Molybdenum-resistant potassium bacteria with molybdenum reduction effect as well as separation and screening method and application of molybdenum-resistant potassium bacteria

Abstract

The invention relates to a molybdenum-resistant potassium bacterium with a molybdenum reduction effect, which is classified and named as bacillus amyloliquefaciens MoTB 79, is preserved in China Center for Type Culture Collection (CCTCC) on April 7, 2024 with a preservation number of CCTCC No.M2024639, and has the advantages that the molybdenum-resistant potassium bacterium has the molybdenum reduction effect, and the molybdenum-resistant potassium bacterium has the molybdenum reduction effect; the invention relates to application of molybdenum-resistant potassium bacteria with a molybdenum reduction effect in soil or waste water polluted by molybdenum ions. The invention relates to application of a molybdenum-resistant potassium bacteria with a molybdenum reduction effect in soil potassium decomposition. The bacillus amyloliquefaciens MoTB 79 has the beneficial effects that the bacillus amyloliquefaciens MoTB 79 can reduce the Mo (VI) adsorbed by the bacillus amyloliquefaciens MoTB 79, and the adsorbed Mo (VI) is reduced into Mo (V) and Mo (IV) with relatively low toxicity, so that the toxicity and ecological harm of the Mo (VI) in soil or a water body are reduced, finally, effective treatment of Mo (VI) pollution is realized, and the bacillus amyloliquefaciens MoTB 79 has a certain potassium dissolving capability while having relatively high Mo (VI) reduction efficiency.

Description

Technical Field

[0001] The invention relates to the technical field of environmental microorganisms, and in particular to a molybdenum-resistant potassium-solubilizing bacterium with molybdenum-reducing function, a separation and screening method and an application thereof. Background Art

[0002] Molybdenum is a multivalent element with various oxidation states from 0 to VI, with Mo(IV) and Mo(VI) being the most stable. Compared with Mo(IV), Mo(VI) is more easily absorbed and more toxic. Long-term exposure to high-concentration molybdenum environments can lead to gout, increase the risk of cardiovascular disease, and even cause cancer in severe cases. It is reported that the molybdenum concentration 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 pond leakage has received increasing attention. Therefore, it is very necessary to repair molybdenum pollution risk areas.

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

[0004] The technical problem to be solved by the present invention is: to overcome the defects of the existing soil molybdenum pollution remediation technology, to provide molybdenum-resistant potassium-dissolving bacteria with molybdenum reduction function, as well as an isolation and screening method and application. The bacteria can tolerate sodium molybdate with a Mo(VI) concentration of up to 100,000 mg / L, can remove 21.66% of Mo(VI) in a BPM culture medium containing 39.55 mg / L Mo(VI) within 48 hours, and can reduce the effective molybdenum content in the soil by 11.14% within 2 days. In addition, the strain can dissolve the insoluble potassium element into a soluble potassium content of 0.14±0.02 mg / L in a potassium-dissolving liquid culture medium. Preliminary studies have shown that the strain of the present invention can provide bacterial species resources for bioremediation of molybdenum-contaminated areas with the problem of low soil potassium utilization.

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

[0006] A molybdenum-resistant potassium-dissolving bacterium with molybdenum-reducing function, whose classification name is Bacillus amyloliquefaciens MoTB 79, and which was deposited in China Center for Type Culture Collection on April 7, 2024 with the deposit number: CCTCC No.M2024639.

