A microbial preparation for saline-alkali soil improvement and a preparation method and application thereof

Abstract

The application discloses a microbial preparation for saline-alkali soil improvement and a preparation method and application thereof, and relates to the technical field of soil improvement, and specifically discloses a microbial preparation for saline-alkali soil improvement, which comprises Bacillus marinus with a preservation number of CGMCC No.28096, Bacillus cereus with a preservation number of CGMCC No.29355, Bacillus subtilis with a preservation number of CICC 25064 and Pseudomonas with a preservation number of CICC 22131, and the ratio of the viable bacterial counts of the Bacillus marinus, the Bacillus cereus, the Bacillus subtilis and the Pseudomonas is (3-5):(1-3):(5-7):(1-3). The microbial preparation can effectively reduce the alkalinity and sodium adsorption ratio of the saline-alkali soil, effectively degrade phosphorus acid tris(2-chloropropyl) ester pollutants, and improve the quality of the saline-alkali soil.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of microbial technology, and particularly relates to a microbial preparation for improving saline-alkali soil, a preparation method and application thereof. BACKGROUND

[0002] Sodium adsorption ratio (SAR) and alkalinity in saline-alkali soil are two key indicators closely related to each other, and they jointly affect the physical, chemical and biological properties of soil, eventually leading to soil degradation and reducing agricultural productivity. Strong sodium ions with high SAR can destroy the physical structure of soil, resulting in clay dispersion, soil hardening, pore blockage, and sharp decrease in water and air permeability and drainage, and increasing the risk of soil erosion. Meanwhile, high alkalinity (high pH and rich in carbonate / bicarbonate) mainly deteriorates the chemical and biological environment of soil, significantly reducing the availability of phosphorus and trace elements such as iron, manganese, zinc and copper, inhibiting the decomposition of organic matter and microbial activity, and possibly directly poisoning the plant roots. More seriously, high alkalinity can promote the precipitation of calcium and magnesium ions into carbonate, which not only reduces the available calcium and magnesium nutrients for plants, but more critically, greatly reduces the concentration of calcium ions in soil solution, thereby maintaining or even exacerbating the high SAR state. The synergistic effect of the two will form a vicious cycle, leading to the collapse of soil physical structure, imbalance of chemical properties, and decline of biological functions, and eventually causing severe soil degradation, extremely low productivity and extremely difficult improvement.

[0003] With the wide application of flame retardants in the plastic, building material and other industrial fields, the organic phosphorus flame retardant tris(2-chloropropyl) phosphate (TCPP) is continuously input into saline-alkali regions through industrial wastewater discharge, electronic waste leaching and atmospheric deposition. Due to the significantly prolonged hydrolysis half-life of TCPP under alkaline conditions (up to 120 days at pH>8.5), and the high salt ion strength (Na ion concentration>0.5M) promoting its adsorption on clay surface by compressing the electric double layer, the residual concentration of TCPP in saline-alkali soil can reach 3-5 times that in non-saline-alkali soil.

[0004] Currently, existing remediation technologies have serious limitations: chemical reduction method is easy to generate strong carcinogenic intermediate epichlorohydrin; phytoremediation is ineffective due to the low transport coefficient of salt-tolerant plants for TCPP; single microbial degradation is inhibited by salt and alkali. Especially under high salt-alkali stress, traditional biochar adsorbents have a 40%-70% decrease in TCPP adsorption capacity due to the blockage of pores by Na ions, and cannot establish an effective pretreatment barrier. In order to protect soil resources, improve the ecological environment and develop regional economy, and achieve sustainable development, it is of great significance to research a microbial preparation for saline-alkali soil for greening, improvement and utilization of saline-alkali regions. SUMMARY

[0005] In order to solve the problems in the prior art, the present application provides a microbial preparation for improving saline-alkali soil, a preparation method and application thereof, which can effectively reduce the alkalinity and sodium adsorption ratio of the saline-alkali soil, and effectively degrade tris(2-chloropropyl) phosphate pollutants, and improve the quality of the saline-alkali soil.

