Extracellular polysaccharide, its preparation method and application

Abstract

The present application belongs to the field of microbial fermentation and flocculation technology, and particularly relates to an extracellular polysaccharide and a preparation method and application thereof. The present application provides an extracellular polysaccharide generated by Paenibacillus mucilaginosus, which is a heteropolysaccharide with high branching; has good viscosity, hydrophilic and hydrophobic regulation ability and typical pseudoplastic fluid behavior, the viscosity linearly increases with the concentration, the contact angle increases with the increase of the concentration, and shows excellent interfacial activity and rheological regulation potential. The results of examples show that the extracellular polysaccharide PM-PS shows significant flocculation effect on various minerals (montmorillonite and kaolin), and can be used as a high-efficiency and environmentally-friendly biological flocculant.

Description

Technical Field

[0001] This invention belongs to the field of microbial fermentation and flocculation technology, specifically relating to an extracellular polysaccharide, its preparation method, and its application. Background Technology

[0002] With the continued acceleration of global industrialization, the discharge of industrial wastewater containing high concentrations of suspended solids (SS) is increasing year by year, seriously disrupting the ecological balance of aquatic bodies and posing a potential threat to human health. Among various wastewater treatment technologies, flocculation treatment is widely used in water environment management and primary wastewater purification due to its advantages such as simple operation, low treatment cost, and excellent pollutant removal efficiency.

[0003] Currently, the mainstream flocculants in the water treatment field are mainly inorganic flocculants and synthetic organic polymer flocculants. Although these traditional chemical flocculants have stable flocculation effects, their practical applications have significant limitations. For example, synthetic organic polymer flocculants (such as polyacrylamide) have poor biodegradability, and long-term residues in the environment can easily cause secondary pollution. Their residual monomers also pose safety risks such as neurotoxicity and carcinogenicity. Aluminum salt inorganic flocculants (such as polyaluminum chloride), with long-term use, easily accumulate metal ions, and prolonged exposure can induce health risks such as Alzheimer's disease. Therefore, developing efficient, safe, and biodegradable green alternatives has become an urgent need in the field of environmental engineering. Summary of the Invention

[0004] The purpose of this invention is to provide an extracellular polysaccharide, its preparation method and application. The extracellular polysaccharide (PM-PS) exhibits significant flocculation effects on a variety of minerals and can be used as a highly efficient and environmentally friendly bioflocculator.

[0005] This invention provides an extracellular polysaccharide, the main chain structure of which is →3)-β-D-Glcp-(1→3)-α-D-Manp-(1→4)-β-D-Galp-(1→4)-α-D-Manp (E)→; The α-D-Manp residues of the main chain are connected to an α-L-Fucp branch at position O-4; the β-D-Galp residues of the main chain are connected to a β-D-Galp branch at position O-4 and a terminal α-D-Manp branch at position O-6.

[0006] As a preferred embodiment, the extracellular polysaccharide has an average molecular weight of 2.5 × 10⁻⁶. 4 Da; The molar ratio of mannose, glucose, galactose and fucose in the extracellular polysaccharide is 28.81:27.18:22.44:9.48.

[0007] This invention also provides a method for preparing the extracellular polysaccharide described above, comprising the following steps: Bacillus subtilis (peptone-like spores) Paenibacillus mucilaginosus The fermentation supernatant of Z1 was subjected to alcohol precipitation to obtain crude polysaccharide; the crude polysaccharide was deproteinized to obtain deproteinized crude polysaccharide; The deproteinized crude polysaccharide was separated by a DEAE-52 cellulose column to obtain the PM-PS1 fraction; the eluent for the DEAE-52 cellulose column was water. The PM-PS1 component was purified using a Sephadex G-200 glucose gel column to obtain the extracellular polysaccharide; the eluent for the Sephadex G-200 glucose gel column was water. The peptone-like Bacillus Z1 is deposited at the China General Microbiological Culture Collection Center, with accession number CGMCC No. 39217.

[0008] As a preferred embodiment, after obtaining the extracellular polysaccharide, the process further includes: dialysis purification.

[0009] The present invention also provides the application of the extracellular polysaccharide described in the above-described scheme or the extracellular polysaccharide prepared by the above-described preparation method in flocculants.

[0010] The present invention also provides a flocculant comprising the extracellular polysaccharide described in the above scheme or the extracellular polysaccharide prepared by the above preparation method.

[0011] As a preferred embodiment, the flocculant also contains metal cations.

[0012] As a preferred embodiment, the concentration of the metal cation is 0.1~1 mmol / L; the metal cation includes Fe. 3+ and / or Al 3+ .

[0013] The present invention also provides the application of the extracellular polysaccharides described in the above-described scheme or the extracellular polysaccharides prepared by the above-described preparation method in wastewater treatment and / or soil improvement.

[0014] As a preferred option, the soil includes saline-alkali soil.

[0015] Beneficial Effects: This invention provides an extracellular polysaccharide with a main chain structure of →3)-β-D-Glcp-(1→3)-α-D-Manp-(1→4)-β-D-Galp-(1→4)-α-D-Manp (E)→; α-L-Fucp branches are attached to the O-4 positions of the α-D-Manp residues in the main chain; β-D-Galp branches are attached to the O-4 positions of the β-D-Galp residues in the main chain, and terminal α-D-Manp branches are attached to the O-6 positions. The extracellular polysaccharide (denoted as PM-PS) of this invention is generated by *Bacillus colloidea* and is a highly branched heteropolysaccharide; it exhibits good viscosity, hydrophilicity / hydrophobicity regulation ability, and typical pseudoplastic fluid behavior. Its viscosity increases linearly with concentration, and its contact angle increases with increasing concentration, demonstrating excellent interfacial activity and rheological regulation potential. The results of the examples show that the extracellular polysaccharide PM-PS exhibits significant flocculation effects on a variety of minerals (such as montmorillonite and kaolinite), and can be used as a highly efficient and environmentally friendly bioflocculator. Attached Figure Description

[0016] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the embodiments will be briefly described below.

[0017] Figure 1 The figures show the growth curves and polysaccharide yield curves of Bacillus colloidis; the left figure is the growth curve, and the right figure is the polysaccharide yield curve. Figure 2 The flowchart shows the extraction process of PM-PS, an extracellular polysaccharide from Bacillus colloidis. Figure 3 Elution curves of PM-PS, an extracellular polysaccharide from Bacillus colloidis; the left figure shows the elution curve of DEAE-52 cellulose column; the right figure shows the elution curve of glucose gel Sephadex G-200 column. Figure 4 The HPGPC chromatogram of extracellular polysaccharide PM-PS from Bacillus colloidis; Figure 5 Infrared spectrum of PM-PS, an extracellular polysaccharide of Bacillus colloidis; Figure 6 The liquid chromatogram of PM-PS, an extracellular polysaccharide of Bacillus colloidis; Figure 7 The image shows the one-dimensional NMR spectrum of PM-PS, an extracellular polysaccharide from Bacillus colloidis; where a is the proton NMR spectrum and b is the carbon NMR spectrum. Figure 8 Two-dimensional NMR spectra of extracellular polysaccharide PM-PS from Bacillus colloidis; where a is TOCSY; b is COSY; c is HSQC; d is HMBC; e is NOESY; and f is DEPT-135. Figure 9 The viscosity and contact angle of PM-PS, an extracellular polysaccharide of Bacillus subtilis; Figure 10 Rheological properties of extracellular polysaccharide PM-PS from Bacillus colloidis; where a~b are the apparent viscosities of polysaccharide aqueous solutions at different concentrations; c~d are the shear stress and shear rate of polysaccharide at different concentrations; e is the viscosity change of 2 g / L polysaccharide at different temperatures; f is the viscosity change of 4 g / L polysaccharide at different temperatures; Figure 11 The flocculation ability of peptone-like Bacillus extracellular polysaccharide PM-PS for different minerals; Figure 12 The effect of Peptone-like Bacillus extracellular polysaccharide PM-PS on mineral particle size and fractal dimension; the left figure shows mineral particle size; the right figure shows fractal dimension. Figure 13 The results show the flocculation effect of PM-PS, an extracellular polysaccharide of Bacillus subtilis, on actual wastewater. Figure 14 The pH results of saline-alkali soil in different treatment groups in Example 8; Figure 15 The electrical conductivity results of saline-alkali soil in different treatment groups in Example 8; Figure 16 The results of alkalinity of saline-alkali soil in different treatment groups in Example 8; Figure 17 The results show the composition of water-stable aggregates in saline-alkali soils from different treatment groups in Example 8.

[0018] Biological Preservation Instructions Bacillus colloidis Z1, classified and named Paenibacillus mucilaginosus It was deposited on June 12, 2026 at the China General Microbiological Culture Collection Center, located at No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC No. 39217. Detailed Implementation

[0019] This invention provides an extracellular polysaccharide, wherein the main chain structure of the extracellular polysaccharide is →3)-β-D-Glcp-(1→3)-α-D-Manp-(1→4)-β-D-Galp-(1→4)-α-D-Manp (E)→; α-L-Fucp side chains are attached to the O-4 positions of the α-D-Manp residues of the main chain; β-D-Galp side chains are attached to the O-4 positions of the β-D-Galp residues of the main chain, and terminal α-D-Manp side chains are attached to the O-6 positions.

[0020] The extracellular polysaccharide (denoted as PM-PS) of this invention is produced by the metabolism of Bacillus collodioni Z1 and is a heteropolysaccharide mainly composed of mannose, glucose, and galactose. The Bacillus collodioni Z1 is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 39217. As one embodiment, the average molecular weight of the extracellular polysaccharide is 2.5 × 10⁻⁶. 4 In one embodiment, the molar ratio of mannose, glucose, galactose, and fucose in the extracellular polysaccharide is 28.81:27.18:22.44:9.48. In this invention, the extracellular polysaccharide has the following backbone: This invention also provides a method for preparing the extracellular polysaccharide described above, comprising the following steps: Bacillus subtilis (peptone-like spores) Paenibacillus mucilaginosus The fermentation supernatant of Z1 was subjected to alcohol precipitation to obtain crude polysaccharide; the crude polysaccharide was deproteinized to obtain deproteinized crude polysaccharide; The deproteinized crude polysaccharide was separated by a DEAE-52 cellulose column to obtain the PM-PS1 fraction; the eluent for the DEAE-52 cellulose column was water. The PM-PS1 component was purified using a Sephadex G-200 glucose gel column to obtain the extracellular polysaccharide; the eluent for the Sephadex G-200 glucose gel column was water. The peptone-like Bacillus Z1 is deposited at the China General Microbiological Culture Collection Center, with accession number CGMCC No. 39217.

[0021] The present invention does not limit the source of the raw materials involved, and all of them can be obtained through conventional commercial methods in the field.

