Phosphorus-solubilizing bacteria and application thereof

Abstract

The invention discloses a phosphate-solubilizing bacterium, which is characterized in that the phosphate-solubilizing bacterium is brevibacterium pseudominum WZU-DH, the phosphate-solubilizing bacterium is preserved in China Center for Type Culture Collection on June 20, 2024, the preservation address is No. 299 on eight road, Wuchang District, Wuhan City, Hubei Province, and the preservation number is CCTCC NO.M20241329; according to the invention, a strain WZU-DH with relatively high phosphorus dissolving capacity is screened from a phosphorus saturated matrix of the constructed wetland, is prepared into a microbial agent, and is inoculated into the phosphorus saturated matrix of the constructed wetland, so that the activation amount of the phosphorus saturated matrix of the constructed wetland can be remarkably increased, the growth of plants is promoted, and the regeneration of the matrix is accelerated.

Description

Technical Field

[0001] The present invention relates to the field of biological and environmental protection technology, and more specifically to a phosphate-dissolving bacterium and an application thereof. Background Art

[0002] Artificial wetlands rely on the synergistic effects of physics, chemistry and biology to enhance the decontamination capacity of natural wetland ecosystems. They have the advantages of low investment, low energy consumption, convenient maintenance and management, and outstanding ecological service functions. They are widely used in domestic sewage, industrial wastewater, surface runoff and watershed water ecological restoration, and have achieved good results. The removal of phosphorus in wetlands mainly relies on adsorption, precipitation, microbial assimilation and plant absorption. Among them, substrate adsorption and precipitation are the main ways to remove phosphorus, accounting for 36.2~87.5%. However, after long-term operation, the adsorption of phosphorus by the substrate gradually tends to saturation, and the accumulation of phosphorus in the wetland is serious, which has a serious impact on the normal operation of the wetland and the removal of pollutants. According to a survey by the US Environmental Protection Agency, about 50% of subsurface artificial wetlands lose their normal functions within 5 years and need to replace the substrate. However, substrate replacement will greatly increase the operating cost and destroy the constructed artificial wetland system. In addition, the treatment and disposal of adsorption saturated substrates may become a new environmental problem. Therefore, substrate phosphorus adsorption saturation has limited the promotion and application of artificial wetlands to a certain extent.

[0003] In summary, activation of phosphorus-saturated substrates as a sustainable treatment method can reduce the material consumption and acquisition costs of periodic substrate replacement. Alternating wet and dry is a more studied activation method with certain short-term effects. However, air drying can only expose small cracks in the organic film material on the surface of the substrate, thereby obtaining limited potential new adsorption sites, and the average removal rate of phosphorus by the substrate is still low. Analysis of phosphorus migration and transformation pathways in wetlands shows that plant harvesting is an effective way to remove and recover phosphorus from wetlands. However, plants cannot directly utilize adsorbed phosphorus in the substrate.

[0004] Microbial agents can mineralize organic phosphorus and dissolve inorganic phosphorus minerals. Inoculating highly efficient phosphate-dissolving bacteria in the phosphorus-saturated matrix of artificial wetlands can convert the insoluble phosphorus adsorbed in the phosphorus-saturated matrix into plant-usable phosphorus through the action of phosphate-dissolving bacteria, thus promoting plant growth. Through effective harvesting, phosphorus can be recovered from the matrix to achieve activation of the phosphorus-saturated matrix, i.e., in-situ regeneration of the phosphorus-saturated matrix. Summary of the invention

[0005] The object of the present invention is to provide a phosphate-dissolving bacterium and its application to solve the above problems. The phosphate-dissolving bacterium provided by the present invention is Bacillus minimus ( Curtobacterium pusillum) WZU-DH can convert unstable phosphorus, aluminum phosphorus, calcium phosphorus, iron phosphorus and other insoluble phosphorus in phosphorus-saturated matrix into soluble phosphorus during the activation of phosphorus-saturated matrix in artificial wetlands.

