Microbial exopolysaccharide for reducing soil porosity, preparation method and application

Abstract

The invention relates to a microbial exopolysaccharide for reducing soil porosity, a preparation method and application. The preparation method comprises the following steps: (1) inoculating microorganisms into a culture medium A, and culturing to obtain a seed solution; inoculating the seed solution into a culture medium B, culturing to obtain fermentation liquor, centrifuging to obtain fermentation liquor supernate, and performing rotary evaporation concentration to obtain concentrated liquor; (2) mixing the concentrated solution with ethanol, and centrifuging to obtain a polysaccharide precipitate; adding water into the polysaccharide precipitate, stirring and dissolving, dialyzing, and carrying out vacuum freeze drying to obtain crude polysaccharide; and (3) preparing the crude polysaccharide into a crude polysaccharide solution, then performing chromatographic separation, gradient elution and vacuum freeze drying, dialyzing, and then performing freeze drying to obtain the microbial exopolysaccharide. The microbial exopolysaccharide is good in rheological property, high in viscosity, simple to prepare, capable of being produced on a large scale, capable of effectively reducing the porosity of media such as soil and quartz sand and capable of being applied to the field of environmental remediation.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, and in particular to a microbial extracellular polysaccharide for reducing soil porosity, a preparation method and an application thereof. Background Art

[0002] Land cultivation, plant root growth, and soil freeze-thaw cycles can lead to the formation of large pores, and pollutants will preferentially migrate to the deep soil layer through these large pores, thereby polluting groundwater. Currently, there are physical, chemical, and biological methods to reduce soil porosity. As a new environmentally friendly and sustainable method, biological methods have great application potential in improving soil properties and reducing soil porosity.

[0003] Polysaccharides are biopolymers composed of monomeric sugar units connected by glycosidic bonds. Most polysaccharides are attached to the cell surface or released into the extracellular medium, thus being defined as microbial exopolysaccharides (EPS). Polysaccharides mostly come from plants (such as starch, cellulose, pectin, etc.), algae (such as alginic acid, agar, rockweed, etc.) and microorganisms (such as bacteria, yeast, mold, etc.), but EPS produced by microorganisms has many advantages: faster production speed than plant production, more energy-saving than microalgae, and EPS produced by different microorganisms have different structures and functional effects. In recent years, scholars at home and abroad have conducted detailed studies on the genus Paenibacillus, and Paenibacillus polymyxa has attracted much attention due to its huge biotechnological potential. However, to date, the effect of microbial exopolysaccharides produced by Paenibacillus polymyxa on soil porosity has not been systematically studied. Therefore, further research and application of microbial exopolysaccharides produced by Paenibacillus polymyxa are needed. Summary of the invention

[0004] In order to solve the above technical problems, the purpose of the present invention is to provide a microbial exopolysaccharide for reducing soil porosity, a preparation method and an application. The microbial exopolysaccharide prepared by the present invention has good rheological properties and high viscosity, a simple preparation process, is suitable for large-scale production, can effectively reduce the porosity of soil, quartz sand and other media, and has great application potential in the field of environmental remediation, especially in the risk control of pollutants in soil and groundwater.

[0005] The technical solution of the present invention to solve the above technical problems is as follows:

[0006] The first object of the present invention is to provide a method for preparing microbial extracellular polysaccharides for reducing soil porosity, characterized in that the preparation method comprises the following steps:

[0007] (1) inoculating the microorganism into culture medium A, and culturing the culture medium under constant temperature and shaking to obtain a seed solution; inoculating the seed solution into culture medium B, and culturing the culture medium under constant temperature and shaking to obtain a fermentation solution, and centrifuging the fermentation solution to obtain a fermentation solution supernatant; and concentrating the fermentation solution supernatant by rotary evaporation to obtain a concentrated solution;

[0008] (2) mixing the concentrated solution with ethanol, standing at low temperature, and centrifuging to obtain a polysaccharide precipitate; adding water to the polysaccharide precipitate, stirring until the precipitate is dissolved, dialyzing for the first time, and then vacuum freeze-drying to obtain a crude polysaccharide;

[0009] (3) The crude polysaccharide is prepared into a crude polysaccharide solution, which is then subjected to chromatography separation, gradient elution, vacuum freeze drying, a second dialysis, and finally freeze drying to obtain microbial extracellular polysaccharides.

