Phosphate solubilizing bacteria and preparation method thereof

Abstract

The invention particularly relates to a phosphate solubilizing bacterium and a preparation method thereof, in particular to a phosphate solubilizing bacterium for improving lead-polluted saline-alkali soil and a preparation method of the phosphate solubilizing bacterium. The preparation method of the phosphate solubilizing bacteria comprises the following steps: respectively inoculating pseudomonas, bacillus megatherium and bacillus velezensis into a seed culture medium for culturing to obtain a first-stage seed solution; inoculating the seed solution into a first-stage seed tank according to the inoculum size, and culturing; inoculating the secondary seed solution into a fermentation tank for culturing, and carrying out segmented fermentation under different pH gradients; centrifuging the phosphate solubilizing bacteria fermentation liquor, and adding a drying protective agent to obtain bacterial slurry; mixing and granulating the bacterial slurry and auxiliary materials to obtain solid particles; and drying and sieving the solid particles. The phosphate solubilizing bacteria prepared by the method can effectively reduce the concentration and pH of lead and soil salinity in the lead-polluted saline-alkali soil, and effectively improve the lead-polluted saline-alkali soil.

Description

Technical Field

[0001] The invention relates to the technical field of soil remediation, and 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 an important part of the ecological environment and a basic natural resource for human survival. With the development of industrial and agricultural production, its degradation is becoming more and more serious. Among them, saline-alkali soil is an important issue of soil degradation. The emergence and development of problems such as unreasonable agricultural irrigation, excessive fertilizer application, vegetation destruction and greenhouse effect have also aggravated the problem of soil salinization. This not only causes resource destruction and agricultural production losses, but also harms the ecological environment and biosphere. With the increasing population, the contradiction between the reduction of cultivated land area and food demand has become increasingly prominent. As an important type of reserve land resource, saline-alkali soil is increasingly valued by governments around the world for its improvement and resource utilization.

[0003] With the development of industrialization, along with the mining of minerals and the discharge of industrial wastewater, heavy metal lead is input through water, causing serious soil pollution, including a part of salinized reserve land. Salt-alkali land improvement technologies include chemical improvement methods, physical improvement methods, plant remediation methods and biological fertilizer improvement methods. The biological fertilizer remediation method is to improve the saline-alkali land by applying biological fertilizers, which has the advantages of low cost, long action time and friendliness to the natural environment. Therefore, it is of great significance to provide a microbial agent and preparation method that can effectively improve lead-contaminated saline-alkali land. 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, and effectively improve the 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: (1) Pseudomonas, Bacillus megaterium, and Bacillus velezinii are inoculated into the same seed culture medium for mixed culture, and the first-level seed solution is obtained after shaking in an incubator; (2) The seed solution obtained in step (1) is inoculated into a primary seed tank at an inoculum volume fraction of 10-20%. The culture conditions of the primary seed tank are as follows: 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 secondary seed solution; (3) The secondary seed liquid obtained in step (2) is inoculated into a fermentation tank at an inoculum amount of 10-20% of the integral fraction, and the culture conditions of the fermentation tank are a temperature of 25-40° C. and a rotation speed of 150-200 rpm, and staged fermentation is performed under different pH gradients 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 to 50-80%, and adding 0.5-15% of 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 them to obtain solid particles; (6) The solid particles obtained in step (5) are vacuum dried and sieved to obtain a phosphate-solubilizing bacteria granule product.

[0006] The mechanism of the present invention using phosphate-solubilizing bacteria to improve saline-alkali soil is as follows: (i) The organic acid secreted by the phosphate-solubilizing bacteria during the metabolic process 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 to promote the absorption and utilization of phosphorus by plants; (ii) Phosphate-solubilizing bacteria can dissolve the insoluble phosphorus source in the soil for plant absorption and utilization, thereby promoting growth; (iii) After applying exogenous phosphate-solubilizing bacteria, the original microbial community structure in the saline-alkali soil will be changed, and the abundance of bacteria related to phosphorus conversion will be increased, thereby improving the physical and chemical properties of the soil through microorganisms. The effects of phosphate-solubilizing bacteria on heavy metals are as follows: (i) Secretion of organic acids, phosphatases and other substances converts insoluble phosphorus into soluble phosphorus, which reacts with heavy metals such as lead to form more stable phosphorus-lead compounds; (ii) Phosphate-solubilizing bacteria absorb and accumulate a portion of heavy metals themselves; (iii) Many metabolites produced by microorganisms can react with heavy metal ions.

