Method for repairing saline-alkali soil by bacillus subtilis with high exopolysaccharide yield

Abstract

The invention belongs to the technical field of soil remediation, and particularly relates to a method for remedying saline-alkali soil by microorganisms, which comprises the following steps: step 1, picking a preservation tube strain, inoculating into an LB liquid culture medium test tube and 5 mL / bottle, and carrying out shake culture overnight at 37 DEG C and 180 rpm; inoculating the seed solution into 250 mL of LB liquid culture solution test tube base according to the inoculum size of 0.1%, carrying out resting culture at 37 DEG C for 48 hours, centrifuging at 5000g for 10 minutes, and adding thalli into 0.1 M of CaCl2 solution. Microbial strains used in the method not only have saline-alkaline tolerance, but also have saline-alkaline land repairing capacity, due to the fact that seawater flows backwards, water and salt are migrated, salt stays on the surface of soil, and the saline-alkaline land is gradually formed by coastal surrounding soil through repeated flowing backwards and sand-sand frosting. The bacillus subtilis with high exopolysaccharide yield in the experiment can promote the generation of soil aggregates, reduce the generation of salt crystals and promote the increase of soil porosity. The EC value of the repaired soil can be reduced by 9.8%; gt; the number of aggregates of 2 mm is increased by 43.45%, and the number of aggregates of 1-2 mm is increased by 25.09%; the aggregate of 0.5 mm to 1 mm and the aggregate of 0.25 mm to 5 mm are reduced by 48.6% and 15.15% respectively; and the porosity is increased by 17.92%.

Description

Technical Field

[0001] The present invention relates to the technical field of soil remediation, and particularly to a method for remediating saline-alkali soil by Bacillus subtilis with high extracellular polysaccharide production. Background Art

[0002] Coastal saline-alkali land refers to the saline-alkalized soil areas near the coastline. Due to their special geographical locations and environmental conditions, these areas are currently facing a series of complex ecological and agricultural problems. The formation of coastal saline-alkali land is mainly affected by factors such as seawater intrusion, groundwater salinization, and unreasonable land use. For a long time, the high salinity and alkalinity in these areas have severely restricted agricultural production and the healthy development of the ecosystem. The high salinity and alkalinity of these lands are the main factors affecting plant growth. High concentrations of soluble salts such as sodium chloride and sodium sulfate in the soil can cause difficulties for plant roots to absorb water, resulting in physiological drought. Even if there is enough water in the soil, plants still show water shortage symptoms. In addition, the high pH value of saline-alkali soil affects the absorption of nutrients by plants, leading to nutrient imbalance and growth stagnation. The problem of soil structure deterioration in coastal saline-alkali land is also very prominent. High salinity can cause the dispersion and hardening of soil particles, forming a dense soil layer that hinders the penetration of water and air. This not only affects the normal growth of plant roots but also exacerbates the problem of soil erosion. Due to poor soil structure and low vegetation coverage, saline-alkali land is easily affected by wind erosion and water erosion, further exacerbating soil degradation. Water resource shortage is another severe challenge faced by coastal saline-alkali land. Groundwater salinization and the lack of fresh water resources limit the possibilities of farmland irrigation and saline-alkali land improvement. At the same time, human activities such as unreasonable irrigation methods and over-reclamation are constantly exacerbating the salinization problem. These problems not only affect agricultural production but also pose a serious threat to the stability and sustainable development of the local ecosystem. Therefore, comprehensive measures need to be taken for scientific management and ecological restoration to improve the soil quality and ecological environment of saline-alkali land and promote the sustainable development of agriculture and the ecosystem.

[0003] Bacillus subtilis shows significant potential in repairing saline-alkali land, mainly by improving soil quality and promoting plant growth through various mechanisms. First, Bacillus subtilis can secrete extracellular polysaccharides and form biofilms, improving soil structure and enhancing soil aeration and water retention. Second, this strain can synthesize plant growth hormones such as indole-3-acetic acid (IAA), promoting plant root development and increasing plant tolerance to saline-alkali environments. In addition, Bacillus subtilis neutralizes alkaline substances in the soil by secreting organic acids, reducing the soil pH value, and dissolving minerals in the soil, making them more easily absorbed by plants. Bacillus subtilis can also inhibit pathogenic microorganisms in the soil through competitive exclusion and antibiotic production, improving the soil microbial community structure and increasing the number of beneficial microorganisms. Through these comprehensive effects, Bacillus subtilis not only significantly improves the physical and chemical properties of saline-alkali soil but also enhances plant growth and productivity, providing an effective solution for the ecological restoration of saline-alkali land and the sustainable development of agriculture.