[0010] The beneficial effects of the present invention are as follows: the present invention provides a method for preparing microbial extracellular polysaccharides by fermentation. The method for preparing microbial extracellular polysaccharides of the present invention has simple process, is easy to operate, and is convenient for large-scale production.

[0011] Based on the above technical solution, the present invention can also be improved as follows.

[0012] Furthermore, the culture medium A comprises the following components: 9.0-12.0 g / L glucose, 1.0-3.0 g / L yeast extract, and 950-1100 mL deionized water; the culture medium B comprises the following components: 9.0-12.0 g / L glucose, 1.0-3.0 g / L yeast extract, and 950-1100 mL deionized water.

[0013] Furthermore, the microorganism in step (1) is Bacillus polymyxa; and the concentrated liquid is 8%-10% of the volume of the supernatant.

[0014] Furthermore, the constant temperature shaking culture of the seed liquid in step (1) is carried out at a temperature of 25-30°C and a culture time of 36-48 hours; the constant temperature shaking culture of the fermentation liquid is carried out at a temperature of 25-30°C and a culture time of 64-72 hours; and the rotary evaporation concentration is carried out at a temperature of 50-60°C.

[0015] Furthermore, the cutoff of the first dialysis is 8000-14000Da, and the time is 3-4 days; the cutoff of the second dialysis is 8000-14000Da, and the time is 2-3 days.

[0016] Furthermore, in step (3), the gradient elution is performed using a NaCl solution, and the concentrations of the NaCl solution are 0 M, 0.1 M, and 0.3 M, respectively.

[0017] The second object of the present invention is to provide a microbial exopolysaccharide for reducing soil porosity, wherein the microbial exopolysaccharide is an α-pyranose polysaccharide composed of mannose and glucose.

[0018] The beneficial effect of the present invention is as follows: the present invention provides a microbial extracellular polysaccharide for reducing soil porosity, the microbial extracellular polysaccharide has good rheological properties and high viscosity, and the microbial extracellular polysaccharide, as one of the biologically active compounds, can effectively reduce the porosity of the soil, thereby reducing the soil permeability coefficient and improving the soil's ability to block and control pollutants.

[0019] Based on the above technical solution, the present invention can also be improved as follows.

[0020] Furthermore, the molar ratio of the mannose to the glucose is 2-3:1, for example, the molar ratio of the mannose to the glucose is 2.05:1.

[0021] Furthermore, the molecular weight of the microbial exopolysaccharide is 7×10 4 ~8×10 4 Da. For example, the molecular weight of the microbial exopolysaccharide is 7.89×10 4 Da.

[0022] The third object of the present invention is to provide an application of microbial exopolysaccharide for reducing soil porosity, and to use the microbial exopolysaccharide in environmental remediation.

[0023] Furthermore, the microbial extracellular polysaccharide is used for risk control of pollutants in soil and groundwater.

[0024] The beneficial effect of the present invention is that the prepared microbial extracellular polysaccharides are innovatively used to improve the porosity of soil media. Microbial extracellular polysaccharides have good rheological properties. They can be dispersed in water to produce thickening or viscosity-increasing effects. In the soil medium, soil particles can be adhered together, thereby reducing the porosity of the soil, thereby improving the soil's barrier effect on pollutants, and can be applied to the risk management of soil and groundwater pollutants. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 These are pictures of microbial extracellular polysaccharide samples prepared in Example 1 of the present invention; wherein (a) is a picture of a physical sample of microbial extracellular polysaccharide; and (b) is a SEM picture of microbial extracellular polysaccharide.

[0026] Figure 2 Spectra of the microbial extracellular polysaccharide prepared in Example 1 of the present invention; wherein (a) is an ultraviolet spectrum of the microbial extracellular polysaccharide; and (b) is a Fourier transform infrared spectrum.

[0027] Figure 3 It is a composition analysis diagram of the microbial extracellular polysaccharide prepared in Example 1 of the present invention; wherein (a) is an ion chromatogram of 12 monosaccharide standards; and (b) is an ion chromatogram of the microbial extracellular polysaccharide.

[0028] Figure 4 The graph is a rheological property graph of the microbial extracellular polysaccharide prepared in Example 1 of the present invention; wherein (a) is the effect of different extracellular polysaccharide concentrations on its apparent viscosity; and (b) is the effect of different initial pH values ​​on the apparent viscosity of the extracellular polysaccharide.