[0007] 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 18~30 hours with shaking, and the rotation speed is controlled at 150~200 rpm to obtain a primary seed solution.

[0008] Furthermore, in step (2), the seed solution obtained in step (1) is inoculated into a primary seed tank at an inoculation amount of 10-20% by volume, and the culture conditions of the primary 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 secondary seed solution.

[0009] 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.

[0010] Further, in step (3), corresponding to different pH gradients, the raw materials and dosages of the salt stress solution feed are: 0.1-0.2 g / L Na2CO3 and 0.08-0.12 g / L NaHCO3 (corresponding to a pH gradient of 6.5-7.2), 0.5-1.5 g / L Na2CO3 and 0.3-0.8 g / L NaHCO3 (corresponding to a pH gradient of 7.2-8.0), 2.5-5.0 g / L Na2CO3 and 2.0-4.0 g / L NaHCO3 (corresponding to a pH gradient of 8.0-8.5), 5.0-7.5 g / L Na2CO3 and 4.0-6.5 g / L NaHCO3 (corresponding to a pH gradient of 8.5-9.0), 7.5-9.5 g / L Na2CO3 and 6.5-8.0 g / L NaHCO3 (corresponding to a pH gradient of 9.0-9.5); The raw materials and dosage of lead supplement are: 0.5~0.8mmol / L Pb(NO3)2 (corresponding pH gradient of 6.5~7.2), 0.8~1.2mmol / L Pb(NO3)2 (corresponding pH gradient of 7.2~8.0), 1.2~1.5mmol / L Pb(NO3)2 (corresponding pH gradient of 8.0~8.5), 1.5~1.8mmol / L Pb(NO3)2 (corresponding pH gradient of 8.5~9.0), 1.8~2.0mmol / L Pb(NO3)2 (corresponding pH gradient of 9.0~9.5).

[0011] Furthermore, the seed tank culture medium in steps (2) and (3) includes the following raw materials: 5-10 g / L glucose, 5-10 g / L sodium acetate, 0.3-0.8 g / L (NH4)SO4, 8-12 g / L Ca3(PO4)2, 8-12 g / L calcium phytate, and 10 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, MgSO4·7H2O 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.

[0012] Furthermore, the fermentation tank culture medium in steps (2) and (3) includes the following raw materials: 5-10 g / L glucose, 5-10 g / L sodium acetate, 0.3-0.8 g / L (NH4)SO4, 8-12 g / L Ca3(PO4)2, 8-12 g / L calcium phytate, and 10 g / L Mn SO4·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%.

[0013] Furthermore, the preparation method of the loaded iron-based modified biochar comprises the following steps: (A1) Collect corn stalks grown in saline-alkali land, remove impurities, wash, dry and crush through a 10-mesh sieve, then soak in 1-1.4 mol / L hydrochloric acid solution for 8-15 h to further remove impurities and maintain the patency of the gaps. Then wash with clean water and dry in an oven at 100-110 °C until the mass is constant. Then put it in a muffle furnace and pass nitrogen at a flow rate of 80-120 mL / min for 25-35 min, then heat 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 100°C and maintained for 3-5 hours respectively, and the biochar was taken out after cooling, and the obtained biochar was ground and passed through a 100-mesh sieve 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 with a sulfuric acid solution, soaking for 24 hours, and drying in an oven at 100-110° C. for 8-12 hours to prepare modified biochar; (A3) adding the modified biochar obtained in step (A2) to a 10-50 mmol / L ferrous phosphate solution, wherein the solid-liquid ratio of the modified biochar to the iron salt solution is 0.01-0.04 g / mL, stirring to uniformly disperse the modified biochar in the solution, introducing nitrogen, and adding dropwise a 100-500 mmol / L NaBH4 solution, wherein the amount of the NaBH4 solution added is equal to the volume of the iron salt solution in a ratio of 1:0.8-1.2; preferably, the amount of the NaBH4 solution added is equal to the volume of the iron salt solution; (A4) The biochar obtained in step (A3) is washed with oxygen-free ethanol, and then stripped by air to volatilize the ethanol, thereby obtaining iron-loaded modified biochar.

