Klebsiella YCTU3162 and its produced extracellular polysaccharide Flocan and its application

Abstract

The present invention is applicable to the field of microbial technology, and provides Klebsiella sp. YCTU3162 and the extracellular polysaccharide Flocan produced by it and its application. Klebsiella sp. YCTU3162 has a deposit number of CCTCC NO: M 20232027 and a deposit date of October 25, 2023. The extracellular polysaccharide Flocan is obtained by the production of Klebsiella sp. YCTU3162. The extracellular polysaccharide Flocan includes glucuronic acid, glucose, mannose and galactose in a molar ratio of 1:2:3:1, and has a pyruvic acid modification group. The production method of the extracellular polysaccharide Flocan in the present invention is simple and easy, with a short fermentation cycle and mild conditions. It has good flocculation activity in synergistic action with metal ions under acidic conditions for simulated kaolin and coal ash wastewater, is biodegradable, has no secondary pollution, and has good economic and social benefits.

Description

Technical Field

[0001] The invention belongs to the technical field of microorganisms, and in particular relates to Klebsiella YCTU3162 and extracellular polysaccharide Flocan produced by the same and application thereof. Background Art

[0002] According to statistics, the carbon emissions of the sewage treatment industry account for about 1% of the total emissions of the whole society, accounting for the largest proportion in the environmental protection industry. In the sewage treatment process, flocculation is a solid-liquid separation technology that separates colloids into clear solutions and solid sediments. At present, common flocculants can be divided into three categories according to their sources: inorganic polymer metal flocculants, chemical synthetic flocculants and biological flocculants. Inorganic flocculants (such as polyaluminum, polyiron, etc.) will have adverse effects on human health and the ecological environment. Chemical synthetic flocculants are mostly polypropylene and polyethylene substances, such as polyacrylamide and polyethyleneimine. Chemical synthetic flocculants are expensive and have more severe safety risks. The residual monomers of polymers have "three-cause" effects (teratogenic, carcinogenic, and mutagenic). It is an inevitable trend for my country to ban the use of chemical synthetic flocculants in water treatment, especially in food fermentation and medicine. As the third generation of flocculants, biological flocculants are polysaccharides or protein substances with excellent properties such as safety, biodegradability and biocompatibility. In addition to using them alone, bioflocculants can be used synergistically with metal ions. Due to the different mechanisms of action of the two, a smaller and safer dose is sufficient to destabilize the colloidal particles.

[0003] Microbial polysaccharides have antioxidant, immunomodulatory, moisture-absorbing and flocculating functions, and are widely used in many industries. A variety of microbial strains have been used to produce polysaccharides, such as Xanthomonascampestris NRRL B-1459 for xanthan gum, Sphingomonas paucimobilis ATCC 31461 for gellan gum, and Streptococcus sp.ID9102 for hyaluronic acid. The development of new microbial polysaccharides is of great significance. However, the prior art does not disclose the production of new extracellular polysaccharides by Klebsiella and their applications, and there is no report on the use of microbial flocculants in conjunction with metal ions to treat wastewater under acidic conditions. Summary of the invention

[0004] The purpose of the present invention is to provide Klebsiella YCTU3162 and the extracellular polysaccharide Flocan produced by it and its application, aiming to solve the problems raised in the above background technology.

[0005] To achieve the above object, the present invention provides the following technical solutions:

[0006] Klebsiella sp.YCTU3162, the preservation unit is China Center for Type Culture Collection, the preservation number is CCTCC NO: M 20232027, and the preservation date is October 25, 2023.

[0007] The exopolysaccharide Flocan is produced by the Klebsiella YCTU3162.

[0008] Furthermore, the structure of the extracellular polysaccharide Flocan is:

[0009]

[0010] The extracellular polysaccharide Flocan comprises glucuronic acid, glucose, mannose and galactose in a molar ratio of 1:2:3:1, and has a pyruvic acid modification group.