[0004] Therefore, it is necessary to provide an efficient method for microbial remediation of saline-alkali soil to solve the above technical problems. Summary of the Invention

[0005] To solve the above technical problems, the present invention provides an efficient method for microbial remediation of saline-alkali soil, which realizes efficient remediation of saline-alkali land by reducing the treatment cost and shortening the remediation cycle, and significantly improves the remediation effect.

[0006] An efficient method for microbial remediation of saline-alkali soil provided by the present invention: Step 1: Pick the strains in the preservation tube and inoculate them into a test tube of LB liquid medium, 5 mL / bottle, and culture them overnight by shaking at 37 °C and 180 rpm; inoculate the seed liquid into 250 mL of LB liquid medium according to an inoculation amount of 0.1%, and statically culture it at 37 °C for 48 h, and then centrifuge it at a rate of 5000 g / min for 10 min with a high-speed centrifuge to obtain the bacterial cells; Step 2: Prepare a 0.1 M CaCl2 solution as a soil binder to prevent the subsequent leaching of the bacterial cells, resuspend the bacterial cells in the CaCl2 solution, adjust the concentration to OD600nm = 0.6 - 0.8, and use an indoor soil column experiment to screen for efficient microbial agents; Step 3: Take the saline-alkali soil, air-dry it naturally, sieve it through a 2 mm sieve, and sterilize it by dry heat at 180 °C for 2 h. Mix the bacterial suspension and the soil in a ratio of 1:1.5, put it into a light-proof airtight container, place it in a constant temperature room, set the indoor temperature to 25 °C from 8 am to 8 pm, and set the indoor temperature to 15 °C from 8 pm to 8 am. After culturing in a cycle for 10 days, take it out and put it into a soil column device, connect a Mariotte bottle for water supply, the initial water volume in each Mariotte bottle is 2 L, and record the water level height of the Mariotte bottle; Step 4: Separate the aggregates by wet sieving method. Place the sample at the top of a 2000μm sieve set (2000, 1000, 500, 250μm), and shake it at a frequency of 30 times per minute for 10 minutes; collect the aggregates at each level, dry them in an oven at 60°C until constant weight, weigh and record them with an electronic balance (accuracy 0.001g); calculate the mean weight diameter (MWD) according to the formula: MWD = Σ(wi×xi) / Σwi, where wi is the weight of the particle size class and xi is the mean value of adjacent sieve pores; repeat the experiment three times and take the arithmetic mean to obtain the quantitative data of the aggregate stability after treatment with the bacterial solution.

[0007] Step 5: Put the soil treated with the bacterial solution into a 100 cm³ core sampler, compact it to the natural bulk density, and record the volume (V); dry it in an oven at 105°C for 24 h until constant weight, weigh the dry soil mass (m), and calculate the bulk density (ρ = m / V); according to the soil particle density (ρs, default 2.65 g / cm³), calculate the total porosity according to the formula: P(%) = [1 - (ρ / ρs)] × 100; measure it in parallel three times and take the arithmetic mean as the quantitative result of the porosity after treatment with the bacterial solution.

[0008] Preferably, the microbial inoculum is Bacillus subtilis with an effective viable cell concentration of 1×10 5 ~9×10 7 CFU / mL.

[0009] Preferably, plow deeply the saline-alkali land in the depth range of 0 - 30 cm, and then add the microbial complex inoculum to the soil, mix it evenly with the soil in the depth of 10 - 20 cm on the surface layer of the saline-alkali land, and balance the soil for 5 days.

[0010] Preferably, among the strains in the preservation tube: the Bacillus subtilis is Bacillus subtilis CGMCC No.8734 Preferably, the microbial inoculum is composed of Bacillus subtilis with an effective viable cell concentration of 1×10 5 ~9×10 7 CFU / mL.