[0006] In order to achieve the above-mentioned target, the present application adopts the following technical solution:

[0007] The microbial preparation for improving saline-alkali soil comprises Bacillus marinus (CGMCC No.28096) Bacillus aquimaris ), Bacillus cereus (CGMCC No.29355) Bacillus cereus ), Bacillus subtilis (CICC 25064) Bacillus subtilis ) and Pseudomonas (CICC 22131) Pseudomonas sp. , and the ratio of the viable bacterial count (CFU) of the Bacillus marinus, the Bacillus cereus, the Bacillus subtilis and the Pseudomonas is (3-5):(1-3):(5-7):(1-3).

[0008] The preparation method of the microbial preparation comprises the following steps:

[0009] S1, the Bacillus marinus, the Bacillus cereus, the Bacillus subtilis and the Pseudomonas are respectively placed in LB culture medium, and are oscillation cultured to prepare seed liquid of each strain;

[0010] S2, the seed liquid of each strain is inoculated into fermentation culture medium to perform fermentation culture, and the Bacillus marinus bacterial liquid, the Bacillus cereus bacterial liquid, the Bacillus subtilis bacterial liquid and the Pseudomonas bacterial liquid are respectively obtained;

[0011] S3, the Bacillus marinus bacterial liquid, the Bacillus cereus bacterial liquid, the Bacillus subtilis bacterial liquid and the Pseudomonas bacterial liquid are mixed to obtain a composite bacterial suspension, and the bacterial slurry is collected by centrifugation, mixed with biochar and corn powder, and dried to obtain a solid microbial preparation.

[0012] Preferably, in the step S1, the components of the LB culture medium are yeast extract 5 g, protein peptone 10 g, NaCl 5 g and water 1 L, pH is 6-8, the culture temperature is 25-35 DEG C, and the culture time is 24-48 h.

[0013] Preferably, in the aforementioned step S2, the components of the fermentation medium are: yeast powder 5.0 g / L, peptone 10.0 g / L, sodium chloride 5.0 g / L, dipotassium hydrogen phosphate 1.0 g / L, potassium dihydrogen phosphate 1.0 g / L, magnesium sulfate 0.2 g / L, ammonium sulfate 0.8 g / L, and calcium chloride 0.02 g / L, pH 6-8, the culture temperature is 25-35 DEG C, and the culture time is 24-48 h; the viable cell count of the Bacillus halodurans liquid, Bacillus cereus liquid, Bacillus subtilis liquid, and Pseudomonas liquid is all 6*10 12 -7*10 12 CFU / mL.

[0014] Preferably, in the aforementioned step S3, the mass ratio of the bacterial slurry, biochar, and corn powder is 1: (1-3): (1-2).

[0015] Application of a microbial preparation in reducing the sodium adsorption ratio and alkalinity in salinized soil.

[0016] Application of a microbial preparation in degrading tris (2-chloropropyl) phosphate pollutants in salinized soil.

[0017] Preferably, the salinized soil has a salt content of 1-10 g / kg and a pH of 8-11.

[0018] The beneficial effects of the present application are that the Bacillus halodurans, Bacillus cereus, Bacillus subtilis, and Pseudomonas in the complex microbial flora can play a good synergistic effect, have strong salt and alkali tolerance, can effectively reduce the sodium adsorption ratio, total salt content, and alkalinity of salinized soil, have excellent degradation performance on tris (2-chloropropyl) phosphate pollutants, effectively improve the organic matter content of salinized soil, improve the soil structure, improve the quality of salinized soil, and help plant growth and improve crop quality. BRIEF DESCRIPTION OF DRAWINGS

[0019] Figure 1 is a repair effect diagram of the microbial preparation on the saline soil under different pH values;

[0020] Figure 2 is a repair effect diagram of the microbial preparation on the saline soil under different salinities. DETAILED DESCRIPTION

[0021] The present application will be specifically described below in combination with the drawings and specific examples.