[0006] In order to achieve the above-mentioned purpose, the present invention specifically adopts the following technical solutions: The present invention provides a phosphate-dissolving bacterium, wherein the phosphate-dissolving bacterium is Bacillus minimus ( Curtobacterium (a little bit) WZU-DH was deposited in the China Center for Type Culture Collection on June 20, 2024, with the deposit address being Wuhan University, Wuhan, China, and the deposit number being CCTCC NO: M20241329.

[0007] The present invention also provides the use of the above-mentioned phosphate-dissolving bacteria in the preparation of a microbial agent with phosphate-dissolving function.

[0008] The present invention also provides a microbial agent with phosphorus-dissolving function, comprising the phosphorus-dissolving bacteria mentioned above.

[0009] The present invention also provides a method for preparing the above-mentioned microbial agent, comprising the steps of inoculating the above-mentioned phosphate-dissolving bacteria into a fermentation medium, and fermenting and culturing to obtain the microbial agent.

[0010] Furthermore, the fermentation medium is LB medium.

[0011] Furthermore, the fermentation culture temperature is 20°C to 30°C, and the time is 12h to 48h.

[0012] The present invention also provides the use of the above-mentioned phosphate-dissolving bacteria or the above-mentioned microbial agent in the regeneration of phosphorus-saturated matrix in artificial wetlands.

[0013] The present invention also provides the use of the above-mentioned phosphate-dissolving bacteria or the above-mentioned microbial agent in promoting plant growth.

[0014] The phosphate-solubilizing bacteria WZU-DH of the present invention, namely Bacillus microplus ( Curtobacterium pusillum) WZU-DH was isolated from the long-term experimental artificial wetland of Wenzhou University. The isolation process was as follows: 10g artificial wetland matrix sample was placed in a 250mL conical flask, 90mL sterile water was added, and the suspension was oscillated to make a gradient bacterial suspension. 0.2mL of the gradient bacterial suspension was applied to the PVK plate medium, and each gradient was repeated 4 times at 30°C for 4 days. The colonies with transparent phosphate-dissolving rings were picked, enriched in LB liquid medium for 12h, and streaked on LB solid medium for culturing. After separation and purification for 3 generations, the microscopic short Bacillus ( Curtobacterium pusillum) , named WZU-DH. The morphological characteristics of the strain WZU-DH are: small colonies, convex in the center, round, opaque, yellow, smooth surface, not easy to be picked up, smelly, and Gram staining is positive.

[0015] Beneficial effects: The present invention screens out a strain WZU-DH with high phosphorus solubilizing ability from the phosphorus saturated matrix of the artificial wetland, prepares the microbial agent, and inoculates it into the phosphorus saturated matrix of the artificial wetland, which can significantly increase the activation amount of the phosphorus saturated matrix of the artificial wetland, promote plant growth, and accelerate the regeneration of the matrix. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0017] Figure 1 The present invention is separated from the microbacterium brevis ( Curtobacterium pusillum) Colony characteristics of WZU-DH on LB solid medium; FIG. 2 is a diagram of the present invention of Bacillus minimus ( Curtobacterium pusillum) Phosphorus solubilizing capacity of WZU-DH (a), the effect of temperature on the phosphorus solubilizing capacity (b), the phosphorus solubilizing capacity of the strain to different phosphorus sources (c), the effect of nitrogen source on the phosphorus solubilizing capacity of the strain (d), and the effect of carbon source on the phosphorus solubilizing capacity of the strain (e); Figure 3 The contents of unstable phosphorus, aluminum phosphorus, iron phosphorus, calcium phosphorus, residual phosphorus and total inorganic phosphorus in the phosphorus-saturated matrix of the artificial wetland after 90 days of experiment; wherein DH is the content of inoculated Bacillus minimus ( Curtobacterium pusillum) WZU-DH treatment, CK is the control treatment without inoculation of bacteria. DETAILED DESCRIPTION

[0018] The present invention will be further explained below in conjunction with the embodiments.

