Biological flocculant for wastewater treatment and application thereof

Abstract

The invention discloses a biological flocculant for wastewater treatment and application of the biological flocculant. The biological flocculant for treating the wastewater is obtained by performing high-density fermentation on bacillus subtilis KC-25226 obtained by performing mutagenesis on a strain screened from a polluted water body by adopting a culture medium such as molasses and the like, and the biological flocculant can be used for treating wastewater from different sources. The bacillus subtilis KC-25226 not only can grow in wastewater environments of different sources, but also has high yield of microbial flocculants and good flocculation effect; in the aspect of wastewater treatment, the effects of COD (Chemical Oxygen Demand) removal rate, ammonia nitrogen removal rate, total phosphorus removal rate and the like are excellent; the biological flocculant provided by the invention obviously reduces wastewater pollution, obviously improves the water quality of polluted water, and has a wide market prospect.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, in particular to a biological flocculant for wastewater treatment and application thereof. Background Art

[0002] Microbial flocculants are metabolites produced by microbial fermentation. These metabolites possess unique polymer properties, primarily composed of active ingredients such as polysaccharides, proteins, cellulose, and nucleic acids. During industrial use, they form flocculations with microorganisms and other components in water, removing and purifying suspended solid particles and bacterial cells. Among inorganic flocculants, aluminum salts pose a risk of developing Alzheimer's disease, osteomalacia, neurological disorders, and microcytic anemia. Iron salts can corrode pipes and equipment, affecting water quality. Organic polymer flocculants, such as polyacrylamide (PAM), produce monomeric hydrolysis products that are toxic. During use, they can cause mutagenic, carcinogenic, and teratogenic effects on livestock and humans and can also lead to secondary environmental pollution. Compared to traditional flocculants, microbial flocculants offer advantages such as safety, low cost, natural biodegradability, short production cycles, turbidity and color removal, and the absence of secondary water pollution during use. They are highly effective, non-toxic, and green water treatment agents.

[0003] However, the following problems still exist in microbial flocculants: First, different microbial flocculants are applicable to different situations, and there is no microbial flocculant that is universally used for precipitation and degradation of various treatment objects; in addition, the microbial fermentation process is complicated, the fermentation liquid is easy to deteriorate and is not conducive to storage and transportation, the production cycle of microbial flocculants is long, the production cost is high, and it is difficult to produce on a large scale. Secondly, the effect of microbial flocculants is more easily affected by environmental factors, so they need to be adjusted and optimized for different treatment objects. The types of existing microbial flocculants mainly include Rhodococcus erythropolis, Bacillus megaterium, Bacillus subtilis, Pseudomonas, Alcaligenes sp., Aspergillus souae AJ7002, etc. At present, the flocculation effect is unstable and is affected by sewage pH, temperature, ionic strength (such as Fe 3 + inhibitory activity) has a significant impact, lacks efficient fermentation, and the fermentation medium is complex. Summary of the Invention

[0004] The object of the present invention is to provide a biological flocculant for wastewater treatment, which is obtained by fermentation of microorganisms that produce bioflocculants. The microorganisms can produce high levels of polysaccharides and proteins, have excellent flocculation effects, and have obvious flocculation effects at low pH. It has high flocculation efficiency for various sewage (such as chemical plant, food processing wastewater, domestic wastewater, pulp wastewater, muddy water, etc.).

[0005] The microorganism is Bacillus subtilis KC-25226, which can effectively utilize crude raw materials such as molasses to ferment and produce microbial flocculants, thereby reducing production costs and being safe and environmentally friendly.

[0006] To achieve the above object, the technical solution adopted by the present invention is as follows:

[0007] A strain KC-25226 that produces a bioflocculant, the strain is classified and named Bacillus subtilis, and was deposited in the China General Microbiological Culture Collection Center on March 3, 2025, at the Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, with the deposit number CGMCC No. 33698.

[0008] The strains are obtained by collecting and isolating a variety of strains capable of producing microbial flocculants from a wastewater treatment pool of a chemical plant in Nanjing, culturing them in a basic culture medium, and then subjecting them to ion beam mutagenesis treatment.

[0009] The strain was obtained by screening using the following method:

[0010] A strain capable of producing microbial flocculants was collected and isolated from a wastewater treatment pool of a chemical plant in Nanjing. Basic culture was carried out using liquid LB medium. After incubation at 35°C for 24 hours, the biomass and amount of flocculants produced were measured simultaneously. A strain with the strongest activity was obtained and named Good-1 as the dominant strain.