[0029] Figure 5 It is the plexiglass column used in the water permeability coefficient experiment.

[0030] Figure 6 This is a diagram showing the effect of the microbial extracellular polysaccharide prepared in Example 1 of the present invention on the water permeability coefficient.

[0031] Figure 7 The three-dimensional pore distribution diagrams of the blank control sand column at different times; among them, (a) is the three-dimensional pore distribution diagram of the sand column at 0h; (b) is the three-dimensional pore distribution diagram of the sand column after 72h; (c) is the three-dimensional pore distribution diagram of the sand column after 144h; (d) is the three-dimensional pore distribution diagram of the sand column after 240h.

[0032] Figure 8 The three-dimensional pore distribution diagrams of the sand column with the microbial extracellular polysaccharide prepared in Example 1 added to the experimental group at different times; among them, (a) is the three-dimensional pore distribution diagram of the sand column at 0h; (b) is the three-dimensional pore distribution diagram of the sand column after 72h; (c) is the three-dimensional pore distribution diagram of the sand column after 144h; (d) is the three-dimensional pore distribution diagram of the sand column after 240h. DETAILED DESCRIPTION

[0033] The principles and features of the present invention are described below, and the examples are only used to explain the present invention and are not used to limit the scope of the present invention. If no specific technology or conditions are specified in the embodiments, the technology or conditions described in the literature in this field or the product instructions are used. If the manufacturer of the reagents or instruments used is not specified, they are all conventional products that can be purchased through regular channels.

[0034] Example 1: Preparation of microbial exopolysaccharides

[0035] (1) Preparation of seed solution: Frozen Paenibacillus polymyxa (purchased from China Industrial Microbiological Culture Collection Administration Center; culture collection number: CICC 22479) was taken out from a -80°C ultra-low temperature freezer and aseptically inoculated into a sterilized culture medium (10.0 g / L glucose, 2.0 g / L yeast extract, 1000 mL deionized water) at an inoculum volume of 1.0%, and cultured in a constant temperature shaking incubator (SHP-350Y; Shanghai Jinghong Experimental Equipment Co., Ltd.) at 30°C and 180 r / min for 48 h to obtain a seed solution;

[0036] (2) Preparation of fermentation broth: The obtained seed liquid was aseptically inoculated into a sterilized culture medium (10.0 g / L glucose, 2.0 g / L yeast extract, 1000 mL deionized water) at a volume of 5%, and cultured in a constant temperature shaking incubator (SHP-350Y; Shanghai Jinghong Experimental Equipment Co., Ltd.) at 30°C and 180 r / min for 72 h. The fermentation broth was collected and then centrifuged in a high-speed refrigerated centrifuge (TDD-5, Xiamen Guoyi Scientific Instrument Co., Ltd.) at a speed of 8000 r / min for 10 min to remove the polymyxa bacillus cells and obtain the fermentation broth supernatant;

[0037] (3) Rotary evaporation concentration: The fermentation supernatant was concentrated in a rotary evaporator (ZFY-1L; Shanghai Kexing Instrument Co., Ltd.) at 55° C. and 120 r / min, and then cooled to room temperature to obtain a concentrate, wherein the volume of the concentrate was 8-10% of the volume of the fermentation supernatant;

[0038] (4) Polysaccharide precipitation: The concentrate was mixed with 95% ethanol at a volume ratio of 1:3, allowed to stand at 8°C for 24 h, and then centrifuged at 6000 r / min for 10 min in a high-speed refrigerated centrifuge (TDD-5; Xiamen Guoyi Scientific Instrument Co., Ltd.) to obtain a polysaccharide precipitate;

[0039] (5) Dialysis purification: Add 100 mL of deionized water to the polysaccharide precipitate after centrifugation, stir until the precipitate is dissolved, and dialyze for 3 days using a dialysis bag with a cutoff of 10,000 Da. During this period, the water is changed every 6 hours. Then, the crude polysaccharide sample is obtained by freeze drying in a vacuum freeze dryer (SJIA-10N, Ningbo Shuangjia Instrument Co., Ltd.) for 24 hours.