[0022] This invention involves fermenting *Bacillus colloides* Z1 to obtain a fermentation broth. The *Bacillus colloides* Z1 described in this invention is deposited at the China General Microbiological Culture Collection Center (CGMCC), with accession number CGMCC No. 39217. In one embodiment, the inoculum amount of *Bacillus colloides* Z1 is 10% (v / v); in another embodiment, the fermentation temperature is 28°C; and the fermentation time is 5 days. The suitable fermentation parameters of this invention are beneficial for increasing the yield of extracellular polysaccharides.

[0023] After obtaining the fermentation broth, the present invention centrifuges the fermentation broth to obtain a fermentation supernatant. As one embodiment, the centrifugation speed is 10000 r / min; the centrifugation time is 5 min. The centrifugation of the present invention can remove bacterial cells, obtaining a supernatant containing extracellular polysaccharides.

[0024] After obtaining the fermentation supernatant, the present invention performs alcohol precipitation on the fermentation supernatant to obtain crude polysaccharide. As one embodiment, the alcohol precipitation includes: adding anhydrous ethanol to the fermentation supernatant until the volume concentration of anhydrous ethanol is 80%. As one embodiment, the alcohol precipitation temperature is 4°C. As one embodiment, after adding anhydrous ethanol, the process further includes: standing. As one embodiment, the standing time is 12-24 h. In a specific embodiment of the present invention, the standing time can be any value within 12-24 h, for example, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 h. As one embodiment, after alcohol precipitation, the process further includes: centrifugation to discard the supernatant, obtaining crude polysaccharide precipitate; the centrifugation speed is 7000 r / min; the centrifugation time is 5 min.

[0025] This invention removes proteins from the crude polysaccharide to obtain deproteinized crude polysaccharide. As one embodiment, the deproteinization method includes the Sevage method. As one embodiment, the deproteinization is performed 3 to 6 times, for example, 3, 4, 5, or 6 times. This invention, by repeating the deproteinization steps, can thoroughly remove protein impurities from the crude polysaccharide. As one embodiment, the Sevage method includes: mixing the crude polysaccharide solution with Sevage reagent, shaking, allowing it to stand, centrifuging, and collecting the supernatant. As one embodiment, the concentration of the crude polysaccharide solution is 20 mg / mL.

[0026] This invention involves mixing the crude polysaccharide solution with Sevage reagent to obtain a mixture; in one embodiment, the volume ratio of the crude polysaccharide solution to Sevage reagent is 1:1. In another embodiment, the mixing method includes shaking; the shaking time is 20-30 min; for example, it can be 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 min. The mixture is then centrifuged, and the supernatant is collected to obtain deproteinized crude polysaccharide.

[0027] After obtaining the deproteinized crude polysaccharide, the present invention separates the deproteinized crude polysaccharide using a DEAE-52 cellulose column to obtain the PM-PS1 fraction; the eluent for the DEAE-52 cellulose column is water. As one embodiment, the water is double-distilled water. In a specific embodiment of the present invention, gradient elution is performed using NaCl solution to sequentially obtain PM-PS1, PM-PS2, PM-PS3, and PM-PS4 fractions; during gradient elution, the eluents are 0, 0.05, 0.1, 0.15, and 0.2 mol / L NaCl solutions, respectively, where 0 mol / L NaCl solution is equivalent to water. When performing gradient elution using NaCl solution, the eluted fraction from the 0 mol / L NaCl solution is collected, thus obtaining the PM-PS1 fraction.

[0028] After obtaining the PM-PS1 component, the present invention purifies the PM-PS1 component by glucose gel Sephadex G-200 column to obtain the extracellular polysaccharide; the eluent of the glucose gel Sephadex G-200 column is water.

[0029] In one embodiment, after obtaining the extracellular polysaccharide, the method further includes dialysis purification. In one embodiment, the dialysis purification includes: placing the extracellular polysaccharide into a dialysis bag for dialysis; in one embodiment, the molecular weight cutoff (MWCO) of the dialysis bag is 8000-14000 Da; and the dialysis time is 48 h. This invention uses a dialysis bag with a MWCO of 8000-14000 Da for dialysis, which can effectively remove small molecule impurities such as salts and monosaccharides from the system, while effectively retaining the extracellular polysaccharide.

[0030] This invention also provides the application of the extracellular polysaccharides described in the above-described scheme or prepared by the above-described method in flocculants. For example, they can be used directly as flocculants or as one of the components in the preparation of flocculants. The extracellular polysaccharides of this invention have good viscosity, hydrophilicity-hydrophobicity regulation ability, and typical pseudoplastic fluid behavior. The viscosity increases linearly with concentration, and the contact angle increases with increasing concentration, exhibiting excellent interfacial activity and rheological regulation potential. The results of the examples show that the extracellular polysaccharide PM-PS exhibits significant flocculation effects on various minerals (montmorillonite and kaolinite), and can be used as a highly efficient and environmentally friendly bioflocculant.

[0031] The present invention also provides a flocculant comprising the extracellular polysaccharide described in the above scheme or the extracellular polysaccharide prepared by the above preparation method.

[0032] In one embodiment, the flocculant further contains a metal cation. In one embodiment, the concentration of the metal cation is 0.5 mmol / L; the metal cation includes Fe.3+ and / or Al 3+ In one embodiment, the metal cation is added in the form of a metal salt. The present invention does not limit the type of metal salt; in one specific embodiment, the metal salt includes FeCl3·6H2O and / or AlCl3·6H2O. After the addition of metal cations to the flocculant, a strong synergistic effect exists between the extracellular polysaccharide (PM-PS) and the high-valence cation. This can effectively overcome the electrostatic repulsion between clay particles through enhanced charge neutralization and bridging, thereby significantly optimizing flocculation performance.

[0033] This invention also provides the application of the extracellular polysaccharides described in the above-described scheme or prepared by the above-described preparation method in wastewater treatment and / or soil improvement. The extracellular polysaccharides of this invention exhibit significant flocculation effects on various minerals, and within a suitable range, can promote the formation of large flocs with dense structure and high stability for wastewater treatment. This invention does not limit the type of wastewater, such as fly ash wastewater, coal washing wastewater, tailings wastewater, and mineral processing wastewater. As one embodiment, the soil includes saline-alkali soil. As one embodiment, the soil improvement includes one of the following: (1) reducing the pH of saline-alkali soil; (2) reducing the electrical conductivity of saline-alkali soil; (3) reducing the alkalinity of saline-alkali soil; (4) promoting the formation of medium-sized water-stable aggregates; wherein the medium particle size is 0.053~0.25 mm. This invention utilizes the extracellular polysaccharide applied to saline-alkali soil, which can effectively reduce the pH of saline-alkali soil, alleviate the degree of soil alkalization, effectively reduce the electrical conductivity of saline-alkali soil, reduce the accumulation of soluble salts in the soil, and has a significant desalination and improvement effect on saline-alkali soil. At the same time, it can also reduce the degree of alkalinity of saline-alkali soil, promote the formation of medium-sized (0.053~0.25 mm) water-stable aggregates, improve the water-stable structure of soil, reduce the proportion of fine particles, and thus improve the structural stability of saline-alkali soil.

[0034] To further illustrate the present invention, the technical solutions provided by the present invention will be described in detail below with reference to the accompanying drawings and embodiments, but these should not be construed as limiting the scope of protection of the present invention.

[0035] The pharmaceuticals and reagents used in this invention: Potassium feldspar, montmorillonite, and other soil minerals were purchased from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). DEAE cellulose DE-52 was purchased from Yingxin Laboratory Equipment Co., Ltd. (Shanghai, China). Dextran G-200 was purchased from Aladdin Biochemical Technology Co., Ltd. (Shanghai, China). All analytical grade chemical reagents used in the experiment were purchased from Comio Chemical Reagent Co., Ltd. (Tianjin, China). The actual saline-alkali soil used was taken from Zhaoyuan County, Daqing City, Heilongjiang Province (125.08′E, 45.52′N).

[0036] Example 1: Growth curves and glycogen production curves of Bacillus colloidis Bacillus subtilis Z1 (CGMCC No. 39217) was inoculated at a 10% (v / v) inoculation rate into sterile seed culture medium (1 L containing: sucrose 10.00 g / L, urea 0.1 g / L, dipotassium hydrogen phosphate 2.00 g / L, magnesium sulfate 0.500 g / L, illite powder 1.00 g / L, calcium carbonate 1.00 g / L, sodium chloride 0.200 g / L, ferric chloride 0.005 g / L). The medium was incubated at 35℃ and 170 r / min for 24 h in a constant temperature shaker. After reaching the logarithmic growth phase, it was inoculated at a 10% (v / v) inoculation rate into fermentation culture medium (1 L containing: sugar 40.00 g / L, urea 0.4 g / L, dipotassium hydrogen phosphate 2.00 g / L, magnesium sulfate 0.500 g / L, illite powder 1.00 g / L). (Calcium carbonate 1.00 g / L, sodium chloride 0.200 g / L, ferric chloride 0.005 g / L), continue shaking culture.

[0037] Determination of the growth curve of *Bacillus colloidis*: 5 mL of seed fermentation broth was inoculated into 200 mL of sterile growth medium (1 L of medium contained: 10.00 g / L sucrose, 2.00 g / L dipotassium hydrogen phosphate, 0.500 g / L magnesium sulfate, 1.00 g / L illite powder, 0.200 g / L sodium chloride, and 0.005 g / L ferric chloride). Bacterial samples were collected at 0 h, 2 h, 4 h, 6 h, 8 h, 12 h, 24 h, 36 h, 48 h, 72 h, and 96 h after culture. The absorbance was measured at 600 nm, and the growth curve was plotted. The results are shown below. Figure 1 As shown in the middle left figure.

[0038] Determination of polysaccharide production curve of Bacillus subtilis: Fermentation broth from the same growth stage as the target bacteria was used for polysaccharide determination. The polysaccharide fermentation broth was centrifuged at 10,000 rpm for 5 min to remove bacterial cells, and the supernatant was collected. Three volumes of anhydrous ethanol were added to the supernatant, mixed well, and incubated overnight at 4°C for alcohol precipitation. Subsequently, the mixture was centrifuged (7,000 rpm, 5 min), the precipitate was collected, and freeze-dried to obtain crude extracellular polysaccharide (EPS) samples. 100 mg of the crude EPS sample was dissolved in 10 mL of deionized water, and the total sugar content was determined using the phenol-sulfuric acid method. The results are as follows: Figure 1 As shown in the middle right figure.