[0011] Good-1 was inoculated into liquid LB seed medium and the cultured Bacillus subtilis was diluted 10-fold to a cell count of 10. 9 / mL, take 0.1mL of bacterial solution and spread it evenly on the plate, wait for it to dry in the air, and then + Ion beam implantation, N + The ion beam implantation dose is (90, 135, 180, 225, 270) × 2.6 × 10 13 N + / cm 2 , N +The ion beam injection energy was 15 keV. After irradiation, the cells were washed with 1 mL of sterile water, diluted 10-fold, and plated onto culture plates. Incubated in an inverted manner at 35°C for 24 hours, individual colonies were picked and shaken for testing. A strain with rapid growth and optimal flocculation was selected and named Bacillus subtilis KC-25226.

[0012] The culture conditions of Bacillus subtilis KC-25226 are:

[0013] The strain is cultured aerobically. The carbon source used to culture the strain can be glucose, trisodium citrate, sodium pyruvate, sodium acetate, molasses, bean dregs hydrolyzate, etc.; the nitrogen source used to culture the strain can be ammonium chloride, ammonium nitrate, sodium nitrate, ammonium sulfate, urea, beef extract, ammonia water, corn steep liquor, fish meal, etc. The optimal culture time of the strain seed liquid is 16 to 36 hours. According to the inoculation amount of 1 to 10% (volume ratio), it is transferred to the fermentation medium, the fermentation temperature is 25 to 42°C, and the fermentation time is 24 to 60 hours. Inorganic salts such as magnesium salts, nitrates, potassium salts, calcium salts, phosphates or hydrochlorides can also be added during the strain culture process.

[0014] The physiological and morphological characteristics of the Bacillus subtilis KC-25226 are:

[0015] Bacillus subtilis KC-25226 grew vigorously on the LB plate. It was clearly observed that the single colonies were uniformly colored, and the spores were oval or columnar, the sporangium was swollen, had flagella, and were milky white in color. The spores were highly heat-resistant, and the colony surface was rough and opaque, dirty white or slightly yellow.

[0016] The invention relates to an application of the strain KC-25226 producing bioflocculant in sewage treatment.

[0017] The Bacillus subtilis KC-25226 is used in fermentation to produce microbial flocculants.

[0018] The Bacillus subtilis KC-25226 produces different ratios of polysaccharides and proteins in fermentation products on different fermentation media. The secretion ratio of polysaccharides and proteins in the fermentation products can be adjusted by adjusting the fermentation conditions, thereby optimizing the flocculation effect of the fermentation products on wastewater from different sources, and having a wide range of applications.

[0019] When Bacillus subtilis KC-25226 is grown in a culture medium with molasses acid hydrolyzate or sodium acetate as the carbon source, the proportion of protease in the fermentation broth is higher, while when grown in a culture medium with glucose as the carbon source, the proportion of exopolysaccharides in the fermentation broth is higher.

[0020] The application comprises the following steps:

[0021] Bacillus subtilis KC-25226 was activated, inoculated with seed solution, cultured for 16 to 36 hours, and then transferred to fermentation medium at an inoculum rate of 1 to 10% (volume ratio). The fermentation temperature was 25 to 42° C. and the fermentation time was 24 to 60 hours.

[0022] The fermentation medium comprises: 5-40 g / L carbon source, 5-40 g / L nitrogen source, 0-6 g / L inorganic salt, 0-3 g / L metal ion, and the rest is water, with a pH of 5.5-8.0.

[0023] The carbon source is at least one of glucose, trisodium citrate, sodium pyruvate, sodium acetate, molasses, bean dregs hydrolyzate, etc.; the nitrogen source is at least one of ammonium chloride, ammonium nitrate, sodium nitrate, ammonium sulfate, urea, beef extract, ammonia water, corn steep liquor, and fish meal; the inorganic salt is any one of magnesium salt, nitrate, potassium salt, calcium salt, phosphate, or hydrochloride, or a combination thereof; and the metal ion is a mixture of 0.01-3 g / L of ferrous sulfate, 0.001-1 g / L of copper sulfate, 0.01-0.5 g / L of zinc sulfate, 0.1-2 g / L of calcium chloride, and 0.01-1 g / L of manganese sulfate.

[0024] The fermentation temperature of the Bacillus subtilis KC-25226 is preferably 37° C., the fermentation time is 48 h, the ventilation ratio is 2.7 v / vm, ​​and the stirring is 300 rpm during the fermentation stage;

[0025] The fermentation liquid after fermentation is processed, the supernatant is collected by centrifugation, and then 1 to 5 times of anhydrous ethanol is added according to the volume ratio, and the liquid is allowed to settle for 12 to 36 hours. The liquid is centrifuged at 4000 rpm, and the precipitate is collected and dried to obtain a crude microbial flocculant.

[0026] A bioflocculant comprises a fermentation product of strain KC-25226.

