the concentration of Mo(VI) was higher than 30000 mg / L, the growth of the strain was inhibited to varying degrees, the logarithmic growth phase was delayed, and the growth rate was significantly lower after entering the logarithmic phase. Comparing the growth rates of the strain with different Mo(VI) concentrations, the results showed that the strain could grow normally in liquid medium with a Mo(VI) concentration of 6000 mg / L, with a growth rate comparable to the control group. The MIC Mo(VI) of the strain could reach 100000 mg / L, indicating that the molybdenum-tolerant potassium-solubilizing bacterium (Bacillus amyloliquefaciens MoTB 79) with molybdenum reduction activity has a strong tolerance to Mo(VI).

[0042] Identification

[0043] The selected strains were sequenced, and the sequencing results were compared with BLAST sequences on the NCB I website, which confirmed them to be Serratia ficariae MoTB 2, as shown in Table 1.

[0044] Table 1

[0045]

[0046]

[0047] Analysis of the Mo(VI) adsorption capacity and post-adsorption conversion behavior of a molybdenum-reducing, potassium-solubilizing bacterium (Bacillus amyloliquefaciens MoTB 79).

[0048] 1. Add molybdenum-resistant potassium-solubilizing bacteria (Bacillus amyloliquefaciens MoTB 79) with molybdenum reduction activity to BPM and LPM liquid media containing Mo(VI) respectively to obtain mixed media. The inoculum amount of molybdenum-resistant potassium-solubilizing bacteria (Bacillus amyloliquefaciens MoTB 79) with molybdenum reduction activity in the mixed media is 1%.

[0049] The formula for BPM medium containing Mo(VI) is: 3000 mg Mo(VI) (sodium molybdate dihydrate), 10.0 g peptone, 5.0 g sodium chloride, 3.0 g beef extract, and 1000 ml water. The pH of the medium is 7.1-7.3, and it is sterilized at 121°C for 20 min.

[0050] The formula for LPM medium containing Mo(VI) is as follows: (NH4)2SO4 3.0g, MgSO4·7H2O 0.5g, NaCl 5.0g, Na2MoO4·2H2O 2.42g, Na2HPO4 0.71g, yeast extract 0.5g, glucose 10.0g, and water 1000ml; the pH of the medium is 7.0, and it is sterilized at 121℃ for 20min.

[0051] 2. A preliminary study was conducted on the Mo(VI) adsorption capacity of the strain using a shake-flask experiment:

[0052] Nine 250ml Erlenmeyer flasks, each containing 100ml of liquid LPM culture medium and numbered No. 1–9, were divided into three groups: flasks 1–3 were group A, flasks 4–6 were group B, and flasks 7–9 were group C. Group A was not inoculated and served as the control group. Flasks B and C were inoculated with 1% OD245 solution. 600 A bacterial culture of molybdenum-tolerant potassium-solubilizing bacteria (Bacillus amyloliquefaciens MoTB 79) with a molybdenum reduction capacity of 1.0 was prepared. Nine bottles were evenly placed in a shaker at 30°C and 150 rpm and cultured. Samples were taken at 24 h and 48 h of culture. The culture medium was centrifuged at 8000 rpm for 10 min. The supernatant was filtered through a 0.22 μm filter membrane, and the concentration of Mo(VI) in the supernatant was determined by ICP-MS. The average value of each group was taken as the experimental data, and their Mo(VI) adsorption rate was calculated.

[0053] As shown in Table 2, in the Mo(VI) adsorption experiment, the molybdenum-resistant potassium-solubilizing bacteria (Bacillus amyloliquefaciens MoTB 79) with molybdenum reduction ability could reduce the concentration of Mo(VI) in the culture medium solution by 8.57 mg / L to 8.90 mg / L, and the removal rate of Mo(VI) in the solution reached 21.66% to 22.50%. The results indicate that the molybdenum-resistant potassium-solubilizing bacteria (Bacillus amyloliquefaciens MoTB 79) with molybdenum reduction ability has a certain removal effect on Mo(VI) in the environment.

[0054] Table 2 Adsorption of Mo(VI) by Bacillus amyloliquefaciens MoTB 79

[0055]

[0056] 3. Take the cells that have been grown for 36 hours in liquid BPM and LPM medium with Mo(VI)-free and Mo(VI) concentration of 3000 mg / L respectively, centrifuge at 2000g for 10 min, discard the supernatant, wash 3 times with distilled water, freeze dry and then perform XPS analysis.

[0057] XPS analysis was performed on the precipitate of molybdenum-resistant potassium-solubilizing bacteria (Bacillus amyloliquefaciens MoTB 79) after adsorption of Mo(VI). Figure 5 It was found that the molybdenum-resistant potassium-solubilizing bacterium (Bacillus amyloliquefaciens MoTB 79), after being cultured in BPM and LPM liquid media containing Mo(VI), was able to reduce 62.54% and 65.75% of the adsorbed Mo(VI) to Mo(V) and Mo(IV), respectively. The results indicate that the molybdenum-resistant potassium-solubilizing bacterium (Bacillus amyloliquefaciens MoTB79) can adsorb Mo(VI) from the environment and reduce it to Mo(V) and Mo(IV), which have relatively lower toxicity.

[0058] like Figure 6 As shown, qualitative and quantitative analysis of the potassium-solubilizing ability of molybdenum-resistant potassium-solubilizing bacteria (Bacillus amyloliquefaciens MoTB 79) with molybdenum reduction activity.

[0059] 1. Preparation of bacterial suspension: A small amount of bacterial cells were picked up with an inoculation loop and inoculated into BPM solid medium for strain activation, while a single colony was picked up and inoculated into 20 ml of BPM liquid medium for bacterial enrichment;

[0060] 2. Inoculation and culture for qualitative analysis of potassium solubilization capacity: The prepared OD 600 A bacterial suspension with a value of 0.8 was spotted at 3 μL onto potassium-solubilizing solid medium. After culturing for 24 hours, the formation of a lysis halo indicates that MoTB 79 has a certain potassium-solubilizing ability.

[0061] 3. Inoculation and culture for quantitative analysis of potassium solubilization capacity: The prepared OD... 600 The bacterial suspension with a value of 0.8 was inoculated into potassium-solubilizing liquid medium at an inoculation rate of 1% of the culture system and cultured at 30℃ and 150rpm for 24h. After the culture was completed, the content of soluble potassium was determined by flame photometry. The content of soluble potassium in the potassium-solubilizing liquid medium was 0.14±0.02mg / L.

[0062] Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Those skilled in the art can make changes, modifications, substitutions and variations to the above embodiments within the scope of the present invention.

Claims

1. A molybdenum-resistant potassium-solubilizing bacterium with molybdenum-reducing activity, characterized in that, Its classification name is: Bacillus amyloliquefaciens ( Bacillus amyloliquefaciens MoTB 79 was deposited on April 7, 2024, at the China Center for Type Culture Collection (CCTCC) with accession number CCTCCNo.M2024639.

2. The application of a molybdenum-resistant potassium-solubilizing bacterium with molybdenum reduction activity as described in claim 1 in reducing the concentration of Mo(VI) in soil or wastewater contaminated with molybdenum ions.

3. The application of a molybdenum-resistant potassium-solubilizing bacterium with molybdenum reduction activity as described in claim 1 in the process of breaking down insoluble potassium into soluble potassium in soil, wherein the insoluble potassium is potassium feldspar.

4. The application according to claim 3, characterized in that, The insoluble potassium element is potassium feldspar.

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