[0011] The production method of the extracellular polysaccharide Flocan comprises the following steps:

[0012] Step 1, inoculating a single colony of Klebsiella YCTU3162 into a seed solution for culturing to obtain a seed solution;

[0013] Step 2, inoculating the seed liquid into the fermentation medium, fermenting to obtain a fermentation liquid, and centrifuging the fermentation liquid to collect the supernatant;

[0014] Step 3, adding 0.1% NaOH to the supernatant obtained in step 2 and mixing, boiling in a boiling water bath, and collecting the supernatant by centrifugation;

[0015] Step 4: Add ethanol to the supernatant obtained in step 3, collect the precipitate by centrifugation, and obtain the extracellular polysaccharide Flocan.

[0016] Further, in step 1, the pH of the seed liquid culture medium is 7, and the seed liquid culture medium is obtained by mixing A and B, wherein the components of liquid A include: NaH2PO4 1g / L, MgSO4·7H2O 0.2g / L, FeSO4 0.0125g / L, CaCl2 0.07g / L, ZnCl20.0075g / L, MnSO4 0.003g / L, yeast powder 4g / L and sucrose 30g / L; the components of liquid B include: NaH2PO4 7.92g / L and Na2HPO4·2H2O23.86g / L;

[0017] The seed liquid culture conditions are: culture temperature is 37°C, rotation speed is 230rpm, and time is 10 to 12 hours.

[0018] Further, in step 2, the pH of the fermentation broth medium is 7, and the fermentation broth medium includes NaH2PO4 0.5 g / L, MgSO4·7H2O 0.8 g / L, FeSO4 0.05 g / L, CaCl2 0.08 g / L, ZnCl2 0.0015 g / L, MnSO4 0.012 g / L, yeast powder 4 / L and sucrose 30 g / L;

[0019] The fermentation liquid culture temperature is 37°C, the rotation speed is 230 rpm, and the time is 30 to 36 hours.

[0020] Furthermore, in step 3, the boiling water bath is used for 10 minutes, the centrifugal speed is 5000 rpm, and the centrifugal time is 10 minutes.

[0021] Furthermore, in step 4, the amount of ethanol added is 3 times the volume of the supernatant, the centrifugal speed is 5000 rpm, and the centrifugal time is 10 min.

[0022] The application of the above-mentioned extracellular polysaccharide Flocan in wastewater flocculation.

[0023] Furthermore, the extracellular polysaccharide Flocan flocculates kaolin and coal ash wastewater in an acidic condition by synergistically acting with metal ion FeCl3.

[0024] Compared with the prior art, the present invention has the following beneficial effects:

[0025] The method for producing the extracellular polysaccharide Flocan by Klebsiella YCTU3162 is simple and easy, with a short fermentation cycle and mild conditions; the extracellular polysaccharide Flocan can be used as a biological flocculant, has good flocculation activity on simulated kaolin and coal ash wastewater in synergistic effect with metal ion FeCl3 under acidic conditions, is biodegradable, has no secondary pollution, and has good economic and social benefits. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 It is a plate colony of the Klebsiella YCTU3162 strain in the present invention.

[0027] Figure 2 The ion chromatograms of the Flocan component determination in the present invention are shown in Figure A. Figure A is a standard monosaccharide spectrum; Figure B is a monosaccharide composition analysis spectrum of Flocan. The labels in the figure are: Man-mannose, GlcA-glucuronic acid, Glc-glucose, Gal-galactose.

[0028] Figure 3 It is the one-dimensional nuclear magnetic resonance spectrum of Flocan in the present invention. Wherein, Figure A is 1 H NMR; Figure B is 13C NMR.

[0029] Figure 4 The two-dimensional nuclear magnetic resonance spectrum of Flocan in the present invention is shown in Figure A, which is COSY, Figure B is HSQC, Figure C is HMBC, Figure D is TOCSY, and Figure E is NOESY.