[0022] Example 1, a microbial preparation for improving salinized soil, comprising:

[0023] Bacillus halodurans ( Bacillus aquimaris ), Bacillus cereus ( Bacillus cereusBacillus subtilis ( Bacillus subtilis ) and Pseudomonas ( Pseudomonas sp. Bacillus subtilis ( ). Bacillus aquimaris It was deposited on August 4, 2023, at the China General Microbiological Culture Collection Center (CGMCC), accession number: CGMCC No. 28096; Bacillus cereus ( Bacillus cereus Bacillus subtilis (CGMCC No. 29355) was deposited on December 18, 2023, at the China General Microbiological Culture Collection Center. Bacillus subtilis The strain was deposited on January 18, 2021, at the China Industrial Microbial Culture Collection Center, with accession number CICC 25064; Pseudomonas ( Pseudomonas sp. The sample was deposited at the China Industrial Microbial Culture Collection Center on February 1, 2008, with accession number CICC 22131. Among them, Bacillus subtilis and Pseudomonas are known species, obtained through purchase. The ratio of viable counts of Bacillus aquatilis, Bacillus cereus, Bacillus subtilis, and Pseudomonas is (3-5):(1-3):(5-7):(1-3).

[0024] Example 2: Determination of the formulation ratio of microbial preparations

[0025] In this invention, all saline-alkali soils were obtained from a saline-alkali land. The pH index was determined using the potentiometric method, the total salt content was determined using the conductivity method, and the SAR was determined using the ion exchange method.

[0026] Each strain was inoculated into 100 mL LB medium (LB medium consists of 5 g yeast extract, 10 g peptone, 5 g NaCl, and 1 L water) and cultured at 30 ℃ and 180 rpm for 24 h to obtain seed culture. The seed cultures were centrifuged at 8000 rpm for 3 min, washed three times with PBS buffer, and diluted to 0.6 with an OD600 value. Five gradients of inoculum amounts (1%, 2%, 3%, 4%, and 5%) were set, with samples without inoculum as blank controls. Three replicates were set for each sample. pH, SAR, and total salt content of saline-alkali soil were measured, and the reduction effect of different inoculum amounts on soil indicators was compared. Based on the optimal results of each single strain inoculum amount, three different inoculum levels were designed, as shown in Table 1. Then, a four-factor, three-level orthogonal experiment was designed based on Table 1, as shown in Table 2.

[0027] Table 1. Orthogonal Design Factors and Levels

[0028]

[0029] Table 2. Orthogonal Experiment Analysis Table

[0030]

[0031] The bacterial suspension was configured according to Tables 1 and 2, and was added into saline-alkali soil in proportion, and saline-alkali soil without the bacterial solution was used as a blank control, and the pH, SAR, total salt content and TCPP pollutant content were measured at different proportions, as shown in Table 3.

[0032] Table 3: Decline effect of each index of saline-alkali soil

[0033]

[0034] As shown in Table 3, the pH, SAR, total salt content and TCPP content of the experimental group 6 have the best decline effect, and the other indexes have little difference, so the best proportion of the composite bacterial population is the experimental group 6, that is, the proportion of Bacillus marinus, Bacillus cereus, Bacillus subtilis and Pseudomonas is 4:3:5:2. The four specific strains are compounded in the application, and unexpected synergistic effect is achieved. Among them, Bacillus marinus has excellent salt tolerance, can create a survival microenvironment for other strains in a high-salt environment, Bacillus subtilis can produce various enzymes and organic acids, which helps to reduce the soil pH and decompose organic matter, and Bacillus cereus and Pseudomonas perform outstanding in degrading TCPP and other organic pollutants, and the four strains jointly act on reducing salinity, degrading pollutants and improving soil, and the effect is much better than that of a single strain.

[0035] The preparation method of the microbial preparation of Example 3 comprises the following specific steps:

[0036] S1, activation and expansion culture of each strain: under the condition of 30 DEG C, each strain is respectively placed in 100 mL of LB culture medium (the components of the LB culture medium are: yeast extract 5 g, protein peptone 10 g, NaCl 5 g and water 1 L), and then is oscillation cultured in a 180 rpm shaker for 24 h, to prepare a seed solution of each strain;