[0027] The preparation method of the biological flocculant comprises activating Bacillus subtilis KC-25226, inoculating seed liquid, culturing for 16 to 36 hours, and then inoculating 1 to 10% of the seed liquid into a fermentation medium. The fermentation temperature is 25 to 42° C. and the fermentation time is 24 to 60 hours to obtain the biological flocculant.

[0028] The fermentation liquid after fermentation was processed, the supernatant was collected by centrifugation, and 5 times of anhydrous ethanol was added according to the volume ratio, and the mixture was allowed to settle for 24 hours. The mixture was centrifuged at 4000 rpm, and the precipitate was collected and dried to obtain a crude microbial flocculant.

[0029] The crude microbial flocculant is directly added to the wastewater to be treated, or the fermentation liquid is added to the wastewater to be treated at a volume ratio of 1 to 8:100 for flocculation treatment.

[0030] Further preferably, the crude microbial flocculant is directly added to the wastewater to be treated in an amount of 1-10% by mass to volume, the fermentation liquid is added to the wastewater to be treated in an amount of 1%-10% by volume, the reaction time is 5-30 minutes, and the pH is 5-12.

[0031] A more specific production method is: after Bacillus subtilis KC-25226 is activated, it is inoculated into a fermentation medium and aerobic fermented at 25-45°C for 24-60 hours.

[0032] The aerobic fermentation medium comprises: 15 g / L molasses acid hydrolyzate, 20 g / L urea, 2 g / L Na2HPO4·3H2O, 2.5 g / L NaH2PO4, 0.5 g / L MgSO4·7H2O, 0.01 g / L ferrous sulfate, 0.002 g / L copper sulfate, 0.005 g / L zinc sulfate, 0.3 g / L calcium chloride, 0.02 g / L manganese sulfate, and the rest is water, with a pH of 7.4.

[0033] The biological flocculant may further include a coagulant aid, and the added amount of the coagulant aid is 1% to 10%.

[0034] The coagulant aid is at least one of potassium chloride, sodium chloride, magnesium chloride, aluminum chloride, ferric chloride, zinc sulfate, and calcium chloride.

[0035] More preferably, the coagulant aid is calcium chloride, and the amount of the coagulant aid added is 2%.

[0036] Beneficial effects: Compared with the prior art, the present invention has the following advantages:

[0037] 1. Compared with other strains, the Bacillus subtilis of the present invention has high metabolic activity, short fermentation cycle, high polysaccharide and protein content in the fermentation product, and strong ability to produce microbial flocculants by fermentation.

[0038] 2. The raw material for cultivation has been changed from glucose to molasses hydrolyzate, which has greatly reduced the cost and can reduce the discharge of waste, thus saving costs and reducing environmental pollution.

[0039] 3. Compared with traditional flocculants, this process has the advantages of safety, low cost, natural degradation, short production cycle, turbidity removal and decolorization, and no secondary pollution to water quality during use.

[0040] 4. The bioflocculant prepared by the present invention has a wide range of applications. DETAILED DESCRIPTION

[0041] The present invention can be better understood by the following examples. It will be readily understood by those skilled in the art that the specific material ratios, process conditions, and results described in the examples are merely illustrative of the present invention and should not and will not limit the present invention as described in detail in the claims.

[0042] Example 1 Mutation Screening and Cultivation of Bacillus subtilis KC-25226

[0043] Five strains of Bacillus subtilis were collected and isolated from a wastewater treatment pool of a chemical plant in Nanjing. They were cultured in liquid LB medium. After culturing at 35°C for 24 hours, the biomass and amount of bioflocculant produced were measured. A strain with the strongest activity was obtained and named Good-1 as the dominant strain.

[0044] Good-1 was inoculated into liquid LB seed medium for cultivation. The cultured Bacillus subtilis solution was diluted 10-fold to a cell count of 10 9 / mL, take 0.1mL of bacterial solution and spread it evenly on the plate, wait for it to dry in the air, and then + Ion beam implantation, N + The ion beam implantation dose is (90, 135, 180, 225, 270) × 2.6 × 10 13 N + / cm 2 , N + The ion beam injection energy was 15 keV. After irradiation, the cells were washed with 1 mL of sterile water, diluted 10-fold, and plated onto culture plates. Incubated in an inverted manner at 35°C for 24 hours, individual colonies were picked and shaken for testing. A strain with rapid growth and optimal flocculation was selected and named Bacillus subtilis KC-25226.

