Composite microbial preparation for water pollution control and preparation method thereof

Abstract

The invention relates to the technical field of environmental treatment, in particular to a composite microbial preparation for water pollution treatment and a preparation method thereof. The composite microbial preparation for water pollution control is prepared from the following components in percentage by mass: 22 to 35 percent of magnetic targeting bacteria-algae symbiont, 5 to 6 percent of sodium carboxymethyl cellulose, 3 to 4 percent of charcoal nano powder, 35 to 40 percent of sodium alginate, 10 to 12 percent of polyvinyl alcohol, 2 to 3 percent of chitosan, 5 to 6 percent of trehalose, 3 to 4 percent of glycerol and 2 to 3 percent of calcium carbonate. The algal-bacterial symbiotic system disclosed by the invention has relatively high bacterial colony density, can efficiently degrade various pollutants in a polluted water body, can realize accurate positioning and enrichment treatment, realizes long-acting self-maintenance purification of water pollution treatment by combining with an algal-bacterial mutual benefit micro-ecological system, and is environment-friendly in all components, low in cost and easy to popularize. The risk of secondary pollution is avoided, and the unification of efficient treatment and environmental safety is realized.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of environmental governance, in particular to a composite microbial preparation for water pollution treatment and a preparation method thereof. BACKGROUND

[0002] Currently, in the face of increasingly serious water body eutrophication problem, microbial treatment reagent has become a key tool in the field of environmental remediation, mainly by putting specific functional microbial inoculants, to specifically degrade organic pollutants, ammonia nitrogen and total nitrogen and other pollutants in water, to achieve the purpose of purifying water quality and restoring ecological health of water body. Compared with traditional physical and chemical methods, microbial treatment method has the advantages of low cost, environmental friendliness and no secondary pollution, and is widely used in water quality improvement engineering of natural river, landscape water body and aquaculture factory.

[0003] However, the existing water pollution treatment microbial reagent still has obvious deficiencies, the traditional bacterial agent has single function, can only target specific pollutants, and is difficult to cope with complex water pollution conditions, and the microbial activity is easily affected by environmental factors, dispersed quickly in water, and has short retention time, resulting in unstable treatment effect and limited duration; although the chemical reinforced treatment reagent has quick effect, it will destroy the ecological balance of water body and cause secondary pollution, therefore, it is urgent to develop a water pollution treatment preparation with high efficiency, long duration and environmental friendliness.

[0004] In view of the above problems, the present application provides a solution. SUMMARY

[0005] The present application aims to provide a composite microbial preparation for water pollution treatment and a preparation method thereof, which can effectively treat complex water pollution and has no toxicity to the environment.