[0019] Example 1 1. Strain Screening (1) Isolation and preservation of strains: Weigh 10 g of artificial wetland zeolite matrix sample and place it in a 250 mL conical flask. Add 90 mL of sterile water and shake at 160 r / min for 30 min. Then let it stand. Use the 10-fold dilution method to prepare 10 -1 -10 -7 CFU / mL bacterial suspension, take 0.2mL of bacterial suspension and spread it on PVK plate medium, repeat each gradient 4 times. Culture at 30℃ for 3 days. Select the colonies with transparent phosphate-dissolving ring, pick them out with an inoculation loop, enrich them in LB liquid medium for 12h, streak culture on LB solid medium, separate and purify for 3 generations, and store them in 4℃ LB slant medium and -80℃ 15% glycerol.

[0020] (2) Initial screening of strains: The strains isolated and stored in LB slant medium were inoculated into sterilized LB liquid medium (50 mL) and enriched in a shaker at 160 r / min and 30°C for 12 h until the logarithmic growth phase to obtain seed liquid. The seed liquid was streaked on LB solid medium. A single colony was picked with a sterilized toothpick and inoculated on a PVK plate medium. The colony was cultured at 30°C for 4 days, and the diameter of the phosphate dissolution zone (D) and colony diameter (d) were measured. The ratio of the phosphate dissolution zone to the colony diameter (D / d) was calculated. Repeat 3 times.

[0021] Through preliminary screening, four phosphate-solubilizing strains were isolated from the artificial zeolite matrix sample and named WZU-DH, WZU-FL, WZU-F and WZU-H. Among them, the D / d value of the WZU-DH strain was the largest, which was 1.81, and the D / d values ​​of the other strains were between 1.29 and 1.50 (see Table 1 for details).

[0022] Table 1 Results of initial screening of phosphate-dissolving bacteria plates

[0023] (3) Rescreening of strains: The WZU-DH, WZU-FL, WZU-F and WZU-H strains obtained above were inoculated into sterilized LB liquid medium for enrichment culture, respectively, until the bacterial content of the bacterial suspension reached 10 7 Take a 250mL conical flask, add 100mL NBRIP liquid medium, inoculate 1mL bacterial suspension (1×10 7 CFU / mL), 30℃, 160r / min, cultured for 4 days, and inoculated with an equal volume of sterile water as control (CK). Each treatment was repeated 4 times. The soluble phosphorus concentration in the culture medium was determined by molybdenum antimony spectrophotometry (see Table 2 for details).

[0024] Table 2 Soluble phosphorus concentration of phosphate-solubilizing bacteria

[0025] As can be seen from Table 2, compared with the control, the soluble phosphorus concentrations in the supernatants of the WZU-DH, WZU-FL, WZU-F, and WZU-H strains were 6.85, 0.81, 1.26, and 1.08 mg / L higher, respectively.

[0026] In the above steps, the formula of each culture medium is as follows: LB solid medium: peptone 10g, yeast powder 5g, NaCl (10g), agar 25g, pH 7.0, water 1000mL. 121℃, sterilize for 20min.

[0027] LB liquid medium: peptone 10g, yeast powder 5g, NaCl (10g), pH 7.0, water 1000mL. 121℃, sterilize for 20min.

[0028] PVK (Pikovskaya) plate medium: glucose 10g, AlPO 4 5g, (NH 4 ) 2 SO 4 0.5 g, NaCl (0.2 g), MgSO 4 7H 2 O0.1g, KCl (0.2g), yeast powder 0.5g, MnSO 4 ·H 2 O0.002g, 0.4% bromophenol blue (pH6.7) 6mL, agar 25g, distilled water 1000mL, pH7.0-7.5. Sterilize at 121℃ for 20min.

[0029] NBRIP (National Botanical Research Institute's Phosphate) liquid medium: glucose 10g, AlPO 4 5g, NaCl (0.2g), MgSO 4 7H 2 O0.25 g, KCl (0.2 g), (NH 4 ) 2 SO 4 0.1g, 1000mL, pH7.0-7.5. 121℃, sterilize for 20min.

