Paenibacillus polymyxa FangSG106 as well as application, product and method thereof

Abstract

The invention relates to flocculation application of paenibacillus polymyxa FangSG106, a product of the paenibacillus polymyxa FangSG106 and a method of the product, and belongs to the technical field of microorganisms. The invention provides a flocculation or water purification application of paenibacillus polymyxa FangSG106 with a preservation number of CGMCC (China General Microbiological Culture Collection Center) No.31655 and an application of the paenibacillus polymyxa FangSG106 in preparation of a biological flocculant or a water purifying agent, further provides the biological flocculant or the water purifying agent based on the paenibacillus polymyxa FangSG106, and also provides a method for removing heavy metals and a water purifying method. The paenibacillus polymyxa FangSG106 disclosed by the invention has broad-spectrum antibacterial activity, and can be used for effectively removing heavy metals and purifying water.

Description

Technical Field

[0001] The invention belongs to the field of microbial technology, in particular to Paenibacillus polymyxa FangSG106 and its application, product and method. Background Art

[0002] Water pollution caused by organic and inorganic pollutants has become an important issue of this era, and the treatment and removal of pollutants in water bodies has become a difficult but necessary task. For the treatment of sewage, there are many methods and treatment technologies such as electrocoagulation, mixed ion exchange materials, membrane filtration, activated carbon, etc., which play an important role in treatment. However, the disadvantages of these methods are that they are either expensive or produce pollutants.

[0003] Compared with inorganic and organic flocculants, naturally occurring bioflocculants are environmentally friendly due to their biodegradable and non-toxic properties, as well as avoiding secondary pollution problems. Bioflocculants are active substances produced by the growth of microorganisms, and are composed of macromolecular polymers such as glycoproteins, polysaccharides, proteins, cellulose and nucleic acids. In recent years, the cultivation of bioflocculants has attracted much attention due to their excellent flocculation activity and biodegradability, which can create a green and healthy environment. Bioflocculants play a key role in treating pollutants in water and wastewater, but low flocculation performance and large dosage have always been the main problems in the development of bioflocculants in actual wastewater treatment. The use of renewable resources to produce new, cost-effective and efficient bioflocculants has become a pressing issue.

[0004] The production of bioflocculants has been committed to finding and screening new bioflocculant microorganisms with high yield, high flocculation activity and low production cost. Microorganisms of the genus Bacillus are generally facultative anaerobes or obligate aerobic bacteria, generally have strong tolerance and are widely distributed in nature. Screening and identifying Bacillus with bioflocculant ability is of great significance.

[0005] In addition, compared with industrial wastewater and domestic wastewater, aquaculture wastewater is mainly composed of nutrients such as carbon, nitrogen, and phosphorus, with fewer toxic metal substances and lower concentrations of nitrogen, phosphorus, SS, and chemical oxygen demand (COD). Therefore, adding microorganisms with bioflocculation ability to water bodies can effectively improve aquaculture wastewater treatment and is conducive to the development of efficient and healthy aquaculture models.

[0006] The use of microorganisms as bioflocculants has been reported, but the survival and reproduction of microorganisms have certain requirements for the water environment. Usually, there may be a large number of harmful microorganisms, pathogenic microorganisms or competing microorganisms of different categories in the use environment of bioflocculants, which requires the microorganisms as the active ingredients of bioflocculants to have certain antibacterial properties in order to better play the role of flocculants.

[0007] Therefore, there is an urgent need to develop new microbial strains that have both broad-spectrum antibacterial properties and flocculation effects. Summary of the invention

[0008] For the purpose of developing a new microbial strain having both broad-spectrum antibacterial properties and flocculation effects, the present invention provides the flocculation use of Paenibacillus polymyxa FangSG106 and its products and methods.

[0009] The technical solution of the present invention is as follows:

[0010] The flocculation or water purification use of Paenibacillus polymyxa FangSG106 with the deposit number of CGMCC No.31655.

[0011] The water purification includes: adsorbing heavy metal ions, inhibiting water pathogens, and improving water quality.

[0012] The heavy metal ions include: Cu 2+ 、Ni 2+ ;

[0013] Preferably, the water pathogens include: Aeromonas vernix, Aeromonas scutellariae, Aeromonas punctatus, Vibrio splendens, and Vibrio anguillarum;

[0014] Preferably, improving water quality includes reducing total nitrogen, total phosphorus, COD and suspended solids in the water.

[0015] The invention discloses a use of Paenibacillus polymyxa FangSG106 with the deposit number of CGMCC No.31655 for preparing a biological flocculant or a water purifier.

[0016] A biological flocculant or water purifier comprises active ingredients, wherein the active ingredients include: Paenibacillus polymyxa FangSG106 with a preservation number of CGMCC No.31655.

[0017] The biological flocculant or water purifier also includes auxiliary materials.

[0018] A method for removing heavy metals, wherein Paenibacillus polymyxa FangSG106 with a preservation number of CGMCC No.31655 is added to a material to be treated containing heavy metals.

[0019] The heavy metals include: Cu 2+ 、Ni 2+ ;

[0020] Preferably, the concentration of Paenibacillus polymyxa FangSG106 in the material to be treated is 5 g / L.