[0040] (6) Crude product separation: The crude polysaccharide sample obtained in step (5) was dissolved in a deionized water solution to prepare a polysaccharide solution with a concentration of 8 mg / mL, and then chromatographically separated by a DEAE cellulose anion exchange column (DEAE-52, Beijing Solebow Technology Co., Ltd.), and gradient eluted with 0 M, 0.1 M and 0.3 M NaCl solutions, respectively, at a flow rate of 1 mL / min, and a tube of sugar-containing eluate was collected every 10 minutes. After vacuum freeze-drying, a microbial extracellular polysaccharide sample was obtained;

[0041] (7) Sample purification: The microbial extracellular polysaccharide sample obtained in step (6) was dissolved in deionized water to prepare a polysaccharide solution with a concentration of 8 mg / mL. The solution was dialyzed for 2 days using a dialysis bag with a cutoff of 10,000 Da and then freeze-dried to obtain a purified polysaccharide solution with a molecular weight of 7.89 × 10 4 ~8×10 4 Da microbial exopolysaccharides.

[0042] Test example

[0043] 1. Apparent performance analysis

[0044] For example Figure 1 The microbial extracellular polysaccharide prepared in Example 1 was subjected to SEM analysis. Figure 1 (b) as shown:

[0045] The microbial extracellular polysaccharide prepared in Example 1 has an ellipsoidal morphology, with particles of different sizes and a smooth surface without wrinkles. In addition to the ellipsoidal particles, a small amount of flake morphology can also be observed.

[0046] 2. Spectroscopic analysis of microbial extracellular polysaccharides

[0047] The microbial extracellular polysaccharide prepared in Example 1 was subjected to ultraviolet spectroscopy analysis and Fourier transform infrared spectroscopy analysis, and the analysis and testing methods are as follows:

[0048] 2.1 UV spectrum analysis

[0049] 2 mg of the microbial exopolysaccharide sample prepared in Example 1 was accurately weighed and dissolved in 8 mL of deionized water to prepare a 0.25 mg / mL microbial exopolysaccharide solution. The wavelength was set to 200-500 nm and ultraviolet analysis was performed using an ultraviolet spectrometer (Model 248; McPherson Inc). The results are shown in FIG. Figure 2 (a) shown.

[0050] 2.2 Fourier transform infrared spectroscopy analysis

[0051] A small amount of microbial extracellular polysaccharide sample was weighed and mixed with KBr at a ratio of 1:10 (w / w), and ground into a powder mixture in a mortar. Then, a small amount of the powder mixture was pressed into a thin sheet in a tablet press (MC-5, Miqi Instrument Equipment Co., Ltd.) using the ATR mode at 4000-500 cm -1 The wavelength was scanned by Fourier infrared spectrometer (MKS2030; MKS Company), and the results were as follows Figure 2 (b) as shown.

[0052] Depend on Figure 2 We can get:

[0053] (1) By Figure 2 (a) It can be seen that no obvious characteristic absorption peaks were found at wavelengths of 260nm and 280nm in the UV spectrum, indicating that the polysaccharide sample does not contain substances such as nucleic acids and proteins and has a high purity; while an obvious characteristic absorption peak was found at 200nm, which is consistent with the UV absorption characteristics of carbohydrates.

[0054] (2) By Figure 2 (b) It can be seen that microbial extracellular polysaccharides are at 3307 cm -1 and 2933cm -1 There are characteristic peaks of sugars at 1740cm, which are the characteristic bands of CH stretching vibration of hydroxyl (-OH) and alkyl; -1 The carbonyl ester (C=O) group present at 1457 cm -1 The stretching vibration of the carboxyl group (COO-) appears at 1379 cm, indicating the presence of glucuronic acid, indicating that the microbial extracellular polysaccharide is an acidic polysaccharide; -1 The characteristic absorption peak at 1045 cm-1 may be caused by the deformation vibration of the CH bond; the stretching vibration of CC and CO in the pyran ring and the stretching vibration of the α-glycosidic bond COC lead to the polysaccharide sample -1 A characteristic absorption peak appeared at , which indicated that the obtained microbial extracellular polysaccharide sample was an acidic polysaccharide containing pyranose residues and each sugar unit was connected by α-glycosidic bonds.