[0039] The results showed that the growth rate of *Bacillus colloidea* was relatively slow during the 0-12 h period, presumably a lag phase, representing the bacteria's adaptation to the new environment. From 12 to 48 h, the bacteria were in the logarithmic growth phase. After 48 h, the absorbance value stabilized, indicating that the bacteria had entered a stationary phase. Extracellular polysaccharide production showed a slow overall increase from 0 to 12 h, followed by a significant increase after 12 h, gradually stabilizing after 48 h, reaching a peak of 17.80 mg / ml. Overall, the polysaccharide yield followed the same growth trend as the bacteria.

[0040] Example 2: Extraction and purification of polysaccharides from Bacillus colloidis In this embodiment, high-purity Bacillus colloidis extracellular polysaccharide (PM-PS) was isolated from the fermentation broth of Bacillus colloidis. A schematic diagram of the polysaccharide extraction and purification method is shown below. Figure 2 As shown, the specific method is as follows: (1) Obtaining crude polysaccharide by alcohol precipitation: The fermentation broth of *Bacillus colloidea* cultured for 5 days in Example 1 was centrifuged (10000 r / min, 5 min) to remove bacterial cells. The supernatant was collected, and anhydrous ethanol was slowly added at 4°C until the final ethanol volume fraction reached 80%. After mixing, the mixture was allowed to stand at 4°C for 12–24 h for ethanol precipitation. After precipitation, the system was centrifuged (7000 r / min, 5 min), the precipitate was collected, and the crude polysaccharide was obtained by freeze-drying.

[0041] (2) Sevage deproteinization The crude polysaccharide precipitate obtained in step (1) was dissolved in distilled water to prepare a polysaccharide solution with a mass concentration of 20 mg / mL. The polysaccharide solution was mixed with Sevage reagent (chloroform: n-butanol = 4:1, v / v) at a volume ratio of 1:1, and the mixture was vigorously shaken for 20-30 min. The supernatant was then collected by centrifugation. The supernatant was then mixed with Sevage reagent again, and the process of mixing the polysaccharide solution with Sevage reagent and centrifuging to collect the supernatant was repeated 3-5 times until no protein appeared, in order to fully remove protein impurities. The deproteinized polysaccharide solution was filtered through a 0.22 μm filter membrane to obtain deproteinized crude polysaccharide, which was then freeze-dried for use in subsequent DEAE-cellulose chromatography.

[0042] (3) DEAE-52 cellulose column purification ① DEAE-52 cellulose pretreatment: The DEAE-52 cellulose exchange resin was soaked in double-distilled water at room temperature for 24 h to allow it to fully swell. Afterward, it was repeatedly washed with double-distilled water to remove suspended cellulose particles. Then, it was soaked in a 0.5 mol / L NaOH solution for 30 min, slowly stirred to allow sedimentation, and then dried under vacuum. It was then washed with double-distilled water until neutral. Next, it was soaked in a 0.5 mol / L HCl solution for 30 min, slowly stirred to allow sedimentation, and then dried under vacuum. This process was repeated several times.

[0043] ② Column Packing: Add an appropriate amount of double-distilled water to the neutralized DEAE-52 cellulose and stir slowly and gently to avoid foaming. Degas the cellulose using an ultrasonic oscillator. Slowly add the completely degassed DEAE-52 cellulose along a glass rod into a 2.6 cm × 30 cm chromatography column. Gently tap the column wall during packing to ensure the packing material settles tightly. After packing, allow to stand at room temperature for 24 hours for later use.

[0044] ③ Sample loading: A peristaltic pump was used for constant flow sample loading. 2.0 g of crude polysaccharide was dissolved in 20 mL of double-distilled water, and the supernatant was collected after centrifugation as the loading solution. Elution was performed sequentially with different concentrations of NaCl solution (0, 0.05, 0.1, 0.15, and 0.2 mol / L) to obtain four fractions, designated PM-PS1, PM-PS2, PM-PS3, and PM-PS4. During elution, the flow rate of the peristaltic pump was controlled at 0.4 mL / min, and 10 mL of eluent was collected from each tube. The eluent was monitored using the phenol-sulfuric acid method until no more polysaccharide was detected. Elution curves were plotted with tube number on the x-axis and absorbance value on the y-axis. The results are shown below. Figure 3 As shown in the left-middle figure, after separation by anion exchange DEAE-cellulose chromatography column, the crude polysaccharide was separated into four fractions, designated PM-PS1, PM-PS2, PM-PS3, and PM-PS4. PM-PS1 was the main elution peak, appearing approximately in tubes 10-20 (10 mL / tube), corresponding to elution conditions of 0 mol / L NaCl (equivalent to water), indicating that this fraction was the unadsorbed major polysaccharide component.

[0045] The eluent from the first elution peak was combined, dialyzed with double-distilled water, and concentrated under reduced pressure for further purification using a Sephadex G-200 column.

[0046] (4) Sephadex G-200 column purification ①Pretreatment of Sephadex G-200: Soak Sephadex G-200 in double-distilled water overnight to allow it to fully swell, and then wash it repeatedly with double-distilled water to remove cellulose suspended particles.

[0047] ② Column Packing: Add an appropriate amount of double-distilled water to the swollen Sephadex G-200 and stir gently and slowly to avoid foaming. Degas the material using an ultrasonic oscillator. Add the completely degassed Sephadex G-200 to a 2.6 cm × 30 cm chromatography column in one go along a glass rod. Gently tap the column wall during packing to ensure the packing material settles tightly. After packing, allow to stand at room temperature for 48 hours for later use.

[0048] ③ Sample loading: The polysaccharide PM-PS1 fraction obtained after purification using a DEAE-52 cellulose column was purified using a Sephadex G-200 elution system. Eluting was performed with water at a constant flow rate of 0.4 mL / min using a peristaltic pump. The collected eluent was analyzed for absorbance using the phenol-sulfuric acid method until no more polysaccharides were detected. An elution curve was plotted with tube number on the x-axis and absorbance value on the y-axis. The results are shown below. Figure 3 As shown in the middle right figure, the curve exhibits a single absorption peak, indicating that the polysaccharide has good homogeneity after purification. The eluents were combined, dialyzed against double-distilled water, concentrated under reduced pressure, and freeze-dried to obtain the pure polysaccharide fraction.

[0049] (5) Dialysis purification The pure polysaccharide component from step (4) was dissolved in water to obtain a polysaccharide solution. This polysaccharide solution was placed in a dialysis bag (molecular weight cutoff (MWCO) of 8000~14000 Da) and dialyzed with deionized water at 4℃ for 48 h to obtain the dialyzed polysaccharide solution. The dialysate was replaced every 8 h during dialysis. Gel permeation chromatography determined the average molecular weight of the obtained polysaccharide to be approximately 2.5016 × 10⁻⁶. 4 Da.

[0050] (6) Reduced pressure concentration The dialysis polysaccharide solution was concentrated under reduced pressure using a rotary evaporator at a water bath temperature of 40-50℃ and a vacuum degree of -0.08 to -0.095 MPa until the volume was reduced to 1 / 5 to 1 / 10 of the original volume, thus obtaining a concentrated solution. This improved the subsequent freeze-drying efficiency and prevented polysaccharide degradation caused by high temperature.

[0051] (7) Freeze-drying The concentrate was pre-frozen at -80℃ for more than 12 hours, and then freeze-dried in a freeze dryer with the cold trap temperature set at -50~-60℃, vacuum degree ≤10 Pa, and drying time 24~48 hours until the sample was completely dried, finally obtaining high-purity extracellular polysaccharide (PM-PS) in white or off-white powder form.

[0052] Example 3 Structural characteristics of purified extracellular polysaccharide (PM-PS) (1) Homogeneity and molecular weight determination The homogeneity of PM-PS was determined using a high-performance gel permeation chromatography (HPGPC, Shimadzu CTO20A, Japan), and the molecular weight of PM-PS was estimated based on the calibration curve. A 0.1 M sodium nitrate buffer was used as the mobile phase, with a 3% (w / v) concentration of sample solvent at a flow rate of 1 mL / min. Calibration was performed using standard samples of dextran with different molecular weights. The results are as follows: Figure 4 As shown.

[0053] The results showed that the polysaccharide exhibited a single and symmetrical chromatographic peak, indicating good homogeneity. Based on calculations using the chromatographic system, its weight-average molecular weight (Mw) was 2.5016 × 10⁻⁶. 4 Da, the number-average molecular weight (Mn) is 1.1468 × 10⁻⁶. 4 Da, peak molecular weight (Mp) is 2.0606 × 10⁻⁶. 4 Therefore, the molecular weight of the polysaccharide is 2.5016 × 10⁻⁶. 4 Da.

[0054] (2) Infrared spectroscopy analysis FT-IR (Fourier Transform Infrared Spectroscopy) is a spectral method that utilizes the changes in molecular dipole moments or charge distribution caused by vibrations. Because different functional groups and chemical bonds in polysaccharides absorb and vibrate at different frequencies, it can be used to detect and analyze the functional groups and chemical bond composition of polysaccharides, such as glycosidic bond configuration, sugar ring size, and sugar residues. The infrared spectrum of PM-PS was determined using a Thermo Fisher Scientific Nicolet IS50 (USA). A small amount of PM-PS sample prepared in Example 2 was thoroughly mixed with an appropriate amount of KBr in a mortar and pressed into a pellet. The pellet was heated at 4000–400 cm⁻¹. -1 The infrared spectrum of the polysaccharide was measured within a certain frequency range, and the functional groups contained in the mycelial polysaccharide were identified by analyzing the peaks at different wavelengths. The results are as follows: Figure 5 As shown.

[0055] The results showed that in the range of 3600–3200 cm -1 The presence of broad, smooth, and strong absorption peaks within the range is likely due to the stretching vibrations of hydrogen bonds within and between sugar molecules, indicating that the polysaccharide contains -OH groups. (2970 cm⁻¹) -1 There is an absorption peak of -CH2 at 1690~1500cm. -1 This is the double bond stretching vibration region, 1600 cm. -1 The absorption peak at 1475~1000 cm⁻¹ is likely due to NH₃ stretching. -1 The absorption peak range is 1020 cm⁻¹, which is caused by in-plane bending vibration of CH or stretching vibration of CO and CX. -1 With 1260 cm -1It should be generated by the stretching vibration of alcohol CO, where 1260 cm -1 The vibration at this point should be caused by the stretching vibration of CO in tertiary alcohols. (1000~650 cm⁻¹) -1 The absorption peaks in the range of 800–950 cm⁻¹ are caused by CH bond bending vibrations. -1 The absorption peaks in the region are usually related to the glycosidic bond configuration, among which, 872 cm⁻¹ -1 The nearby absorption peaks suggest the possible presence of β-glycosidic bonds in the sample. PM-PS, due to its high hydrogen bond density, is expected to exhibit high flocculation performance.