[0006] To achieve the above-mentioned purpose, the present application adopts the following technical scheme: a composite microbial preparation for water pollution treatment consists of the following components in mass percentage: 22-35% magnetic targeting algal symbiont, 5-6% sodium carboxymethyl cellulose, 3-4% biochar nanopowder, 35-40% sodium alginate, 10-12% polyvinyl alcohol, 2-3% chitosan, 5-6% trehalose, 3-4% glycerol and 2-3% calcium carbonate. The preparation method of the magnetic targeting algal symbiont comprises the following steps: A1: high-density expansion of bacillus subtilis, bacillus amyloliquefaciens, bacillus thuringiensis, nitrifying bacteria, denitrifying bacteria, rhodopseudomonas palustris, saccharomyces cerevisiae, chlorella and scenedesmus in the fermentation tank, and then centrifugation at 4 DEG C and 10000 rpm to obtain bacillus subtilis slurry, denitrifying bacteria slurry, rhodopseudomonas palustris slurry, nitrifying bacteria slurry, saccharomyces cerevisiae slurry, chlorella slurry and scenedesmus slurry; A2: Bacillus subtilis slurry, Bacillus amyloliquefaciens slurry, Bacillus thuringiensis slurry, denitrifying bacteria slurry, Rhodopseudomonas palustris slurry, nitrifying bacteria slurry, Saccharomyces cerevisiae slurry, Chlorella vulgaris algal slurry and Scenedesmus algal slurry were transferred to a reaction kettle, the stirrer speed was set to 500 rpm, the temperature was 4℃, and the mixture was stirred for 5 min to obtain a primary bacteria-algae slurry; then trehalose and glycerol were added to the primary bacteria-algae slurry, the stirrer temperature and speed were unchanged, and the stirring was repeated for 60 min to obtain a composite bacteria-algae slurry; A3: Amino-functionalized superparamagnetic magnetite nanoparticles were accurately weighed using an analytical balance, then the amino-functionalized superparamagnetic magnetite nanoparticles were added to sterile phosphate buffer to obtain a magnetic nanoparticle suspension, the magnetic nanoparticle suspension was placed in an ice water bath, then the ultrasonic instrument power was set to 500 W, the ice water bath magnetic nanoparticle suspension was treated for 2 s and paused for 3 s for 10-15 min to obtain an activated magnetic nanoparticle suspension; A4: The composite bacteria-algae slurry and the activated magnetic nanoparticle suspension were transferred to a vacuum homogenizer, then active zeolite powder, hydroxypropyl starch and potassium dihydrogen phosphate were added, the vacuum homogenizer temperature was set to 4℃, the speed was set to 200 rpm, and the mixture was homogenized under-0.05 MPa to-0.08 MPa vacuum conditions for 30 min, then it was transferred to a 4℃ refrigerator and left to stand for 30-45 min to obtain a mature slurry; A5: The mature slurry was extruded into a fine strip through a 0.8 mm screen extrusion granulator, then transferred to a high-speed rotary granulator, and rounded into spherical wet granules with a particle size of 0.5-1 mm, the spherical wet granules were laid flat on a tray, the tray was placed in a-45℃ ultra-low temperature freezer for pre-freezing for 4h to obtain pre-frozen symbiotic granules; A6: The pre-frozen symbiotic granules were transferred to a freeze-drying chamber, the temperature was set to-25℃, the pressure was set to 10 Pa, the primary drying was carried out for 24 h, then the temperature was set to 25℃, the pressure was set to 10 Pa, and the secondary drying was carried out for 6 h to obtain dry symbiotic granules; A7: The dry symbiotic granules were crushed in a turbo mill, then sieved using a vibrating sieve separator, and the dry symbiotic granules that passed through the 80 mesh but were retained on the 120 mesh screen were collected to obtain magnetic targeting bacteria-algae symbionts, which were vacuum packaged and stored at 4℃ under low temperature; Furthermore, in step A2, the mass ratio of Bacillus subtilis sludge, denitrifying bacteria sludge, Rhodopseudomonas palustris sludge, nitrifying bacteria sludge, Saccharomyces cerevisiae sludge, Chlorella pulveratum sludge, and Scenedesmus stenoptera sludge is 8:4:2:20:12:8:6:24:16; the mass ratio of primary bacterial-algal sludge, trehalose, and glycerol in step A2 is 100:10:5; the mass ratio of aminated superparamagnetic iron oxide nanoparticles and sterile phosphate buffer in step A3 is 1:20; and the mass ratio of composite bacterial-algal sludge, activated magnetic nanoparticle suspension, activated zeolite powder, hydroxypropyl starch, and potassium dihydrogen phosphate in step A4 is 100:200:20:15:2. Furthermore, a method for preparing a compound microbial agent for water pollution treatment includes the following steps: B1: Weigh sodium alginate and add it to deionized water, then transfer it to a 45-50℃ water bath. Set the stirrer speed to 500 rpm and stir to dissolve for 15 minutes. Let it stand for 10 minutes to obtain sodium alginate gel. Weigh polyvinyl alcohol and add it to hot water at 85-90℃. Set the stirrer speed to 2000 rpm and stir to dissolve for 15 minutes to obtain polyvinyl alcohol solution. B2: Weigh chitosan and add it to a 1% acetic acid solution. Set the stirrer speed to 500 rpm and stir for 10 minutes to dissolve the chitosan in acetic acid solution. Weigh biochar nanopowder and add it to 100 ml of deionized water. Disperse the biochar using ultrasonication at 400 W for 10 minutes to obtain a biochar dispersion. B3: Add the magnetically targeted algal symbiont, sodium carboxymethyl cellulose, biochar dispersion, trehalose, glycerol and calcium carbonate into a vacuum homogenizer. Set the vacuum homogenizer temperature to 25℃, the rotation speed to 800-1000 rpm, and the vacuum degree to -0.07 MPa. Vacuum mix for 15-20 min to obtain the active mixture. B4: Add sodium alginate solution, polyvinyl alcohol solution and chitosan acetic acid solution to the active mixture in sequence, set the speed of the vacuum homogenizer to 1200-1500 rpm and the vacuum degree to -0.09 MPa, and stir for 30-45 min to obtain the composite gel slurry; B5: Transfer the composite gel slurry to a pressure dripping pellet mill. Set the pressure of the pressure dripping pellet mill to 0.3 MPa and the nozzle orifice diameter to 0.8 mm. Drip the composite gel slurry into a curing tank containing 4% calcium chloride solution. The composite gel slurry drips into the curing tank to form solid gel microspheres. Let the solid gel microspheres stand and cure in the curing tank for 30 minutes to obtain wet-process formulation microspheres. B6: The wet-process formulation microspheres are retrieved, rinsed three times with deionized water, and then transferred to a fluidized bed dryer. The inlet air temperature is set to 35℃, and the drying time is 45-60 minutes. After drying, the microspheres are transferred to a vibrating sieve to collect uniform particles that pass through 20 mesh but remain at 40 mesh to obtain the water pollution treatment compound microbial preparation. The water pollution treatment compound microbial preparation is vacuum-packed and sealed in an aluminum foil composite bag and stored at 4℃.