[0055] 3. Identification and analysis of monosaccharide composition of microbial extracellular polysaccharides

[0056] The monosaccharide composition of the microbial extracellular polysaccharide prepared in Example 1 was identified, and the identification steps were as follows:

[0057] (1) Monosaccharide standards and preparation: 12 monosaccharide standards were prepared into 10 mg / mL standard solutions, and each monosaccharide standard solution was further prepared with 5 mg / mL standard solution as a standard. The 12 monosaccharides are: mannose, ribose, rhamnose, glucuronic acid, galacturonic acid, N-acetyl-glucosamine, glucose, N-acetyl-galactosamine, galactose, xylose, arabinose and fucose;

[0058] (2) Preparation of microbial exopolysaccharide hydrolyzate: Weigh 2.0 mg of the microbial exopolysaccharide sample prepared in Example 1 into an ampoule, add 2.0 mL of 2.0 mol / L trifluoroacetic acid (TFA) and mix well, seal with nitrogen, and place in an oil pan at 120° C. for hydrolysis for 4 h. After cooling, add distilled water and evaporate to dryness, then wash with methanol and spin dry, repeat three times to remove residual TFA, and finally add 1 mL of deionized water to obtain 2 mg / mL of microbial exopolysaccharide hydrolyzate.

[0059] (3) Derivatization of monosaccharides: 500 μL of the standard monosaccharide mixture prepared in step (1) and the above-mentioned microbial extracellular polysaccharide hydrolyzate were taken separately, and 500 μL of 0.3 mol / L NaOH solution was added to mix well, and then 500 μL of 0.5 mol / L PMP-methanol solution was added. The mixture was placed in a 70°C water bath for 30 min, cooled, and then 500 μL of 0.3 mol / L HCl solution was added. Finally, 1 mL of chloroform was added and extracted three times. The upper water sample was taken, filtered through a 0.45 μm water membrane, and then analyzed by high performance liquid chromatography. The results are shown as follows: Figure 3 As shown, Figure 3 Conduct analysis.

[0060] The specific analysis method is as follows: the retention time of each monosaccharide absorption peak in the microbial extracellular polysaccharide is compared with the retention time of the 12 monosaccharide standard peaks to determine the monosaccharide composition of the microbial extracellular polysaccharide; according to the molar concentration and peak area of ​​the monosaccharide standard and internal standard, the molar mass of different monosaccharides in the sample is calculated, and then the molar ratio of the monosaccharides is calculated. Among them, 1 is mannose, 2 is ribose, 3 is rhamnose, 4 is glucuronic acid, 5 is galacturonic acid, 6 is N-acetyl-glucosamine, 7 is glucose, 8 is N-acetyl-galactosamine, 9 is galactose, 10 is xylose, 11 is arabinose, and 12 is fucose.

[0061] Depend on Figure 3 It can be seen that the monosaccharide peaks of microbial exopolysaccharides are mainly 1 and 7, corresponding to mannose and glucose, with a molar ratio of 2.05: 1. This indicates that microbial exopolysaccharides are an acidic heteropolysaccharide.

[0062] 4. Rheological properties analysis of microbial extracellular polysaccharides:

[0063] In this test example, the concentration and initial pH value of the microbial extracellular polysaccharide prepared in Example 1 were measured to detect its apparent viscosity to analyze its rheological properties, as shown below:

[0064] 4.1 Effect of microbial extracellular polysaccharide concentration on apparent viscosity

[0065] A certain amount of microbial extracellular polysaccharide was dispersed in deionized water at room temperature to prepare microbial extracellular polysaccharide solutions with concentrations of 0.2%, 0.4%, 0.6%, 0.8%, and 1.0% (w / w), respectively. The apparent viscosity of the microbial extracellular polysaccharide dispersion was then measured at rotation speeds of 0.5 rpm, 1.0 rpm, 2.0 rpm, 4.0 rpm, 5.0 rpm, 10.0 rpm, 20.0 rpm, 50.0 rpm, 60.0 rpm, and 100.0 rpm. The results are shown in FIG. Figure 4 (a) shown.

[0066] 4.2 Effect of initial pH value on apparent viscosity

[0067] The microbial exopolysaccharide prepared in Example 1 was first prepared into a microbial exopolysaccharide dispersion with a concentration of 0.8%. Then, the initial pH values ​​of the microbial exopolysaccharide dispersion were adjusted to 1.5, 3.5, 7.5 and 9.5 respectively using a 1.0 mol / L NaOH solution and a 1.0 mol / L HCl solution. The apparent viscosity of the microbial exopolysaccharide dispersion was measured at rotation speeds of 0.5 rpm, 1.0 rpm, 2.0 rpm, 4.0 rpm, 5.0 rpm, 10.0 rpm, 20.0 rpm, 50.0 rpm, 60.0 rpm and 100.0 rpm, respectively. The results are shown in FIG. Figure 4 (b) as shown.

