Molybdenum-resistant phosphate solubilizing bacteria with molybdenum reduction effect as well as separation and screening method and application thereof

Abstract

The invention relates to a molybdenum-resistant phosphate solubilizing bacterium with a molybdenum reduction effect, which is classified and named as Serratia filicata MoTB 2, is preserved in China Center for Type Culture Collection (CCTCC) on April 7, 2024 with a preservation number of CCTCC NO.M2024638, and has the advantages that the molybdenum-resistant phosphate solubilizing bacterium can be used for preparing the molybdenum-resistant phosphate solubilizing bacterium, and the molybdenum-resistant phosphate solubilizing bacterium can be used for preparing the molybdenum-resistant phosphate solubilizing bacterium; the invention relates to application of a molybdenum-resistant phosphate solubilizing bacterium with a molybdenum reduction effect in soil or wastewater polluted by molybdenum ions. The invention relates to application of a molybdenum-resistant phosphate solubilizing bacterium with a molybdenum reduction effect in dissolving insoluble phosphate. The serratia marcescens MoTB 2 has the beneficial effects that the serratia marcescens MoTB 2 can be used for reducing Mo (VI adsorbed by the serratia marcescens MoTB 2 into Mo (V) and Mo (IV) which are relatively low in toxicity, so that the toxicity and ecological harm of the Mo (VI in soil or a water body are reduced, and insoluble phosphate in the soil can be activated while relatively high Mo (VI reduction efficiency is achieved.

Description

Technical Field

[0001] The invention relates to the technical field of environmental microorganisms, and in particular to a molybdenum-resistant phosphate-solubilizing bacterium with molybdenum reduction 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 phosphate-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 22.28% 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 9.40% within 2 days. In addition, the strain can dissolve insoluble phosphates in an inorganic phosphorus liquid culture medium to a soluble phosphorus content of 17.00±2.04 mg / L, and can provide bacterial strain resources for bioremediation of molybdenum-contaminated areas with insufficient soil phosphorus supply.

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

[0006] A molybdenum-resistant phosphate-solubilizing bacterium with molybdenum reducing function, whose classification name is Serratia ficaria MoTB 2, and which was deposited in China Center for Type Culture Collection on April 7, 2024 with the deposit number: CCTCC NO.M2024638.

[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-B l ue, and the absorbance of the obtained product Mo-B l ue at 865nm was 6.3416.

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

[0012] S1. Take rhizosphere soil samples of pioneer plants in the molybdenum tailings area, make them into soil suspensions, and then dilute them in a gradient manner and streak them in beef extract peptone solid medium containing high concentration of Mo(VI) for screening and separation, so as to isolate the 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 molybdenum-resistant strains, and using the formation of blue colonies after 24 hours of culture as the basis for initial screening to screen 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 phosphate-dissolving effect of the strain with the strongest molybdenum-reducing ability to determine the molybdenum-resistant phosphate-dissolving 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 scheme, the present invention also provides a molybdenum-resistant phosphate-solubilizing bacteria with molybdenum reducing function for use in soil or wastewater contaminated with molybdenum ions.

[0019] Based on the above technical scheme, the present invention also provides a use of molybdenum-resistant phosphate-solubilizing bacteria with molybdenum reducing function in dissolving insoluble phosphates.

[0020] Furthermore, the poorly soluble phosphate is tricalcium phosphate.

[0021] Further, the sparingly soluble phosphates are dissolved until the soluble phosphorus content reaches 17.00±2.04 mg / L.

[0022] The beneficial effects of the present invention are:

[0023] Serratia MoTB 2 was identified by 16S rDNA sequencing, and its molybdenum reduction ability and phosphorus solubility were determined. In the study of Mo(VI) adsorption and transformation in the environment, Serratia MoTB 2 was able to reduce the Mo(VI) adsorbed by itself, and the reduced Mo(VI) accounted for 60.30% to 76.62%. The adsorbed Mo(VI) was reduced to Mo(V) and Mo(IV) with relatively low toxicity. It can still survive in solutions or soils with high molybdenum content, and can effectively control the migration and transformation process of Mo(VI), Mo(V) and Mo(IV) 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. Within 48 hours, 39.55 mg / L Mo(VI) can be removed from soil or water. The invention can remove 22.28% of Mo(VI) in the BPM medium containing Mo(VI), reduce the effective molybdenum content in the soil by 9.40% within 2 days, and can dissolve sparingly soluble phosphate to improve the utilization rate of phosphorus in the soil. The strain can dissolve sparingly soluble phosphate in an inorganic phosphorus liquid medium until the soluble phosphorus content reaches 17.00±2.04 mg / L, indicating that the Serratia MoTB 2 in the invention has a high molybdenum reduction efficiency and a certain phosphorus dissolving ability. The maximum molybdenum tolerance concentration of Serratia MoTB 2 is 100000 mg / L. The strain can provide bacterial strain resources for bioremediation of molybdenum-contaminated areas with insufficient soil phosphorus supply, is environmentally friendly, is a strain with good application prospects, and is of great significance to the development of bioremediation technology. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 This is a photo of the screening results of Serratia MoTB 2 plate in the present invention;