[0030] Figure 5 In the figure, Figure A shows the turbidity removal rate of kaolin suspension by Flocan alone under different pH conditions in the present invention; Figure B shows the turbidity removal rate of coal ash suspension by Flocan alone under different pH conditions.

[0031] Figure 6 The experimental results of the flocculation activity of the simulated kaolin suspension by the synergistic effect of Flocan and FeCl3 of the present invention are shown in Figure A. Figure A is the flocculation result of the kaolin suspension at 1-2-4-8min; Figure B is the flocculation result of the HF+Fe experimental group at 1min; Figure C is the statistical diagram of turbidity removal rate; Figure D is the particle size distribution diagram of flocculated particles; Figure E is a SEM photo.

[0032] Figure 7 The experimental results of the flocculation activity of the simulated coal ash suspension by the synergistic effect of Flocan and FeCl3 of the present invention are shown in Figure A. Figure A is the flocculation result of the coal ash suspension at 1-2-3-5min; Figure B is the flocculation result of the HF+Fe experimental group at 1min; Figure C is the statistical diagram of turbidity removal rate; Figure D is the particle size distribution diagram of flocculated particles; Figure E is a SEM photo. DETAILED DESCRIPTION

[0033] In order to make the purpose, technical solution and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0034] The specific implementation of the present invention is described in detail below in conjunction with specific embodiments.

[0035] The Klebsiella sp. YCTU3162 provided in one embodiment of the present invention is deposited in the China Center for Type Culture Collection, with a deposit number of CCTCC NO: M 20232027, a deposit date of October 25, 2023, and a deposit address of Wuhan University, Luojia Mountain, Bayi Road, Wuchang District, Wuhan City, Hubei Province, China.

[0036] The extracellular polysaccharide Flocan provided in one embodiment of the present invention is produced by the Klebsiella YCTU3162.

[0037] As a preferred embodiment of the present invention, the extracellular polysaccharide Flocan comprises glucuronic acid, glucose, mannose and galactose in a molar ratio of 1:2:3:1, and has a pyruvic acid modification group.

[0038] An embodiment of the present invention provides a method for producing the extracellular polysaccharide Flocan, comprising the following steps:

[0039] Step 1, inoculating a single colony of Klebsiella YCTU3162 into a seed solution for culturing to obtain a seed solution;

[0040] Step 2, inoculating the seed liquid into the fermentation medium, fermenting to obtain a fermentation liquid, and centrifuging the fermentation liquid to collect the supernatant;

[0041] Step 3, adding 0.1% NaOH to the supernatant obtained in step 2 and mixing, boiling in a boiling water bath, and collecting the supernatant by centrifugation;

[0042] Step 4: Add ethanol to the supernatant obtained in step 3, collect the precipitate by centrifugation, and obtain the extracellular polysaccharide Flocan.

[0043] As a preferred embodiment of the present invention, in step 1, the pH of the seed liquid culture medium is 7, and the seed liquid culture medium is obtained by mixing A and B (1:1, v:v), and the components of liquid A include: NaH2PO4

[0044] 1g / L, MgSO4·7H2O 0.2g / L, FeSO4 0.0125g / L, CaCl2 0.07g / L, ZnCl20.0075g / L, MnSO4 0.003g / L, yeast powder 4g / L and sucrose 30g / L; Liquid B ingredients include: NaH2PO4 7.92g / L and Na2HPO4·2H2O23.86g / L;

[0045] The seed liquid culture conditions are: culture temperature is 37°C, rotation speed is 230rpm, and time is 10 to 12 hours.

[0046] As a preferred embodiment of the present invention, in step 2, the pH of the fermentation broth medium is 7, and the fermentation broth medium includes NaH2PO4 0.5 g / L, MgSO4·7H2O 0.8 g / L, FeSO4 0.05 g / L, CaCl2 0.08 g / L, ZnCl2 0.0015 g / L, MnSO4 0.012 g / L, yeast powder 4 / L and sucrose 30 g / L;

[0047] The fermentation liquid culture temperature is 37°C, the rotation speed is 230 rpm, and the time is 30 to 36 hours.