[0045] The strain is cultured aerobically. Carbon sources for culturing the strain include glucose, trisodium citrate, sodium pyruvate, sodium acetate, molasses, and soybean dregs hydrolyzate. Nitrogen sources for culturing the strain include ammonium chloride, ammonium nitrate, sodium nitrate, ammonium sulfate, urea, beef extract, ammonia water, corn steep liquor, and fish meal. The optimal cultivation time for the strain's seed solution is 16 to 36 hours. The inoculum is inoculated at 1 to 10% and then transferred to a fermentation medium at a temperature of 28 to 42°C for 24 to 60 hours. Inorganic salts such as magnesium salts, nitrates, potassium salts, calcium salts, phosphates, or hydrochlorides may also be added during the cultivation process.

[0046] Bacillus subtilis grows vigorously on LB plates, with single colonies clearly visible and uniformly colored. Spores are oval or columnar, with enlarged cysts, flagella, and a milky white color. The spores are highly heat-resistant. The colony surface is rough and opaque, with a dirty white or slightly yellowish color.

[0047] Example 2 Fermentation of Bacillus subtilis KC-25226

[0048] Bacillus subtilis KC-25226 was activated and inoculated with seed solution (10 g / L peptone, 5 g / L yeast powder, 10 g / L sodium chloride, 5 g / L glucose, pH 7), cultured for 20 h, and then transferred to fermentation medium at an inoculum size of 3%, and fermented at 35°C for 48 h. During the fermentation stage, the ventilation ratio was 1.5 v / vm and the stirring was 180 rpm.

[0049] The fermentation medium comprises: 15 g / L molasses acid hydrolyzate, 20 g / L urea, 2 g / L Na2HPO4·3H2O, 2.5 g / L NaH2PO4, 0.5 g / L MgSO4·7H2O, 0.01 g / L ferrous sulfate, 0.002 g / L copper sulfate, 0.005 g / L zinc sulfate, 0.3 g / L calcium chloride, 0.03 g / L manganese sulfate, and the remainder water, with a pH of 7.4. (The molasses acid hydrolyzate is prepared by adding 4% sulfuric acid by volume to molasses, reacting at 150°C for 4 to 6 hours, and adjusting the pH to 7.)

[0050] The fermentation broth after fermentation was processed, and the supernatant was collected by centrifugation. Then, 5 times of anhydrous ethanol was added according to the volume ratio, and the mixture was allowed to settle for 24 hours. The mixture was centrifuged at 4000 rpm, and the precipitate was collected and dried to obtain a crude microbial flocculant. The crude microbial flocculant was added to a kaolin suspension (98 mL of a kaolin suspension with a concentration of 8 g / L) at an addition amount of 2% (mass-to-volume ratio), and 2 g of the crude microbial flocculant was added. The mixture was stirred for 10 minutes and allowed to stand for 30 minutes. The flocculation rate reached 97.6%.

[0051] Example 3 Flocculation effect of wastewater treated after fermentation of Bacillus subtilis KC-25226

[0052] Bacillus subtilis KC-25226 was activated and inoculated with a seed solution (10 g / L peptone, 5 g / L yeast powder, 10 g / L sodium chloride, 5 g / L glucose, pH 7). The culture was carried out for 20 hours, and the inoculum size was 3% and the cells were transferred to a fermentation medium. The culture was carried out at 35° C. for 48 hours. During the fermentation stage, the aeration ratio was 1.5 v / vm and the stirring was 180 rpm. The fermentation medium contained 15 g / L molasses acid hydrolyzate, 20 g / L urea, 2 g / L Na2HPO4·3H2O, 2.5 g / L NaH2PO4, 0.5 g / L MgSO4·7H2O, 0.01 g / L ferrous sulfate, 0.002 g / L copper sulfate, 0.005 g / L zinc sulfate, 0.3 g / L calcium chloride, and the remainder was water. The pH was 7.4. The fermentation liquid after fermentation was treated, and the supernatant was collected by centrifugation. Then, 5 times of anhydrous ethanol was added according to the volume ratio, and the mixture was allowed to settle for 24 hours. The mixture was centrifuged at 4000 rpm, and the precipitate was collected and dried to obtain a crude microbial flocculant. The crude product was added to wastewater at an addition amount of 2% (mass volume ratio), the pH was 7.0, the mixture was stirred for 10 minutes, and the mixture was allowed to stand for 30 minutes. The flocculation effect of the wastewater treated after fermentation reached 98.5%.