[0030] 2. Strain identification: (1) Morphological identification: The phosphate-dissolving strain WZU-DH was inoculated into LB solid medium and cultured at 30°C for 2 days. The colony morphology was observed and recorded: the colonies were small, with a raised center, round, opaque, yellow, smooth surface, not easy to be picked up, and had a bad smell (see attached figure for details). Figure 1 ).

[0031] A single colony was picked out from LB solid medium and inoculated into LB liquid medium, cultured at 160 r / min and 30°C for 12 h, and Gram staining was performed. The morphological characteristics of the bacteria were observed using an optical microscope (OLympus BH-2, Japan). The strain was Gram-positive.

[0032] (2) Molecular identification: The preserved WZU-DH strain was inoculated into sterilized LB liquid medium for enrichment culture for 12 hours. Take 1 mL of bacterial solution, add it to a 1.5 mL centrifuge tube, centrifuge it at room temperature at 8000r / min for 1 minute, discard the supernatant, and collect the bacteria. The Ezup column bacterial genomic DNA extraction kit (Shenggong, SK8255) was used for extraction and purification according to the instructions. The genomic DNA of the purified phosphate-solubilizing strain WZU-DH was extracted and amplified using the bacterial 16S rDNA universal primer 27F / 1492R (Shenggong, Synthesis Department) to establish a PCR amplification system. PCR amplification conditions: 95℃ pre-denaturation for 10 minutes, 94℃ denaturation for 30 seconds, 57℃ annealing for 30 seconds, 72℃ extension for 90 seconds, denaturation, annealing and extension for 30 cycles, and 72℃ extension for 10 minutes before the reaction was terminated. The PCR amplification product was detected by 1% agarose electrophoresis and sequenced by a bioengineering company after purification. The DNA sequence obtained by sequencing was input into NBCI and compared with all sequences in the NBCI database using the Blast program. The results showed that it was identical to Bacillus minimus ( Curtobacterium pusillum ) with a similarity of 99.5%.

[0033] The 16S rDNA nucleotides of the phosphate-solubilizing bacteria strain WZU-DH are: Example 2 Phosphate dissolving properties of the phosphate-dissolving bacteria WZU-DH of the present invention: The strains preserved in LB slant medium were selected and placed in LB liquid medium, cultured at 30°C and 160 rpm for 12 h, 5 mL of the culture solution was taken and diluted with 0.1% saline to make the bacterial suspension contain 10 7 CFU / mL. Take 1mL of the above bacterial suspension and inoculate it into 250mL conical flasks containing 100mL NBRIP liquid culture medium, and culture it at 30℃ and 160r / min. Take samples every 4 days in the early stage, and take samples every day after 12 days to determine the soluble phosphorus concentration. The experiment was repeated 3 times, and the sterile NBRIP liquid culture medium without inoculation of strains was used as a control. The soluble phosphorus concentration in the culture medium was determined by the molybdenum antimony anti-spectrophotometric method.

[0034] (1) Phosphate solubilization ability of WZU-DH: The soluble phosphorus concentration in the culture medium increased first and then decreased over time. On the 14th day, the soluble phosphorus concentration in the culture medium increased to 15.18 mg / L, and then gradually decreased. On the 16th day, it dropped to 14.99 mg / L (see Table 3 and attached figure for details). Figure 2a ).

[0035] Table 3

[0036] (2) Effect of temperature on the phosphorus solubilization ability of the phosphorus-dissolving strain WZU-DH: Compare the phosphorus solubilization ability of the strain at 5℃, 10℃, 20℃, 30℃, and 40℃. Take 1mL of the above bacterial suspension and inoculate it into 100mL of NBRIP liquid culture medium respectively. Culture it at 5℃, 10℃, 20℃, 30℃, and 40℃ at 160r / min for 8 days. Take samples and measure the soluble phosphorus concentration in the culture medium. Each group of experiments was repeated 3 times, and the sterile NBRIP liquid culture medium without inoculation of strains was used as the control group. The results showed that when cultured at 30℃, the strain had the highest phosphorus solubility of 11.55mg / L, followed by 20℃, with a phosphorus solubility of 5.39mg / L. Under the conditions of 5℃, 10℃, and 40℃, the phosphorus solubility of the strain was significantly lower than that of the other two groups, at 1.33-1.73mg / L (see Table 4 and attached figure for details). Figure 2b ).