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

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

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

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

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

[0012] S1. Take the rhizosphere soil of pioneer plants in the molybdenum tailings area, make it into a soil suspension, and then dilute it in a gradient manner and streak it in a beef extract peptone solid medium containing a high concentration of Mo (VI) to screen and isolate molybdenum-resistant bacteria, so as to isolate surviving strains that are tolerant to Mo (VI);

[0013] S2. After the colonies grow, streak the colonies on the plate surface several times to purify the strains to obtain molybdenum-resistant strains;

[0014] S3, using low-phosphomolybdate solid medium to culture the molybdenum-resistant bacteria, using the formation of blue colonies after 24 hours of culture as the basis for initial screening, and screening out strains with molybdenum-reducing ability;

[0015] S4. Quantitative analysis of strains with molybdenum reducing ability using LPM liquid culture medium is performed to screen out strains with stronger molybdenum reducing ability;

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

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

[0018] Based on the above technical solution, the present invention also provides a molybdenum-resistant potassium-dissolving bacteria with molybdenum reducing function for use in soil or wastewater contaminated with molybdenum ions.

[0019] Based on the above technical solution, the present invention also provides a molybdenum-resistant potassium-solubilizing bacteria with molybdenum reducing function for use in soil potassium-solubilizing.

[0020] Furthermore, molybdenum-resistant potassium-dissolving bacteria with molybdenum-reducing function dissolve the insoluble potassium element to a soluble potassium content of 0.14±0.02 mg / L.

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

[0022] Compared with the prior art, the present invention has the following beneficial technical effects: Bacillus amyloliquefaciens MoTB 79 is identified by 16S rDNA sequence sequencing, and the molybdenum reduction ability and potassium dissolving performance of Bacillus amyloliquefaciens MoTB 79 are determined. In the study of Mo(VI) adsorption and conversion in the environment, Bacillus amyloliquefaciens MoTB 79 can reduce the Mo(VI) adsorbed by itself, and the proportion of reduced Mo(VI) can reach 62.54% to 65.75%. The adsorbed Mo(VI) is reduced to Mo(V) and Mo(IV) with relatively low toxicity. 79 can still survive in solutions or soils with high Mo(VI) content, and can effectively control the migration and transformation process of Mo(VI), Mo(IV) and Mo(V) in soil or water, reduce the toxicity and ecological hazards of Mo(VI) in soil or water, and ultimately achieve effective control of Mo(VI) pollution. While having a high Mo(VI) reduction efficiency, it also has a certain potassium-solubilizing ability. Bacillus amyloliquefaciens MoTB 79 can dissolve insoluble potassium feldspar and improve the utilization rate of potassium in the soil. It is environmentally friendly and is a strain with good application prospects. It is of great significance to the development of bioremediation technology. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 This is a photo of the screening results of Bacillus amyloliquefaciens MoTB 79 plate in the present invention;

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

[0025] Figure 3 is the growth curve of Bacillus amyloliquefaciens MoTB 79 in the present invention at different initial Mo(VI) concentrations;

[0026] Figure 4 It is a spectrum scanning diagram of the product Mo-B lue of the reduction of Mo(VI) by Bacillus amyloliquefaciens MoTB 79 in LPM liquid culture medium in the present invention;

[0027] Figure 5 The XPS test results of the Bacillus amyloliquefaciens MoTB 79 after adsorbing BPM and LPM culture media containing Mo(VI) in the present invention;

[0028] Figure 6 This is a characteristic diagram of the potassium-solubilizing effect of Bacillus amyloliquefaciens MoTB 79 in the present invention. DETAILED DESCRIPTION

[0029] The principles and features of the present invention are described below in conjunction with the accompanying drawings. The examples given are only used to explain the present invention and are not used to limit the scope of the present invention.

[0030] A molybdenum-resistant potassium-solubilizing bacterium with molybdenum reducing function, which is classified and named as Bacillus amyloliquefaciens MoTB 79, and was deposited with the China Center for Type Culture Collection on April 7, 2024 with the deposit number: CCTCC No.M2024639, and the deposit address is: Wuhan University, Wuhan, China.