[0025] Figure 2 is the phylogenetic tree of Serratia MoTB 2 in the present invention;

[0026] Figure 3 is the growth curve of Serratia MoTB 2 at different initial molybdenum concentrations in the present invention;

[0027] Figure 4 It is a spectrum scanning diagram of the product Mo-B lue of the reduction of Mo(VI) by Serratia MoTB 2 in LPM liquid culture medium in the present invention;

[0028] Figure 5The XPS test results of the Serratia MoTB2 after adsorbing the BPM and LPM culture media containing Mo(VI) in the present invention;

[0029] Figure 6 This is a characteristic diagram of the phosphorus dissolving effect of Serratia MoTB 2 in the present invention. DETAILED DESCRIPTION

[0030] 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.

[0031] A molybdenum-resistant phosphate-solubilizing bacterium with molybdenum reducing function, which is classified and named as Serratia ficaria MoTB 2, and was deposited with the China Center for Type Culture Collection on April 7, 2024 with the deposit number: CCTCC NO.M2024638, and the deposit address is: Wuhan University, Wuhan, China.

[0032] 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. The bacterial growth rate is significantly reduced in BPM liquid culture medium containing Mo(VI) exceeding 60000 mg / L. The maximum tolerated Mo(VI) concentration is 100000 mg / L, and the test environment is: liquid culture medium.

[0033] Serratia ficaria MoTB 2 can reduce the Mo(VI) adsorbed by itself, and the reduced Mo(VI) accounts for 60.30% to 76.62%. The adsorbed Mo(VI) is reduced to Mo(V) and Mo(IV) with relatively low toxicity.

[0034] The bacteria reduced Mo(VI) to generate Mo-B l ue, and the absorbance of the obtained product Mo-B l ue at 865nm was 6.3416. The test environment was: LPM liquid culture medium containing Mo(VI) source.

[0035] like Figure 1 , Figure 2 As shown, strain screening:

[0036] Take rhizosphere soil samples of pioneer plants in the molybdenum tailings area, for example, collect soil samples from the rhizosphere of pioneer plants in the molybdenum tailings area in Luanchuan, Henan Province, add 5.0g of the soil sample to be tested into a triangular flask containing 45ml of sterile water, and culture it in a shaking incubator at 30℃ and 150r / min for 30min to obtain a soil suspension;

[0037] 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;

[0038] Screening of strains with molybdenum reducing properties:

[0039] The above 142 strains 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 reducing ability were screened out. Then, the strains with molybdenum reducing ability were inoculated into a 250 ml conical flask filled with 100 ml liquid LPM culture medium at an inoculation amount of 1%, and three repeated shaking flasks were set up for the strains. The sterile LPM liquid culture medium was used as a control, and the culture was carried out at 30°C and 150 rpm. After 24 hours, the supernatant was subjected to a full spectrum scan using an ultraviolet-visible spectrophotometer, and the molybdenum blue production ability was quantitatively analyzed by measuring the absorbance of the supernatant at 865 nm, and a strain with the strongest molybdenum reducing ability was screened out. Its full spectrum scanning spectrum, i.e., the molybdenum blue scanning spectrum of the reduction product and the reading at a wavelength of 865 nm, i.e., the Mo(VI) reduction effect, are shown in FIG. Figure 4 As shown;

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

[0041] 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.

[0042] Figure 3The obtained Serratia MoTB 2 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, respectively. The growth curve of the strain after 30 hours of culture was shown. It can be seen from the figure that when the Mo(VI) concentration was 3000 mg / L and 6000 mg / L, Mo(VI) had no obvious 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 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 6000 mg / L, which was equivalent to the growth rate of the control group. The MIC Mo(VI) can reach 100,000 mg / L, indicating that Serratia MoTB 2 has a strong tolerance to Mo(VI).