[0021] A water purification method adopts Paenibacillus polymyxa FangSG106 with a preservation number of CGMCC No.31655 to purify water.

[0022] The concentration was 1×10 8 CFU / mL of Paenibacillus polymyxa FangSG106 was put into the water to be purified.

[0023] The beneficial effects of the present invention are as follows:

[0024] The Paenibacillus polymyxa FangSG106 provided by the present invention with a deposit number of CGMCC No.31655 has been confirmed to have both broad-spectrum antibacterial properties and flocculation ability, with a high flocculation rate, and can effectively inhibit water pathogens, purify water quality and remove heavy metals. The antibacterial spectrum of Paenibacillus polymyxa FangSG106 includes: Rhizoctonia solani, rice blast fungus, sclerotinia, Fusarium oxysporum, Fusarium oxysporum cucumber transformation, cabbage wilt fungus, gray mold fungus, Aeromonas verticillasteri, Aeromonas sedata, Aeromonas punctata, Vibrio splenici and Vibrio anguillarum. The flocculation rate of Paenibacillus polymyxa FangSG106 is as high as 97.34% and the spore rate is as high as 98.59%. It can purify water quality by effectively reducing total nitrogen, total phosphorus, COD, and suspended solids in water bodies, and can reduce Cu 2+ and Ni 2+ The adsorption rate of equal heavy metal ions is over 59%, with good effect and good application prospect.

[0025] The deposit information of Paenibacillus polymyxa FangSG106 of the present invention is as follows:

[0026] Deposit number: CGMCC No.31655

[0027] Storage date: August 12, 2024

[0028] Classification name: Paenibacillus polymyxa

[0029] Depository: China National Microbiological Culture Collection Administration General Microbiology Center

[0030] Deposit address: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 This is the inhibitory effect of Paenibacillus polymyxa FangSG106 of Experimental Example 2 of the present invention on seven pathogenic fungal strains: (a) Rhizoctonia solani, (b) Magnaporthe oryzae, (c) Sclerotinia sclerotiorum, (d) Fusarium oxysporum, (e) Fusarium oxysporum cucumber transformed type, (f) Fusarium wilt, and (g) Botrytis cinerea. The upper figure is the CK group and the lower figure is the strain FangSG106 group.

[0032] Figure 2 This is a microscopic morphological picture of the spores of Paenibacillus polymyxa FangSG106 in Experimental Example 4 of the present invention.

[0033] Figure 3 This is a graph showing the experimental results of the effect of environmental pH on the flocculation activity of strain FangSG106 in Experimental Example 5 of the present invention.

[0034] Figure 4 This is a graph showing the experimental results of the effect of ambient temperature on the flocculation activity of strain FangSG106 in Experimental Example 6 of the present invention. DETAILED DESCRIPTION

[0035] The present invention is further described in detail below with reference to specific embodiments, experimental examples and drawings, but the embodiments of the present invention are not limited thereto.

[0036] 1. Sources of biological materials

[0037] (i) The pathogenic fungi used in Experimental Example 2 of the present invention: Rhizoctonia solani, Sclerotinia sclerotiorum and Fusarium oxysporum f. sp. conglutinans were kindly donated by the National Key Laboratory of Microbiology, Huazhong Agricultural University.

[0038] Pathogenic fungi: Magnaporthe oryzae and Fusarium oxysporum were kindly donated by Beijing Academy of Agriculture and Forestry Sciences.

[0039] Pathogenic fungi: Fusarium oxysporum cucumber transformation type ACCC39326 (Fusarium oxysporum) and Botrytis cinerea ACCC37347 (Botrytis cinerea) were purchased from China Agricultural Microbiological Culture Collection Center.

[0040] Pathogenic bacteria: Pseudomonas syringae, Agrobacterium tumefaciens, Acidovorax citrulli and Xanthomonas citrisubsp. citri were isolated and obtained in our laboratory in the early stage. Clostridioides difficile ATCC43593 was purchased from Guangdong Microbial Culture Collection Center. Serratia marcescens was donated by the Institute of Agricultural Resources and Agricultural Zoning, Chinese Academy of Agricultural Sciences.

[0041] The applicant promises to distribute the above-mentioned strain to the public within 20 years from the filing date of the present invention for the purpose of verifying the technical effects of the present invention.

[0042] (ii) Aeromonas veronii, Aeromonas jandaei, Aeromonas punctata, Vibriosplendidus and Vibrio anguillarum of Experimental Example 10 of the present invention were isolated and obtained from diseased fish tissues or water environment samples in the early stage of this laboratory.

[0043] Aeromonas veronii was isolated and obtained by the applicant's laboratory from diseased fish tissues or water environment samples in the early stage. It is the Aeromonas veronii recorded in the article "Chen F, Sun J, Han Z, et al. Isolation, Identification and Characteristics of Aeromonas veronii From Diseased Crucian Carp (Carassiusauratus gibelio). Front Microbiol, 2019, 10: 2742." The applicant promises to release it to the public within 20 years from the date of application of the present invention for the purpose of verifying the technical effect of the present invention.