[0068] Depend on Figure 4 We can get:

[0069] (1) By Figure 4 (a) It can be seen that with the increase of rotation speed, the apparent viscosity of microbial extracellular polysaccharide dispersions of different concentrations decreased significantly. At the same rotation speed, the viscosity of the dispersion gradually increased with the increase of microbial extracellular polysaccharide concentration, which indicates that the microbial extracellular polysaccharide dispersion has non-Newtonian flow behavior and exhibits shear thinning phenomenon.

[0070] (2) Changes in the initial pH value can cause ionic interactions to cause structural modifications or composition changes to cause conformational changes. Figure 4(b) It can be seen that at all pH values, the microbial extracellular polysaccharide dispersion exhibits pseudoplasticity, and both acidity (pH = 1.5-3.5) and alkalinity (pH = 7.5-9.5) affect the apparent viscosity of the dispersion. When the initial pH value is 3.5, the viscosity of the microbial extracellular polysaccharide dispersion is the highest. As the initial pH value decreases to 1.5, the viscosity of the microbial extracellular polysaccharide dispersion decreases significantly. When the initial pH value increases from 7.5 to 9.5, the viscosity of the microbial extracellular polysaccharide dispersion decreases. Under strong alkaline conditions, the hydroxyl groups on the polymer chain are ionized, destroying the intramolecular and intermolecular hydrogen bonds, weakening the hydrogen bonds, and resulting in a decrease in the viscosity of the dispersion.

[0071] 5. Analysis on the effect of microbial extracellular polysaccharides on reducing the water permeability of quartz sand medium.

[0072] The water permeability test of this test example is as follows: Figure 5 The experiment was carried out in a 30cm long, 10cm inner diameter organic glass column as shown, and the organic glass column was filled with 80-120 mesh quartz sand that was dried after acid cleaning with a height of 10cm. Three groups of experiments were set up, namely, a deionized water filling liquid seepage column, a fermentation medium filling liquid seepage column, and a microbial extracellular polysaccharide filling liquid seepage column. The deionized water, fermentation medium, and microbial extracellular polysaccharide in the three groups were filled in the column by wet method. After filling, a peristaltic pump was used to drip deionized water into the column from top to bottom at a speed of 1mL / s, and the water height in the column was kept at 20cm. At the same time, the volume of water flowing out in 5 minutes was measured, and the water permeability coefficient in different sand columns was tested at different times. The obtained structure is shown in the figure Figure 6 shown.

[0073] Depend on Figure 6 It can be seen that the water permeability coefficient of the column with microbial extracellular polysaccharide added is the lowest, followed by the column with fermentation medium added, and the worst water permeability coefficient is the blank group, and with the increase of time, the water permeability coefficients of the three sand columns gradually decrease. This shows that microbial extracellular polysaccharides can stick quartz sand particles together, block internal pores, and thus reduce its water permeability coefficient.

[0074] 6. Analysis of the effect of microbial extracellular polysaccharides on reducing the porosity of quartz sand media.

[0075] This test was conducted in a 3 cm high, 2 cm inner diameter organic glass column, the top of the glass column was open, and the bottom was evenly distributed with round holes of 4 mm in diameter. Before filling in the quartz sand, a piece of filter paper was placed at the bottom to prevent the quartz sand from flowing out.

[0076] A blank group and an experimental group were set up. The blank group was not treated in any way. The experimental group had microbial extracellular polysaccharides added to the organic glass column. The blank group and the experimental group were cured at 25°C and then cured for different time periods (0h, 72h, 144h and 240h). X-ray computed tomography (X-CT) was then used to scan the organic glass columns of the blank group and the experimental group at different curing times, and the porosity was analyzed using VG Studio software. The obtained structure is shown in the figure below. Figure 7 , 8 shown.