[0037] Table 4

[0038] (3) Phosphate solubilization ability of the phosphate-dissolving strain WZU-DH for different phosphorus sources: calcium phosphate, aluminum phosphate and iron phosphate were used as the sole phosphorus sources. Take 1 mL of the above bacterial suspension and inoculate it into 100 mL of NBRIP liquid culture medium with different phosphorus sources, culture it at 30°C and 160 r / min for 8 days, take samples, and measure the soluble phosphorus concentration in the culture medium. Each group of experiments was repeated 3 times, and the sterile NBRIP liquid culture medium with different phosphorus sources corresponding to the strain that was not inoculated was used as the control group. The results showed that the phosphate-dissolving strain WZU-DH had the strongest phosphate solubilization ability for calcium phosphate, with a soluble phosphorus concentration of 161.21 mg / L in the culture medium, followed by aluminum phosphate (11.55 mg / L), and the worst phosphate solubilization ability for iron phosphate was 7.82 mg / L (see Table 5 and attached figure for details). Figure 2c ).

[0039] Table 5

[0040] (4) Effect of nitrogen source on the phosphorus solubilization ability of the phosphorus-dissolving strain WZU-DH: Using ammonium sulfate, potassium nitrate, ammonium nitrate, and peptone as nitrogen sources, 1 mL of the above bacterial suspension was taken and inoculated into 100 mL of NBRIP liquid culture medium with different nitrogen sources. The culture was carried out at 30°C and 160 r / min for 8 days, and samples were taken to measure the soluble phosphorus concentration in the culture medium. Each group of experiments was repeated 3 times, and the sterile NBRIP liquid culture medium with different nitrogen sources corresponding to the strain that was not inoculated was used as the control group. The results showed that when ammonium sulfate was the only nitrogen source, the phosphorus solubilization amount of the phosphorus-dissolving strain WZU-DH was the highest at 11.55 mg / L, followed by potassium nitrate (6.31 mg / L). When peptone and ammonium nitrate were the only nitrogen sources, the phosphorus solubilization ability of the strain was poor (see Table 6 and attached figure for details). Figure 2d ).

[0041] Table 6

[0042] (5) Effect of carbon source on the phosphorus solubilization ability of the phosphorus-dissolving strain WZU-DH: Glucose, sucrose, starch, and lactose were selected as carbon sources, and 1 mL of the above bacterial suspension was taken and inoculated into 100 mL of NBRIP liquid culture medium with different carbon sources. The culture was carried out at 30°C and 160 r / min for 8 days, and samples were taken to measure the soluble phosphorus concentration in the culture medium. Each group of experiments was repeated 3 times, and the sterile NBRIP liquid culture medium with different carbon sources corresponding to the strain that was not inoculated was used as the control group. The results showed that when glucose was used as the only carbon source, the phosphorus solubilization amount of the phosphorus-dissolving strain WZU-DH was the highest, which was 11.55 mg / L, followed by sucrose, which was 8.09 mg / L. However, when starch and lactose were used as the only carbon sources, the phosphorus solubilization effect was the worst, which was 4.15 and 4.30 mg / L, respectively (see Table 7 and attached figures for details). Figure 2e ).