[0044] Aeromonas jandaei was isolated and obtained by the applicant's laboratory from diseased fish tissues or water environment samples in the early stage. It is the Aeromonas jandaei recorded in the article "Chen M, Xue M, Chen J, et al. Isolation, identification and characterization of Aeromonas jandaei from diseased Chinese soft-shell turtles. J Fish Dis. 2024, 47(4): e13919." The applicant promises to release it to the public within 20 years from the date of application of the present invention for the purpose of verifying the technical effect of the present invention.

[0045] Aeromonas punctata was isolated by the applicant's laboratory from diseased fish tissues or water environment samples in the early stage. It is the Aeromonas punctata recorded in the article "Yingna, Wang Yuan, Qin Bo, et al. Isolation, identification and drug sensitivity test of pathogenic bacteria of discus fish enteritis [J]. Fisheries Science and Technology Information, 2020, 47(05): 245-248." The applicant promises to release it to the public within 20 years from the date of application of this invention to verify the technical effect of the invention.

[0046] Vibrio splendidus was isolated and obtained by the applicant's laboratory from diseased fish tissues or water environment samples in the early stage. It is the Vibrio splendidus recorded in the article "Ningning Liu, Shanshan Zhang, Weiwei Zhang, et al. Vibrio sp. 33a potential bacterial antagonist of Vibrio splendidus pathogenic to sea cucumber (Apostichopus japonicus). Aquaculture, 2017, 470: 68-73." The applicant promises to release it to the public within 20 years from the date of application of the present invention for the purpose of verifying the technical effect of the present invention.

[0047] Vibrio anguillarum was isolated and obtained by the applicant's laboratory from diseased fish tissues or water environment samples in the early stage. It is the Vibrio anguillarum recorded in the article "Yang N, Sun C. The Inhibition and Resistance Mechanisms of Actinonin, Isolated from Marine Streptomyces sp. NHF165, against Vibrio anguillarum. Front Microbiol. 2016, 13; 7: 1467." The applicant promises to release it to the public within 20 years from the date of application of this invention for the purpose of verifying the technical effect of the invention.

[0048] 2. Experimental culture medium

[0049] Seed culture medium formula: glucose 10g, K2HPO4 0.3g, MgSO4 7H2O 0.5g, CaCO3 3g, NaCl0.3g, K2SO4 0.3g, yeast powder 1g, distilled water 1L, pH 7.0-7.2, sterilize at 121℃ for 20min for use.

[0050] Optimized culture medium formula: sucrose 8g, glucose 4g, corn syrup powder 6g, K2HPO4 0.3g, MgSO47H2O 0.5g, NaCl 0.3g, K2SO4 0.3g, yeast powder 0.75g, ammonium sulfate 0.75g, distilled water 1L, pH 7.0-7.2, sterilize at 121℃ for 20min for use.

[0051] TSA, TSB and 2216E culture media were purchased from Qingdao Haibo Biotechnology Co., Ltd.

[0052] Nickel ion mother solution: Use sterile water to prepare nickel ion mother solution with a concentration of 1g / L, adjust the pH to 7, and sterilize at 121℃ for 20min for use.

[0053] Copper ion stock solution: Use sterile water to prepare a copper ion stock solution with a concentration of 1g / L, adjust the pH to 7, and sterilize at 121℃ for 20min for use.

[0054] Group 1 Example: Flocculation or water purification use of FangSG106 of the present invention

[0055] This group of embodiments provides the flocculation or water purification use of Paenibacillus polymyxa FangSG106 with a deposit number of CGMCC No.31655.

[0056] In some embodiments, the water purification includes: adsorbing heavy metal ions, inhibiting water pathogens, and improving water quality.

[0057] In a specific embodiment, the heavy metal ions include: Cu 2+ 、Ni 2+ ;

[0058] Preferably, the water pathogens include: Aeromonas vernix, Aeromonas scutellariae, Aeromonas punctatus, Vibrio splendens, and Vibrio anguillarum;

[0059] Preferably, improving water quality includes reducing total nitrogen, total phosphorus, COD and suspended solids in the water.

[0060] The second group of embodiments, use of FangSG106 of the present invention for preparing biological flocculants or water purifiers This group of embodiments provides the use of Paenibacillus polymyxa FangSG106 with a deposit number of CGMCC No.31655 for preparing biological flocculants or water purifiers.

[0061] In some embodiments, the active ingredient of the bioflocculant or water purifier includes: Paenibacillus polymyxa FangSG106 with a deposit number of CGMCC No.31655.

[0062] In a further embodiment, the biological flocculant or water purifier further includes: auxiliary materials.

[0063] In a more specific embodiment, the excipient is selected from: solvents, propellants, solubilizers, cosolvents, emulsifiers, colorants, adhesives, disintegrants, fillers, lubricants, wetting agents, osmotic pressure regulators, stabilizers, glidants, flavoring agents, preservatives, suspending agents, coating materials, fragrances, anti-adhesives, integrities, penetration enhancers, pH regulators, buffers, plasticizers, surfactants, foaming agents, defoamers, thickeners, inclusion agents, humectants, absorbents, diluents, flocculants, deflocculating agents, filter aids, release retardants, etc.

