Phosphorus solubilizing bacteria and method for preparing the same

Abstract

The application relates to a phosphorus solubilizing bacterium and a preparation method thereof, in particular to a phosphorus solubilizing bacterium for improving a lead-polluted saline-alkali soil and a preparation method thereof. The preparation method of the phosphorus solubilizing bacterium comprises the following steps: inoculating Pseudomonas, Bacillus megaterium and Bacillus velezensis into seed culture medium respectively to obtain first-stage seed liquid; inoculating the seed liquid into a first-stage seed tank to culture; inoculating second-stage seed liquid into a fermentation tank to culture, and performing segmented fermentation under different pH gradients; centrifuging the phosphorus solubilizing bacterium fermentation liquid, adding a dry protection agent to obtain bacterium slurry; mixing the bacterium slurry and auxiliary materials to granulate to obtain solid particles; and drying the solid particles and screening. The phosphorus solubilizing bacterium prepared by the method can effectively reduce the concentration of lead, pH and soil salinity in the lead-polluted saline-alkali soil, and effectively improve the lead-polluted saline-alkali soil.

Description

Technical Field

[0001] The present invention relates to the technical field of soil remediation, in particular to a phosphate-solubilizing bacterium and a preparation method thereof, and in particular to a phosphate-solubilizing bacterium for improving lead-contaminated saline-alkali land and a preparation method thereof. Background Art

[0002] Soil is a vital component of the ecological environment and a fundamental natural resource on which human survival depends. With the development of industrial and agricultural production, its degradation is becoming increasingly severe. Saline-alkali soils are a major cause of soil degradation. The emergence and development of problems such as irrational agricultural irrigation, excessive fertilizer application, vegetation destruction, and the greenhouse effect have also exacerbated soil salinization. This not only causes resource destruction and agricultural production losses, but also harms the ecological environment and biosphere. As the contradiction between population growth, shrinking arable land, and food demand becomes increasingly prominent, saline-alkali soils, as a key reserve land resource, are receiving increasing attention from governments worldwide for their improvement and resource utilization.

[0003] With the development of industrialization, along with mineral mining and the discharge of industrial wastewater, heavy metal lead is introduced through water, causing serious soil pollution, including some salinized reserve land. Salt-alkali land improvement technologies include chemical improvement, physical improvement, phytoremediation, and biofertilizer improvement. Biofertilizer remediation is to improve saline-alkali land by applying biofertilizer, which has the advantages of low cost, long action time, and environmental friendliness. Therefore, it is of great significance to provide a microbial agent that can effectively improve lead-contaminated saline-alkali land and a preparation method. Summary of the Invention

[0004] In order to overcome the shortcomings and deficiencies in the prior art, the purpose of the present invention is to provide a phosphate-solubilizing bacteria and a preparation method for improving lead-contaminated saline-alkali land. The phosphate-solubilizing bacteria prepared by this method can effectively reduce the lead concentration, pH and soil salinity in lead-contaminated saline-alkali land, effectively improving lead-contaminated saline-alkali land.

[0005] In order to solve the above technical problems, the present invention adopts the following technical solution: a preparation method of phosphate-solubilizing bacteria for improving lead-contaminated saline-alkali land, comprising the following steps:

[0006] (1) Pseudomonas, Bacillus megaterium, and Bacillus velezensis were inoculated into the same seed culture medium for mixed culture, and the mixture was shaken in an incubator to obtain a first-level seed solution;

[0007] (2) The seed solution obtained in step (1) is inoculated into a first-level seed tank at an inoculum volume fraction of 10-20%. The culture conditions of the first-level seed tank are temperature 25-40°C, rotation speed 150-200 rpm, pH 6.5-7.5, dissolved oxygen 20-50%, and culture for 18-30 hours to obtain a second-level seed solution;

[0008] (3) The secondary seed liquid obtained in step (2) is inoculated into a fermentation tank at an inoculum amount of 10-20% of the total volume fraction. The culture conditions of the fermentation tank are a temperature of 25-40°C and a rotation speed of 150-200 rpm. The fermentation is carried out in stages under different pH gradients to obtain a fermentation liquid.

[0009] (4) centrifuging the phosphate-solubilizing bacteria fermentation liquid obtained in step (3), adjusting the solid content of the bacterial liquid to 50-80%, and adding 0.5-15% of a drying protective agent to the adjusted bacterial liquid to obtain a bacterial slurry;

[0010] (5) mixing the bacterial slurry obtained in step (4) with auxiliary materials and granulating the mixture to obtain solid particles;

[0011] (6) The solid particles obtained in step (5) are vacuum dried and sieved to obtain a phosphate-solubilizing bacteria granule product.