[0048] As a preferred embodiment of the present invention, in step 3, the boiling time in the boiling water bath is 10 minutes, the centrifugal speed is 5000 rpm, and the centrifugal time is 10 minutes.

[0049] As a preferred embodiment of the present invention, in step 4, the amount of ethanol added is 3 times the volume of the supernatant, the centrifugal speed is 5000 rpm, and the centrifugal time is 10 min.

[0050] An embodiment of the present invention provides the use of the extracellular polysaccharide Flocan in wastewater flocculation.

[0051] As a preferred embodiment of the present invention, the extracellular polysaccharide Flocan flocculates kaolin and coal ash wastewater under acidic conditions (pH 1-7) by synergistically acting with metal ions FeCl3.

[0052] Example 1, Screening, Isolation and Strain Identification of Klebsiella YCTU3162:

[0053] Take soil from a certain place in the Yancheng tidal flat, dissolve 1g of soil sample in liquid culture medium, obtain suspension by low-speed centrifugation, dilute and spread on solid culture medium, culture at 37℃ for 2-3 days, and after the colonies grow, pick single colonies with sticky polysaccharides on the surface according to the morphological characteristics of the colonies, dilute and spread on new solid culture medium, culture at 37℃, pick single colonies for dilution and spread after the colonies grow, and repeat this process until the colonies grown on the solid culture medium have a single morphology. The screening culture medium is: KH2PO4 1g / L, CaCl2 0.1g / L, MgCl2·6H2O 0.3g / L, FeSO4 0.0125g / L, KNO3 2g / L, Sucrose 20g / L, pH 7, and solid culture medium needs to add 15g / L agar. The culture medium is sterilized at 121℃ for 20min.

[0054] Morphological and physiological and biochemical tests were performed on a single strain. The colonies were round and smooth in surface. The strain secreted a large amount of extracellular polysaccharides and produced no pigment. Figure 1 The left picture shows a single colony on the flat culture medium, and the right picture shows a large amount of white mucopolysaccharide produced.

[0055] The 16S rRNA gene sequence of the strain was determined, as shown in the sequence table (SEQ ID NO.1), and the 16S rRNA gene sequence of the strain was compared with the gene sequence in the GenBank database for homology and the results were analyzed to determine the species of the bacteria at the molecular biological level. After Blast comparison, the similarity with Klebsiella quasipneumoniae sp.T-3-1 was 99.93%, and the sequence similarity with Klebsiella quasipneumoniae strain BB-301 was as high as 99.86%, belonging to the genus Klebsiella, and named Klebsiella sp.YCTU3162.

[0056] Example 2: Production and purification method of the exopolysaccharide Flocan produced by Klebsiella YCTU3162, the specific steps are as follows:

[0057] Inoculate a single colony of Klebsiella YCTU3162 into a seed solution for cultivation to obtain a seed solution;

[0058] (1) Preparation of seed solution: The seed solution culture medium is prepared by mixing A and B (1:1, v:v): A solution composition: NaH2PO4 1g / L, MgSO4·7H2O 0.2g / L, FeSO4 0.0125g / L, CaCl2 0.07g / L, ZnCl2 0.0075g / L, MnSO4 0.003g / L, yeast powder 4g / L and sucrose 30g / L; B solution composition: NaH2PO4 7.92g / L and Na2HPO4·2H2O 23.86g / L, pH 7. The culture medium is sterilized at 121℃ for 20min;

[0059] (2) inoculating a single bacterial colony into the culture medium sterilized in step (1), and culturing at a temperature of 37° C. and a rotation speed of 230 rpm for 10 to 12 hours to obtain a seed culture solution;