[0064] According to the content of the present invention, for different needs in actual production applications, combined with conventional technical means in the field of drug preparation (for example, "Encyclopedia of Preparation Technology", "Drug Preparation Technology", "Research and Application of Microbial Agent Technology", etc.), those skilled in the art can select and formulate the above-mentioned excipients, and prepare the polymyxa bacillus FangSG106 with a deposit number of CGMCC NO.31655 into different dosage forms, such as powders, tablets, gels, sprays, granules, capsules, liquids, etc.

[0065] In a specific embodiment, the dosage form of the product is selected from: one or more of powder, tablet, gel, spray, granule, capsule, liquid.

[0066] The third group of embodiments, the bioflocculant or water purifier of the present invention

[0067] This group of embodiments provides a biological flocculant or water purifier. All embodiments of this group have the following common features: the biological flocculant or water purifier includes an active ingredient, and the active ingredient includes: Paenibacillus polymyxa FangSG106 with a deposit number of CGMCC No.31655.

[0068] In a further embodiment, the biological flocculant or water purifier further includes: auxiliary materials.

[0069] In a more specific embodiment, the excipient is selected from: solvents, propellants, solubilizers, cosolvents, emulsifiers, colorants, adhesives, disintegrants, fillers, lubricants, wetting agents, osmotic pressure regulators, stabilizers, glidants, flavoring agents, preservatives, suspending agents, coating materials, fragrances, anti-adhesives, integrities, penetration enhancers, pH regulators, buffers, plasticizers, surfactants, foaming agents, defoamers, thickeners, inclusion agents, humectants, absorbents, diluents, flocculants, deflocculating agents, filter aids, release retardants, etc.

[0070] According to the content of the present invention, for different needs in actual production applications, combined with conventional technical means in the field of drug preparation (for example, "Encyclopedia of Preparation Technology", "Drug Preparation Technology", "Research and Application of Microbial Agent Technology", etc.), those skilled in the art can select and formulate the above-mentioned excipients, and prepare the polymyxa bacillus FangSG106 with a deposit number of CGMCC NO.31655 into different dosage forms, such as powders, tablets, gels, sprays, granules, capsules, liquids, etc.

[0071] In a specific embodiment, the dosage form of the product is selected from: one or more of powder, tablet, gel, spray, granule, capsule, liquid.

[0072] The fourth group of embodiments, the method for removing heavy metals of the present invention

[0073] This group of embodiments provides a method for removing heavy metals. All embodiments in this group have the following common features: Paenibacillus polymyxa FangSG106 with a deposit number of CGMCC No.31655 is added to the material to be treated containing heavy metals.

[0074] In a specific embodiment, the heavy metals include: Cu 2+ 、Ni 2+ ;

[0075] Preferably, the concentration of Paenibacillus polymyxa FangSG106 in the material to be treated is 5 g / L.

[0076] The fifth group of embodiments, the water purification method of the present invention

[0077] This group of embodiments provides a water purification method. All embodiments in this group have the following common features: using Paenibacillus polymyxa FangSG106 with a preservation number of CGMCC No.31655 to purify water.

[0078] In a specific embodiment, the concentration is 1×10 8 CFU / mL of Paenibacillus polymyxa FangSG106 was put into the water to be purified.

[0079] Experimental Example 1: Isolation and Identification of Paenibacillus polymyxa

[0080] The specific separation and purification steps are as follows:

[0081] (1) Weigh 5 g of soil sample and add it to 45 ml of sterile water. Incubate it in a constant temperature shaker at 30 °C and 200 rpm for 30 min. After standing for 20 min, obtain a sample suspension.

[0082] (2) 1.8 mL of the soil suspension supernatant obtained in step (1) was transferred to a 2 mL cryovial and treated in an 80°C water bath for 10 min;

[0083] (3) Pipette 100 μL of the sample suspension obtained in step (2) after water bath, dilute it in a 10-fold gradient, select a suitable gradient and evenly spread it on the LB solid culture medium plate, and incubate it inverted in a 30°C incubator for 48 h;

[0084] (4) picking a single colony grown on the LB solid culture medium plate in step (3), and repeatedly streaking and purifying the strain on the LB solid culture medium plate to obtain a purified Bacillus strain;

[0085] (5) Liquid culture of the purified monoclonal culture strain in step (4) was cultured, the cells were collected, genomic DNA was extracted, and the 16SrDNA fragment was amplified using the universal primers 27F and 1492R described in paragraph 58 of Chinese invention patent ZL202210478937.4. The PCR amplification product was detected by agarose gel electrophoresis, and the PCR product was sequenced. The PCR reaction system: 10×buffer 10μL, 10mM dNTP 2μL, 1μL of each upper and lower primer, 2μL of DNA template, 0.5μL of Taq enzyme, and 34μL of ddH2O. Pre-denaturation at 95℃ for 10min; then 94℃ for 30s, 60℃ for 30s, and 72℃ for 1min for a total of 35 cycles, and then extended at 72℃ for 5min. The PCR product was detected by gel electrophoresis and sent to Wuhan Jinkairui Biotechnology Co., Ltd. for sequencing. The identified gene sequence was compared in the NCBI database using the BLAST tool.