[0043] Identification

[0044] The strain obtained by screening was sequenced, and the sequencing results were compared by BLAST sequence comparison on the NCBI website, and it was determined to be Serratia ficar ia MoTB 2, as shown in Table 1;

[0045] Table 1

[0046]

[0047]

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

[0049] 1. Adding Serratia ficaria MoTB 2 to the BPM and LPM liquid culture media containing Mo(VI) respectively to obtain a mixed culture medium, wherein the inoculation amount of Serratia ficaria MoTB 2 in the mixed culture medium is 1%;

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

[0051] The formula of the medium of LPM containing Mo(VI) is: (NH4)2SO4 3.0g, MgSO4·7H2O 0.5g, NaCl 5.0g, Na2MoO4·2H2O 2.42g, Na2HPO4 0.71g, yeast extract powder 0.5g, glucose 10.0g, water 1000ml; pH of the medium is 7.0, sterilized at 121℃ for 20min;

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

[0053] Nine 250ml triangular flasks are filled with 100ml of liquid LPM culture medium, numbered No.1 to 9, and divided into three groups:

[0054] No. 1 to 3 are group A, No. 4 to 6 are group B, and No. 7 to 9 are group C;

[0055] Group A was not inoculated and served as the control group. Group B and Group C were inoculated with 1% OD 600 =1.0, 9 bottles were evenly placed in a shaker at 30°C and 150rpm for culture, samples were taken at 24h and 48h of culture, the culture solution was centrifuged at 8000rpm for 10min, the supernatant was filtered through a 0.22um filter membrane, the Mo 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;

[0056] As can be seen from Table 2, in the Mo(VI) adsorption experiment, Serratia MoTB 2 can reduce the Mo(VI) concentration 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 show that Serratia MoTB 2 has a certain removal effect on Mo(VI) in the environment;

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

[0058]

[0059]

[0060] 3. Take the cells grown for 36 h in BPM and LPM liquid 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;

[0061] XPS analysis of the Serratia MoTB2 bacterial precipitate after adsorption of Mo(VI) revealed Figure 5It can be seen that after being cultured in BPM and LPM liquid culture media containing Mo(VI), Serratia MoTB 2 can reduce 76.62% and 60.30% of the adsorbed Mo(VI) to Mo(V) and Mo(IV), respectively. The results show that Serratia MoTB 2 can adsorb Mo(VI) in the environment and reduce it to relatively less toxic Mo(V) and Mo(IV).

[0062] Qualitative and quantitative analysis of the phosphate solubilization ability of Serratia marcescens MoTB 2

[0063] 1. Preparation of bacterial suspension: Use an inoculation loop to pick a small amount of Serratia MoTB 2 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;

[0064] 2. Inoculation and cultivation for qualitative analysis of phosphate solubilization capacity: 600 The value is 0.8. 3ul of bacterial suspension is spotted on inorganic phosphorus solid culture medium. After 24h of culture, a lysis halo is formed, indicating that Serratia MoTB 2 has a certain ability to dissolve phosphorus. Figure 6 As shown;

[0065] 3. Inoculation and culture for quantitative analysis of phosphate solubilization capacity: 600 The bacterial suspension with a value of 0.8 was inoculated into the inorganic phosphorus liquid culture medium according to 1% inoculation amount of the culture system, and cultured at 30°C, 150rpm for 24 hours. After the culture was completed, the content of soluble phosphate was determined by molybdenum antimony anti-spectrophotometry. In the inorganic phosphorus liquid culture medium, the insoluble phosphate was dissolved to reach a soluble phosphate content of 17.00±2.04mg / L.

[0066] 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 phosphate-solubilizing bacteria having molybdenum reducing function, characterized in that: Its classification name is: Serratia ficaria MoTB 2, and on April 7, 2024, it was deposited in: China Center for Type Culture Collection with the deposit number: CCTCC NO.M2024638.

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

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

4. The molybdenum-resistant phosphate-solubilizing bacteria having molybdenum-reducing function according to any one of claims 1 to 3, characterized in that: The bacteria reduced Mo(VI) to generate Mo-Blue, and the absorbance of the obtained product Mo-Blue at 865nm was 6.3416.

5. A method for separating and screening molybdenum-resistant phosphate-solubilizing bacteria having molybdenum reducing function as claimed in any one of claims 1 to 4, characterized in that: The steps include: S1. Take rhizosphere soil samples of pioneer plants in the molybdenum tailings area, make them into soil suspensions, and then dilute them in a gradient manner and streak them in beef extract peptone solid medium containing high concentration of Mo(VI) for screening and separation, so as to isolate the 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 molybdenum-resistant strains, and using the formation of blue colonies after 24 hours of culture as the basis for initial screening to screen 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 phosphate-dissolving effect of the strain with the strongest molybdenum-reducing ability to determine the molybdenum-resistant phosphate-dissolving bacteria with molybdenum-reducing function.

6. The separation and screening method according to claim 4, 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 phosphate-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-tolerant phosphate-solubilizing bacteria having molybdenum-reducing function as claimed in any one of claims 1 to 4 in dissolving sparingly soluble phosphates.

9. The use according to claim 8, characterized in that: The sparingly soluble phosphate is tricalcium phosphate.

10. The use according to claim 8 or 9, characterized in that: Dissolve sparingly soluble phosphates until the soluble phosphorus content reaches 17.00±2.04mg / L.

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