[0053] Table 1 Flocculation effect of wastewater treated after fermentation

[0054]

[0055] Example 4 Effects of different Bacillus subtilis strains on polysaccharide and extracellular protein production

[0056] Bacillus subtilis KC-25226, Bacillus subtilis 13932, Bacillus subtilis 168, Bacillus subtilis BNCC359371, and Bacillus subtilis BNCC190341 were activated, inoculated with seed solution (10 g / L peptone, 5 g / L yeast extract, 10 g / L sodium chloride, 5 g / L glucose, pH 7), cultured for 20 h, and then transferred to fermentation medium at an inoculum size of 3%. The culture was fermented at 37° C. for 48 h. During the fermentation stage, the aeration ratio was 1.8 v / vm and the stirring was 200 rpm. The fermentation medium contained 15 g / L molasses acid hydrolyzate, 25 g / L urea, and Na2HPO4·

[0057] The fermentation broth consisted of 2 g / L 3H2O, 2.5 g / L NaH2PO4, 0.3 g / L MgSO4·7H2O, 0.02 g / L ferrous sulfate, 0.001 g / L copper sulfate, 0.002 g / L zinc sulfate, 0.4 g / L calcium chloride, and the remainder was water. The pH was 7.4. The fermentation broth was tested to examine the effects of different production strains on growth, exopolysaccharide production, and protease production.

[0058] From Table 2, we can see that Bacillus subtilis KC-25226 has the best growth, the highest exopolysaccharide yield, reaching 5.5 g / L, and has a better ability to produce protease.

[0059] Table 2 Effects of different production strains on growth, production of exopolysaccharides and proteases

[0060]

[0061] Example 5 Effect of different flocculation conditions on flocculation effect

[0062] Bacillus subtilis KC-25226 was activated and inoculated with a seed solution (10 g / L peptone, 5 g / L yeast powder, 10 g / L sodium chloride, 5 g / L glucose, pH 7). The culture was carried out for 24 hours, and then the culture was transferred to a fermentation medium with an inoculum size of 3%. The culture was fermented at 35° C. for 36 hours. During the fermentation stage, the aeration ratio was 1.5 v / vm and the stirring was 350 rpm. The fermentation medium contained 18 g / L molasses acid hydrolyzate, 26 g / L urea, 2.5 g / L Na2HPO4·3H2O, 1.5 g / L NaH2PO4, 0.32 g / L MgSO4·7H2O, 0.03 g / L ferrous sulfate, 0.002 g / L copper sulfate, 0.006 g / L zinc sulfate, 0.2 g / L calcium chloride, and the remainder was water. The pH was 7.4. The fermentation broth was processed, centrifuged, and the supernatant was collected. Five times the volume of anhydrous ethanol was then added, and the mixture was allowed to settle for 24 hours. The mixture was then centrifuged at 4000 rpm, and the precipitate was collected and dried to obtain a crude microbial flocculant. This crude microbial flocculant was then added to a kaolin suspension (98 mL of an 8 g / L kaolin suspension) at a mass volume ratio of 1 to 7%. The reaction was stirred for 10 minutes at a temperature of 35°C and a pH of 6.5, and the mixture was allowed to stand for 30 minutes. The flocculation effect of the microbial flocculant at various addition levels was investigated, and the results are shown in Table 6.

[0063] Under the same fermentation conditions, the fermentation broth was processed, centrifuged to obtain the supernatant, and then 5 times the volume of anhydrous ethanol was added. The mixture was allowed to settle for 24 hours and then centrifuged at 4000 rpm. The precipitate was collected and dried to obtain a crude microbial flocculant. The crude product was added to a kaolin suspension (98 mL of an 8 g / L kaolin suspension) at a mass volume ratio of 2% (mass volume ratio). The pH was set at 7, and the stirring reaction time was 10 minutes. The reaction temperatures were 20°C, 25°C, 28°C, 30°C, 35°C, 37°C, 42°C, 50°C, 60°C, 70°C, and 80°C, respectively, and the mixture was allowed to settle for 30 minutes. The effects of different temperatures on the flocculation effect were investigated, and the results are shown in Table 3.

[0064] Under the same fermentation conditions, the fermented broth was processed, centrifuged, and the supernatant was collected. Five times the volume of anhydrous ethanol was added, and the mixture was allowed to settle for 24 hours. The mixture was then centrifuged at 4000 rpm, and the precipitate was collected and dried to obtain a crude microbial flocculant. The crude product was then added to a kaolin suspension (98 mL of an 8 g / L kaolin suspension) at a 2% (mass-volume) addition rate. 2% of various coagulants (magnesium chloride, zinc sulfate, and calcium chloride) were added (mass-volume ratio). No coagulant was added as a control. The reaction was stirred for 10 minutes, the reaction pH was 7, the temperature was 37°C, and the mixture was allowed to settle for 30 minutes. The effects of various coagulants on the flocculation performance were investigated, and the results are shown in Table 5.