[0060] (3) Preparation of fermentation broth: The fermentation broth medium is NaH2PO4 0.5 g / L, MgSO4·7H2O0.8 g / L, FeSO40.05 g / L, CaCl2 0.08 g / L, ZnCl2 0.0015 g / L, MnSO4 0.012 g / L, yeast powder 4 / L and sucrose 30 g / L, with a pH of 7. Sterilize the medium at 121°C for 20 min;

[0061] (4) adding the seed culture solution obtained in step (2) to the sterilized culture medium in step (3), and fermenting at a temperature of 37° C. and a rotation speed of 230 rpm for 30 to 36 hours to obtain a fermentation solution;

[0062] (5) centrifuging the fermentation broth obtained in step (4) to collect the supernatant, adding 0.1% NaOH to mix, boiling in a boiling water bath for 10 min, and centrifuging at 5000 rpm for 10 min to collect the supernatant;

[0063] (6) Add 3 volumes of ethanol to the supernatant obtained in step (5), centrifuge at a speed of 5000 rpm for 10 min, and precipitate to obtain pure Flocan polysaccharide.

[0064] Example 3: The structure of the exopolysaccharide Flocan produced by Klebsiella YCTU3162 is analyzed as follows:

[0065] (1) The molecular weight of Flocan was determined to be 2736 KDa by gel permeation chromatography.

[0066] (2) Flocan was hydrolyzed with trifluoroacetic acid, washed with methanol, dissolved in sterile water, and then analyzed by ion chromatography. The same components appeared at the same position. Figure 2 It can be seen that Flocan is an acidic heteropolysaccharide composed of glucuronic acid, glucose, mannose, and galactose in a molar ratio of 1:2:3:1.

[0067] (3) Flocan was partially hydrolyzed with trifluoroacetic acid, prepared with D2O to 80 mg / ml, and NMR analysis was performed using TMSP as an internal standard. The one-dimensional NMR spectrum of Flocan is shown in Figure 3 Flocan's 2D NMR spectrum can be found at Figure 4 .

[0068] Specifically speaking, 1 In H NMR, the chemical shift of the α anomeric proton is at δ5-6ppm, AE is the α conformation, the chemical shift of the β anomeric proton is at δ4-5ppm, F and G are attributed to the β conformation, and the chemical shift of δ1.47ppm is attributed to -CH3 of pyruvate. 13 In C NMR, there are 7 signals between δ96-106ppm, indicating that Flocan has 7 anomeric carbons, and the chemical shift δ27.41ppm is attributed to -CH3 of pyruvic acid, and δ178.08 is attributed to -COOH of glucuronic acid and pyruvic acid. The chemical shifts of sugar residues and the connection mode of monosaccharide residues were further analyzed by HSQC, COSY, TOCSY, HMBC and NOESY. Combining the results of one-dimensional and two-dimensional NMR and literature and database prediction analysis, the chemical shifts of Flocan are summarized in Table 1. The bold chemical shifts in the table are the substitution sites of sugar residues.

[0069] Table 1 Flocan 1 H NMR and 13 C NMR chemical shift (ppm)

[0070]

[0071]

[0072] Combined with the above analysis, it can be determined that the structure of the extracellular polysaccharide Flocan produced by Klebsiella YCTU3162 is:

[0073]

[0074] Example 4: Determination of the flocculation activity test (turbidity removal rate) of the extracellular polysaccharide Flocan produced by Klebsiella YCTU3162. Kaolin suspension and coal ash suspension were used as simulated wastewater to measure the flocculation activity of the single Flocan polysaccharide under different pH conditions:

[0075] Prepare Flocan solution (1g / L) in advance. For kaolin suspension, prepare 20mL of kaolin suspension (mass concentration is 4g / L), add 100μL of polysaccharide solution for flocculation experiment. Adjust different initial pH, measure the absorbance of the initial kaolin suspension, stir magnetically (stir at 150rpm for 2min, let stand for 10min), and measure the change of absorbance of the supernatant. Measure the absorbance of the flocculation system at 550nm to calculate the turbidity removal rate.