[0031] The bacteria grow well in an environment with a Mo(VI) content of 3000 mg / L, wherein the test environment is: solid or liquid culture medium, in particular: the growth rate is fast in a liquid BPM culture medium with a Mo(VI) content of 3000 mg / L, and the bacterial growth rate is significantly reduced in a liquid BPM culture medium with a Mo(VI) content of more than 60000 mg / L, the maximum tolerance Mo(VI) concentration is 100000 mg / L, and the test environment is: a liquid culture medium containing Mo(VI);

[0032] Bacillus amyloliquefaciens MoTB 79 can reduce the Mo(VI) adsorbed by itself, and the reduced Mo(VI) accounts for 62.54% to 65.75%.

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

[0034] like Figure 1 , Figure 2 As shown, strain screening:

[0035] Take rhizosphere soil samples of pioneer plants in the molybdenum tailings area, for example, from the rhizosphere soil samples of pioneer plants in the molybdenum tailings area in Luanchuan, Henan Province, add 5.0 g of the soil sample to be tested into a triangular flask containing 45 ml of sterile water, and incubate it in a shaking incubator at 30°C and 150 r / min for 30 minutes to obtain a soil suspension;

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

[0037] Screening of molybdenum-resistant bacteria with molybdenum-reducing properties:

[0038] The above 142 molybdenum-resistant bacteria were inoculated in a low phosphomolybdate (LPM) solid culture medium, and the formation of blue colonies (phosphomolybdenum blue reaction) after culturing for 24 hours was used as the basis for preliminary screening, and 44 strains with molybdenum reduction ability were screened out. Then, the strains with molybdenum reduction ability were inoculated into a 250ml conical flask filled with 100ml liquid LPM culture medium at an inoculation amount of 1%, and three repeated shaking flasks were set up for the strains. Sterile LPM liquid culture medium was used as a control, and cultured at 30°C and 150rpm. After 24 hours, the supernatant was fully spectrally scanned with a UV-visible spectrophotometer, and its molybdenum blue production ability was quantitatively analyzed by measuring the absorbance of the supernatant at 865nm, and a strain with the strongest molybdenum reduction ability was screened out. Its full spectrum scanning spectrum, i.e., the reduced product molybdenum blue scanning spectrum and the reading at a wavelength of 865nm, i.e., the Mo(VI) reduction effect, are shown in FIG. Figure 4 As shown;

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

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

[0041] Figure 3The obtained molybdenum-reducing and molybdenum-resistant potassium-solubilizing bacteria (Bacillus amyloliquefaciens MoTB79) were inoculated into culture media with initial Mo(VI) concentrations of 0, 3000, 6000, 9000, 15000, 30000, 60000, 70000, 80000, 90000, and 100000 mg / L, respectively. The growth curve of the molybdenum-resistant potassium-solubilizing bacteria (Bacillus amyloliquefaciens MoTB79) with molybdenum reduction after 30 hours of cultivation showed that when the Mo(VI) concentration was 300 When the concentration of Mo(VI) was 0mg / L and 6000mg / L, Mo(VI) had no obvious inhibitory effect on the growth of the strain. However, when the concentration of Mo(VI) was higher than 30000mg / L, the growth of the strain was inhibited to varying degrees, the logarithmic growth phase was delayed, and the growth was significantly lower after entering the logarithmic phase. At the same time, the growth rates of the strains with different Mo(VI) concentrations were compared. The results showed that the strain could grow normally in the liquid culture medium with a Mo(VI) concentration of 6000mg / L, which was equivalent to the growth rate of the control group. The MIC Mo(VI) of the strain could reach 100000mg / L, indicating that the molybdenum-resistant potassium-solubilizing bacteria (Bacillus amyloliquefaciens MoTB 79) with molybdenum reduction function had a strong tolerance to Mo(VI).