[0065] Bacillus subtilis KC-25226 was activated and inoculated with a seed solution (10 g / L peptone, 5 g / L yeast powder, 10 g / L sodium chloride, 5 g / L glucose, pH 7). The culture was carried out for 24 h, and then the culture was transferred to a fermentation medium with an inoculum size of 3%. The culture was fermented at 35°C for 36 h. During the fermentation stage, the aeration ratio was 1.5 v / vm and the stirring was 350 rpm. The fermentation medium contained 18 g / L molasses acid hydrolyzate, 26 g / L urea, 2.5 g / L Na2HPO4·3H2O, 1.5 g / L NaH2PO4, 0.32 g / L MgSO4·7H2O, 0.03 g / L ferrous sulfate, 0.002 g / L copper sulfate, 0.006 g / L zinc sulfate, 0.2 g / L calcium chloride, and the remainder was water. The fermentation broth was processed, centrifuged, and the supernatant was collected. Then, 5 times the volume of anhydrous ethanol was added, and the mixture was allowed to settle for 24 hours. The mixture was centrifuged at 4000 rpm, and the precipitate was collected and dried to obtain a crude microbial flocculant. The crude product was added to a kaolin suspension (98 mL of an 8 g / L kaolin suspension) at a 2% addition rate (mass-volume ratio). 2% calcium chloride solution of different coagulants (mass-volume ratio) was added, and the pH was adjusted to 6, 7, 8, 9, 10, 11, and 12, respectively. The reaction was stirred for 10 minutes, the reaction temperature was 37°C, and the mixture was allowed to settle for 30 minutes. The effect of different pH values ​​on the flocculation effect was investigated, and the results are shown in Table 4.

[0066] Under the same fermentation conditions, the fermented broth was treated by centrifugation, the supernatant was collected, and 5 times the volume of anhydrous ethanol was added. The mixture was allowed to settle for 24 hours. The mixture was then centrifuged at 4000 rpm, the precipitate was collected and dried, and a crude microbial flocculant was added to 98 mL of a kaolin suspension (8 g / L) at a pH of 7. The reaction was stirred for 0, 5, 10, 25, and 30 minutes at 37°C, and the mixture was allowed to settle for 30 minutes. The effects of different reaction times on the flocculation effect were investigated.

[0067] Table 3 shows that the flocculant produced by the bacteria has excellent thermal stability and can maintain a flocculation efficiency of over 80% between 20°C and 80°C. Table 4 shows that the pH change of the flocculation system affects the microbial flocculant. The flocculation effect under acidic conditions is significantly higher than that under alkaline conditions. At a pH of 4-8, the flocculation rate reaches over 90%, indicating that the flocculant has better wastewater treatment capabilities under low pH conditions. Table 5 shows that different types of coagulants have different flocculation effects on the flocculants produced by the microbial flocculant-producing bacteria. Metal ions can strengthen the binding force between microbial flocculant molecules and suspended particles, enhancing bridging and electrical neutralization. Calcium chloride is the most effective coagulant. The flocculant has the best flocculation effect when no coagulant is added. Coagulants do not have a significant coagulant-aiding effect in this patented process. Table 6 shows that the flocculation effect is more pronounced with the addition of more flocculants. The flocculation effect reaches over 90% when the microbial flocculant produced by the strain is added at a level of 3-6%.

[0068] Table 3 Effect of different temperatures on flocculation effect

[0069]

[0070]

[0071] Table 4 Effect of different pH on flocculation effect

[0072]

[0073] Table 5 Effect of different coagulants on flocculation effect

[0074]

[0075] Table 6 Effect of different addition amounts on flocculation effect

[0076]

[0077]

[0078] Example 6 Comparative test of microbial flocculants and traditional flocculants in wastewater

[0079] Bacillus subtilis KC-25226 was activated and inoculated with a seed solution (10 g / L peptone, 5 g / L yeast powder, 10 g / L sodium chloride, 5 g / L glucose, pH 7). The culture was carried out for 24 hours, and then the culture was transferred to a fermentation medium with an inoculum size of 3%. The culture was fermented at 35° C. for 26 hours. During the fermentation stage, the aeration ratio was 2.0 v / vm and the stirring was 400 rpm. The fermentation medium contained 20 g / L molasses acid hydrolyzate, 28 g / L urea, 1.5 g / L Na2HPO4·3H2O, 2.5 g / L NaH2PO4, 0.35 g / L MgSO4·7H2O, 0.04 g / L ferrous sulfate, 0.002 g / L copper sulfate, 0.003 g / L zinc sulfate, 0.2 g / L calcium chloride, and the remainder was water. The pH was 7.4. The fermentation broth was processed, centrifuged, and the supernatant was collected. Five times the volume of anhydrous ethanol was added, and the mixture was allowed to settle for 24 hours. The mixture was then centrifuged at 4000 rpm, and the precipitate was collected and dried to obtain a crude microbial flocculant. This was then added to wastewater at a 2% mass-to-volume ratio. The mixture was stirred for 10 minutes, reacted at a pH of 6.8, and allowed to stand for 30 minutes at 37°C. The effects of the microbial flocculant and traditional flocculants on flocculation in wastewater were investigated.