[0076] The turbidity removal rate calculation formula is: turbidity removal rate = (A0-A1) / A0×100%; where A0 is the absorbance of the initial kaolin suspension, and A1 is the absorbance of the suspension in the experimental group.

[0077] For the coal ash suspension, 20 mL of coal ash suspension (mass concentration of 10 g / L) was prepared, and 100 μL of polysaccharide solution was added for flocculation experiment. Different initial pH values ​​were adjusted, magnetic stirring was performed (stirring at 150 rpm for 2 min, standing for 10 min), the change of absorbance was measured in time, and the turbidity removal rate was calculated.

[0078] See also Figure 5 It can be seen that in kaolin (Figure A) and coal ash suspension (Figure B), the addition of extracellular polysaccharides can achieve a turbidity removal rate of more than 70% at pH 1-7; the turbidity removal rate is lower under alkaline conditions.

[0079] Example 5: Determination of flocculation activity test (turbidity removal rate) of the synergistic effect of the exopolysaccharide Flocan produced by Klebsiella YCTU3162 and the metal ion FeCl3, using kaolin suspension and coal ash suspension as simulated wastewater;

[0080] Two concentrations of polysaccharide solutions (1g / L-LF and 2g / L-HF) were prepared in advance. For the kaolin suspension, 20mL of kaolin suspension (mass concentration of 4g / L) was prepared. 100μL of extracellular polysaccharide and 200μL FeCl3 solution (50g / L-Fe) were added to different groups for flocculation experiments. The kaolin suspension without flocculant was used as the control. Under acidic conditions, magnetic stirring (stirring at 150rpm for 2min, standing for 10min), taking pictures to record the phenomenon, and measuring the change of absorbance in time. The absorbance of the control and flocculation system was measured at 550nm to calculate the turbidity removal rate. During the measurement process, the particle size distribution of the flocculated suspension was measured, the flocculated particles were collected, and the surface morphology was photographed by SEM after freeze-drying.

[0081] Figure 6 This is a graph showing the flocculation results of kaolin suspension simulated wastewater at pH 3. As the flocculation time increases (1-2-4-8 min), the suspension becomes clearer. The HF+Fe experimental group becomes clearer after 8 min (Fig. A). Large flocculated particles are clearly seen in the high-dose polysaccharide and FeCl3 (HF+Fe) experimental group at 1 min (Fig. B). After 8 min, the turbidity removal rate can reach 99.37% (Fig. C). The particle size (Fig. D) and SEM (Fig. E) of the flocculated particles are shown in the figure, indicating that the flocculated particles are enlarged. The synergistic effect of Flocan and FeCl3 of the present invention accelerates the flocculation rate and has good flocculation activity for the simulated kaolin wastewater.

[0082] For the coal ash suspension, 20 mL of coal ash suspension (mass concentration of 10 g / L) was prepared, and 100 μL of extracellular polysaccharide and 200 μL of FeCl3 solution (50 g / L) were added to different groups for flocculation experiments, with the coal ash suspension without flocculant as the control. Under acidic conditions, magnetic stirring (stirring at 150 rpm for 2 min, standing for 10 min), taking pictures to record the phenomenon, and measuring the change in absorbance in time. The absorbance of the control and flocculation system was measured at 550 nm to calculate the turbidity removal rate. During the measurement process, the particle size distribution of the flocculation suspension was measured, the flocculated particles were collected, and the surface morphology was photographed by SEM after freeze-drying.