[0011] Compared with the related technology, a method for microbial remediation of saline-alkali land soil with high extracellular polysaccharide production provided by the present invention has the following beneficial effects: The microbial strains used in the present invention not only have the ability to tolerate salinity and alkalinity, but also have the ability to promote the stress resistance and growth promotion of rice. Since it is very difficult for rice to survive in saline-alkali land and the yield is very low, the saline-alkali-tolerant rice used in this experiment. Applying this strain can improve the salinity of the soil and increase the rice yield, and the rice roots and rhizomes will loosen the soil and improve the soil air permeability, further improving the living environment for the strain. The EC value of the soil after the combined remediation of saline-alkali-tolerant rice and microorganisms can be reduced by up to 9.8%. Description of the Drawings

[0012] Figure 1 Schematic diagram of experimental data of soil EC value in Example 1 of a method for repairing saline-alkali soil with a high extracellular polysaccharide-producing microorganism provided by the present invention; Figure 2 Schematic diagram of comparison of soil aggregates in Example 2 of a method for repairing saline-alkali soil with a high extracellular polysaccharide-producing microorganism provided by the present invention; Figure 3 Schematic diagram of comparison of soil porosity in Example 2 of a method for repairing saline-alkali soil with a high extracellular polysaccharide-producing microorganism provided by the present invention. Detailed implementation manners

[0013] The technical solutions of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are some, but not all, of the embodiments of the present invention. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention.

[0014] The present invention provides a strain of Bacillus subtilis, and the Bacillus subtilis NJWGYHYH20130799 was deposited on January 17, 2014 at the General Microbiological Center of the China Committee for Culture Collection of Microorganisms, located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with the activity of survival and the deposit number of CGMCC No. 8734. Examples

[0015] A method for repairing saline-alkali soil with a high extracellular polysaccharide-producing microorganism: Step 1: Pick the strains in the preservation tube and inoculate them into a test tube of LB liquid medium, 5 mL per bottle, and shake and culture overnight at 37°C and 180 rpm; inoculate the seed liquid into a 250 mL test tube of LB liquid culture medium at an inoculation amount of 0.1%, and rest at 37°C for 48 h to prepare a strain fermentation broth.

[0016] Step 2: Centrifuge the fermentation broth in a refrigerated high-speed centrifuge at 4°C and 5000g for 10 minutes, remove the supernatant, aspirate 200 μL of 1.5 M NaCl solution to resuspend the cells, and then centrifuge at 4°C and 8000g for 10 minutes, and retain the supernatant, which is the extracellular matrix component, and freeze it in a -20°C refrigerator.

[0017] Step 3: Add 10 μL of proteinase K at 20 mg / mL to the extracellular matrix components extracted in Step 2, and react at 70 °C for ten minutes. Weigh precisely 0.1000 g of glucose dried to constant weight at 98 - 100 °C, dissolve it in water, add 0.5 mL of hydrochloric acid, and make up the volume to 100 mL in a volumetric flask. This is a 1 mg / mL glucose solution. Weigh precisely 1.0000 g and dilute it with water to 20 mL. This is a 5% phenol solution. Mix 20 μL of the separated ECM with 20 μL of 5% phenol in a 96-well plate, add 98% concentrated sulfuric acid, incubate at 25 °C for 10 minutes. Each sample is processed in triplicate. Measure the concentration at 492 nm using a Multiskan SkyHigh full-wavelength microplate reader. Glucose is used as the standard. Make a glucose standard curve to calculate the concentration of exopolysaccharide produced by the strain.

[0018] Step 4: Air-dry the soil sample and pass it through a 10-mesh sieve, then sterilize it at 180 °C for 2 hours. Weigh 200 g of the soil sample and place it in a container, spray 10 mL of the activated bacterial solution, and repeat this process 10 times to ensure that the microorganisms are evenly distributed in the soil. Finally, cover the soil surface with plastic wrap and place it in a dark place to promote the reproduction of microorganisms. In the plexiglass soil column device, sequentially pack filter paper, a 40-mesh filter screen, 5 cm of quartz sand, 35 cm of soil sample, 5 cm of quartz sand, a 40-mesh filter screen, and filter paper from bottom to top. Connect a Mariotte bottle and fill it with deionized water, open the valve and record the liquid level drop rate until it stabilizes, then close the valve and keep it for 3 days to stabilize the soil column state. Prepare simulated rainwater with a pH of 5.6, add specific amounts of NaCl, CaSO4, MgSO4, NaHCO3, and CaCO3, and place it in a sealed container for standby. Each leaching is 120 mL, record the change in the liquid level of the Mariotte bottle every 1 hour, collect the filtrate, conduct leaching every three days, and continue for 15 days to complete one experimental cycle. After the experiment, measure the EC values of the soil at heights of 10 cm, 20 cm, 30 cm, and 40 cm respectively.