[0012] The mechanism of the present invention using phosphate-solubilizing bacteria to improve saline-alkali soil is as follows: (1) The organic acids secreted by the phosphate-solubilizing bacteria during their metabolism can lower the pH in the soil, reduce the soil's adsorption of phosphorus, promote the growth of plants in saline-alkali soil, or combine with cations in the soil to form compounds, promoting the absorption and utilization of phosphorus by plants; (2) Phosphate-solubilizing bacteria can dissolve insoluble phosphorus sources in the soil for plant absorption and utilization, thereby promoting growth; (3) The application of exogenous phosphate-solubilizing bacteria will change the original microbial community structure in the saline-alkali soil, increase the abundance of bacteria related to phosphorus conversion, and thus improve the physical and chemical properties of the soil through microorganisms. The effects of phosphate-solubilizing bacteria on heavy metals are as follows: (1) They secrete substances such as organic acids and phosphatases to convert insoluble phosphorus into soluble phosphorus, which reacts with heavy metals such as lead to form more stable phosphorus-lead compounds; (2) Phosphate-solubilizing bacteria themselves absorb and accumulate a portion of heavy metals; (3) Many metabolites produced by microorganisms can react with heavy metal ions.

[0013] Furthermore, in step (1), Pseudomonas, Bacillus megaterium, and Bacillus velezensis are inoculated into the same seed culture medium at a weight ratio of 0.5~2:0.5~2:0.5~2, respectively, for mixed culture, and cultured in an incubator at a temperature of 25~40°C for 18~30h with shaking, and the rotation speed is controlled at 150~200rpm to obtain a first-level seed solution.

[0014] Furthermore, in step (2), the seed solution obtained in step (1) is inoculated into a first-level seed tank at an inoculum volume fraction of 10-20%. The culture conditions of the first-level seed tank are temperature 25-40°C, rotation speed 150-200 rpm, pH 6.5-7.5, dissolved oxygen 20-50%, and culture for 18-30 hours to obtain a second-level seed solution.

[0015] Furthermore, in step (3), the pH gradient is 6.5-7.2, 7.2-8.0, 8.0-8.5, 8.5-9.0, 9.0-9.5, and the culture time in each stage is 3-6 h.

[0016] Furthermore, in step (3), corresponding to different pH gradients, the raw materials and dosages of salt stress solution feed are: 0.1~0.2g / L Na2CO3 and 0.08~0.12g / LNaHCO3 (corresponding to pH gradient of 6.5~7.2), 0.5~1.5g / L Na2CO3 and 0.3~0.8g / LNaHCO3 (corresponding to pH gradient of 7.2~8.0), 2.5~5.0g / L Na2CO3 and 2.0~4.0g / LNaHCO3 (corresponding to pH gradient of 8.0~8.5), 5.0~7.5g / L Na2CO3 and 4.0~6.5g / LNaHCO3 (corresponding to pH gradient of 8.5~9.0), 7.5~9.5g / L Na2CO3 and 6.5~8.0g / LNaHCO3 (corresponding to pH gradient of 9.0~9.5);

[0017] The raw materials and dosage of lead supplement are: 0.5~0.8mmol / L Pb(NO3)2 (corresponding to pH gradient of 6.5~7.2), 0.8~1.2mmol / L Pb(NO3)2 (corresponding to pH gradient of 7.2~8.0), 1.2~1.5mmol / L Pb(NO3)2 (corresponding to pH gradient of 8.0~8.5), 1.5~1.8mmol / L Pb(NO3)2 (corresponding to pH gradient of 8.5~9.0), and 1.8~2.0mmol / L Pb(NO3)2 (corresponding to pH gradient of 9.0~9.5).

[0018] Furthermore, the seed tank culture medium in steps (2) and (3) includes the following raw materials: glucose 5~10 g / L, sodium acetate 5~10 g / L, (NH4)SO4 0.3~0.8 g / L, Ca3(PO4)2 8~12 g / L, calcium phytate 8~12 g / L, MnSO4·H2O 0.01~0.05 g / L, FeSO4·7H2O 0.01~0.05 g / L, sodium molybdate 0.2~0.5 g / L, MgSO4·7H2O 0.01~0.05 g / L, NaCl 0.1~0.5 g / L, KCl 0.1~0.5 g / L, loaded iron-based modified biochar 10~20 wt%, Pb(NO3)2 0.1~1.0 g / L.