[0014] 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.

[0015] Furthermore, in step (4), after centrifuging the phosphate-solubilizing bacteria fermentation liquid, 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.

[0016] 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.

[0017] The present invention also provides a phosphate-solubilizing bacterium, which is prepared by the above-mentioned preparation method.

[0018] The beneficial effects of the present invention are: (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 the method can effectively reduce the lead concentration, pH and soil salinity in the lead-contaminated saline-alkali land, thereby effectively improving the lead-contaminated saline-alkali land.

[0019] (2) During the fermentation process of phosphate-solubilizing bacteria, pH, salinity and lead concentration gradient fermentation are adopted, and the prepared phosphate-solubilizing bacteria can maintain the ability to degrade salinity and lead.

[0020] (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.

[0021] (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 bacterial 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

[0022] In order to facilitate the understanding of those skilled in the art, the present invention is further described below in conjunction with embodiments, and the contents mentioned in the implementation modes are not intended to limit the present invention.

[0023] 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: (1) Inoculating Pseudomonas, Bacillus megaterium, and Bacillus velezinii 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 culturing in an incubator at a temperature of 25-40° C. with shaking for 18-30 hours and controlling the rotation speed at 150-200 rpm to obtain a primary seed solution; (2) The seed solution obtained in step (1) is inoculated into a primary seed tank at an inoculum volume fraction of 10-20%. The culture conditions of the primary seed tank are as follows: 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 secondary seed solution; (3) The secondary seed liquid obtained in step (2) is inoculated into a fermentation tank at an inoculum amount of 10-20% of the integral fraction, and the culture conditions of the fermentation tank are a temperature of 25-40° C. and a rotation speed of 150-200 rpm, and staged fermentation is performed under different pH gradients 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 to 50-80%, and adding 0.5-15% of 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 them to obtain solid particles; (6) The solid particles obtained in step (5) are vacuum dried and sieved to obtain a phosphate-solubilizing bacteria granule product.

[0024] 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 the salt stress liquid feed are: 0.1-0.2 g / L Na2CO3 and 0.08-0.12 g / L NaHCO3, 0.5-1.5 g / L Na2CO3 and 0.3-0.8 g / L NaHCO3, 2.5-5.0 g / L Na2CO3 and 2.0-4.0 g / L NaHCO3, 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.

[0025] 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 SO4·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)2 0.1~1.0g / L.

[0026] 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, 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%.

[0027] In the present invention, compared with the fermentation tank culture medium, the seed tank culture medium is additionally added with 0.1-1.0 g / L Pb(NO3)2.

[0028] In some embodiments of the present invention, the method for preparing the loaded iron-based modified biochar comprises the following steps: (A1) Collect corn stalks grown in saline-alkali land, remove impurities, wash, dry and crush through a 10-mesh sieve, then soak in 1-1.4 mol / L hydrochloric acid solution for 8-15 h to further remove impurities and maintain the patency of the gaps. Then wash with clean water and dry in an oven at 100-110 °C until the mass is constant. Then put it in a muffle furnace and pass nitrogen at a flow rate of 80-120 mL / min for 25-35 min, then heat 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 100°C and maintained for 3-5 hours respectively, and the biochar was taken out after cooling, and the obtained biochar was ground and passed through a 100-mesh sieve 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 with a sulfuric acid solution, soaking for 24 hours, and drying in an oven at 100-110° C. for 8-12 hours to prepare 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; (A3) adding the modified biochar obtained in step (A2) to a 10-50 mmol / L ferrous phosphate solution, wherein the solid-liquid ratio of the modified biochar to the iron salt solution is 0.01-0.04 g / mL, stirring to uniformly disperse the modified biochar in the solution, introducing nitrogen, and adding dropwise a 100-500 mmol / L NaBH4 solution, wherein the volume ratio of the NaBH4 solution to the iron salt solution is 1:0.8-1.2; (A4) The biochar obtained in step (A3) is washed with oxygen-free ethanol, and then stripped by air to volatilize the ethanol, thereby obtaining iron-loaded modified biochar.