[0042] Identification

[0043] The strains obtained by screening were sequenced, and the sequencing results were compared by BLAST sequence comparison on the NCB I website, and were determined to be Serratiafi caria MoTB 2, as shown in Table 1;

[0044] Table 1

[0045]

[0046]

[0047] Analysis of Mo(VI) adsorption capacity and conversion behavior after adsorption by molybdenum-tolerant potassium-solubilizing bacteria (Bacillus amyloliquefaciens MoTB 79) with molybdenum reduction

[0048] 1. Adding molybdenum-reducing and potassium-dissolving bacteria (Bacillus amyloliquefaciens MoTB 79) to the BPM and LPM liquid culture media containing Mo(VI) to obtain a mixed culture medium, wherein the inoculation amount of the molybdenum-reducing and potassium-dissolving bacteria (Bacillus amyloliquefaciens MoTB 79) in the mixed culture medium is 1%;

[0049] The formula of 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, 1000 ml water, pH of the medium is 7.1-7.3, sterilized at 121°C for 20 min;

[0050] The formula of LPM medium containing Mo(VI) is: (NH 4 ) 2 SO 4 3.0 g, MgSO 4 7H 2 O 0.5g, NaCl 15.0g, Na 2 MoO 4 ·2H 2 O 2.42g, Na 2 HPO 4 0.71g, yeast extract powder 0.5g, glucose 10.0g, water 1000ml; pH of the culture medium is 7.0, sterilized at 121℃ for 20min;

[0051] 2. Preliminary study on the Mo(VI) adsorption capacity of the strain using shake flask test:

[0052] Nine 250ml triangular flasks were filled with 100ml of liquid LPM medium, numbered No. 1 to 9, and divided into three groups: No. 1 to 3 were group A, No. 4 to 6 were group B, and No. 7 to 9 were group C. Group A was not inoculated and was the control group. Group B and C were inoculated with 1% OD 600 =1.0, 9 bottles were evenly placed in a shaker at 30°C and 150 rpm for culture, samples were taken at 24h and 48h of culture, the culture solution was centrifuged at 8000 rpm for 10 min, the supernatant was filtered through a 0.22 um filter membrane, the Mo(VI) concentration in the supernatant was determined by I CP-MS, the average value of each group was taken as the experimental data, and their Mo(VI) adsorption rates were calculated;

[0053] As shown in Table 2, in the adsorption experiment of Mo(VI), the molybdenum-resistant potassium-solubilizing bacteria (Bacillus amyloliquefaciens MoTB 79) with molybdenum reduction can 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 is 21.66% to 22.50%. The results show that the molybdenum-resistant potassium-solubilizing bacteria (Bacillus amyloliquefaciens MoTB 79) with molybdenum reduction 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 grown for 36 h in liquid BPM and LPM medium without Mo(VI) or with Mo(VI) concentration of 3000 mg / L, centrifuge at 2000 g for 10 min, discard the supernatant, and then wash with distilled water for 3 times, freeze-dry and then perform XPS measurement;

[0057] XPS analysis of the cell precipitate of the molybdenum-tolerant potassium-solubilizing bacteria (Bacillus amyloliquefaciens MoTB 79) after adsorption of Mo(VI) showed that Figure 5 It can be seen that after being cultured in BPM and LPM liquid culture media containing Mo(VI), the molybdenum-resistant potassium-solubilizing bacteria (Bacillus amyloliquefaciens MoTB 79) with molybdenum reduction function can reduce 62.54% and 65.75% of the adsorbed Mo(VI) to Mo(V) and Mo(IV), respectively. The results show that the molybdenum-resistant potassium-solubilizing bacteria (Bacillus amyloliquefaciens MoTB79) with molybdenum reduction function can adsorb Mo(VI) in the environment and reduce it to relatively less toxic Mo(V) and Mo(IV).

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

[0059] 1. Preparation of bacterial suspension: Use an inoculation loop to pick a small amount of bacteria and inoculate it into BPM solid medium for strain activation, and pick a single colony and inoculate it into 20 ml BPM liquid medium for strain enrichment;

[0060] 2. Inoculation and cultivation for qualitative analysis of potassium dissolving ability: 600 The value is 0.8. 3 μ l of bacterial suspension is spotted on potassium-solubilizing solid medium. After 24 hours of culture, a dissolution halo is formed, indicating that MoTB 79 has a certain potassium-solubilizing ability.