[0019] After measurement, compared with the soil without adding the bacterial solution, the EC values of the soil at heights of 10 cm, 20 cm, 30 cm, and 40 cm decreased by 4.69%, 8.86%, 5.08%, and 9.8% respectively. Example

[0020] In another example, Step 1: Pick the strain from the preservation tube and inoculate it into a test tube of LB liquid medium, 5 mL per bottle, and shake it overnight at 37 °C and 180 rpm; inoculate the seed liquid into 250 mL of LB liquid medium at an inoculation amount of 0.1%, and statically culture it at 37 °C for 48 h, then centrifuge it at a rate of 5000 g / min for 10 min using a high-speed centrifuge to obtain the bacterial cells.

[0021] Step 2: Prepare 0.1M CaCl2 solution as a soil binder to prevent the subsequent leaching of bacteria, resuspend the bacteria in the CaCl2 solution, adjust the concentration to OD600nm = 0.6 - 0.8, and use indoor soil column experiments to screen for efficient microbial agents.

[0022] Step 3: Take saline-alkali soil, air-dry it naturally, sieve it through a 2mm sieve, sterilize it at 180°C in a dry heat oven for 2 hours, mix the bacterial suspension and soil in a ratio of 1:1.5, put it into a light-proof airtight container, place it in a constant temperature room, set the indoor temperature to 25°C from 8 am to 8 pm, and set the indoor temperature to 15°C from 8 pm to 8 am, and culture it cyclically for 5 days.

[0023] Step 4: Use the wet sieving method to separate aggregates. Place 100g of the sample on top of a 2000μm sieve set (2000, 1000, 500, 250μm), shake it at a frequency of 30 times per minute for 10 minutes. Collect aggregates at each level, dry them at 60°C to a constant weight, and weigh and record them with an electronic balance (accuracy 0.001g). Calculate the mean weight diameter (MWD) according to the formula: MWD = Σ(wi×xi) / Σwi, where wi is the weight of the particle size grade and xi is the average value of adjacent sieve pores. Repeat the experiment three times and take the arithmetic mean to obtain quantitative data on the stability of the aggregates after treatment with the bacterial solution.

[0024] The experimental results show that compared with the soil without the addition of the bacterial solution, the aggregates >2mm and 1 - 2mm in the experimental group increased by 43.45% and 25.09% respectively; the aggregates 0.5mm - 1mm and 0.25 - 5mm decreased by 48.6% and 15.15% respectively.

[0025] Step 5: Put the soil treated with the bacterial solution into a 100 cm³ core cutter, compact it to the natural bulk density, and record the volume (V); dry it in an oven at 105°C for 24 hours to a constant weight, weigh the dry soil mass (m), and calculate the bulk density (ρ = m / V); according to the soil particle density (ρs, default 2.65 g / cm³), calculate the total porosity according to the formula: P(%) = [1 - (ρ / ρs)] × 100; measure it in parallel three times and take the arithmetic mean as the quantitative result of the porosity after treatment with the bacterial solution.

[0026] The experimental results show that compared with the soil without the addition of the bacterial solution, the porosity of the experimental group increased by 17.92%.

[0027] Among them, the microbial agent is composed of Bacillus subtilis with an effective viable cell concentration of 1×10 5 ~9×10 7 CFU / mL.

[0028] Among them, deep plowing is carried out on the saline-alkali land in the depth range of 0-30 cm, and then a microbial complex bacterium agent is added to the soil and mixed evenly with the soil at a depth of 10-20 cm in the surface layer of the saline-alkali land, and the soil is balanced for 5 days.

[0029] Among them, in the preservation tube strain: the Bacillus subtilis is Bacillus subtilis CGMCC No.8734.

[0030] The above are only the embodiments of the present invention, and do not limit the patent scope of the present invention accordingly. Any equivalent structure or equivalent process transformation made by using the content of the specification and drawings of the present invention, or directly or indirectly applied in other related technical fields, shall be equally included in the patent protection scope of the present invention.