[0019] Furthermore, the fermentation tank culture medium in steps (2) and (3) includes the following raw materials: glucose 5~10 g / L, sodium acetate 5~10 g / L, (NH4)SO4 0.3~0.8 g / L, Ca3(PO4)2 8~12 g / L, calcium phytate 8~12 g / L, MnSO4·H2O 0.01~0.05 g / L, FeSO4·7H2O 0.01~0.05 g / L, sodium molybdate 0.2~0.5 g / L, MgSO4·7H2O 0.01~0.05 g / L, NaCl 0.1~0.5 g / L, KCl 0.1~0.5 g / L, and 10~20 wt% of loaded iron-based modified biochar.

[0020] Furthermore, the preparation method of the loaded iron-based modified biochar comprises the following steps:

[0021] (A1) Corn stalks grown in saline-alkali land were collected, impurities removed, washed, dried, and crushed through a 10-mesh sieve. They were then soaked in a 1-1.4 mol / L hydrochloric acid solution for 8-15 h to further remove impurities and maintain pore patency. They were then rinsed with clean water and dried in an oven at 100-110°C until the mass was constant. They were then placed in a muffle furnace and introduced with nitrogen at a flow rate of 80-120 mL / min for 25-35 min. The stalks were then heated at 8-12°C / min. ~1 The temperature was raised to 280-320 °C, 480-520 °C and 680-720 °C at a rate of 1000 °C and maintained for 3-5 h respectively. The biochar was taken out after cooling and then ground and passed through a 100-mesh sieve to obtain biochar.

[0022] (A2) soaking the biochar obtained in step (A1) in a trace element solution at a concentration of 20-50 g / L, adjusting the pH to 7.2-8.6 with a sulfuric acid solution, soaking for 24 hours, and then drying in an oven at 100-110° C. for 8-12 hours to produce modified biochar;

[0023] (A3) adding the modified biochar obtained in step (A2) to a 10-50 mmol / L ferrous phosphate solution at a solid-to-liquid ratio of the modified biochar to the iron salt solution of 0.01-0.04 g / mL, stirring until the modified biochar is evenly dispersed in the solution, introducing nitrogen, and adding dropwise a 100-500 mmol / L NaBH4 solution at a ratio of the volume of the NaBH4 solution to the volume of the iron salt solution of 1:0.8-1.2; preferably, the amount of the NaBH4 solution added is the same as the volume of the iron salt solution;

[0024] (A4) The biochar obtained in step (A3) is washed with oxygen-free ethanol, and then stripped to volatilize the ethanol, thereby obtaining iron-loaded modified biochar.

[0025] Furthermore, in step (A2), the trace element solution includes the following raw materials: 0.1-1.0 g / L of cobalt chloride, 3-10 g / L of manganese sulfate, 0.2-3 g / L of anhydrous copper sulfate, 0.2-0.5 g / L of sodium molybdate, and 0.5-5 g / L of zinc sulfate.

[0026] Furthermore, in step (4), after the phosphate-solubilizing bacteria fermentation liquid is centrifuged, the solid content of the bacterial liquid is adjusted to 50-80%, and 0.5-15% of a drying protective agent is added to the adjusted bacterial liquid to obtain a bacterial slurry; the drying protective agent is at least one of galactose, trehalose, sucrose, lactose, raffinose, glycerol, dimethyl sulfoxide and gelatin.

[0027] Furthermore, in step (5), the bacterial slurry and the auxiliary material are mixed and granulated in a weight ratio of 1:0.5-3; the auxiliary material is at least one of wood ash and vermiculite powder.

[0028] The present invention also provides a phosphate-solubilizing bacterium, which is prepared by the above-mentioned preparation method.

[0029] The beneficial effects of the present invention are:

[0030] (1) The present invention provides a method for preparing phosphate-solubilizing bacteria for improving lead-contaminated saline-alkali land. The phosphate-solubilizing bacteria prepared by this method can effectively reduce the lead concentration, pH and soil salinity in lead-contaminated saline-alkali land, thereby effectively improving lead-contaminated saline-alkali land.

[0031] (2) During the fermentation process of phosphate-solubilizing bacteria, pH, salinity and lead concentration gradient fermentation are adopted, and the phosphate-solubilizing bacteria prepared can maintain the ability to degrade salinity and lead.

[0032] (3) The iron-based modified biochar is loaded with corn straw grown in saline-alkali land to maintain the salinity of the biochar, so that the phosphate-solubilizing bacteria in the subsequent fermentation can always adapt to the salinity.