[0056] (3) Determination of monosaccharide composition The monosaccharide composition of PM-PS was determined by HPLC (Aglient 1100, USA). 2.0 mg of PM-PS prepared in Example 2 was placed in a 10 mL hydrolysis tube, and 2 mL of 4 M trifluoroacetic acid (TFA) solution was added and mixed. The tube was sealed with N2 (10 L / min, 1 min) and hydrolyzed in a 100 °C oven for 2 h. After cooling, 1 mL of the hydrolysate was dried under N2 in a 70 °C water bath to remove TFA. This process was repeated several times. Then, 1 mL of 0.3 M NaOH solution was added to fully dissolve the residue, followed by 1 mL of 0.5 M PMP methanol solution for derivatization. The reacted sample was diluted 10-fold with ultrapure water before analysis, and the results were corrected using a monosaccharide standard solution. The results are shown below. Figure 6 As shown.

[0057] Comparing the peak times of various standard monosaccharides in the mixed standard (peaks 1 to 14 for L-guluronic acid, D-mannuronic acid, D-mannose, D-glucosamine, D-ribose, L-rhamnose, D-glucuronic acid, D-galacturonic acid, D-galactosamine, D-glucose, D-galactose, D-xylose, L-arabinose, and L-fucose), it can be seen that Bacillus colloidis polysaccharide is a heteropolysaccharide composed of mannose (Man), glucose (Glc), galactose (Gal), and fucose (Fuc). The molar ratio of these four monosaccharides is 28.81:27.18:22.44:9.48, with mannose and glucose being the main constituent monosaccharides. Clearly, mannose is the most abundant monosaccharide, while glucose has a lower proportion than mannose. These four monosaccharides account for 96.82% of the total mass of the polysaccharide, while the uronic acid content is negligible. This result is consistent with previous studies (Mu, J., Zhou, H., Chen, Y., Yang, G., Cui, 17-24. http: / / dx.doi.org / 10.1016 / j.carbpol.2018.01.036; Okaiyeto, K., Nwodo, UU, Mabinya, LV, Okoh, AI, 2014. Evaluation of the flocculation potential and characterization of bioflocculant produced by Micrococcus sp. Leo. AppliedBiochemistry and Microbiology 50(6), 601-608. The results (http: / / dx.doi.org / 10.1134 / s000368381406012x) are consistent, indicating that the polysaccharide of Bacillus colloidis is mainly composed of glucan and mannan.

[0058] (4) Nuclear magnetic resonance analysis A small amount of the polysaccharide PM-PS prepared in Example 2 was dissolved in an appropriate amount of heavy water, and after multiple freeze-drying and deuteration exchange processes, it was analyzed in a nuclear magnetic resonance spectrometer (Bruker Avance III, Germany) at an analysis frequency of 500.13 Hz. 1 H-spectral analysis was performed at an analysis frequency of 125.75 MHz. 13 C-spectral analysis. Two-dimensional spectra, including HSQC, HMBC, COSY, and TOCSY, are used to resolve direct hydrogen-carbon correlations, long-range correlations, and hydrogen-hydrogen coupling relationships. All spectra are processed using built-in software for Fourier transform, phase correction, baseline correction, and peak assignment. The results are as follows: Figure 7 As shown.

[0059] (5) Methylation analysis To determine the glycosidic bond linkages of monosaccharide residues in polysaccharide PM-PS, methylation analysis was performed on the purified PM-PS samples. First, approximately 3–5 mg of lyophilized polysaccharide was fully methylated under anhydrous conditions using a methylation reagent. After the reaction, the mixture was quenched with distilled water, and the organic phase was extracted with chloroform. The extract was evaporated to dryness, and 2 M trifluoroacetic acid (TFA) was added. Hydrolysis was carried out at 120 °C for 1–2 h to cleave the methylated polysaccharide into methylated monosaccharide derivatives. The hydrolysis products were evaporated to dryness and reduced with sodium borohydride (or borane), followed by acetylation with acetic anhydride to obtain partially methylated glycosyl acetates (PMAAs). The obtained derivatives were dissolved in chloroform and analyzed by gas chromatography-mass spectrometry (GC-MS). GC-MS used capillary column separation, and each PMAA was identified based on retention time and characteristic fragment ions. The results are shown below. Figure 8 As shown in Table 1. By analyzing the methylation and acetylation positions of each derivative, the substitution patterns of the corresponding monosaccharide residues were determined, and the connection modes of the polysaccharide backbone and branch structures were inferred accordingly.

[0060] Based on the above one-dimensional and two-dimensional NMR spectra and methylation results, the main chain structure, branching points and connection modes of the polysaccharide were comprehensively analyzed, and the structure of the isolated and purified extracellular polysaccharide PM-PS was deduced.

[0061] like Figure 7 As shown in Figure a, polysaccharides 1The 1H-NMR spectrum can be divided into three main regions: the anomeric proton region (δH 4.5–5.5), the intracyclic proton region (δH 3.1–4.5), and the alkyl region (δH 1.2–2.3). The anomeric region shows significant signals at 5.83, 5.34, and 5.16 ppm, indicating the presence of an α-pyranose structure, possibly derived from glucose, mannose, galactose, or fucose. The intracyclic proton region shows multiple overlapping peaks at 3.3–4.0 ppm, suggesting a complex branched structure in the polysaccharide, primarily composed of glucose or galactose, accompanied by a high-field signal partially derived from mannose. The peaks in the alkyl region at 1.56, 1.47, and 1.36 ppm are attributed to methyl or short-chain alkyl substitution structures, while signals at 1.92 and 1.87 ppm suggest the presence of acetyl structures. The polysaccharide… 13 C-NMR spectrum ( Figure 7 (b) can also be divided into several structural regions: anomeric carbon region (δC 95-110), cyclic carbon region (δC 50-85), alkyl carbon region (δC 15-25), and carbonyl carbon region (δC 165-180). Angiomeric carbon signals corresponding to α-glycosidic bonds were observed at 102.13 and 100.82 ppm, while the 50-85 ppm range represented C2-C5 cyclic carbon signals with hydroxyl groups. The chemical shifts of the alkyl region were consistent with those of the methyl and methylene carbons, while the signals appearing in the carbonyl region at 181.75, 173.88, and 169.30 ppm indicated the presence of carboxyl and acetylated structures in the molecule.

[0062] Polysaccharide structure through one-dimensional ( 1 H: Figure 7 a; 13 C: Figure 7 b; DEPT-135, Figure 8 (f) Two-dimensional homokernel correlation (TOCSY: Figure 8 a, COSY: Figure 8 b, NOESY: Figure 8 (e) and heteronuclear related (HSQC: Figure 8 c, HMBC: Figure 8 (d) The results were obtained through comprehensive analysis of the spectra. HSQC showed that most of the carbon and hydrogen signals of the polysaccharide fell within the range of 60–110 ppm. 13 C) and 3.50~5.50ppm ( 1 H) range. According to Figure 8 As shown in Table 1, various monosaccharide residues with different linkage modes can be identified. Seven anodic cross-peaks (labeled AG) were observed in the HSQC spectrum, and their anodic configurations could be inferred from diagnostic chemical shifts. Based on the cross-peaks in the two-dimensional spectrum, the complete spin system of the seven pyranose residues was determined, and the spin configurations of each residue were finally completed. 1 H / 13All C values ​​were assigned, and the results are shown in Table 2.

[0063] Table 1. Methylation results of polysaccharides

[0064] Table 2 Signal Attribution of Polysaccharide Residues

[0065] By integrating the attribution information and linkage sites of each residue, a branched backbone of the repeating structural unit was constructed. Residue G (β-D-Galp) is located at the core of the structure, and its C-4 is linked to residue E (main chain terminal α-D-Manp). This linkage is confirmed by the HMBC correlation peak and the deshielding drift of residue E at C-4, indicating that it is a reducing α-D-mannopyranose residue. In addition to C-4, residue G is also linked to residue F (α-D-Manp) at C-3. This (1→3) bond is supported by the HMBC cross peak between residue G at C-3 and residue F at H-1 and the deshielding drift of F at C-3. Furthermore, the HMBC cross peak between residue G at C-6 and residue D (β-D-Galp) at H-1 further confirms another (1→6) bond, indicating that C-6 is the second branch point, which is completely consistent with the characteristic of residue D as a terminal monosaccharide in methylation analysis.

[0066] Residue F is linked to C-3 of residue G via its anodic position, simultaneously serving as a secondary branch point of the repeating unit. Both C-3 and C-4 are substituted, with the demasking drift of C-4 and the α-anodic shift of residue B (α-D-Manp) indicating that residue B is linked to O-4 of F, forming a side chain. The HMBC cross-peak between C-3 of F and H-1 of residue C (β-D-Glcp) further confirms another (1→3) bond, making residue C the terminal unit of another side chain. Combined with the 2,4,6-trimethylglucol detected by methylation analysis, residue C is confirmed to be a non-reducing terminal glucose.

[0067] Therefore, residue B is a 1,4-disubstituted mannose residue, with its anomeric carbon linked to the main chain residue F, and its C-4 linked to the fucose side chain. This is completely consistent with the corresponding 2,3,6-tri-O-methylmannitol (a typical 1,4-bonded derivative) in the methylation analysis.

[0068] Based on all structural information, this polysaccharide has a main chain of →3)-β-D-Glcp (C)-(1→3)-α-D-Manp (F)-(1→4)-β-D-Galp (G)-(1→4)-α-D-Manp (E)→; wherein, α-L-Fucp (A) side chain is attached to the O-4 position of the main chain residue F, β-D-Galp (D) side chain is attached to the O-3 position of G, and terminal α-D-Manp (B) side chain is attached to the O-6 position of the main chain residue G. Its skeleton is as follows: Example 4 Chemical properties of extracellular polysaccharide PM-PS The extracellular polysaccharide PM-PS isolated and purified in Example 2 was prepared into PM-PS solutions with concentrations of 0, 4, 8, 12, 16 and 20 g / L, and the chemical properties of the PM-PS were determined.

[0069] (1) Viscosity The thickening capacity of extracellular polysaccharide PM-PS solutions at different concentrations (0, 4, 8, 12, 16, and 20 g / L) was measured using a rotational viscometer (Brookfield DV3T, USA). Measurements were taken at a constant temperature of 25°C using a No. 61 rotor at a rotation speed of 100 rpm. Results are shown below. Figure 9 As shown in Table 3.

[0070] (2) Hydrophilicity The contact angles of extracellular polysaccharide PM-PS solutions at different concentrations (0, 4, 8, 12, 16, and 20 g / L) were measured using a contact angle meter (Powereach JC2000D3M, China). 5 μL of each concentration was added to a clean glass slide for measurement; the results are shown below. Figure 9 As shown in Table 3.

[0071] Table 3. Thickening ability and hydrophilicity / hydrophobicity of Bacillus polysaccharide solutions of different concentrations.