[0029] 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.

[0030] 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.

[0031] Example 1 This embodiment provides a method for preparing iron-based modified biochar, comprising the following steps: (1) Corn stalks grown in saline-alkali land were collected, impurities were removed, washed, dried, and crushed to pass through a 10-mesh sieve. They were 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. They were then washed with clean water and dried in an oven at 105 °C until the mass was constant. They were then placed in a muffle furnace and nitrogen was introduced at a flow rate of 100 mL / min for 30 min. -1 The temperature was raised to 300 ℃, 500 ℃ and 700 ℃ at a rate of 100 °C and maintained for 4 h respectively. After cooling, the biochar was taken out and ground and passed through a 100-mesh sieve to obtain biochar. (2) The biochar obtained in step (1) is immersed in a trace element solution at a concentration of 30 g / L, and the pH is adjusted to 8.0 with a sulfuric acid solution. After immersion for 24 hours, it is dried in an oven at 105°C for 10 hours to prepare modified biochar. The trace element solution includes 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; (3) Add the modified biochar obtained in step (2) to a 30 mmol / L ferrous phosphate solution, with the solid-liquid ratio (g / mL) of the modified biochar to the iron salt solution being 0.03, stir until the modified biochar is evenly dispersed in the solution, introduce nitrogen, and add 300 mmol / L NaBH4 solution dropwise, with the amount of NaBH4 solution added being the same as the volume of the iron salt solution; (4) The biochar obtained in step (3) is washed with oxygen-free ethanol, and then stripped by air to volatilize the ethanol, thereby obtaining iron-loaded modified biochar.

[0032] Example 2 This embodiment provides a method for preparing phosphate-solubilizing bacteria for improving lead-contaminated saline-alkali land, comprising the following steps: (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, for mixed culture, and cultured in an incubator at a temperature of 30° C. with shaking for 24 hours, and the rotation speed was controlled at 180 rpm to obtain a primary seed solution; the seed solution culture medium was LB medium; (2) The seed solution obtained in step (1) is inoculated into a primary seed tank at an inoculum volume fraction of 20%. The culture conditions of the primary seed tank are as follows: temperature 30°C, rotation speed 180 rpm, pH 7, dissolved oxygen 40%, and culture for 24 hours to obtain a secondary seed solution; (3) The secondary seed liquid obtained in step (2) is inoculated into a fermenter at an inoculum amount of 20% of the integral fraction. The culture conditions of the fermenter are a temperature of 30° C. and a rotation speed of 180 rpm. The fermentation is carried out in stages under different pH gradients to obtain a fermentation liquid. The pH gradient is 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, and the culture time for each stage is 3.5, 3.5, 5.5, 5.5, and 6 h, respectively. 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, 2.0mmol / L Pb(NO3)2; (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; (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.

[0033] (6) The solid particles obtained in step (5) are vacuum dried at a drying temperature of 40° C. for a drying time of 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.

[0034] 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, Mn S O4·H2O 0.03g / L, FeSO4·7H2O 0.03g / L, sodium molybdate 0.3g / L, MgSO4·7H2O 0.03g / L, NaCl 0.3g / L, KCl 0.3g / L, loaded iron-based modified biochar 15wt%, 0.8g / L Pb(NO3)2.

[0035] 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, Mn SO4·H2O 0.03g / L, FeSO4·7H2O 0.03g / L, sodium molybdate 0.3g / L, MgSO4·7H2O 0.03g / L, NaCl 0.3g / L, KCl 0.3g / L, loaded iron-based modified biochar 15wt%.

[0036] This embodiment also provides a phosphate-solubilizing bacteria, which is prepared by the above preparation method.