[0086] After PCR molecular identification, a total of 200 strains of Bacillus polymyxa were obtained, and the strains were preserved in ultra-low temperature glycerol tubes in the strain resource bank of Wuhan Weikang Probiotics Research Institute.

[0087] Experimental Example 2: Primary screening of antagonistic ability of Paenibacillus polymyxa and secondary screening of broad-spectrum antibacterial ability

[0088] (1) The plate confrontation method was used to determine the antagonistic effect and perform the initial screening of strains. Starting from the glycerol tube, 200 strains of Paenibacillus polymyxa were activated on LB solid culture medium plates and cultured in a constant temperature incubator at 30°C for 48 hours to grow single colonies. Four different Paenibacillus polymyxa colonies were taken from each plate and inoculated on a PDA solid plate. Then, the activated Rhizoctonia solani and Fusarium oxysporum cucumber transformation pathogenic fungi plates were taken out, and a round bacterial cake was punched out with a 5 mm diameter puncher and transferred to the center of the PDA culture medium plate. The distance between each indicator fungus and each Paenibacillus polymyxa was equal, and the plate without Paenibacillus polymyxa was used as the blank control. The inoculated plate was inverted and cultured in a 28°C incubator. After the blank control was fully grown on the plate, the results were observed. The presence of an inhibition zone was observed, the strain number was recorded, and the size of the inhibition zone was measured. The 14 strains with the highest inhibition rate were screened from the 200 strains, and further antagonistic strains were screened.

[0089] Bacterial inhibition rate (%) = (control pathogen diameter - treated pathogen diameter) / control pathogen diameter x 100%

[0090] (2) According to the screening results of step (1) of Experimental Example 2, 14 strains of Paenibacillus polymyxa were selected for broad-spectrum antibacterial ability screening using the plate confrontation method, and the experiment was repeated three times. Among them, Paenibacillus polymyxa FangSG106 had the strongest broad-spectrum antibacterial ability, as shown in Tables 1 and Figure 1As shown, the inhibition rate of the fungus against seven pathogenic fungi was higher than 60%, and the inhibition rates against Rhizoctonia solani, Sclerotinia sclerotiorum, Magnaporthe grisea, Fusarium oxysporum, Fusarium oxysporum cucumber transformed type, Fusarium wilt and Botrytis cinerea were 67.53±1.12%, 62.08±1.35%, 70.13±1.33%, 70.29±1.67%, 72.66±0.53%, 71.15±0.67% and 66.66±0.33%, respectively.

[0091] Table 1. Antibacterial results of FangSG106 strain against seven pathogenic fungi

[0092]

[0093]

[0094] (3) In addition, the Oxford cup method was used to detect the ability of the polymyxa bacillus FangSG106 strain to inhibit six pathogenic bacteria. The antibacterial experimental method is as follows:

[0095] Preparation of suspensions of four plant pathogenic bacteria: Pseudomonas syringae, Agrobacterium tumefaciens, Acidovorax citrinum and Xanthomonas citri var. citri were inoculated into LB liquid medium at a 2% inoculum, respectively, and cultured in a shaking incubator at 30°C and 220 rpm for 24 h. The bacterial suspension concentration was adjusted to 10 8 CFU / mL is reserved.

[0096] Preparation of Clostridium difficile suspension: C. difficile was inoculated into a BHI blood plate containing 5% defibrinated sheep blood and cultured at 37°C for 16-20 hours. The concentration of the bacterial suspension was adjusted to 10 8 CFU / mL.

[0097] Preparation of Serratia marcescens suspension: Serratia marcescens was inoculated into LB liquid medium at a 2% inoculum, and cultured at 37°C, 250 r / min, and the bacterial suspension concentration was adjusted to 10 8 CFU / mL.

[0098] Cool the LB solid medium (BHI solid medium for Clostridium difficile) to about 50°C and mix the pathogenic bacteria into the medium in a certain proportion so that the number of viable pathogenic bacteria is around 10 6 CFU / mL order of magnitude, and then quickly poured into a plate with an Oxford cup placed in advance. After the culture medium cooled and solidified, the Oxford cup was taken out, 150 μL of Paenibacillus polymyxa FangSG106 fermentation liquid was added to the well, and the plates were placed in a constant temperature incubator at the corresponding temperature for 24-48 hours, and then the diameter of the inhibition zone was measured.

[0099] The antibacterial results are shown in Table 2. Paenibacillus polymyxa FangSG106 has a certain antibacterial ability against the six pathogenic bacteria.

[0100] Table 2. Antibacterial results of Paenibacillus polymyxa FangSG106 against six pathogenic bacteria

[0101]

[0102] Experimental Example 3: Comparison of flocculation ability of Paenibacillus polymyxa

[0103] Prepare a 4g / L kaolin suspension, take 93ml of kaolin suspension and place it in a 100mL colorimetric tube, add 2mL of 1.0% calcium chloride solution and 5mL of strain fermentation liquid to prepare a mixture. Mix thoroughly by turning upside down, let stand at room temperature for 10 minutes, and observe whether flocculation occurs. If flocculation occurs, it indicates that the strain has the potential to produce flocculants, so it should be retained and rescreened; if there is no flocculation, it should be discarded.