[0072] Figure 9 The black curve in the middle describes the thickening ability of the extracellular polysaccharide PM-PS solution as a function of concentration. The linear regression equation of torque-concentration obtained by fitting the data is: y = 0.0396x + 0.0150 (R²). 2=0.9969). The results showed that within the measured concentration range (0–12 g / L), the thickening ability of the extracellular polysaccharide PM-PS increased approximately linearly with increasing polysaccharide concentration. In the low concentration range (0–4 g / L), the thickening ability was low, the polysaccharide molecules were uniformly dispersed in the solution, the intermolecular interactions were weak, and no obvious network structure had yet formed. When the concentration increased to 4–8 g / L, the thickening ability continued to increase linearly, indicating that the interactions between polysaccharide molecules strengthened, but a relatively uniform distribution was still maintained. At this stage, the polysaccharide molecules gradually formed a relatively loose network structure. When the concentration continued to rise, reaching the 8–12 g / L range, the thickening ability still maintained a linear growth trend, indicating that within this concentration range, the polysaccharide molecules gradually formed a more complex network structure, but the strengthening of interactions did not exceed the linear range. In summary, the linearly increasing thickening ability indicates that during the concentration increase process, the polysaccharide molecules formed an effective interconnected structure, which helps to capture and aggregate suspended particles. Therefore, in practical applications, the thickening ability of the solution can be optimized by adjusting the polysaccharide concentration, thereby achieving the desired flocculation effect.

[0073] Figure 9 The red curve shows the contact angle of polysaccharide solutions at different concentrations, and its trend with concentration. Different curve fitting methods were used to fit the data, with the exponential model y = -80.2044e... -x / 8.1446+104.0082 (R 2=0.9890) provides a concise and efficient fit, and its physical meaning is clearer. Therefore, the decay exponential model is used to characterize the linear regression equation of contact angle-concentration. The results show that in the low concentration range (0~2 g / L), the contact angle of the solution increases significantly: from 24.12˚ to 43.21˚, indicating that polysaccharide molecules form a preliminary adsorption layer at the solid-liquid interface, and the wettability of the solution is significantly reduced. When the concentration increases to 2~6 g / L, the contact angle increases further, but the rate of increase slows down. This indicates that the hydrophobicity of the solution increases, and polysaccharide molecules form a denser layered structure at the solid-liquid interface. This structure increases the surface tension of the droplets at the interface, thereby further increasing the contact angle. When the concentration continues to rise, in the range of 6~12 g / L, the increase in contact angle tends to level off, from 68.42˚ to 84.20˚. This indicates that the polysaccharide molecules at the interface may be close to saturation, forming the densest covering layer. At this point, further increases in concentration have limited effect on the contact angle. In summary, the increasing contact angle reflects the degree of polysaccharide molecule adsorption at the solid-liquid interface. This adsorption enhances the interfacial stability of the solution and helps improve flocculation. In the low to medium concentration range, the gradual adsorption of polysaccharide molecules forms an effective interfacial layer, promoting particle capture and sedimentation. At high concentrations, polysaccharide adsorption reaches saturation, further enhancing its effectiveness as a flocculant. However, excessively high concentrations of polysaccharides may lead to overly compact floc structures or the formation of colloids, which could negatively impact sedimentation efficiency.

[0074] (3) Rheological properties The rheological properties of extracellular polysaccharide PM-PS solutions were tested using a rotational rheometer (Thermo Fisher HAAKE MARS60, USA) with a P60 / Ti rotor (60 mm diameter, titanium). Two ml samples were taken each time and allowed to stand for 5 minutes on the sample stage before measurement to eliminate shear history and achieve temperature equilibrium.

[0075] Concentration-related shear test: Different concentrations (0.2–12 g / L) of extracellular polysaccharide PM-PS aqueous solutions were measured at 25°C within a shear rate range of 0.01–100 s⁻¹. -1 The apparent viscosity and shear stress within the sample were determined. Steady-state shear data were fitted using a power-law model and a Carreau model. Power-law model: in: This represents shear stress (Pa). This represents the consistency coefficient (Pa·sn). The shear rate is represented by s⁻¹, and n represents the flow property index (flow index, dimensionless).

[0076] Carreau model: Where: η represents the apparent viscosity (Pa·s), η0 represents the zero shear rate viscosity or zero tangential viscosity (Pa·s), and λ represents the time constant (s). Shear rate (s) -1 ), m is a dimensionless exponent related to polymer concentration.

[0077] Temperature-dependent shear test: The shear rates of extracellular polysaccharide PM-PS aqueous solutions at concentrations of 2 g / L and 4 g / L were measured at different temperatures (25, 35, 45, 55, and 65 °C) within the range of 0.01–100 s⁻¹. -1 The apparent viscosity within the solution. The temperature-dependent shear rheological behavior of the polysaccharide solution was fitted using the Arrhenius equation: Where: η represents apparent viscosity (Pa·s), A represents frequency factor (dimensionless), Ea represents flow activation energy (kJ / mol), R represents gas constant (8.314 J / mol·K), and T represents thermodynamic temperature (K).

[0078] Figure 10 Figure a shows that at 25°C, 0.01 s -1 ~100 s -1 The effect of different concentrations of extracellular polysaccharide PM-PS aqueous solutions on the apparent viscosity (η) under shear rate (γ) was investigated. Overall, within the concentration range of 0.2–12.0 g / L, the viscosity of the polysaccharide solution decreased with increasing shear rate, exhibiting shear-thinning behavior, indicating that the polysaccharide solution is a pseudoplastic non-Newtonian fluid. This shear-thinning effect was particularly pronounced at high concentrations. Fitting a power-law model revealed that the parameter n was less than 1, further confirming the pseudoplastic characteristics of the polysaccharide. This behavior may be due to weakened interchain interactions and increased chain orientation under shear force, leading to enhanced system fluidity.

[0079] Figure 10 Figure b shows the relationship between shear stress and shear rate for polysaccharides of different concentrations under the same conditions. With increasing shear rate, the shear stress exhibits a non-linear increase, displaying obvious yield stress characteristics. This indicates that the polysaccharide system possesses a certain degree of viscoelasticity, possibly due to the partial disruption of the entanglement and cross-linking between polysaccharide molecules at high shear rates, leading to an enhanced stress response. This result is consistent with classical rheological theory and fits the description of yield stress behavior in colloidal systems.

[0080] Figure 10 The viscosity changes of 2 g / L and 4 g / L polysaccharides at different temperatures (20, 30, 40, 50, and 60 °C) were investigated in sections c to d. With increasing temperature, the viscosity of both polysaccharide concentrations decreased significantly, consistent with the viscosity-temperature relationship described by the Arrhenius equation. Data fitting revealed that the activation energy of the high-concentration polysaccharide was slightly higher than that of the low-concentration polysaccharide, indicating that the high-concentration system is more sensitive to temperature changes. Increased temperature increases the energy of molecular motion, reduces the strength of hydrogen bonds between polysaccharide chains, and thus decreases the viscosity of the system. Furthermore, the viscosity change of the high-concentration polysaccharide was greater than that of the low-concentration system, possibly because the intermolecular forces are stronger in the high-concentration system, making the effect of temperature changes on its fluidity more significant.

[0081] Example 5 Evaluation of the flocculation activity of extracellular polysaccharide PM-PS In this example, kaolin (particle size 4.90 μm, Zhonglian Chemical Reagent Co., Ltd., Tianjin) and montmorillonite (particle size 14.74 μm, Maclean Chemical Reagent Co., Ltd., Shandong) were used as model minerals to evaluate the flocculation performance of the extracellular polysaccharide (PM-PS) isolated and prepared in Example 2.

[0082] Mineral powders (kaolin and montmorillonite) were dispersed in deionized water at a mass concentration of 1 g / L and stirred continuously for 12 h under magnetic stirring to fully hydrate them and form stable mineral suspensions (kaolin suspension and montmorillonite suspension) for later use.

[0083] Flocculation experiments were conducted in glass beakers (inner diameter 110 mm, height 140 mm, total volume 1000 mL) placed on a thermostatic magnetic stirrer (HMS-1-2L, Labshark, China). The prepared kaolin and montmorillonite suspensions were divided into 8 groups, as follows: PM-PS group: The extracellular polysaccharide PM-PS prepared in Example 2 was added to the mineral suspension to obtain a flocculation system; wherein the amount of PM-PS added was 30 mg / L.

[0084] Na + Group: The operation is the same as the PM-PS group, except that Na is also added to the flocculation system. + A NaCl solution is prepared by dissolving NaCl in deionized water. The amount added is adjusted to ensure that the NaCl concentration is within the specified range. + The final concentration in the flocculation system was 0.5 mmol / L.

[0085] K + Group: The operation is the same as the PM-PS group, the difference being that K is also added to the flocculation system. +A KCl solution was prepared by dissolving KCl in deionized water, and the amount added was adjusted to achieve the desired KCl concentration. + The final concentration in the flocculation system was 0.5 mmol / L.

[0086] Ca 2+ Group: The operation is the same as the PM-PS group, the difference being that Ca is also added to the flocculation system. 2+ A CaCl2 solution was prepared by dissolving CaCl2·2H2O in deionized water. The amount added was adjusted to make the CaCl2 solution more suitable for the desired concentration. 2+ The final concentration in the flocculation system was 0.5 mmol / L.

[0087] Mg 2+ Group: The operation is the same as the PM-PS group, the difference being that Mg is also added to the flocculation system. 2+ A MgCl2 solution was prepared by dissolving MgCl2·6H2O in deionized water. The amount added was adjusted to ensure that the MgCl2 solution was of the correct concentration. 2+ The final concentration in the flocculation system was 0.5 mmol / L.

[0088] Fe 3+ Group: The operation is the same as the PM-PS group, the difference being that Fe is also added to the flocculation system. 3+ FeCl3 solution was prepared by dissolving FeCl3·6H2O in deionized water. The amount added was adjusted to make Fe... 3+ The final concentration in the flocculation system was 0.5 mmol / L.

[0089] Al 3+ Group: Operation is the same as the PM-PS group, the difference being that Al is also added to the flocculation system. 3+ AlCl3·6H2O was dissolved in deionized water to prepare an AlCl3 solution. The amount added was adjusted to make Al... 3+ The final concentration in the flocculation system was 0.5 mmol / L.

[0090] CK group: The procedure was the same as the PM-PS group, except that no extracellular polysaccharides were added.

[0091] The pH of the flocculation system in all treatment groups was adjusted to 7.0–7.5, and all reagents used were of analytical grade.