[0037] Example 3 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:

[0038] Comparative Example 1 The difference between this comparative example and Example 3 is that in the preparation method of phosphate-solubilizing bacteria in Comparative Example 1, no iron-based modified biochar was added, and the obtained phosphate-solubilizing bacteria microbial biomass was 4.2×10 9 cfu / g, the obtained phosphate-solubilizing bacteria were applied to the lead-contaminated saline-alkali land, the dosage was 4kg / mu, and the changes of soil indicators at different action times are shown in Table 2 below:

[0039] 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.

[0040] The above specific embodiments are further explanations of the technical solutions and beneficial effects of the present invention, and are not limitations of the implementation methods. For those skilled in the art, any obvious replacements that do not depart from the concept of the present invention are within the protection scope of the present invention.

Claims

1. A method for preparing phosphate-solubilizing bacteria for improving lead-contaminated saline-alkali land, characterized in that: The steps include: (1) Pseudomonas, Bacillus megaterium, and Bacillus velezinaceus are inoculated into the same seed culture medium for mixed culture, and a primary seed solution is 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 culturing, maintaining the fermentation under different pH gradients for segmented fermentation, and obtaining 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 them to obtain solid particles; (6) The solid particles obtained in step (5) are vacuum dried and sieved to obtain a phosphate-solubilizing bacteria granule product.

2. The method for preparing phosphate-solubilizing bacteria for improving lead-contaminated saline-alkali land according to claim 1, characterized in that: In step (1), Pseudomonas, Bacillus megaterium and Bacillus velez 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 a rotation speed controlled at 150-200 rpm to obtain a primary seed solution.

3. The method for preparing phosphate-solubilizing bacteria 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 primary seed tank at an inoculation amount of 10-20% by volume. The culture conditions of the primary seed tank are as follows: 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 secondary seed solution.

4. The method for preparing phosphate-solubilizing bacteria for improving lead-contaminated saline-alkali land according to claim 1, characterized in that: 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.

5. The method for preparing phosphate-solubilizing bacteria for improving lead-contaminated saline-alkali land according to claim 1, characterized in that: 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, MgSO4·7H2O 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.

6. The method for preparing phosphate-solubilizing bacteria for improving lead-contaminated saline-alkali land according to claim 1, characterized in that: 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, Mn S O4·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%.

7. The method for preparing phosphate-solubilizing bacteria for improving lead-contaminated saline-alkali land according to any one of claims 5 and 6, characterized in that: The preparation method of the loaded iron-based modified biochar comprises the following steps: (A1) After collecting corn stalks grown in saline-alkali land and removing impurities, the corn stalks were washed, dried, crushed and sieved, and then soaked in a hydrochloric acid solution. After being cleaned with clean water, the corn stalks were placed in an oven and dried until the mass was constant. The corn stalks were then placed in a muffle furnace, and nitrogen was introduced. The temperature was raised to 280-320°C, 480-520°C and 680-720°C and maintained for 3-5 h respectively. After cooling, the corn stalks were taken out to obtain biochar. The obtained biochar was 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 prepare modified biochar; (A3) adding the modified biochar obtained in step (A2) to a 10-50 mmol / L ferrous phosphate solution, wherein the solid-liquid ratio of the modified biochar to the iron salt solution is 0.01-0.04 g / mL, stirring to uniformly disperse the modified biochar in the solution, introducing nitrogen, and adding a 100-500 mmol / L NaBH4 solution, wherein the volume ratio of the NaBH4 solution to the iron salt solution is 1:0.8-1.2; (A4) The biochar obtained in step (A3) is washed with oxygen-free ethanol, and then stripped by air to volatilize the ethanol, thereby obtaining iron-loaded modified biochar.

8. The method for preparing phosphate-solubilizing bacteria for improving lead-contaminated saline-alkali land according to claim 1, characterized in that: In step (4), after centrifuging the phosphate-solubilizing bacteria fermentation liquid, 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.

9. The method for preparing phosphate-solubilizing bacteria 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.

10. A phosphate-solubilizing bacteria, characterized in that: The phosphate-solubilizing bacteria is prepared by the preparation method of any one of claims 1 to 9.

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