[0043] Table 7

[0044] Example 3 Application of the phosphate-dissolving strain WZU-DH of the present invention in activation of phosphorus-saturated matrix in artificial wetlands (1) The phosphorus adsorption saturated artificial wetland zeolite matrix samples were placed in the experimental barrel (upper diameter 200mm×lower diameter 185mm×height 175mm), and the mass of each barrel of zeolite was about 3400g. Canna indica with good growth and uniform growth was selected, and after being washed with tap water and deionized water to remove impurities, it was moved to the barrel. The planting density of canna was 64 plants / m 2 . After the plants grow normally, inoculate the enriched solution of the phosphate-solubilizing strain WZU-DH at a 1% inoculation rate. Add twice, 17 mL each time, with an interval of 7 days. The uninoculated strain is used as the control. Set up 6 parallels for each treatment, and calculate the average value. Among them, the preparation method of the enriched solution of the phosphate-solubilizing strain WZU-DH is as follows: inoculate the preserved WZU-DH strain into the sterilized LB liquid culture medium, and enrich and culture it at 30°C for 12 hours.

[0045] (2) Canna lilies were harvested 90 days after the experiment, and the plant height, biomass, root length, and root volume of each treatment were measured. The contents of labile phosphorus, aluminum phosphorus, iron phosphorus, calcium phosphorus, residual phosphorus, and total inorganic phosphorus in the zeolite matrix samples of the artificial wetland were also determined.

[0046] The results showed that the plant height, longest root length, root volume and biomass of the group added with the phosphate-dissolving strain WZU-DH of the present invention were 74.7%, 62.5%, 42.0% and 65.4% higher than those of the control group, respectively (see Table 7), and the unstable phosphorus, aluminum phosphorus, iron phosphorus, calcium phosphorus, residual phosphorus and total inorganic phosphorus contents in the artificial wetland zeolite matrix samples were 58.7%, 31.3%, 60.2%, 34.5%, 41.9% and 39.8% lower than those of the control group, respectively (see Table 8 and the attached figure for details). Figure 3 ).

[0047] Table 7 Plant growth traits

[0048] Table 8

[0049] Note: In Table 8, aluminum phosphorus refers to aluminum-bound phosphorus, iron phosphorus refers to iron-bound phosphorus, and calcium phosphorus refers to calcium-bound phosphorus.

[0050] In summary, the phosphate-dissolving strain WZU-DH of the present invention can convert insoluble inorganic phosphate into soluble phosphorus that can be directly absorbed and utilized by plants. After 8 days of cultivation, the phosphate-dissolving capacity of calcium phosphate is 161.21 mg / L. Curtobacterium pusillum ) has the ability to dissolve phosphorus, and for the first time combined phosphorus-dissolving bacteria with the activation of phosphorus-saturated adsorption matrix in artificial wetlands, providing a certain reference for the application research of phosphorus-dissolving bacteria and an effective method for the in situ regeneration of phosphorus-saturated matrix in artificial wetlands.

[0051] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited by the description of the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.

[0052] The above are only preferred embodiments of the present invention and are not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A phosphate-dissolving bacterium, characterized in that: The phosphate-dissolving bacteria is Bacillus minimus ( Curtobacterium pusillum WZU-DH was deposited in the China Center for Type Culture Collection on June 20, 2024, with the deposit address at No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province, and the deposit number is CCTCC NO.M20241329.

2. Use of the phosphate-dissolving bacteria as claimed in claim 1 in the preparation of a microbial agent with phosphate-dissolving effect.

3. A microbial agent with phosphate dissolving effect, characterized in that: Including the phosphate-dissolving bacteria described in claim 1.

4. A method for preparing the microbial agent according to claim 3, characterized in that: The method comprises the steps of inoculating the phosphate-dissolving bacteria as claimed in claim 1 into a fermentation medium and fermenting and culturing the medium to obtain the microbial agent.

5. The preparation method according to claim 4, characterized in that: The fermentation medium is LB medium.

6. The preparation method according to claim 4, characterized in that: The fermentation culture temperature is 20°C to 30°C, and the fermentation culture time is 12h to 48h.

7. Use of the phosphate-dissolving bacteria according to claim 1 or the microbial agent according to claim 3 in activating phosphorus-saturated substrates in artificial wetlands.

8. Use of the phosphate-dissolving bacteria according to claim 1 or the microbial agent according to claim 3 in promoting plant growth.

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