[0092] Place a standard magnetic stir bar in the center of the bottom of the beaker. The sample was 10 mm × 30 mm in diameter, and the stirring speed was adjusted digitally to meet different shear conditions. The system temperature was controlled at 25 ± 1℃ during the experiment to minimize the impact of temperature fluctuations on the results. A fixed sampling tube (6 mm inner diameter) was vertically installed inside the beaker, with its inlet located approximately 6 cm below the liquid surface. The flocculation experiment employed a staged control method to record the entire process of particle structure changes. The entire experiment lasted 15 minutes and included three stages: rapid stirring (300 rpm, 0–2 min), slow stirring (120 rpm, 2–10 min), and static settling (0 rpm, 10–15 min).

[0093] The flocculation efficiency (FE, %) after 5 minutes of settling is used as the main indicator for evaluating optimal conditions. The flocculation efficiency is calculated based on the turbidity reduction rate, and the formula is as follows: In the formula: A0 is the absorbance of the control solution; A1 is the absorbance of the supernatant after the sample has been flocculated and allowed to stand for 5 minutes.

[0094] Test results as follows Figure 11 As shown in Table 4.

[0095] Table 4. Flocculation ability of Bacillus subtilis polysaccharides on different minerals.

[0096] according to Figure 11 It was found that in the control group (CK) without any flocculant, the flocculation efficiencies of kaolinite and montmorillonite were low, at 10.66% and 43.28%, respectively. When polysaccharide was added alone, the flocculation rates of the two clay minerals significantly increased to 67.96% and 83.18%, respectively, preliminarily confirming the good flocculation activity of the polysaccharide. Further investigation into the synergistic effect of cations and polysaccharides revealed that the added cations promoted the flocculation effect to varying degrees, exhibiting a clear correlation between ion valence states: in the monovalent cation system, Na... + and K + It has little effect on the flocculation rate; however, the addition of divalent cations exhibits stronger coagulation aid ability, further increasing the flocculation rate to between 70% and 80%. The most significant improvement occurs in the high-valence metal ion system, Fe... 3+ Al 3+ The addition of [specific ingredient] significantly improved the flocculation effect, especially Fe [specific ingredient]. 3+With the synergistic effect of polysaccharides, the flocculation rates of kaolinite and montmorillonite reached 95.34% and 97.77%, respectively, tending towards complete sedimentation. This indicates a strong synergistic effect between polysaccharides and high-valence cations, which may effectively overcome the electrostatic repulsion between clay particles through enhanced charge neutralization and bridging, thereby significantly optimizing flocculation performance.

[0097] To further evaluate the flocculation performance of the polysaccharide (PM-PS) prepared in this study, it was subjected to Fe... 3+ The flocculation rates of kaolinite (95.34%) and montmorillonite (97.77%) under synergistic effect were compared with those of other typical bio-flocculators reported in the literature, and the results are shown in Table 5.

[0098] Table 5. Flocculation effect of different bioflocculators

[0099] The results showed that the extracellular polysaccharide PM-PS exhibited extremely excellent flocculation activity with the assistance of high-valence metal ions, and its highest flocculation efficiency (97.77%) not only exceeded that of most common Bacillus species (such as...) Bacillus aryabhattai PSK1's 94.56% and Bacillus megaterium PL8 (90.23%) and Klebsiella spp. Klebsiella sp. ZZ-3 (94.5%) is a flocculant that produces flocculants with performance comparable to the top-performing flocculants currently reported in the literature. Bacillus licheniformis NJ3 (97.30%) and Bacillus subtilis Compared to 35A (96.57%), it still exhibits stronger settling ability. Even when processing structurally stable kaolinite particles, PM-PS combined with Fe... 3+ The subsequent flocculation performance was also superior to that of, etc. Rhizopus sp. M9 (92.71%) and Pseudomonas sp. (91.5%) and other microbial flocculants. This comparative result strongly demonstrates the great application potential and technological advantages of this polysaccharide bioflocculant in treating clay mineral wastewater. Its highly efficient sedimentation performance under the synergistic effect of charge neutralization and high-valence ions makes it a highly competitive environmentally friendly water purification material.

[0100] Example 6: Aggregation ability of extracellular polysaccharide PM-PS for different mineral particles The flocculation experiment was conducted strictly in time segments to capture the entire process of particle structure evolution. The entire experimental period was 15 minutes, divided into four stages: premixing, rapid stirring, slow stirring, and static sedimentation, as follows: Before the reaction began, a premixing stage was set up, in which the kaolin suspension was slowly stirred at 60 rpm for approximately 0.5 minutes under constant temperature conditions (25±1℃) to ensure uniform dispersion and stability of the system. Subsequently, the first group of samples was collected, and their particle size distribution and surface charge were measured using a laser particle size analyzer (SYNC, Microtrac, USA) and a Zeta potentiometer (ZS90, Malvern, UK). Before premixing, Fe... 3+ The final concentration of the flocculation system was 0.5 mmol / L. After adding different doses (1, 2, 4, 8, 16, 24, 32, 40 mg / L) of the extracellular polysaccharide PM-PS, a rapid stirring phase (0–2 min) was immediately initiated, with the stirring speed increased to 300 rpm to simulate the initial flocculation environment under high shear conditions, promoting rapid particle collisions and micro-cluster formation. Samples were collected at 2 min to capture the characteristic responses of the structural mutation process and early aggregation behavior. Then, a slow stirring phase (2–10 min) was initiated, with the stirring rate adjusted to 120 rpm to simulate shear-induced structural reconstruction and re-aggregation. Samples were collected at 4, 6, 8, and 10 min to monitor floc size evolution and surface electrical changes. Finally, the reaction system entered a settling phase (10–15 min), and stirring was immediately stopped, allowing the suspended flocs to settle naturally under gravity. The system naturally settled under gravity and formed a certain degree of stratification. To ensure the representativeness of the final samples, the entire system was gently mixed 15 minutes before sampling to ensure thorough and uniform distribution of flocs and supernatant. The mixture was then collected through sampling tubes to characterize the overall particle size distribution and structural stability at the end of the settling period. The test results are as follows: Figure 12 As shown in Tables 6 and 7.

[0101] Table 6. Effect of extracellular polysaccharide PM-PS on kaolin particle size

[0102] Table 7 Effect of extracellular polysaccharide PM-PS on montmorillonite particle size

[0103] In the kaolin solution, as the polysaccharide concentration gradually increased, the volume average particle size (D[4,3]) of the flocs in the system also showed a continuous increasing trend, indicating that the flocculation process was significantly affected by the concentration. Specifically, when the concentration was 1.0 mg / L, the floc particle size did not increase significantly, and this dose failed to significantly break the original colloidal stability of the system. However, when the concentration was increased to 2.0 mg / L, the particle size briefly jumped to 6.32 µm after 2 min, and then rapidly decreased and stabilized at a level similar to that of 1.0 mg / L (5.31 µm), indicating that although this concentration triggered the initial aggregation of particles, the structure had not yet been stabilized. The significant changes in D[4,3] mainly occurred in the medium and high concentration treatment groups (4.0~24.0 mg / L). In these groups, the particle size increased rapidly during the rapid stirring stage and remained at a high level in the subsequent stages. Specifically, a significant increase in particle size was observed at a concentration of 16.0 mg / L, with the treated group reaching a particle size of 14.61 µm within 2 minutes and eventually stabilizing at 12.70 µm. The 32.0 mg / L group exhibited even stronger flocculation capacity, with the maximum particle size increasing to 20.00 µm and stabilizing at 16.08 µm. However, when PM-PS increased to 40.0 mg / L, although the initial particle size rapidly increased to 39.39 µm, it subsequently dropped rapidly, with the final settling stage D[4,3] decreasing to 19.14 µm. While still higher than the stable value of the 32.0 mg / L group, the significant decrease indicates substantial instability in the floc structure during shearing and redistribution. This phenomenon suggests the coexistence of larger flocs and finer particles in the system, enhancing structural heterogeneity. This instability may stem from chain entanglement, bridging, or adsorption saturation caused by excessive EPS, leading to a looser floc structure, increased internal porosity, and consequently reduced resistance to shear failure. Therefore, although the 40.0 mg / L group exhibits a certain advantage in apparent particle size, its floc structural integrity and mechanical stability may be lower than the medium concentration treatment group, ultimately resulting in potentially suboptimal flocculation. This complex behavior will be further analyzed in conjunction with other structural indicators to examine the relationship between particle size growth and structural stability.

[0104] Overall, as the polysaccharide concentration increased, the fractal dimension (Df) gradually rose, reaching a peak at 32.0 mg / L (2.10). While Df also showed an upward trend at 40.0 mg / L, the value dropped back to (1.60), similar to the level at 2.0 mg / L (1.62). This anomaly corresponds to the rapid increase and subsequent sharp decrease in particle size. Notably, considering the performance at different concentrations, the fractal dimension exhibited a typical window characteristic with increasing polysaccharide concentration. Df increased slowly between 1.0 and 16.0 mg / L, reaching its maximum at 32.0 mg / L, while at 40.0 mg / L, Df approached the low-to-medium concentration level. This suggests that when excessive polysaccharide is added, some of the excess polymer chains in the system cannot effectively participate in the internal structure construction of the flocs. Instead, they entangle or coat the particle surface, making the floc surface appear larger, but the internal structure does not actually become more compact, and may even become looser.

[0105] The evolution of volume-average particle size in montmorillonite suspension systems is highly sensitive to polysaccharide dosage. For example... Figure 12As shown, the initial particle sizes (D[4,3]) of each treatment were approximately 14–15 μm, indicating similar initial particle sizes. With the progress of flocculation, the particle size increase in the low-dose group (1.0–4.0 mg / L) was relatively limited. At 15 min, the D[4,3] values ​​were 16.80, 24.23, and 30.54 μm, respectively, showing only a slight increase from the original particle size. This indicates that the number of polysaccharide molecules was insufficient to form an effective spatial network structure, and only a small number of small flocs were generated after particle collisions. The particle size increase was more significant in the medium-dose group (8.0 mg / L), with D[4,3] gradually increasing from 14.75 μm to 59.57 μm. This shows that the polysaccharide could form a certain degree of adsorption bridging on the montmorillonite surface, promoting the formation of relatively stable aggregates, but the overall aggregation scale was still limited. Within the dose range of 16.0–24.0 mg / L, the aggregation process of montmorillonite particles was the most intense. In the 16.0 mg / L group, the particle size increased rapidly from 14.84 μm to 123.57 μm within 2 min, then slowly increased to 66.13 μm at 10 min; it slightly decreased to 160.44 μm at 15 min, indicating that at this dose, the polysaccharide could quickly achieve effective bridging and netting between particles, forming relatively stable large flocs. The response of the 24.0 mg / L group was even stronger, with D[4,3] reaching 165.77 μm at 2 min, a peak value of 276.70 μm at 10 min, and a slight decrease to 256.51 μm at 15 min, indicating that this concentration range is the optimal flocculation range for the montmorillonite system: the polysaccharide chains can not only fully neutralize and compress the electric double layer, but also construct a dense three-dimensional network structure through multi-point adsorption and spatial bridging, significantly amplifying particle collision and aggregation efficiency. When the dose was further increased to 32.0~40.0 mg / L, the increase in particle size actually weakened. The 32.0 mg / L group stabilized at approximately 143–160 μm in size between 2 and 10 min, without exhibiting the sustained amplification seen at moderate concentrations. The 40.0 mg / L group maintained a particle size of only 80–89 μm throughout the process, significantly lower than the optimal dosage group. This "overdose instability" phenomenon suggests that when the polysaccharide concentration exceeds a certain threshold, excessive adsorption causes the particle surface to re-acquire the same charge or form a thick swelling layer, weakening effective bridging and trapping effects, thus partially inhibiting the formation of large flocs. In summary, polysaccharides exhibit a typical dose-dependent effect on montmorillonite particle aggregation: low doses are insufficient to form effective structures, moderate doses gradually increase the effect, 16.0–24.0 mg / L is the optimal range for forming large-scale flocs, while excessive addition causes partial deagglomeration or refinement of the floc structure. This pattern provides a basis for determining the optimal dosage range in practical applications.

