Molybdenum-tolerant bacteria with molybdenum reduction effect, isolation and screening method and application

Abstract

The application relates to a molybdenum-tolerant phosphorus-dissolving bacterium with molybdenum reduction, which is named Serratia ficaria MoTB 2, and is preserved in the China Center for Type Culture Collection (CCTCC) on April 7, 2024, with a preservation number of CCTCC NO.M2024638. The application also relates to application of the molybdenum-tolerant phosphorus-dissolving bacterium with molybdenum reduction in soil or wastewater containing molybdenum ion pollution, and application of the molybdenum-tolerant phosphorus-dissolving bacterium with molybdenum reduction in dissolving insoluble phosphate. The Serratia ficaria MoTB 2 can reduce self-adsorbed Mo (VI) into Mo (V) and Mo (IV) with relatively low toxicity, so as to reduce the toxicity and ecological harm of Mo (VI) in soil or water body, and can activate insoluble phosphate in soil while having high Mo (VI) reduction efficiency.

Description

Technical Field

[0001] This invention relates to the field of environmental microbiology, specifically to a molybdenum-resistant phosphate-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 molybdenum-resistant phosphate-solubilizing bacteria with molybdenum reduction capabilities, as well as isolation and screening methods and applications. This bacterium can tolerate sodium molybdate with a Mo(VI) concentration of up to 100,000 mg / L. Within 48 hours, it can remove 22.28% of Mo(VI) from BPM medium containing 39.55 mg / L Mo(VI), and within 2 days, it can reduce the content of available molybdenum in the soil by 9.40%. Furthermore, in inorganic phosphorus liquid medium, the strain can dissolve insoluble phosphates to a soluble phosphorus content of 17.00 ± 2.04 mg / L, which can provide bacterial resources for the bioremediation of molybdenum-polluted areas with insufficient soil phosphorus supply.

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

[0006] A molybdenum-resistant phosphate-solubilizing bacterium with molybdenum-reducing activity, classified as Serratiaficaria MoTB 2, was deposited at the China Center for Type Culture Collection on April 7, 2024, with accession number CCTCC NO.M2024638.

[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 reduced Mo(VI) to generate Mo-Bluue, and the absorbance of the product Mo-Bluue at 865 nm was 6.3416.

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

[0012] S1. Take rhizosphere soil samples of pioneer plants in the molybdenum tailings area, prepare 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, so as to isolate surviving strains that are tolerant 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. Molybdenum-resistant strains were cultured in low phosphorus molybdate solid medium. The formation of blue colonies after 24 hours of culture was used as the initial screening criterion to screen out strains with molybdenum reduction capabilities.

[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 phosphorus-solubilizing effect of the strain with the strongest molybdenum reducing ability to identify molybdenum-resistant phosphate-solubilizing bacteria with molybdenum reducing ability.

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

[0018] Based on the above technical solution, the present invention also provides an application of molybdenum-resistant phosphorus-solubilizing bacteria with molybdenum reduction function 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 phosphate-solubilizing bacteria with molybdenum reduction activity in the dissolution of insoluble phosphates.

[0020] Furthermore, the insoluble phosphate is tricalcium phosphate.

[0021] Furthermore, the insoluble phosphate was dissolved to a soluble phosphorus content of 17.00±2.04 mg / L.

[0022] The beneficial effects of this invention are:

[0023] Serratia marcescens MoTB 2 was identified by 16S rDNA sequencing. Its molybdenum reducing capacity and phosphorus solubility were also measured. In the study of Mo(VI) adsorption and transformation in the environment, Serratia marcescens MoTB 2 was able to reduce its own adsorbed Mo(VI), with a reduction rate of 60.30%–76.62%. The adsorbed Mo(VI) was reduced to the relatively less toxic Mo(V) and Mo(IV), which can still survive in solutions or soils with high molybdenum content. This effectively controls the migration and transformation of Mo(VI), Mo(V), and Mo(IV) in soil or water, reducing the toxicity and ecological harm of Mo(VI) in soil or water, and ultimately achieving effective remediation of Mo(VI) pollution. Within 48 hours, it can reduce the concentration of Mo(VI) at 39.55 mg / L... Mo(VI) removal in BPM medium was 22.28%, reducing the available molybdenum content in the soil by 9.40% within 2 days. Serratia marcescens MoTB 2 can dissolve insoluble phosphates to improve phosphorus utilization in the soil. In inorganic phosphorus liquid medium, this strain dissolved insoluble phosphates to a soluble phosphorus content of 17.00±2.04 mg / L, indicating that Serratia marcescens MoTB 2 in this invention has both high molybdenum reduction efficiency and a certain phosphorus-dissolving ability. The maximum molybdenum tolerance concentration of Serratia marcescens MoTB 2 is 100,000 mg / L. This strain can provide bacterial resources for bioremediation in molybdenum-contaminated areas with insufficient soil phosphorus supply. It is environmentally friendly and a strain with good application prospects, which is of great significance to the development of bioremediation technology. Attached Figure Description