[0061] 3. Inoculation and culture for quantitative analysis of potassium dissolving capacity: 600 The bacterial suspension with a value of 0.8 was inoculated into the potassium-dissolving liquid culture medium at 1% of the inoculum size of the culture system, and cultured at 30°C, 150rpm for 24 hours. After the culture was completed, the content of soluble potassium was determined by flame photometry. In the potassium-dissolving liquid culture medium, the insoluble potassium element was dissolved into a soluble potassium content of 0.14±0.02 mg / L.

[0062] Although the embodiments of the present invention have been shown and described above, it is to be understood that the above embodiments are exemplary and are not to be construed as limitations of the present invention. A person skilled in the art may change, modify, replace and vary the above embodiments within the scope of the present invention.

Claims

1. A molybdenum-resistant potassium-solubilizing bacterium having a molybdenum-reducing effect, It is characterized in that Its classification name is Bacillus amyloliquefaciens MoTB 79, and on April 7, 2024, it was deposited in the China Center for Type Culture Collection with the deposit number: CCTCC No. M2024639.

2. The molybdenum-resistant potassium-solubilizing bacteria having molybdenum-reducing function according to claim 1, It is characterized in that The bacteria grow well in an environment with a Mo(VI) content of 3000 mg / L.

3. A molybdenum-resistant potassium-solubilizing bacterium having a molybdenum-reducing effect according to claim 1 or 2, It is characterized in that The maximum tolerance concentration of Mo(VI) of this bacterium is 100000 mg / L.

4. A molybdenum-resistant potassium-solubilizing bacterium having a molybdenum-reducing effect according to any one of claims 1 to 3, It is characterized in that The bacteria reduces Mo(VI) to generate Mo-Blue, and the absorbance of the product Mo-Blue at 865nm can reach 4.0997.

5. A method for separating and screening molybdenum-resistant potassium-solubilizing bacteria having molybdenum-reducing function as claimed in any one of claims 1 to 4, It is characterized in that The steps include: S1. Take the rhizosphere soil of pioneer plants in the molybdenum tailings area, make it into a soil suspension, and then dilute it in a gradient manner and streak it in a beef extract peptone solid medium containing a high concentration of Mo (VI) to screen and isolate molybdenum-resistant bacteria, so as to isolate surviving strains that are tolerant to Mo (VI); S2. After the colonies grow, streak the colonies on the plate surface several times to purify the strains to obtain molybdenum-resistant strains; S3, using low-phosphomolybdate solid medium to culture the molybdenum-resistant bacteria, using the formation of blue colonies after 24 hours of culture as the basis for initial screening, and screening out strains with molybdenum-reducing ability; S4. Quantitative analysis of strains with molybdenum reducing ability using LPM liquid culture medium is performed to screen out strains with stronger molybdenum reducing ability; S5. Characterize the potassium-solubilizing effect of the obtained molybdenum-reducing resistant strain to obtain molybdenum-resistant potassium-solubilizing bacteria with molybdenum-reducing function.

6. The separation and screening method according to claim 4, It is characterized in that The Mo(VI) source added to the beef extract peptone solid culture medium in S1 is sodium molybdate, and the Mo(VI) concentration is 3000 mg / L.

7. Use of the molybdenum-resistant potassium-solubilizing bacteria having molybdenum-reducing function as claimed in any one of claims 1 to 4 in soil or wastewater contaminated with molybdenum ions.

8. Use of the molybdenum-resistant potassium-solubilizing bacteria having molybdenum-reducing function as claimed in any one of claims 1 to 4 in soil potassium-solubilizing.

9. The use according to claim 8, It is characterized in that Molybdenum-resistant potassium-dissolving bacteria with molybdenum-reducing function dissolve the insoluble potassium element to a soluble potassium content of 0.14±0.02 mg / L.

10. The use according to claim 9, It is characterized in that The insoluble potassium element is potassium feldspar.

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