[0077] Depend on Figure 7 , Figure 8 It can be seen that the porosity of the sand column gradually decreases with the increase of time. After 240 hours, the porosity of the sand column in the blank group only decreased by 12.52%, while the porosity of the sand column with microbial extracellular polysaccharides added decreased by 68.20% after 240 hours. Microbial extracellular polysaccharides adhere to the surface of the medium particles, and their high viscosity can stick the medium particles together, thereby reducing the porosity between the medium particles.

[0078] In summary, the microbial extracellular polysaccharide prepared by the present invention is a high molecular weight heteropolysaccharide composed of mannose and glucose, has good rheological properties, high viscosity, simple preparation process, suitable for large-scale production, and can effectively reduce the porosity of media such as soil and quartz sand. It has great application potential in the field of environmental remediation, especially in the risk control of pollutants in soil and groundwater.

[0079] Although the embodiments of the present invention have been shown and described above, it is to be understood that the above embodiments are exemplary and are not to be construed as limitations of the present invention. A person skilled in the art may change, modify, replace and vary the above embodiments within the scope of the present invention.

Claims

1. A method for preparing microbial extracellular polysaccharides for reducing soil porosity, characterized in that: The preparation method comprises the following steps: (1) inoculating the microorganism into culture medium A, and culturing the culture medium under constant temperature and shaking to obtain a seed solution; inoculating the seed solution into culture medium B, and culturing the culture medium under constant temperature and shaking to obtain a fermentation solution, and centrifuging the fermentation solution to obtain a fermentation solution supernatant; and concentrating the fermentation solution supernatant by rotary evaporation to obtain a concentrated solution; (2) mixing the concentrated solution with ethanol, standing at low temperature, and centrifuging to obtain a polysaccharide precipitate; adding water to the polysaccharide precipitate, stirring until the precipitate is dissolved, dialyzing for the first time, and then vacuum freeze-drying to obtain a crude polysaccharide; (3) The crude polysaccharide is prepared into a crude polysaccharide solution, which is then subjected to chromatography separation, gradient elution, vacuum freeze drying, a second dialysis, and finally freeze drying to obtain microbial extracellular polysaccharides.

2. The method for preparing microbial extracellular polysaccharides for reducing soil porosity according to claim 1, characterized in that: The culture medium A comprises the following components: 9.0-12.0 g / L glucose, 1.0-3.0 g / L yeast extract, and 950-1100 mL deionized water; the culture medium B comprises the following components: 9.0-12.0 g / L glucose, 1.0-3.0 g / L yeast extract, and 950-1100 mL deionized water.

3. The method for preparing microbial exopolysaccharide for reducing soil porosity according to claim 1, characterized in that: The microorganism in step (1) is Bacillus polymyxa; and the concentrated liquid is 8%-10% of the volume of the supernatant.

4. The method for preparing microbial exopolysaccharide for reducing soil porosity according to claim 1, characterized in that: The constant temperature shaking culture of the seed liquid in step (1) is 25-30°C for 36-48h; the constant temperature shaking culture of the fermentation liquid is 25-30°C for 64-72h; and the rotary evaporation concentration is performed at 50-60°C.

5. The method for preparing microbial exopolysaccharide for reducing soil porosity according to claim 1, characterized in that: The cutoff of the first dialysis is 8000-14000Da, and the time is 3-4 days; the cutoff of the second dialysis is 8000-14000Da, and the time is 2-3 days.

6. The method for preparing microbial extracellular polysaccharides for reducing soil porosity according to claim 1, characterized in that: The gradient elution in step (3) is performed using a NaCl solution, and the concentrations of the NaCl solution are 0 M, 0.1 M, and 0.3 M, respectively.

7. A microbial exopolysaccharide for reducing soil porosity, characterized in that: The microbial extracellular polysaccharide prepared by the preparation method according to any one of claims 1 to 6 is an α-pyranose polysaccharide composed of mannose and glucose.

8. The microbial exopolysaccharide for reducing soil porosity according to claim 7, characterized in that: The molar ratio of the mannose to the glucose is 2 to 3:

1.

9. The microbial exopolysaccharide for reducing soil porosity according to claim 8, characterized in that: The molecular weight of the microbial exopolysaccharide is 7×10 4 ~8×10 4 Da.

10. An application of microbial extracellular polysaccharide for reducing soil porosity, characterized in that: The microbial extracellular polysaccharide according to any one of claims 7 to 9 is used in environmental remediation.

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