[0024] Figure 1 This is a photograph of the screening results of Serratia marcescens MoTB 2 plates in this invention;

[0025] Figure 2 This is the phylogenetic tree of Serratia moTB 2 in this invention;

[0026] Figure 3 The growth curves of Serratia marcescens MoTB 2 at different initial molybdenum concentrations in this invention are shown.

[0027] Figure 4 This is a spectral scan of Mo-Bluue, the product of the reduction of Mo(VI) by Serratia moriformis MoTB2 in LPM liquid medium in this invention.

[0028] Figure 5The XPS results are as follows: Serratia marcescens MoTB 2 adsorbs Mo(VI) into BPM and LPM culture media.

[0029] Figure 6 This is a diagram illustrating the phosphorus-solubilizing effect of Serratia marcescens MoTB 2 in this invention. Detailed Implementation

[0030] 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.

[0031] A molybdenum-resistant phosphate-solubilizing bacterium with molybdenum-reducing activity, classified as Serratiaficaria MoTB 2, was deposited on April 7, 2024, at the China Center for Type Culture Collection (CCTCC NO.M2024638), Wuhan University, Wuhan, China.

[0032] The bacteria grew well in an environment with a Mo(VI) concentration of 3000 mg / L. The test environment was solid or liquid culture medium. The growth rate of the bacteria decreased significantly in BPM liquid culture medium with a Mo(VI) concentration of more than 60000 mg / L. The maximum tolerated Mo(VI) concentration was 100000 mg / L, and the test environment was liquid culture medium.

[0033] Serratia ficaria MoTB 2 can reduce its own adsorbed Mo(VI), with the reduction rate reaching 60.30% to 76.62%. The adsorbed Mo(VI) is reduced to Mo(V) and Mo(IV), which have relatively low toxicity.

[0034] The bacterium reduces Mo(VI) to produce Mo-Bluue. The absorbance of the product Mo-Bluue at 865 nm is 6.3416. The test environment is LPM liquid medium containing Mo(VI) source.

[0035] like Figure 1 , Figure 2 As shown, the strains were screened:

[0036] Take soil samples from the rhizosphere of pioneer plants in the molybdenum tailings area. For example, collect soil samples from the rhizosphere of pioneer plants in the molybdenum tailings area of ​​Luanchuan, Henan. Add 5.0g of the soil sample to be tested to an Erlenmeyer flask containing 45ml of sterile water and incubate it in a shaker at 30℃ and 150r / min for 30min to obtain a soil suspension.

[0037] 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.

[0038] Screening for strains with molybdenum-reducing activity:

[0039] The 142 strains 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 reduction capabilities. Then, these molybdenum-reducing strains were inoculated at a 1% inoculation 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 capacity by measuring the absorbance at 865 nm. One strain with the strongest molybdenum reduction capacity 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;

[0040] Molybdenum-resistant growth curve test of molybdenum-reducing phosphate-solubilizing bacteria (Serratia marcescens MoTB 2)

[0041] 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.

[0042] Figure 3To investigate the growth of *Serratia marcescens* strain MoTB 2, the strain was inoculated into culture media with initial Mo(VI) concentrations of 0, 3000, 6000, 9000, 15000, 30000, 60000, 70000, 80000, 90000, and 100000 mg / L. The growth curves of the strain after 30 hours of cultivation were shown in the figure. As can be seen from the figure, when the Mo(VI) concentration was 3000 mg / L and 6000 mg / L, Mo(VI) had no significant inhibitory effect on the growth of the strain. However, when the Mo(VI) concentration 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. Furthermore, comparing the growth rates of the strain at different Mo(VI) concentrations, the results showed that the strain could grow normally in liquid culture medium with a Mo(VI) concentration of 6000 mg / L, with a growth rate comparable to the control group. The strain's MIC (Minimum Intake, Cholesterol, and Chloride) was also observed. Mo(VI) levels can reach 100,000 mg / L, indicating that Serratia marcescens MoTB 2 has strong tolerance to Mo(VI).

[0043] Identification

[0044] The selected strains were sequenced, and the sequencing results were compared with BLAST sequences on the NCBI website, which identified them as Serratia ficariae MoTB 2, as shown in Table 1.