[0033] (4) During the fermentation process of phosphate-solubilizing bacteria, iron-loaded modified biochar is added. On the one hand, the iron-loaded modified biochar can serve as a carrier for the growth and reproduction of microorganisms. On the other hand, during the use of the microbial agent product, zero-valent iron can reduce the bioavailability and mobility of lead in the soil, providing mild growth conditions for the long-term restoration of phosphate-solubilizing bacteria. Phosphate-solubilizing bacteria change the valence state of lead through their own metabolism, further reducing the toxicity of lead. DETAILED DESCRIPTION

[0034] In order to facilitate understanding by those skilled in the art, the present invention will be further described below with reference to the embodiments. The contents mentioned in the embodiments are not intended to limit the present invention.

[0035] In some embodiments of the present invention, a method for preparing phosphate-solubilizing bacteria for improving lead-contaminated saline-alkali land comprises the following steps:

[0036] (1) Pseudomonas, Bacillus megaterium, and Bacillus velezensis were inoculated into the same seed culture medium at a weight ratio of 0.5-2:0.5-2:0.5-2, respectively, for mixed culture, and cultured in an incubator at a temperature of 25-40°C for 18-30 hours with a rotation speed controlled at 150-200 rpm to obtain a first-level seed solution;

[0037] (2) The seed solution obtained in step (1) is inoculated into a first-level seed tank at an inoculum volume fraction of 10-20%. The culture conditions of the first-level seed tank are temperature 25-40°C, rotation speed 150-200 rpm, pH 6.5-7.5, dissolved oxygen 20-50%, and culture for 18-30 hours to obtain a second-level seed solution;

[0038] (3) The secondary seed liquid obtained in step (2) is inoculated into a fermentation tank at an inoculum amount of 10-20% of the total volume fraction. The culture conditions of the fermentation tank are a temperature of 25-40°C and a rotation speed of 150-200 rpm. The fermentation is carried out in stages under different pH gradients to obtain a fermentation liquid.

[0039] (4) centrifuging the phosphate-solubilizing bacteria fermentation liquid obtained in step (3), adjusting the solid content of the bacterial liquid to 50-80%, and adding 0.5-15% of a drying protective agent to the adjusted bacterial liquid to obtain a bacterial slurry;

[0040] (5) mixing the bacterial slurry obtained in step (4) with auxiliary materials and granulating the mixture to obtain solid particles;

[0041] (6) The solid particles obtained in step (5) are vacuum dried and sieved to obtain a phosphate-solubilizing bacteria granule product.

[0042] In some embodiments of the present invention, in step (3), the pH gradient is 6.5-7.2, 7.2-8.0, 8.0-8.5, 8.5-9.0, 9.0-9.5, and the culture time for each stage is 3-6 h; corresponding to different pH gradients, the raw materials and dosages of salt stress solution feed are: 0.1-0.2 g / L Na2CO3 and 0.08-0.12 g / LNaHCO3, 0.5-1.5 g / LNa2CO3 and 0.3-0.8 g / LNaHCO3, 2.5-5.0 g / L Na2CO3 and 2.0-4.0 g / LNaHCO3, 5.0-7.5 g / L Na2CO3 and 4.0~6.5g / LNaHCO3, 7.5~9.5g / LNa2CO3 and 6.5~8.0g / LNaHCO3; the raw materials and dosage of lead supplement are 0.5~0.8mmol / L Pb(NO3)2, 0.8~1.2mmol / L Pb(NO3)2, 1.2~1.5mmol / L Pb(NO3)2, 1.5~1.8mmol / L Pb(NO3)2, 1.8~2.0mmol / LPb(NO3)2 respectively.

[0043] In some embodiments of the present invention, the seed tank culture medium in steps (2) and (3) includes the following raw materials: glucose 5-10 g / L, sodium acetate 5-10 g / L, (NH4)SO4 0.3-0.8 g / L, Ca3(PO4)2 8-12 g / L, calcium phytate 8-12 g / L, Mn S O4·H2O 0.01~0.05g / L, FeSO4·7H2O 0.01~0.05g / L, sodium molybdate 0.2~0.5g / L, MgSO4·7H2O0.01~0.05g / L, NaCl 0.1~0.5g / L, KCl 0.1~0.5g / L, loaded iron-based modified biochar 10~20wt%, Pb(NO3)20.1~1.0g / L.

[0044] In some embodiments of the present invention, the fermentation tank culture medium in steps (2) and (3) includes the following raw materials: glucose 5~10 g / L, sodium acetate 5~10 g / L, (NH4)SO4 0.3~0.8 g / L, Ca3(PO4)2 8~12 g / L, calcium phytate 8~12 g / L, MnSO4·H2O 0.01~0.05 g / L, FeSO4·7H2O 0.01~0.05 g / L, sodium molybdate 0.2~0.5 g / L, MgSO4·7H2O 0.01~0.05 g / L, NaCl 0.1~0.5 g / L, KCl 0.1~0.5 g / L, and 10~20 wt% of loaded iron-based modified biochar.