[0106] From the perspective of fractal dimension, polysaccharide concentration significantly affects the spatial structure and compactness of montmorillonite flocs. Initially, the fractal dimensions of all treatments were approximately 1.24–1.30, indicating that the system was dominated by dispersed particles and loose small flocs, and the floc skeleton had not yet formed. The fractal dimension of the low-dose treatment (1.0 mg / L) remained almost between 1.25 and 1.30 throughout the reaction process, reaching only 1.25 at 15 min, indicating that the amount of polysaccharide at this point was insufficient to drive significant structural reorganization, and only limited adhesion and loose aggregation occurred between particles. As the dose increased to 2.0–4.0 mg / L, the fractal dimension increased significantly over time: under the 2.0 mg / L condition, it gradually increased to approximately 1.82 in the range of 2–10 min, while the 4.0 mg / L treatment reached a maximum value of 2.06 at 15 min, which was the highest level among all treatments. Combined with the aforementioned particle size results, this indicates that moderate doses of polysaccharide not only promote floc size increase but also drive the evolution of particles from a loose, branching structure to a more compact, denser three-dimensional framework, which is beneficial for forming large flocs with strong mechanical stability. At higher doses (8.0–16.0 mg / L), the fractal dimension was also significantly higher than the initial level, but slightly lower overall than the 4 mg / L treatment. At 8 mg / L, the fractal dimension stabilized between 1.44 and 1.71, and although the structure was more compact than the control, it still retained some branches and pores; the 16 mg / L treatment maintained around 1.71–1.76 between 4 and 15 min, indicating that after excessive polysaccharide coverage of the particle surface, some bridging points were replaced by monolayer adsorption, resulting in the flocs maintaining a relatively loose structure while increasing in size. This trend was more pronounced when excessive dosage was added (24~40 mg / L): the fractal dimension reached a local peak of about 1.92 at 8 min under the condition of 24.0 mg / L, and then dropped to 1.63; the fractal dimensions of the treatments of 32.0 mg / L and 40 mg / L were stable in the range of 1.70~1.88 and 1.55~1.72, respectively, and then dropped slightly in the later stage. This suggests that high concentration of polysaccharides promotes rapid bridging and aggregation in the early stage, and in the later stage, due to the reversal of surface charge or the thickening of the swelling layer, the floc structure tends to be loose and locally deagglomerated.

[0107] In summary, the evolution of fractal dimension reveals a typical dose-dependent effect of polysaccharides on the aggregate structure of montmorillonite particles: low doses result in limited structural recombination, while moderate doses (especially 4.0 mg / L) significantly improve floc density, forming compact and stable large flocs; excessively high doses cause the flocs to regress from a dense to a looser network structure. This result corroborates the particle size variation pattern, indicating the existence of an optimal polysaccharide dosage range that balances floc size and density, providing important reference for optimizing dosage settings in practical flocculation processes.

[0108] Example 7: Application of polysaccharides in practical wastewater improvement To evaluate the application potential of extracellular polysaccharide PM-PS in actual wastewater, fly ash wastewater from a power plant in Harbin, Heilongjiang Province was selected as the test object. Its main water quality components are shown in Table 8.

[0109] Table 8 Chemical composition of fly ash wastewater

[0110] Fe was determined using the model system in Example 6. 3+ The final concentration in the flocculation system was 0.5 mmol / L. Based on the optimal dosage of extracellular polysaccharide PM-PS (d0 = 32.9 mg / L), dosage gradients ranging from 0.6 to 2.4 d0 (0.4, 0.6, 0.8, 1.0, 1.2, 1.4, 1.6, 1.8, 2.0, 2.2, and 2.4 d0) were established for testing. After mixing and standing for 5 min, the absorbance (OD) of the supernatant was measured at 550 nm. 500 (HITACHI UV-3010 UV-Vis spectrophotometer, Japan). Flocculation activity (FA) is calculated based on the percentage reduction in wastewater turbidity, using the following formula: FA = [(AB) / A] × 100%; where A is the OD of the supernatant after natural settling of the original wastewater. 500 Value, B is the OD of the supernatant obtained after adding PM-PS. 500 value.

[0111] Test results as follows Figure 13 As shown in Table 9, the flocculation effect of polysaccharides in fly ash wastewater varies significantly with the dosage. When the dosage is in the range of 0.6–1.6 d0, the flocculation efficiency remains at a high level (>80%). Specifically, the flocculation efficiency reaches a peak of 91.51% at 0.8 d0; and it still maintains a high efficiency of 90.8% at the simulated optimal dosage of 1.0 d0, indicating that this range represents the optimal flocculation range.

[0112] As the dosage continued to increase, the flocculation efficiency began to decline: it dropped to 72.57% at 2.0 d0 and further to 61.28% at 2.4 d0. This trend is consistent with the simulation results, indicating that accurate control of the dosage is key to achieving optimal floc formation. Compared with the kaolin model system, the optimal dosage of polysaccharide in actual wastewater shifted down to approximately 0.8 d0. This downward trend suggests that the polyvalent metal ions and inorganic components in fly ash wastewater partially neutralized the charge on the particle surface, reduced electrostatic repulsion, and synergistically exerted a flocculation effect with polysaccharide, thus requiring a slightly lower dosage than the pure soil model system.

[0113] Overall, this natural polysaccharide flocculant exhibits excellent flocculation effects in fly ash wastewater, demonstrating good application potential in the treatment of fly ash settling tanks and related industrial wastewater. More importantly, its flocculation efficiency remains above 80% over a wide dosage range, which is a significant advantage for practical engineering operations. Further research can be conducted to verify its suitability under various water chemistry conditions (pH, coexisting pollutants), the long-term stability of the floc structure, and large-scale field tests.

[0114] Table 9. Flocculation of Bacillus subtilis polysaccharides in actual wastewater.

[0115] Peptone-like Bacillus ( Paenibacillus mucilaginosus The extracellular polysaccharide (PM-PS) produced is a heteropolysaccharide composed of mannose (Man), glucose (Glc), galactose (Gal), and fucose (Fuc). The molar ratio of these four monosaccharides is 28.81:27.18:22.44:9.48, and the molecular weight is approximately 2.5 × 10⁻⁶. 4 Da. Its chemical properties exhibit good viscosity, hydrophilicity / hydrophobicity adjustment ability, and typical pseudoplastic fluid behavior. The viscosity increases linearly with concentration, and the contact angle increases with increasing concentration, demonstrating excellent interfacial activity and rheological control potential.

[0116] In terms of flocculation and agglomeration capabilities, PM-PS exhibits significant flocculation effects on a variety of minerals (montmorillonite and kaolinite), especially on Fe. 3+ The optimal flocculation efficiency, reaching 97.77%, is achieved in its presence, surpassing most commercially available bioflocculants. Its ability to aggregate mineral particles exhibits a dose-dependent effect; low to medium concentrations (e.g., 16–32.0 mg / L) promote the formation of large, dense, and stable flocs, while excessively high concentrations may lead to a loose structure. In actual fly ash wastewater treatment, PM-PS achieved a flocculation efficiency exceeding 90% at 0.8–1.0 times the optimal simulated dosage, demonstrating promising prospects for industrial application.

[0117] Example 8: Application of polysaccharides in the improvement of saline-alkali soil To further confirm whether the effect of Bacillus colloidis on soil aggregates originates from polysaccharides, saline-alkali soil samples taken from Lindian, Daqing, were passed through a 0.053 mm sieve and randomly divided into 4 groups: Bacterial culture treatment group: 50 mL of Bacillus subtilis fermentation broth was added to 250 g of saline-alkali soil and mixed thoroughly to achieve a viable bacterial count of 3.38 × 10⁻⁶ in the soil. 9 CFU / kg is denoted as the bacterial culture group.

[0118] Polysaccharide treatment group: 1.25 g of polysaccharide prepared in Example 2 was added to 250 g of saline-alkali soil and 50 mL of deionized water was added and mixed thoroughly so that the amount of polysaccharide added to the soil was 5 g / kg, which was recorded as the polysaccharide group.

[0119] Gel fertilizer treatment group: 0.25 g of gelatinous Bacillus spore-forming fertilizer (produced by Dongnong Tongfu Agricultural Technology Development Co., Ltd., registration certificate number: Microbial Fertilizer (2016) No. 1803, effective viable count ≥1.0×10⁻⁶) was added to 250 g of saline-alkali soil. 9 Add CFU / g and then add 50 mL of deionized water and mix thoroughly. This is recorded as the microbial fertilizer group.

[0120] Control group: 250 g of saline-alkali soil was mixed with 50 mL of deionized water, and this was recorded as CK.

[0121] After each treatment, the saline-alkali soil was placed in petri dishes and cultured under constant temperature conditions. During the culture period, deionized water was added regularly to maintain the soil moisture content at approximately 60% of field capacity. The culture temperature was 25±2℃, the relative humidity was 60%~70%, the light / dark cycle was 12 h / 12 ​​h, and the light intensity was 3000~5000 lx. After 180 days of culture, the amount of water-stable aggregates was measured and recorded for each particle size fraction. Meanwhile, the pH, electrical conductivity, and alkalinity of soil samples at different time points (30, 60, 90, 120, 150, and 180 days) were measured to explore the ameliorative effect of polysaccharides on saline-alkali soil. The method is described in [He Xiaodong, Miao Cong, Li Xiaoran, et al. Study on aggregate characteristics of saline soil in Yinbei area of ​​Ningxia [J]. Soil Bulletin, 2025, 56(4):996-1005.DOI:10.19336 / j.cnki.trtb.2024081801.] The results are as follows. Figures 14-17 As shown in Tables 10 to 12.