[0104] Inoculate each strain retained in the initial screening into the optimized culture medium and culture it in a shaker at 30°C and 220rpm for 72h. Take 5mL of the bacterial solution in a 100mL colorimetric tube, and then add 93ml of kaolin suspension and 2mL of 1.0% calcium chloride solution to prepare a mixture. Shake the mixture thoroughly at room temperature using an oscillator at 180rpm for 3min, gently pour it into a test tube, and then let it settle for 10min. Carefully draw the supernatant and measure the optical density at a wavelength of 550nm using a UV-visible spectrophotometer. Use uninoculated liquid culture medium instead of the strain fermentation liquid as the control group. Calculate the flocculation activity using the following formula:

[0105] Flocculation rate = (AB) / A×100%

[0106] A is the optical density of the control experimental sample at 550 nm, B is the optical density of the experimental sample at 550 nm, and each experiment was repeated 3 times.

[0107] Table 3. Top 5 strains in terms of flocculation activity

[0108]

[0109] The experimental results are shown in Table 3. The flocculation rates of the five dominant strains were 87.16%, 90.88%, 91.36%, 93.16% and 97.88%, respectively. The repeated experimental results showed that the FangSG106 strain had stable flocculation activity and a high flocculation rate, and was further used in subsequent experiments and sent for preservation.

[0110] The deposit information of this strain is as follows:

[0111] Deposit number: CGMCC No.31655

[0112] Storage date: August 12, 2024

[0113] Classification name: Paenibacillus polymyxa

[0114] Depository: China National Microbiological Culture Collection Administration General Microbiology Center

[0115] Deposit address: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

[0116] Experimental Example 4: Evaluation of the spore rate of Paenibacillus polymyxa FangSG106

[0117] (1) Paenibacillus polymyxa FangSG106 was inoculated into a solid culture medium and placed in a constant temperature incubator at 30°C for 48 hours for activation. The activated single colony was picked and inoculated into a shake flask filled with a seed culture medium for culture. The shake flask was filled with 30 mL / 250 mL of liquid and cultured at 30°C and 220 rpm for 8 hours to obtain a seed solution. The seed solution was transferred to a fermenter filled with an optimized culture medium at a 5% inoculation rate for fermentation culture. Samples were taken at different culture time points for microscopic examination to observe the spore formation of the strain. Examples of the microscopic examination results of the strain are shown in FIG. Figure 2 As shown, the fermentation was stopped after culturing at 30° C. for 32 h to 36 h to obtain a fermentation broth.

[0118] (2) The fermented liquid obtained in (1) is centrifuged at 12000 rpm and then the bacterial sludge is collected, and the bacterial powder is obtained by spray drying. The inlet air temperature of the spray drying tower is 158-162°C, the outlet air temperature is 80-84°C, the vacuum degree in the spray drying tower is -0.01Mpa to -0.02MPa, and the bacterial powder is obtained by spray drying.

[0119] (3) Take 0.1 g of the Bacillus polymyxa powder sample and add it to 9.9 g of sterile physiological saline with glass beads containing 0.5% Tween 80, and shake and homogenize it on a shaker at 250 rpm at 20°C for 30 minutes to obtain an initial bacterial suspension. Take 2 mL of the homogenized initial bacterial suspension into a freezing tube and immerse it in a water bath for 10 minutes at 80°C. Take out the bacterial solution after the water bath and cool it to room temperature with ice water to obtain the sample test solution. The test solution is diluted ten times with sterile physiological saline, and the appropriate dilution is selected for counting, and the spore rate of the bacterial powder is calculated.

[0120] Table 4. Spore rate of strains

[0121]

[0122] The experimental results are shown in Table 4. The spore rate of Paenibacillus polymyxa FangSG106 can reach 98.59%.

[0123] Experimental Example 5: Effect of medium pH on flocculation activity of strain FangSG106

[0124] The activated cultured FangSG106 strain seed solution was inoculated into the optimized culture medium with pH values ​​of 3, 4, 5, 6, 7, 8, 9, 10 and 11 at a ratio of 2%, and cultured in a shaking incubator at 30°C and 220 rpm for 72 h. The flocculation activity of the strain was determined using the same method as in Experimental Example 3 to determine the optimal pH value required for FangSG106 to produce flocculants.

[0125] The experimental results are as follows Figure 3 As shown, the results showed that the strain had a wide pH applicable range. In the range of pH 3-7, the flocculation rate of strain FangSG106 gradually increased, and the highest flocculation rate of the strain was 97.34% at pH 7. As the pH value continued to increase, the flocculation rate of the strain gradually decreased.

[0126] Experimental Example 6: Effect of culture temperature on flocculation ability of strain FangSG106

[0127] The activated cultured FangSG106 strain seed solution was inoculated into an optimized culture medium with a pH value of 7 at a ratio of 2%, and cultured in a constant temperature shaker at 20°C, 30°C, 35°C, 40°C, 50°C, and 60°C at 220 rpm for 72 h. The flocculation activity of the strain was determined using the same method as in Experimental Example 3 to determine the optimal culture temperature required for FangSG106 to produce flocculants.