[0045] In the present invention, compared with the fermentation tank culture medium, the seed tank culture medium is additionally supplemented with 0.1-1.0 g / L Pb(NO3)2.

[0046] In some embodiments of the present invention, the method for preparing the loaded iron-based modified biochar comprises the following steps:

[0047] (A1) Corn stalks grown in saline-alkali land were collected, impurities removed, washed, dried, and crushed through a 10-mesh sieve. They were then soaked in a 1-1.4 mol / L hydrochloric acid solution for 8-15 h to further remove impurities and maintain pore patency. They were then rinsed with clean water and dried in an oven at 100-110°C until the mass was constant. They were then placed in a muffle furnace and introduced with nitrogen at a flow rate of 80-120 mL / min for 25-35 min. The stalks were then heated at 8-12°C / min. ~1 The temperature was raised to 280-320 °C, 480-520 °C and 680-720 °C at a rate of 1000 °C and maintained for 3-5 h respectively. The biochar was taken out after cooling and then ground and passed through a 100-mesh sieve to obtain biochar.

[0048] (A2) soaking the biochar obtained in step (A1) in a trace element solution at a concentration of 20-50 g / L, adjusting the pH to 7.2-8.6 with a sulfuric acid solution, soaking for 24 hours, and then drying in an oven at 100-110° C. for 8-12 hours to produce modified biochar; the trace element solution comprises the following raw materials: 0.1-1.0 g / L cobalt chloride, 3-10 g / L manganese sulfate, 0.2-3 g / L anhydrous copper sulfate, 0.2-0.5 g / L sodium molybdate, and 0.5-5 g / L zinc sulfate;

[0049] (A3) adding the modified biochar obtained in step (A2) to a 10-50 mmol / L ferrous phosphate solution at a solid-to-liquid ratio of the modified biochar to the ferric salt solution of 0.01-0.04 g / mL, stirring until the modified biochar is evenly dispersed in the solution, introducing nitrogen, and adding a 100-500 mmol / L NaBH4 solution dropwise, at a volume ratio of the NaBH4 solution to the ferric salt solution of 1:0.8-1.2;

[0050] (A4) The biochar obtained in step (A3) is washed with oxygen-free ethanol, and then stripped to volatilize the ethanol, thereby obtaining iron-loaded modified biochar.

[0051] In some embodiments of the present invention, in step (4), after the phosphate-solubilizing bacteria fermentation broth is centrifuged, the solid content of the bacterial broth is adjusted to 50-80%, and 0.5-15% of a drying protective agent is added to the adjusted bacterial broth to obtain a bacterial slurry; the drying protective agent is at least one of galactose, trehalose, sucrose, lactose, raffinose, glycerol, dimethyl sulfoxide and gelatin.

[0052] In some embodiments of the present invention, in step (5), the bacterial slurry and the auxiliary material are mixed and granulated in a weight ratio of 1:0.5-3; the auxiliary material is at least one of wood ash and vermiculite powder.

[0053] Example 1

[0054] This embodiment provides a method for preparing iron-loaded modified biochar, comprising the following steps:

[0055] (1) The corn stalks grown in saline-alkali land were collected, impurities were removed, washed, dried and crushed to pass through a 10-mesh sieve, and then soaked in a 1.2 mol / L hydrochloric acid solution for 10 h to further remove impurities and maintain the patency of the gaps. After that, they were washed with clean water and dried in an oven at 105 °C until the mass was constant. Then, they were placed in a muffle furnace and nitrogen was introduced at a flow rate of 100 mL / min for 30 min, and then heated at 10 °C·min -1 The temperature was raised to 300 °C, 500 °C and 700 °C at a rate of 100 °C and maintained for 4 h respectively. After cooling, the biochar was taken out and the obtained biochar was ground and passed through a 100-mesh sieve to obtain biochar;

[0056] (2) The biochar obtained in step (1) was immersed in a trace element solution at a concentration of 30 g / L, and the pH was adjusted to 8.0 with a sulfuric acid solution. After immersion for 24 hours, the modified biochar was dried in an oven at 105°C for 10 hours. The trace element solution included the following raw materials: 0.3 g / L cobalt chloride, 5.0 g / L manganese sulfate, 1.2 g / L anhydrous copper sulfate, 0.4 g / L sodium molybdate, and 3.0 g / L zinc sulfate.

[0057] (3) The modified biochar obtained in step (2) was added to a 30 mmol / L ferrous phosphate solution, with a solid-liquid ratio (g / mL) of the modified biochar to the iron salt solution of 0.03. The modified biochar was stirred to be uniformly dispersed in the solution, nitrogen was introduced, and a 300 mmol / L NaBH4 solution was added dropwise. The amount of NaBH4 solution added was the same as the volume of the iron salt solution.