[0122] Table 10 pH values ​​of different treatment groups for saline-alkali soil

[0123] Table 11 EC values ​​of different treatment groups for saline-alkali soil

[0124] Table 12 Alkalinity of different treatment groups of saline-alkali soil

[0125] Figure 14The results showed that different treatments had varying degrees of impact on the pH of saline-alkali soil during the 180-day incubation period. The control group (CK) showed relatively small overall pH changes, maintaining around 8.98 from an initial 9.07, indicating that the alkalinity of saline-alkali soil is relatively stable under natural conditions and unlikely to improve significantly in the short term. In contrast, the polysaccharide treatment group, the bacterial solution treatment group, and the gelatinous fertilizer treatment group all exhibited varying degrees of alkalinity-reducing effects. Specifically, the soil pH in the polysaccharide treatment group gradually decreased from 9.01 at day 30 to 8.45 at day 150, and reached 8.83 at day 180; the bacterial solution treatment group decreased from 9.11 to 8.59; and the gelatinous fertilizer treatment group showed the most significant decrease, with its pH dropping from an initial 8.54 to 8.19, and remaining lower than other treatment groups throughout the entire incubation period. These results indicate that polysaccharides derived from Bacillus colloidis and their related preparations can effectively reduce the pH of saline-alkali soil and alleviate soil alkalization. Polysaccharides themselves can improve saline-alkali soil to a certain extent, while the microbial fertilizer treatment has a more significant effect, which is speculated to be related to the continuous secretion of extracellular polysaccharides by the strain in the soil, promoting the formation of aggregates and improving the soil microenvironment. With the extension of the cultivation time, the soil pH of each treatment group showed an overall downward trend, indicating that Bacillus colloidis and its extracellular polysaccharides have a sustained improving effect on saline-alkali soil.

[0126] Figure 15The results showed that different treatments significantly affected the electrical conductivity (EC) of saline-alkali soil during the 180-day incubation period. The control group (CK) showed relatively small changes in soil EC, consistently remaining between 0.93 and 0.97 mS / cm, indicating that the salinity of saline-alkali soil is relatively stable under natural conditions, and salt ions are difficult to reduce spontaneously. In contrast, the polysaccharide treatment group, the bacterial culture treatment group, and the gelatinous fertilizer treatment group all showed a trend of decreasing soil EC. Specifically, the EC in the polysaccharide treatment group decreased from 0.84 mS / cm at 30 days to 0.74 mS / cm at 180 days, indicating that extracellular polysaccharides can effectively reduce the content of soluble salt ions in the soil. The bacterial culture treatment group had a lower EC in the early stages of incubation, fluctuated slightly in the later stages, but still decreased to 0.79 mS / cm at 180 days. The gelatinous fertilizer treatment group showed the most significant change, with its EC gradually decreasing from an initial 1.09 mS / cm to 0.74 mS / cm, exhibiting the largest decrease. The results indicate that polysaccharides derived from Bacillus colloidis and their related preparations can effectively reduce the electrical conductivity of saline-alkali soil and decrease the accumulation of soluble salts in the soil, demonstrating a significant desalination and improvement effect on saline-alkali soil. Among these, the gelatinous fertilizer treatment was more effective than polysaccharide and bacterial solution treatments alone, suggesting that it may improve the microenvironment of saline-alkali soil and reduce the degree of salt damage by continuously secreting extracellular polysaccharides, promoting the formation of soil aggregates, and enhancing the salt ion fixation capacity. At 30 days of cultivation, the organic matter content (EC) of the bacterial fertilizer treatment group was slightly higher than other treatment groups, which is related to the promotion of soil mineral weathering and ion exchange by the microbial activity in the bacterial fertilizer. Organic acids released during bacterial metabolism can promote the dissolution of Ca, Mg, and K ions in the soil, while also promoting the entry of exchangeable Na ions into the soil solution, resulting in a temporary increase in EC in the early stages of cultivation. As the cultivation time increased, soluble salts were gradually fixed, migrated, or leached, and EC continuously decreased, indicating that the bacterial fertilizer has a long-term desalination and improvement effect on saline-alkali soil.

[0127] Figure 16The results showed that different treatments significantly affected the saline-alkali soil alkalinity (ESP) during the 180-day culture period. The control group (CK) showed relatively small changes in ESP, decreasing only from 42.24% at 30 days to 40.17% at 180 days, indicating that the alkalization state of saline-alkali soil is relatively stable under natural conditions and difficult to recover spontaneously. In contrast, the polysaccharide treatment group, the bacterial solution treatment group, and the microbial fertilizer treatment group all showed significant effects in reducing soil alkalinity. Specifically, the polysaccharide treatment group saw its ESP gradually decrease from 32.00% to 27.06%, indicating that extracellular polysaccharides can effectively reduce the degree of soil exchange-oriented alkalization; the bacterial solution treatment group saw a decrease from 38.30% to 25.43%, indicating that *Bacillus colloidea* could continuously improve the soil alkalization environment during its growth; the microbial fertilizer treatment group showed the most significant reduction, with its ESP decreasing from 32.69% to 22.76%, and remaining lower than other treatment groups throughout the entire culture period. The results indicate that extracellular polysaccharides derived from *Bacillus colloidea* and related preparations can effectively reduce the alkalinity of saline-alkali soil and alleviate soil alkali damage. The microbial fertilizer treatment showed the best effect, which is speculated to be related to the strain's continuous secretion of extracellular polysaccharides, promoting soil particle cementation, improving the ion exchange environment, and enhancing soil structural stability. With prolonged culture time, the alkalinity of all treatment groups showed a continuous decreasing trend, indicating that *Bacillus colloidea* and its extracellular polysaccharides have a good and sustainable improvement effect on saline-alkali soil and have application potential.

[0128] Figure 17The results showed that different treatments significantly affected the composition of water-stable aggregates in saline-alkali soil during the 180-day cultivation period. In the control group (CK), the content of large aggregates larger than 0.25 mm remained consistently low, increasing only slowly from 0.90% at 30 days to 1.86% at 180 days, while the proportion of fine particles smaller than 0.053 mm remained consistently high, indicating that saline-alkali soil has a weak ability to form aggregate structures under natural conditions. In contrast, the polysaccharide treatment group, the bacterial solution treatment group, and the gelatinous fertilizer treatment group all significantly promoted the formation of large-diameter aggregates. Specifically, the content of aggregates larger than 0.25 mm increased from 2.73% to 9.85% in the polysaccharide treatment group, from 1.36% to 6.92% in the bacterial solution treatment group, and the gelatinous fertilizer treatment group showed the most significant increase, from 2.91% to 12.31%, significantly higher than the control group. Meanwhile, the proportion of aggregates in the 0.053–0.25 mm particle size range was significantly higher in all treatment groups than in the control group. The polysaccharide and gelatinous fertilizer treatment groups stabilized at approximately 53.26% and 52.37% respectively in the later stages of cultivation, indicating that extracellular polysaccharides can promote the formation of medium-sized, water-stable aggregates. Conversely, the content of particles smaller than 0.053 mm gradually decreased with cultivation time. In the control group, the proportion decreased only from 71.45% to 64.07%, while in the polysaccharide, bacterial solution, and gelatinous fertilizer treatment groups, it decreased to 36.88%, 47.60%, and 35.31% respectively, indicating that fine soil particles gradually cemented together to form relatively stable large aggregate structures. These results demonstrate that extracellular polysaccharides derived from *Bacillus colloidea* can significantly promote aggregate formation in saline-alkali soils, improve soil water stability, reduce the proportion of fine particles, and thus improve the structural stability of saline-alkali soils. Among them, the treatment with gelatinous fertilizer showed the best effect, which is speculated to be related to the continuous secretion of extracellular polysaccharides by the strain, the enhancement of interparticle bridging, and the promotion of soil cementation. This indicates that the polysaccharide and its related preparations have good application potential in the improvement of saline-alkali soil.

[0129] In summary, the structural characteristics, physicochemical properties, and comprehensive performance of peptone-like Bacillus polysaccharides in mineral flocculation, wastewater treatment, and soil improvement demonstrate that they possess significant theoretical value and practical application potential as a highly efficient and environmentally friendly bioflocculator.

[0130] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. An extracellular polysaccharide, characterized in that, The main chain structure of the extracellular polysaccharide is →3)-β-D-Glcp-(1→3)-α-D-Manp-(1→4)-β-D-Galp-(1→4)-α-D-Manp (E)→; The α-D-Manp residues of the main chain are connected to an α-L-Fucp branch at position O-4; the β-D-Galp residues of the main chain are connected to a β-D-Galp branch at position O-4 and a terminal α-D-Manp branch at position O-6.

2. The extracellular polysaccharide according to claim 1, characterized in that, The extracellular polysaccharide has an average molecular weight of 2.5 × 10⁻⁶. 4 Da; The molar ratio of mannose, glucose, galactose and fucose in the extracellular polysaccharide is 28.81:27.18:22.44:9.

48.

3. The method for preparing the extracellular polysaccharide according to claim 1 or 2, characterized in that, Includes the following steps: Bacillus subtilis (peptone-like spores) Paenibacillus mucilaginosus The fermentation supernatant of Z1 was subjected to alcohol precipitation to obtain crude polysaccharide; the crude polysaccharide was deproteinized to obtain deproteinized crude polysaccharide; The deproteinized crude polysaccharide was separated by a DEAE-52 cellulose column to obtain the PM-PS1 fraction; the eluent for the DEAE-52 cellulose column was water. The PM-PS1 component was purified using a Sephadex G-200 glucose gel column to obtain the extracellular polysaccharide; the eluent for the Sephadex G-200 glucose gel column was water. The peptone-like Bacillus Z1 is deposited at the China General Microbiological Culture Collection Center, with accession number CGMCC No. 39217.

4. The preparation method according to claim 3, characterized in that, After obtaining the extracellular polysaccharide, the process also includes: dialysis purification.

5. The application of the extracellular polysaccharide according to claim 1 or 2 or the extracellular polysaccharide prepared by the preparation method according to claim 3 or 4 in flocculants.

6. A flocculant, characterized in that, The flocculant includes the extracellular polysaccharide as described in claim 1 or 2, or the extracellular polysaccharide prepared by the preparation method described in claim 3 or 4.

7. The flocculant according to claim 6, characterized in that, The flocculant also contains metal cations.

8. The flocculant according to claim 7, characterized in that, The concentration of the metal cation is 0.1~1 mmol / L; the metal cation includes Fe. 3+ and / or Al 3+ .

9. The application of the extracellular polysaccharide according to claim 1 or 2 or the extracellular polysaccharide prepared by the preparation method according to claim 3 or 4 in wastewater treatment and / or soil improvement.

10. The application according to claim 9, characterized in that, The soil includes saline-alkali soil.

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