[0128] The experimental results are as follows Figure 4 As shown, the results showed that when strain FangSG106 was cultured at 30°C, the flocculation activity of the fermentation broth was the highest, and as the temperature continued to rise, the flocculation activity of strain FangSG106 gradually decreased.

[0129] Experimental Example 7: Extraction and purification of bioflocculant

[0130] The activated cultured FangSG106 strain seed liquid was inoculated into an optimized medium with a pH value of 7 at a ratio of 2%, and placed in a constant temperature shaker at 30°C and 220rpm for 72h, and the fermentation broth was collected for use. The fermentation broth was centrifuged at 4000rpm for 20min, the bacteria were removed, and the supernatant was collected. The supernatant was mixed with sterile distilled water at a ratio of 1:1 (v / v) and centrifuged at 4000rpm for 20min to remove insoluble substances. Then, the supernatant was mixed with ethanol at a ratio of 1:2 (v / v), and placed at 4°C for 12 hours, after which the precipitate was extracted. The precipitate was collected by centrifugation and vacuum dried. The crude extract was weighed, dissolved in 100mL of distilled water, and a mixed solution of one volume of chloroform and n-butanol (5:2, v / v) was added. The mixture was fully stirred and allowed to stand at room temperature for 12 hours. Finally, the precipitate was vacuumed, dried, and dissolved in deionized water to obtain a pure bioflocculant.

[0131] Experimental Example 8: Heavy Metal Ion Tolerance of Paenibacillus polymyxa FangSG106

[0132] (1) The method for preparing FangSG106 strain seed solution and fermentation solution is the same as step (1) of Experimental Example 4;

[0133] (2) Strain Cu 2+ Tolerance: FangSG106 strain seed solution was inoculated at a ratio of 2% into a 250 mL flask containing 50 mL of optimized culture medium. Cu 2+ The mother solution makes the Cu 2+ The final concentrations were 20, 50, 100, 150, 200, and 300 mg / L, respectively. The cultures were then cultured in a shaker at 30°C and 220 rpm for 48 h to observe whether the strains grew in the culture medium.

[0134] (3) Strain Ni 2+ Tolerance: FangSG106 strain seed solution was inoculated at a ratio of 2% into a 250 mL flask containing 50 mL of optimized culture medium. Ni 2+ The mother solution makes the Ni 2+ The final concentrations were 20, 50, 100, 150, 200, and 300 mg / L, respectively. The cultures were then cultured in a shaker at 30°C and 220 rpm for 48 h to observe whether the strains grew in the culture medium.

[0135] Table 5. Heavy metal ion tolerance of Paenibacillus polymyxa FangSG106 strain

[0136]

[0137] “﹢” means it can grow; “﹣” means it cannot grow or grows very slowly

[0138] The tolerance of FangSG106 strain is shown in Table 5. The experimental results show that the strain can tolerate nickel ions below 150 mg / L and copper ions below 200 mg / L.

[0139] Experimental Example 9: Application of Paenibacillus polymyxa FangSG106 in heavy metal treatment

[0140] (1) The method for preparing FangSG106 strain seed solution and fermentation broth was the same as step (1) of Experimental Example 4. The fermentation broth was centrifuged at 10,000 rpm for 10 min to collect the bacterial cells, which were then resuspended by pipetting with PBS and washed and centrifuged three times. The bacterial precipitate was collected for later use.

[0141] (2) Strain Cu 2+ Adsorption capacity: adjust the Cu in water solution 2+ The final concentration was 100 mg / L, the pH was 7, the bacterial concentration was adjusted to 5 g / L, the mixed system was placed in a shaker at 30°C and 220 rpm for incubation, and samples were taken after 24 hours of incubation. The supernatant was centrifuged at 10000 rpm for 10 minutes and the Cu was determined by flame atomic absorption spectrometry. 2+ The final concentration was calculated and the removal rate was calculated.

[0142] (3) Strain Ni 2+ Adsorption capacity: Adjust the Ni content in water solution 2+ The final concentration was 100 mg / L, the pH was 7, the bacterial concentration was adjusted to 5 g / L, the mixed system was placed in a shaker at 30°C and 220 rpm for incubation, and samples were taken after 24 hours of incubation. The supernatant was centrifuged at 10000 rpm for 10 minutes and the Ni content was determined by flame atomic absorption spectrometry. 2+ The final concentration was calculated and the removal rate was calculated.

[0143] Table 6. Adsorption capacity of heavy metal ions by Paenibacillus polymyxa FangSG106 strain

[0144]

[0145] The experimental results are shown in Table 6. The FangSG106 strain has a strong inhibitory effect on 100 mg / L Cu 2+ and Ni 2+ All of them have good adsorption effect. After 24h treatment, the adsorption of Cu 2+ The adsorption rate of Ni 2+ The adsorption rate was 66.88%, indicating that the strain has good application prospects in the treatment of environmental heavy metal pollution.

[0146] Experimental Example 10: Antibacterial ability of Paenibacillus polymyxa FangSG106 against water pathogens

[0147] The Oxford cup method was used to detect the ability of Paenibacillus polymyxa FangSG106 to inhibit five aquatic pathogenic bacteria. The antibacterial experimental method is as follows:

[0148] (1) Preparation of aquatic pathogenic bacteria suspension:

[0149] Aeromonas vernix, Aeromonas spp. and Aeromonas punctatus were inoculated into TSB liquid medium at 2% inoculation volume, respectively, and cultured at 37°C and 220 rpm in a shaking incubator for 24 h. The bacterial solution concentration was adjusted to 10 8 CFU / mL is reserved.