[0058] (4) The biochar obtained in step (3) is washed with oxygen-free ethanol, and then stripped to volatilize the ethanol, thereby obtaining the loaded iron-based modified biochar.

[0059] Example 2

[0060] This embodiment provides a method for preparing phosphate-solubilizing bacteria for improving lead-contaminated saline-alkali land, comprising the following steps:

[0061] (1) Pseudomonas aeruginosa, Bacillus megaterium, and Bacillus velezensis were inoculated into the same seed culture medium at a weight ratio of 1:1:1, respectively, and mixed cultured. The culture was shaken in an incubator at a temperature of 30°C for 24 hours, and the rotation speed was controlled at 180 rpm to obtain a first-level seed solution; the seed solution culture medium was LB medium;

[0062] (2) The seed solution obtained in step (1) was inoculated into a first-level seed tank at an inoculum volume fraction of 20%. The culture conditions of the first-level seed tank were as follows: temperature 30°C, rotation speed 180 rpm, pH 7, dissolved oxygen 40%, and cultured for 24 hours to obtain a second-level seed solution.

[0063] (3) The secondary seed liquid obtained in step (2) was inoculated into a fermentation tank at an inoculum amount of 20% of the total volume fraction. The culture conditions of the fermentation tank were a temperature of 30°C and a rotation speed of 180 rpm. The fermentation was carried out in stages under different pH gradients to obtain fermentation liquid. The pH gradient was 6.5-7.2, 7.2-8.0, 8.0-8.5, 8.5-9.0, and 9.0-9.5, with a total of 5 stages. The culture time for each stage was 3.5, 3.5, 5.5, 5.5, and 6 h, respectively.

[0064] Corresponding to different pH gradients, the salt stress solution feeds were 0.1g / L Na2CO3 and 0.1g / LNaHCO3, 0.7g / LNa2CO3 and 0.5g / LNaHCO3, 4.5g / L Na2CO3 and 3.0g / LNaHCO3, 7.0g / L Na2CO3 and 5.5g / LNaHCO3, 8.5g / L Na2CO3 and 7.5g / LNaHCO3; the lead feeds were 0.6mmol / L Pb(NO3)2, 1.0mmol / L Pb(NO3)2, 1.3mmol / L Pb(NO3)2, 1.6mmol / L Pb(NO3)2, and 2.0mmol / L Pb(NO3)2;

[0065] (4) centrifuging the phosphate-solubilizing bacteria fermentation liquid obtained in step (3), adjusting the solid content of the bacterial liquid to 60%, and adding 10 wt % of a drying protective agent to the adjusted bacterial liquid to obtain a bacterial slurry; the drying protective agent is composed of galactose, trehalose, sucrose and glycerol in a weight ratio of 1:1:1:1;

[0066] (5) The bacterial slurry obtained in step (4) and auxiliary materials are mixed and granulated in a weight ratio of 1:2 to obtain solid particles; the auxiliary materials are composed of wood ash and vermiculite powder in a weight ratio of 2:1.

[0067] (6) The solid particles obtained in step (5) were vacuum dried at a drying temperature of 40°C for 12 h, and passed through a 2.0 mm sieve to obtain a phosphate-solubilizing bacteria granule product with a particle size of ≤2.0 mm.

[0068] Furthermore, the seed tank culture medium in steps (2) and (3) includes the following raw materials: glucose 6.0 g / L, sodium acetate 6.0 g / L, (NH4)SO4 0.5 g / L, Ca3(PO4)2 10 g / L, calcium phytate 10 g / L, MnSO4·H2O 0.03 g / L, FeSO4·7H2O 0.03 g / L, sodium molybdate 0.3 g / L, MgSO4·7H2O 0.03 g / L, NaCl 0.3 g / L, KCl 0.3 g / L, loaded iron-based modified biochar 15 wt%, and 0.8 g / L Pb(NO3)2.

[0069] Furthermore, the fermentation tank culture medium in steps (2) and (3) includes the following raw materials: glucose 6.0 g / L, sodium acetate 6.0 g / L, (NH4)SO4 0.5 g / L, Ca3(PO4)2 10 g / L, calcium phytate 10 g / L, MnSO4·H2O 0.03 g / L, FeSO4·7H2O 0.03 g / L, sodium molybdate 0.3 g / L, MgSO4·7H2O 0.03 g / L, NaCl 0.3 g / L, KCl 0.3 g / L, and 15 wt% of loaded iron-based modified biochar.