[0037] S2, inoculating each strain seed solution into a fermentation tank of fermentation medium (the components of the fermentation medium are: yeast powder 5.0 g / L, protein peptone 10.0 g / L, sodium chloride 5.0 g / L, potassium hydrogen phosphate 1.0 g / L, potassium dihydrogen phosphate 1.0 g / L, magnesium sulfate 0.2 g / L, ammonium sulfate 0.8 g / L and calcium chloride 0.02 g / L) at a volume ratio of 1%, pH 6, 30 DEG C, and performing expansion culture;

[0038] S3, fermentation of each strain: the expanded culture products of each strain were respectively taken and subjected to secondary fermentation culture in a fermenter under the conditions of 30℃ and pH 6-8, and the fermentation medium (the components of the fermentation medium were: yeast powder 5.0 g / L, peptone 10.0 g / L, sodium chloride 5.0 g / L, potassium phosphate dibasic 1.0 g / L, potassium phosphate monobasic 1.0 g / L, magnesium sulfate 0.2 g / L, ammonium sulfate 0.8 g / L, and calcium chloride 0.02 g / L) to obtain Bacillus halodurans bacterial liquid, Bacillus cereus bacterial liquid, Bacillus subtilis bacterial liquid, and Pseudomonas bacterial liquid, and the viable cell count of each obtained bacterial liquid was 6×10 12 -7×10 12 CFU / mL;

[0039] S4, preparation of microbial preparation: 40 g of Bacillus halodurans bacterial liquid, 30 g of Bacillus cereus bacterial liquid, 50 g of Bacillus subtilis bacterial liquid, and 20 g of Pseudomonas bacterial liquid were mixed to obtain a composite bacterial suspension, and the bacterial slurry was collected by centrifugation at 4000 rpm; the bacterial slurry was mixed with biochar and corn powder at a ratio of 1:2:1, and low-temperature drying at 35℃ was performed to obtain a solid bacterial agent.

[0040] Example 4, repair effect of microbial preparation on saline-alkali soil under different pH values

[0041] The pH of the saline-alkali soil was adjusted to 8, 9, 10, 11, and 12, respectively, and the microbial preparation prepared in Example 3 was added to the soil at a mass ratio of 5%, and after 15 days of plowing, the pH, SAR, total salt content, and the degradation rate of phosphorus acid tris(2-chloropropyl) ester were measured, and the results are shown in Figure 1 .

[0042] As can be seen from Figure 1 , the microbial preparation has the best repair effect under the conditions of pH 8.0-10.0, and the degradation rate of phosphorus acid tris(2-chloropropyl) ester is higher than 78%, and the degradation rates of pH, SAR, total salt content, and phosphorus acid tris(2-chloropropyl) ester content all decrease with the increase of pH value, and when the pH value is 11, the degradation rates of pH, SAR, total salt content, and phosphorus acid tris(2-chloropropyl) ester can still reach 13.6%, 41.2%, 52.6%, and 62.5%, respectively, indicating that the microbial preparation has excellent alkali resistance. When the pH value is 8.0, the degradation rate of phosphorus acid tris(2-chloropropyl) ester is as high as 96.4%, and the degradation rate is greatly affected by the pH value. The microbial preparation not only can reduce the pH value of saline-alkali soil, but also can improve the degradation rate of phosphorus acid tris(2-chloropropyl) ester while reducing the pH value. This pH adaptive regulation mechanism enables the microbial preparation to simultaneously play the dual functions of microenvironment improvement and pollutant degradation in saline-alkali soil, thereby improving the repair effect on saline-alkali soil.

[0043] Example 5, effect of microbial preparation on saline-alkali soil at different salinity

[0044] The saline-alkali soil with total salt content of 1.0, 3.0, 5.0, 7.0, 8.0, 9.0, 10.0, 11.0 g / kg and pH 8 was selected, and the microbial preparation prepared in Example 3 was added into the soil at a mass ratio of 5%, and after 15 days of plowing, the decrease rates of pH, SAR, total salt content and phosphoric acid tris (2-chloropropyl) ester content were determined, and the results are shown in Table 2. Figure 2