Claims

1. A method for microbial remediation of saline-alkali soil with high extracellular polysaccharide production, characterized in that Step 1: Pick the strains in the preservation tube and inoculate them into a test tube of LB liquid medium, 5 mL per bottle, and culture them overnight with shaking at 37 °C and 180 rpm; inoculate the seed liquid into a 50 mL test tube of LB liquid culture medium at an inoculation amount of 0.1%, and let it stand at 37 °C for 48 h to prepare the strain fermentation broth, and then take 50 mL of the fermentation broth of each strain. Step 2: Centrifuge the fermentation broth in a refrigerated high-speed centrifuge at 4 °C and 5000 g for 10 minutes, discard the supernatant, aspirate 200 μL of 1.5 M NaCl solution to resuspend the cells, and then centrifuge at 4 °C and 8000 g for 10 minutes, and retain the supernatant, which is the extracellular matrix component, and freeze it in a -20 °C refrigerator. Step 3: Add 10 μL of 20 mg / mL proteinase K to the extracellular matrix component extracted in Step 2, react at 70 °C for ten minutes. Weigh accurately 0.1000 g of glucose dried to constant weight at 98 - 100 °C, dissolve it in water, add 0.5 mL of hydrochloric acid, and make up the volume to 100 mL in a volumetric flask, which is a 1 mg / mL glucose solution. Weigh accurately 1.0000 g and dilute it with water to 20 mL, which is a 5% phenol solution. Mix 20 μL of the separated ECM with 20 μL of 5% phenol in a 96-well plate, add 98% concentrated sulfuric acid, incubate at 25 °C for 10 minutes, and perform three parallel treatments for each sample. Use a Multiskan SkyHigh full-wavelength microplate reader to measure the concentration at 492 nm. Glucose is used as the standard. Make a glucose standard curve to calculate the concentration of extracellular polysaccharide produced by the strain. Step 4: Air-dry the soil sample and pass it through a 10-mesh sieve, and sterilize it at 180 °C for 2 hours. Weigh 200 g of the soil sample and place it in a container, spray 10 mL of the activated bacterial solution, and repeat this process 10 times to ensure that the microorganisms are evenly distributed in the soil. Finally, cover the soil layer surface with plastic wrap and place it in a dark place to promote the reproduction of microorganisms. In an acrylic soil column device, filter paper, 40-mesh filter screen, 5 cm of quartz sand, 35 cm of soil sample, 5 cm of quartz sand, 40-mesh filter screen, and filter paper are filled from bottom to top in sequence. Connect a Mariotte bottle and fill it with deionized water, open the valve to record the liquid level drop rate, and close the valve after it stabilizes, and keep it for 3 days to stabilize the soil column state. Prepare simulated rainwater with a pH of 5.6, add a specific amount of NaCl, CaSO4, MgSO4, NaHCO3, and CaCO3, and load it into a sealed container for standby. Each leaching is 120 mL, record the change of the Mariotte bottle liquid level every 1 hour, collect the filtrate, and perform leaching every three days for 15 days to complete an experimental cycle. Step 5: Separate the aggregates by wet sieving method. Place the sample at the top of the 2000 μm sieve set (2000, 1000, 500, 250 μm), and shake it at a frequency of 30 times per minute for 10 min. Collect the aggregates at each level, dry them at 60 °C until constant weight, and weigh and record them with an electronic balance (accuracy 0.001 g). Calculate the mean weight diameter (MWD) according to the formula: MWD = Σ(wi×xi) / Σwi, where wi is the weight of the particle size class and xi is the mean value of adjacent sieve pores. Repeat the experiment three times and take the arithmetic mean to obtain the quantitative data of the aggregate stability after the treatment with the bacterial solution; Step 6: Put the soil treated with the bacterial solution into a 100 cm³ core sampler, compact it to the natural bulk density, and record the volume (V); dry it in an oven at 105 °C for 24 h until constant weight, weigh the dry soil mass (m), and calculate the bulk density (ρ = m / V); according to the soil particle density (ρs, default 2.65 g / cm³), calculate the total porosity according to the formula: P(%) = [1−(ρ / ρs)]×100; measure it in parallel three times and take the arithmetic mean as the quantitative result of the porosity after the treatment with the bacterial solution.

2. The method for microbial remediation of saline-alkali soil with high extracellular polysaccharide production according to claim 1, characterized in that, The microbial inoculant is composed of Bacillus subtilis with an effective viable bacteria concentration of 1×105 - 9×107 CFU / mL.

3. A method for microbial remediation of saline-alkali soil with high extracellular polysaccharide production according to claim 1, characterized in that, Deep plow the saline-alkali land in the depth range of 0 - 30 cm, then add the microbial inoculant to the soil, mix it evenly with the soil in the 10 - 20 cm depth of the saline-alkali land surface layer, and equilibrate the soil for 5 days.

4. A method for microbial remediation of saline-alkali soil with high extracellular polysaccharide production according to claim 1, characterized in that, Among the strains in the preservation tube: Bacillus subtilis is Bacillus subtilis CGMCC No.8734.

5. A method for microbial remediation of saline-alkali soil with high extracellular polysaccharide production according to claim 4, characterized in that, The microbial inoculant is composed of Bacillus subtilis with an effective viable bacteria concentration of 1×105 - 9×107 CFU / mL.

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