[0070] This embodiment also provides a phosphate-solubilizing bacterium, which is prepared by the above preparation method.

[0071] Example 3

[0072] In this example, the phosphate-solubilizing bacteria obtained in Example 2 were applied to the lead-contaminated saline-alkali land, and the obtained phosphate-solubilizing bacteria had a microbial biomass of 6.7×10 9 cfu / g, the dosage is 4kg / mu, and the changes in soil indicators at different action times are shown in Table 1 below:

[0073]

[0074] Comparative Example 1

[0075] The difference between this comparative example and Example 3 is that in the preparation method of the phosphate-solubilizing bacteria in Comparative Example 1, no iron-based modified biochar was added, and the obtained phosphate-solubilizing bacteria biomass was 4.2×10 9 cfu / g, the obtained phosphate-solubilizing bacteria were applied to lead-contaminated saline-alkali land at a dosage of 4 kg / mu. The changes in soil indicators at different action times are shown in Table 2 below:

[0076]

[0077] The phosphate-solubilizing bacteria prepared by the method for preparing phosphate-solubilizing bacteria for improving lead-contaminated saline-alkali land can effectively reduce the lead concentration, pH and soil salinity in the lead-contaminated saline-alkali land, and has a good improvement effect on the lead-contaminated saline-alkali land.

[0078] The above specific embodiments are further explanations of the technical solutions and beneficial effects of the present invention, and are not intended to limit the implementation methods. For those skilled in the art, any obvious substitutions that do not depart from the concept of the present invention are within the scope of protection of the present invention.

Claims

1. A method for preparing a phosphate-solubilizing bacteria granule product for improving lead-contaminated saline-alkali land, characterized by: The steps include: (1) Pseudomonas, Bacillus megaterium, and Bacillus velezensis were inoculated into the same seed culture medium for mixed culture, and the first-level seed solution was obtained after shaking culture in an incubator; (2) inoculating the seed solution obtained in step (1) into a primary seed tank for cultivation; (3) inoculating the secondary seed liquid obtained in step (2) into a fermentation tank for cultivation, maintaining the fermentation under different pH gradients for segmented fermentation to obtain a fermentation liquid; (4) centrifuging the phosphate-solubilizing bacteria fermentation liquid obtained in step (3), adjusting the solid content of the bacterial liquid, and adding a drying protective agent to the adjusted bacterial liquid to obtain a bacterial slurry; (5) mixing the bacterial slurry obtained in step (4) with auxiliary materials and granulating the mixture to obtain solid particles; (6) vacuum drying the solid particles obtained in step (5) and sieving to obtain a phosphate-solubilizing bacteria granule product; The fermentation tank culture medium in step (3) includes the following raw materials: glucose 5~10g / L, sodium acetate 5~10g / L, (NH4)SO4 0.3~0.8g / L, Ca3(PO4)2 8~12g / L, calcium phytate 8~12g / L, MnSO4·H2O 0.01~0.05g / L, FeSO4·7H2O 0.01~0.05g / L, sodium molybdate 0.2~0.5g / L, MgSO4·7H2O 0.01~0.05g / L, NaCl 0.1~0.5g / L, KCl 0.1~0.5g / L, and 10~20wt% of loaded iron-based modified biochar; In step (3), the culture conditions of the fermentation tank are as follows: temperature 25-40°C, rotation speed 150-200 rpm, pH gradient 6.5-7.2, 7.2-8.0, 8.0-8.5, 8.5-9.0, 9.0-9.5, and culture time for each stage is 3-6 h; In step (3), corresponding to different pH gradients, the raw materials and dosages of salt stress solution feed are: 0.1~0.2g / L Na2CO3 and 0.08~0.12g / L NaHCO3, corresponding to a pH gradient of 6.5~7.2; 0.5~1.5g / L Na2CO3 and 0.3~0.8g / L NaHCO3, corresponding to a pH gradient of 7.2~8.0; 2.5~5.0g / L Na2CO3 and 2.0~4.0g / L NaHCO3, corresponding to a pH gradient of 8.0~8.5; 5.0~7.5g / L Na2CO3 and 4.0~6.5g / L NaHCO3, corresponding to a pH gradient of 8.5~9.0; 7.5~9.5g / L Na2CO3 and 6.5~8.0g / L NaHCO3, corresponding to a pH gradient of 9.0~9.5; The raw materials and dosage of the lead supplement are: 0.5~0.8mmol / L Pb(NO3)2, corresponding to a pH gradient of 6.5~7.2; 0.8~1.2mmol / L Pb(NO3)2, corresponding to a pH gradient of 7.2~8.0; 1.2~1.5mmol / L Pb(NO3)2, corresponding to a pH gradient of 8.0~8.5; 1.5~1.8mmol / L Pb(NO3)2, corresponding to a pH gradient of 8.5~9.0; 1.8~2.0mmol / L Pb(NO3)2, corresponding to a pH gradient of 9.0~9.