[0045] It can be seen from Figure 2 that the microbial preparation has good salt tolerance, and when the total salt content is 10 g / kg, the decrease rates of pH, SAR, total salt content and phosphoric acid tris (2-chloropropyl) ester content can still reach 8.6%, 40.2%, 45.1% and 86.3% respectively, and the microbial preparation can be well applied in saline-alkali soil. When the total salt content is 1 g / kg, the degradation rate of phosphoric acid tris (2-chloropropyl) ester is as high as 96.7%, and the degradation rate decreases with the increase of total salt content, so it can be known that the salt in the soil will inhibit the process of microbial preparation degrading phosphoric acid tris (2-chloropropyl) ester, and the microbial preparation can reduce the total salt content of saline-alkali soil, which is beneficial to improve the degradation rate of phosphoric acid tris (2-chloropropyl) ester to a certain extent.

[0046] Example 6, effect of microbial preparation on actual saline-alkali land

[0047] The microbial preparation prepared in Example 3 was applied to a saline-alkali land, after plowing and watering for 3 days in spring, organic fertilizer was added and stirred, and after 15 days, the land was leveled and the microbial preparation was applied. The specific degradation results are shown in Table 4, wherein the effect is the percentage of the difference between the test point data and the blank reference point data and the blank reference point data.

[0048] Table 4 Influence of microbial preparation provided by the present application on saline-alkali soil

[0049]

[0050] It can be seen from the results in Table 4 that after the saline-alkali land is treated with the microbial preparation, the condition of the saline-alkali land can be effectively improved.

[0051] The above shows and describes the basic principles, main features and advantages of the present application. Those skilled in the art should understand that the above examples do not limit the present application in any form, and any technical solutions obtained by equivalent substitution or equivalent transformation fall within the protection scope of the present application.​

Claims

1. The application of a microbial preparation, characterized in that, Microbial agents are used to degrade tris(2-chloropropyl) phosphate pollutants in saline-alkali soils and to reduce the sodium adsorption ratio and alkalinity in saline-alkali soils, wherein the salinity of the saline-alkali soil is 1-10 g / kg and the pH is 8-11. The microorganisms in the microbial agents are a combination of the following four strains: *Bacillus marinum* with preservation number CGMCC No. 28096. Bacillus aquimaris Bacillus cereus with accession number CGMCC No. 29355 Bacillus cereus Bacillus subtilis with accession number CICC 25064 Bacillus subtilis ) and Pseudomonas with accession number CICC 22131 ( Pseudomonas sp. The ratio of viable bacteria of *Bacillus cereus*, *Bacillus subtilis*, and *Pseudomonas* is (3-5):(1-3):(5-7):(1-3); the microbial preparation is a solid microbial preparation using biochar and corn flour as carriers.

2. The application of the microbial preparation according to claim 1, characterized in that, The method for preparing the microbial preparation includes the following steps: S1. Bacillus cereus, Bacillus subtilis, and Pseudomonas aeruginosa were placed in LB medium and cultured with shaking to prepare seed culture for each strain. S2. The seed liquids of each strain were inoculated into the fermentation medium and fermented to obtain Bacillus marinum, Bacillus cereus, Bacillus subtilis and Pseudomonas aeruginosa bacterial liquids, respectively. S3. Mix the bacterial suspensions of Bacillus cereus, Bacillus subtilis, and Pseudomonas aeruginosa to obtain a compound bacterial suspension. Centrifuge to collect the bacterial sludge. Mix the bacterial sludge with biochar and corn flour, and dry to obtain a solid microbial preparation.

3. The application of the microbial preparation according to claim 2, characterized in that, In step S1, the LB culture medium consists of: 5 g yeast extract, 10 g peptone, 5 g NaCl and 1 L water, pH 6-8, culture temperature 25-35℃, and culture time 24-48 h.

4. The application of the microbial preparation according to claim 2, characterized in that, In step S2, the viable count of Bacillus marinum, Bacillus cereus, Bacillus subtilis, and Pseudomonas aeruginosa bacterial solutions is 6 × 10⁻⁶. 12 -7×10 12 CFU / mL.

5. The application of the microbial preparation according to claim 2, characterized in that, In step S3, the mass ratio of the fungal mud, biochar and corn flour is 1:(1-3):(1-2).

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