[0083] Figure 7The figure is a flocculation result of simulated wastewater of coal ash suspension at pH 3. As the flocculation time increases (1-2-3-5min), the suspension can be clearly seen to be clarified. Coal ash wastewater settles faster than kaolin wastewater. The HF+Fe experimental group is clarified after 5min (Figure A). Large flocculated particles are clearly seen in the high-dose polysaccharide and FeCl3 (HF+Fe) experimental group at 1min (Figure B). The turbidity removal rate can reach 99.44% after 5min (Figure C). The particle size (Figure D) and SEM (Figure E) of the flocculated particles indicate that the flocculated particles increase after the addition of the flocculant. The Flocan of the present invention and FeCl3 work together to accelerate the flocculation rate and have good flocculation activity for simulated coal ash wastewater.

[0084] The above are only preferred embodiments of the present invention. It should be pointed out that, for those skilled in the art, several modifications and improvements can be made without departing from the concept of the present invention. These should also be regarded as the protection scope of the present invention. These will not affect the effect of the implementation of the present invention and the practicality of the patent.

Claims

1. Klebsiella sp.YCTU3162, the depository is China Center for Type Culture Collection, the deposit number is CCTCC NO: M 20232027, and the deposit date is October 25, 2023.

2. A method for producing extracellular polysaccharide Flocan, characterized in that: The following steps are involved: Step 1, inoculating a single colony of Klebsiella sp. YCTU3162 described in claim 1 into a seed liquid culture medium for culturing to obtain a seed liquid; Step 2, inoculating the seed liquid into the fermentation medium, fermenting to obtain a fermentation liquid, and centrifuging the fermentation liquid to collect the supernatant; Step 3, adding 0.1% NaOH to the supernatant obtained in step 2 and mixing, boiling in a boiling water bath, and collecting the supernatant by centrifugation; Step 4, adding ethanol to the supernatant obtained in step 3, collecting the precipitate by centrifugation, and obtaining the extracellular polysaccharide Flocan; In the step 1, the pH of the seed liquid culture medium is 7, and the seed liquid culture medium is obtained by mixing A and B, wherein the components of liquid A include: NaH2PO4 1g / L, MgSO4·7H2O 0.2g / L, FeSO4 0.0125g / L, CaCl20.07g / L, ZnCl2 0.0075g / L, MnSO4 0.003g / L, yeast powder 4g / L and sucrose 30g / L; the components of liquid B include: NaH2PO4 7.92g / L and Na2HPO4·2H2O23.86g / L; The seed solution culture conditions were as follows: culture temperature was 37°C, rotation speed was 230 rpm, and time was 10 to 12 h; In the step 2, the pH of the fermentation broth medium is 7, and the fermentation broth medium includes 0.5 g / L NaH2PO4, 0.8 g / L MgSO4·7H2O, 0.05 g / L FeSO4, 0.08 g / L CaCl2, 0.0015 g / L ZnCl2, 0.012 g / L MnSO4, 4 g / L yeast powder and 30 g / L sucrose; The fermentation liquid culture temperature is 37°C, the rotation speed is 230rpm, and the time is 30 to 36 hours.

3. The method according to claim 2, characterized in that In step 3, the boiling water bath is used for 10 min, the centrifugal speed is 5000 rpm, and the centrifugal time is 10 min.

4. The method according to claim 2, characterized in that: In step 4, the amount of ethanol added is 3 times the volume of the supernatant, the centrifugal speed is 5000 rpm, and the centrifugal time is 10 min.

5. An extracellular polysaccharide Flocan prepared according to the method according to any one of claims 2 to 4, characterized in that: The structure of the extracellular polysaccharide Flocan is: The extracellular polysaccharide Flocan comprises glucuronic acid, glucose, mannose and galactose in a molar ratio of 1:2:3:1, and has a pyruvic acid modification group.

6. Use of the extracellular polysaccharide Flocan according to claim 5 in wastewater flocculation.

7. The use according to claim 6, characterized in that: The extracellular polysaccharide Flocan flocculates kaolin and coal ash wastewater in an acidic condition by synergistically acting with the metal ion FeCl3.

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