5.

2. The method for preparing the phosphate-solubilizing bacteria granule product for improving lead-contaminated saline-alkali land according to claim 1, characterized in that: In step (1), Pseudomonas, Bacillus megaterium, and Bacillus velezensis are inoculated into a seed culture medium at a weight ratio of 0.5-2:0.5-2:0.5-2, and cultured in an incubator at a temperature of 25-40°C for 18-30 hours with shaking and a rotation speed controlled at 150-200 rpm to obtain a first-level seed solution.

3. The method for preparing the phosphate-solubilizing bacteria granule product for improving lead-contaminated saline-alkali land according to claim 1, characterized in that: In step (2), the seed solution obtained in step (1) is inoculated into a first-level seed tank at an inoculum volume fraction of 10-20%. The culture conditions of the first-level seed tank are temperature 25-40°C, rotation speed 150-200 rpm, pH 6.5-7.5, dissolved oxygen 20-50%, and culture for 18-30 hours to obtain a second-level seed solution.

4. The method for preparing the phosphate-solubilizing bacteria granule product for improving lead-contaminated saline-alkali land according to claim 1, characterized in that: The seed tank culture medium in step (2) includes the following raw materials: glucose 5~10g / L, sodium acetate 5~10g / L, (NH4)SO4 0.3~0.8g / L, Ca3(PO4)2 8~12g / L, calcium phytate 8~12g / L, MnSO4·H2O 0.01~0.05g / L, FeSO4·7H2O 0.01~0.05g / L, sodium molybdate 0.2~0.5g / L, MgSO4·7H2O 0.01~0.05g / L, NaCl 0.1~0.5g / L, KCl 0.1~0.5g / L, loaded iron-based modified biochar 10~20wt%, Pb(NO3)2 0.1~1.0g / L.

5. The method for preparing the phosphate-solubilizing bacteria granule product for improving lead-contaminated saline-alkali land according to any one of claims 1 and 4, characterized in that: The preparation method of the loaded iron-based modified biochar comprises the following steps: (A1) Corn stalks grown on saline-alkali land were collected, impurities removed, washed, sun-dried, crushed, and sieved. The stalks were then immersed in a hydrochloric acid solution, rinsed with clean water, and dried in an oven until the mass remained constant. The stalks were then placed in a muffle furnace, purged with nitrogen, and heated to 280-320°C, 480-520°C, and 680-720°C for 3-5 hours, respectively. The stalks were cooled, removed, and ground and sieved to obtain biochar. (A2) soaking the biochar obtained in step (A1) in a trace element solution at a concentration of 20-50 g / L, adjusting the pH to 7.2-8.6, and drying in an oven to produce modified biochar; (A3) adding the modified biochar obtained in step (A2) to a 10-50 mmol / L ferrous phosphate solution at a solid-to-liquid ratio of the modified biochar to the ferric salt solution of 0.01-0.04 g / mL, stirring until the modified biochar is evenly dispersed in the solution, introducing nitrogen, and adding a 100-500 mmol / L NaBH4 solution at a volume ratio of the NaBH4 solution to the ferric salt solution of 1:0.8-1.2; (A4) The biochar obtained in step (A3) is washed with oxygen-free ethanol, and then stripped to volatilize the ethanol, thereby obtaining iron-loaded modified biochar.

6. The method for preparing the phosphate-solubilizing bacteria granule product for improving lead-contaminated saline-alkali land according to claim 1, characterized in that: In step (4), after the phosphate-solubilizing bacteria fermentation liquid is centrifuged, the solid content of the bacterial liquid is adjusted to 50-80%, and 0.5-15% of a drying protective agent is added to the adjusted bacterial liquid to obtain a bacterial slurry; the drying protective agent is at least one of galactose, trehalose, sucrose, lactose, raffinose, glycerol, dimethyl sulfoxide and gelatin.

7. The method for preparing the phosphate-solubilizing bacteria granule product for improving lead-contaminated saline-alkali land according to claim 1, characterized in that: In step (5), the bacterial slurry and the auxiliary material are mixed and granulated in a weight ratio of 1:0.5-3; the auxiliary material is at least one of wood ash and vermiculite powder.

8. A phosphate-solubilizing bacteria granule product, characterized in that: The phosphate-solubilizing bacteria granule product is prepared by the preparation method of any one of claims 1 to 7.

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