[0080] As shown in Table 7, the flocculation rate of microbial flocculants was higher than that of PAC and PAM, reaching 96.8%, and the removal rates of COD, ammonia nitrogen and total phosphorus were significantly higher than those of PAC and PAM.

[0081] Table 7 Comparative test of microbial flocculants and traditional flocculants in wastewater

[0082]

[0083] Example 7 Effect of different fermentation media on the flocculation effect of microbial flocculants

[0084] Bacillus subtilis KC-25226 was activated and inoculated with a seed solution (10 g / L peptone, 5 g / L yeast powder, 10 g / L sodium chloride, 5 g / L glucose, pH 7). The culture was carried out for 20 h. The culture was then transferred to different fermentation media at an inoculum size of 3% and fermented at 35°C for 38 h. During the fermentation stage, the aeration ratio was 2.5 v / vm and the stirring was 500 rpm. The fermentation media were as follows: ① 25 g / L molasses acid hydrolyzate, 32 g / L urea, 1.5 g / L Na2HPO4·3H2O, 2.5 g / L NaH2PO4, MgSO4·

[0085] 7H2O 0.4g / L, ferrous sulfate 0.03g / L, copper sulfate 0.002g / L, zinc sulfate 0.003g / L, calcium chloride 0.3g / L, the rest is water, pH=7.4.

[0086] ② Glucose 25g / L, ammonium nitrate 32g / L, Na2HPO4·3H2O 1.5g / L, NaH2PO4 2.5g / L, MgSO4·7H2O 0.4g / L, ferrous sulfate 0.02g / L, copper sulfate 0.005g / L, zinc sulfate 0.001g / L, calcium chloride 0.3g / L, and the rest is water, pH=7.4.

[0087] ③Sodium acetate 25g / L, corn steep liquor 32g / L, Na2HPO4·3H2O 2.6g / L, NaH2PO4 1.4g / L, MgSO4·7H2O 0.22g / L, ferrous sulfate 0.05g / L, copper sulfate 0.004g / L, zinc sulfate 0.002g / L, calcium chloride 0.3g / L, and the rest is water, pH=7.4.

[0088] The fermentation broth after fermentation was processed, the supernatant was collected by centrifugation, and 5 times of anhydrous ethanol was added according to the volume ratio. The mixture was allowed to settle for 24 hours and centrifuged at 4000 rpm. The precipitate was collected and dried to obtain a crude microbial flocculant. The crude product was added to a kaolin suspension (98 mL of a kaolin suspension with a concentration of 8 g / L) at an addition amount of 2% (mass-to-volume ratio). The reaction pH was 7.0, the stirring reaction time was 10 minutes, the reaction temperature was 37°C, and the mixture was allowed to stand for 30 minutes.

[0089] Table 8 shows that different fermentation media produce different exopolysaccharide and protease capacities, yet the flocculation effect consistently exceeds 90%. Bacillus subtilis KC-25226 produces varying ratios of polysaccharide to protein in its fermentation products when grown in different fermentation media. This ratio can be adjusted by adjusting fermentation conditions, thereby optimizing the fermentation product's ability to enhance wastewater flocculation, demonstrating a wide range of applications.

[0090] Table 8 Effects of different production strains on growth, production of exopolysaccharides and proteases

[0091]

[0092] Example 8 Flocculation effect of microbial flocculants on wastewater from different sources

[0093] Bacillus subtilis KC-25226 was activated and inoculated with a seed solution (10 g / L peptone, 5 g / L yeast extract, 10 g / L sodium chloride, 5 g / L glucose, pH 7). The culture was carried out for 20 h, and then the cells were transferred to a fermentation medium at an inoculum size of 3%. The culture was fermented at 35° C. for 38 h. During the fermentation stage, the aeration ratio was 2.5 v / vm and the stirring was 500 rpm. The fermentation medium contained: ① 22 g / L molasses acid hydrolyzate, 28 g / L urea, 1.3 g / L Na2HPO4·3H2O, 2.3 g / L NaH2PO4, 0.5 g / L MgSO4·7H2O, 0.02 g / L ferrous sulfate, 0.003 g / L copper sulfate, 0.002 g / L zinc sulfate, 0.4 g / L calcium chloride, and the remainder was water. The pH was 7.4.