[0150] Vibrio splendens and Vibrio anguillarum were inoculated into 2216E liquid culture medium at 2% inoculation volume, and cultured in a shaking incubator at 25°C and 220 rpm for 24 h. The bacterial solution concentration was adjusted to 10 8 CFU / mL is reserved.

[0151] (2) Antibacterial ability test:

[0152] The corresponding solid culture medium for pathogens (TSA solid culture medium for Aeromonas virginiana, Aeromonas scutellariae and Aeromonas punctata, 2216E solid culture medium for Vibrio splendens and Vibrio anguillarum) was cooled to about 50°C, and the pathogens were mixed into the culture medium in a certain proportion so that the number of viable bacteria of the pathogens was 10 6 CFU / mL order of magnitude, and then quickly poured into a plate with an Oxford cup placed in advance. After the culture medium cooled and solidified, the Oxford cup was taken out, 150 μL of Paenibacillus polymyxa FangSG106 fermentation liquid was added to the well, and the plates were placed in a constant temperature incubator at the corresponding temperature for 24-48 hours, and then the diameter of the inhibition zone was measured.

[0153] The antibacterial results are shown in Table 7. Paenibacillus polymyxa FangSG106 has a certain antibacterial ability against the five pathogenic bacteria.

[0154] Table 7. Antibacterial results of Paenibacillus polymyxa FangSG106 against five pathogenic bacteria

[0155]

[0156] Experimental Example 11: Application of Paenibacillus polymyxa FangSG106 in aquaculture wastewater

[0157] Aquaculture wastewater samples were collected and placed in an experimental aquarium. The fermentation broth of strain FangSG106 was prepared according to the method of Experimental Example 4. The concentration of the bacterial broth was adjusted to 1×10 8CFU / mL, was added to aquarium water samples at a ratio of 1:1000 (V / V) to evaluate its impact on wastewater, and COD, total nitrogen, and total phosphorus contents in water were measured using a water quality analyzer, a total nitrogen analyzer, and a molybdenum antimony counterspectrophotometer, and the suspended solids content was determined according to the national standard method.

[0158] Table 8. Water environment index parameters at different time points

[0159]

[0160] The experimental results are shown in Table 8. The water quality of aquaculture wastewater was significantly improved after treatment with strain FangSG106. After 30 days of treatment, the COD content of aquaculture environmental wastewater decreased from the initial 36.84 mg / L to 15.77 mg / L, the total nitrogen content decreased from the initial 5.63 mg / L to 3.37 mg / L, the total phosphorus content decreased from the initial 0.47 mg / L to 0.29 mg / L, and the suspended matter content decreased from the initial 30.36 mg / L to 13.83 mg / L. It can be seen that strain FangSG106 can solve the problem of accumulation of harmful substances in aquaculture water and has great potential for development as a bioflocculant.

Claims

1. Use of Paenibacillus polymyxa FangSG106 with a deposit number of CGMCC No.31655 for flocculation or water purification.

2. The flocculation or water purification use of Paenibacillus polymyxa FangSG106 with a deposit number of CGMCC No.31655 according to claim 1, characterized in that: The water purification includes: adsorbing heavy metal ions, inhibiting water pathogens, and improving water quality.

3. The flocculation or water purification use of Paenibacillus polymyxa FangSG106 with a deposit number of CGMCC No.31655 according to claim 2, characterized in that: The heavy metal ions include: Cu 2+ 、Ni 2+ ; And / or, the water pathogens include: Aeromonas vernix, Aeromonas scutellariae, Aeromonas punctatus, Vibrio splendens, and Vibrio anguillarum; And / or, the improving water quality includes: reducing total nitrogen, total phosphorus, COD, and suspended solids in the water body.

4. Use of Paenibacillus polymyxa FangSG106 with a deposit number of CGMCC No.31655 in preparing bioflocculants or water purifiers.

5. A bioflocculant or water purifier, comprising an active ingredient, characterized in that: The active ingredients include: Paenibacillus polymyxa FangSG106 with a preservation number of CGMCC No.31655.

6. A bioflocculant or water purifier according to claim 5, characterized in that: Also includes: Auxiliary materials.

7. A method for removing heavy metals, characterized in that: Paenibacillus polymyxa FangSG106 with a deposit number of CGMCC No.31655 is added to the material to be treated containing heavy metals.

8. A method for removing heavy metals according to claim 7, characterized in that: The heavy metals include: Cu 2+ 、Ni 2+ ; And / or, the concentration of Paenibacillus polymyxa FangSG106 in the material to be treated is 5 g / L.

9. A water purification method, characterized in that: Paenibacillus polymyxa FangSG106 with a preservation number of CGMCC No.31655 was used for water purification.

10. A water purification method according to claim 9, characterized in that: The concentration was 1×10 8 CFU / mL of Paenibacillus polymyxa FangSG106 was put into the water to be purified.

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