[0045] Table 1

[0046]

[0047]

[0048] Analysis of the adsorption capacity of Serratia ficaria MoTB 2 for Mo(VI) and the transformation behavior after adsorption

[0049] 1. Serratia ficaria MoTB 2 was added to BPM and LPM liquid media containing Mo(VI) respectively to obtain mixed media. The inoculum amount of Serratia ficaria MoTB 2 in the mixed media was 1%.

[0050] The formula for BPM culture medium containing Mo(VI) is as follows: Mo(VI) 3000mg (sodium molybdate dihydrate), peptone 10.0g, sodium chloride 5.0g, beef extract 3.0g, and water 1000ml; the pH of the culture medium is 7.1-7.3, and it is sterilized at 121℃ for 20min.

[0051] The formulation of LPM 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, water 1000ml; the pH of the medium is 7.0, and it is sterilized at 121℃ for 20min.

[0052] 2. Preliminary study on the Mo(VI) adsorption capacity of Serratia marcescens MoTB 2 using shake-flask experiments.

[0053] Nine 250ml Erlenmeyer flasks, each containing 100ml of liquid LPM culture medium and numbered No. 1–9, are divided into three groups:

[0054] Numbers 1-3 are Group A, numbers 4-6 are Group B, and numbers 7-9 are Group C;

[0055] Group A was not vaccinated and served as the control group. Groups B and C were each vaccinated with 1% OD. 600 Nine bottles containing 1.0% Serratia marcescens MoTB 2 were evenly placed in a shaker at 30℃ and 150 rpm and cultured. Samples were taken at 24h and 48h of culture. The culture was centrifuged at 8000 rpm for 10 min, and the supernatant was filtered through a 0.22 μm filter membrane. The Mo concentration 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 rates were calculated.

[0056] As shown in Table 2, in the Mo(VI) adsorption experiment, Serratia marcescens MoTB 2 can reduce the concentration of Mo(VI) in the culture medium solution by 5.86 mg / L to 8.81 mg / L, and the removal rate of Mo(VI) in the solution is 14.82% to 22.28%. The results indicate that Serratia marcescens MoTB 2 has a certain removal effect on Mo(VI) in the environment.

[0057] Table 2 Adsorption of Mo(VI) by Serratia marcescens MoTB 2

[0058]

[0059]

[0060] 3. Take the cells that have been grown for 36 hours in BPM and LPM liquid 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.

[0061] XPS analysis was performed on the Serratia marcescens MoTB 2 cell precipitate after adsorption of Mo(VI). Figure 5It was found that after being cultured in liquid culture media containing Mo(VI) in BPM and LPM medium, Serratia mossica MoTB 2 was able to reduce 76.62% and 60.30% of the adsorbed Mo(VI) to Mo(V) and Mo(IV), respectively. The results indicate that Serratia mossica MoTB 2 can adsorb Mo(VI) from the environment and reduce it to Mo(V) and Mo(IV), which have relatively low toxicity.

[0062] Qualitative and quantitative analysis of the phosphorus-solubilizing ability of Serratia marcescens MoTB 2

[0063] 1. Preparation of bacterial suspension: A small amount of Serratia moTB 2 was 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 strain enrichment.

[0064] 2. Inoculation and culture for qualitative analysis of phosphorus solubility: The prepared OD... 600 A bacterial suspension with a concentration of 0.8 μL was spotted onto an inorganic phosphorus solid medium. After 24 hours of incubation, the formation of a lysing halo indicates that *Serratia marcescens* MoTB 2 possesses a certain phosphorus-solubilizing ability. Figure 6 As shown;

[0065] 3. Inoculation and culture for quantitative analysis of phosphorus solubility: The OD prepared above... 600 The bacterial suspension with a value of 0.8 was inoculated into inorganic phosphorus 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 phosphate was determined by the molybdenum antimony spectrophotometric method. The content of soluble phosphate in the inorganic phosphorus liquid medium was 17.00±2.04mg / L.

[0066] 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-tolerant phosphorus solubilizing bacteria having molybdenum reducing action, characterized by, The classification name is: Serratia marcescens (Serratia marcescens) Serratia ficaria ) MoTB 2, on April 7, 2024, with the preservation number: CCTCC NO: M2024638, preserved in: China Center for Type Culture Collection.

2. The use of the molybdenum-tolerant phosphorus solubilizing bacteria with molybdenum reducing effect according to claim 1 for reducing the concentration of Mo(Ⅵ) in soil or wastewater contaminated with molybdenum ions.

3. The use of the molybdenum-tolerant phosphorus solubilizing bacteria with molybdenum reducing effect according to claim 1 for solubilizing tricalcium phosphate.

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