[0094] ② Glucose 22g / L, ammonium nitrate 28g / L, Na2HPO4·3H2O 1.6g / L, NaH2PO4 2.2g / L, MgSO4·7H2O 0.4g / L, ferrous sulfate 0.02g / L, copper sulfate 0.003g / L, zinc sulfate 0.002g / L, calcium chloride 0.3g / L, and the rest is water, pH=7.4.

[0095] ③ Sodium acetate 22g / L, corn steep liquor 28g / L, Na2HPO4·3H2O 2.6g / L, NaH2PO4 1.0g / L, MgSO4·7H2O 0.25g / L, ferrous sulfate 0.02g / L, copper sulfate 0.004g / L, zinc sulfate 0.003g / L, calcium chloride 0.3g / L, and the remainder water, pH = 7.4. The fermentation broth was treated by centrifugation to remove the supernatant. Then, 5 times the volume of anhydrous ethanol was added, and the mixture was allowed to settle for 24 hours. The mixture was centrifuged at 4000rpm, and the precipitate was collected and dried to obtain a crude microbial flocculant. The crude product was added to wastewater at a dosage of 2% (mass-to-volume ratio). The reaction pH was 7, the stirring reaction time was 10 minutes, the reaction temperature was 37°C, and the mixture was allowed to settle for 30 minutes. The effect of the microbial flocculant on the flocculation effect in different wastewaters was investigated.

[0096] Table 9 shows that the flocculation rates of microbial flocculants in treating pulp wastewater, food processing wastewater, and chemical plant wastewater are 95.4%, 94.2%, and 91.5%, respectively. The COD removal rates are 87.5%, 91.7%, and 85.4%, respectively. The ammonia nitrogen removal rate reaches more than 85%, and the total phosphorus removal rate reaches more than 80%.

[0097] Table 9 Flocculation effect of microbial flocculants in different wastewaters

[0098]

[0099] The technical solutions provided by the embodiments of the present invention are introduced in detail above. Specific examples are used herein to illustrate the principles and implementation methods of the embodiments of the present invention. The description of the above embodiments is only applicable to help understand the principles of the embodiments of the present invention. At the same time, for those skilled in the art, according to the embodiments of the present invention, there may be changes in the specific implementation methods and application scopes. In summary, the contents of this specification should not be understood as limiting the present invention.

Claims

1. A strain KC-25226 producing a bioflocculant, characterized in that: The strain is classified as Bacillus subtilis ( Bacillus subtilis ), has been deposited in the General Microbiology Center of China Culture Collection Administration of Microorganisms, and its deposit number is CGMCC No.33698.

2. The bioflocculant-producing strain KC-25226 according to claim 1, characterized in that The strain can produce a biological flocculant by fermenting molasses, and the biological flocculant has an obvious flocculation effect at a low pH.

3. Use of the bioflocculant-producing strain KC-25226 according to claim 1 in sewage treatment.

4. A bioflocculant, characterized in that: The bioflocculant comprises the fermentation product of the strain KC-25226 according to claim 1.

5. A bioflocculant according to claim 4, characterized in that: Bacillus subtilis ( Bacillus subtilis ) KC-25226 was activated, seed liquid was added, cultured for 16-36 h, and then transferred to fermentation medium at an inoculation rate of 1-10%. The fermentation temperature was 25-42 °C and the fermentation time was 24 h-60 h to obtain a bioflocculant.

6. A bioflocculant according to claim 5, characterized in that: The fermentation medium is composed of 5-40 g / L carbon source, 5-40 g / L nitrogen source, 0-6 g / L inorganic salt, 0-3 g / L metal ion, and the rest is water, with a pH of 5.5-8.

0.

7. A bioflocculant according to claim 6, characterized in that: The fermentation medium comprises: 15 g / L molasses acid hydrolyzate, 20 g / L urea, 2 g / L Na2HPO4•3H2O, 2.5 g / L NaH2PO4, 0.5 g / L MgSO4•7H2O, 0.01 g / L ferrous sulfate, 0.002 g / L copper sulfate, 0.005 g / L zinc sulfate, 0.3 g / L calcium chloride, 0.02 g / L manganese sulfate, and the rest is water, with a pH of 7.

4.

8. The bioflocculant according to claim 6, characterized in that: The fermentation liquid was processed, the supernatant was collected by centrifugation, and 5 times of anhydrous ethanol was added according to the volume ratio. The liquid was allowed to settle for 24 hours and centrifuged at 4000 rpm. The precipitate was collected and dried to obtain a crude bioflocculant.

9. Use of the bioflocculant according to any one of claims 4 to 8 in sewage treatment.

10. The use according to claim 9, characterized in that Add bioflocculant to the wastewater to be treated at a dosage of 1%~10%.

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