[0007] Furthermore, the mass ratio of sodium alginate to deionized water in step B1 is 4:50; the mass ratio of polyvinyl alcohol to hot water in step B1 is 1:10; and the mass ratio of chitosan to 1% acetic acid solution in step B2 is 1:50. In summary, due to the adoption of the above technical solution, the beneficial effects of the present invention are as follows: 1: This invention utilizes a complete pollutant degradation chain composed of bacteria and microalgae to simultaneously and efficiently convert key pollutants such as ammonia nitrogen, organic matter, total nitrogen, and total phosphorus in water into harmless substances rather than simply adsorbing them. The overall purification efficiency far exceeds that of traditional single-function adsorbents and chemical agents. 2: This invention can achieve precise navigation and enrichment through an external magnetic field, directly delivering active ingredients to the core pollution area, greatly improving the utilization rate of the preparation, while forming a self-oxygenated and self-circulating micro-ecosystem, solving the problem of traditional bacterial agents failing due to lack of oxygen and nutrients, and realizing the upgrade of water pollution control from short-term intervention to long-term steady state. 3. All ingredients in this product are safe materials with good biocompatibility. Its mechanism of action is ecological regulation rather than chemical poisoning. It competitively inhibits harmful algae by restoring the beneficial microbial community in the water, thereby improving water quality from the source and ensuring a high degree of environmental safety in the treatment process. It is suitable for sensitive water areas such as landscapes and aquaculture. Detailed Implementation

[0008] To make the technical means, creative features, objectives and effects of this invention easier to understand, the invention will be further described below with reference to specific embodiments.

[0009] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0010] Example 1 1: High-density expansion culture of Bacillus subtilis, Bacillus amyloliquefaciens, Bacillus thuringiensis, nitrifying bacteria, denitrifying bacteria, Rhodopseudomonas palustris, Saccharomyces cerevisiae, Chlorella vulgaris, and Scenedesmus was carried out in a fermenter, and then centrifuged at 4°C and 10,000 rpm to obtain Bacillus subtilis bacterial sludge, denitrifying bacteria bacterial sludge, Rhodopseudomonas palustris bacterial sludge, nitrifying bacteria bacterial sludge, Saccharomyces cerevisiae bacterial sludge, Chlorella vulgaris algae sludge, and Scenedesmus algae sludge. 2: Transfer 80g of Bacillus subtilis sludge, 40g of Bacillus amyloliquefaciens sludge, 20g of Bacillus thuringiensis sludge, 200g of denitrifying bacteria sludge, 120g of Rhodopseudomonas palustris sludge, 80g of nitrifying bacteria sludge, 60g of Saccharomyces cerevisiae sludge, 240g of Chlorella vulgaris sludge, and 160g of Scenedesmus spp. sludge to a reaction vessel. Set the stirrer speed to 500 rpm and the temperature to 4℃. Stir for 5 minutes to obtain 1kg of primary bacterial and algal sludge. Then add 100g of trehalose and 50g of glycerol to the primary bacterial and algal sludge. Keep the stirrer temperature and speed constant and repeat stirring for 60 minutes to obtain 1.1kg of composite bacterial and algal sludge. 3: Accurately weigh 100g of aminated superparamagnetic iron oxide nanoparticles using an analytical balance. Then, add the aminated superparamagnetic iron oxide nanoparticles to 2kg of sterile phosphate buffer to obtain a magnetic nanoparticle suspension. Place the magnetic nanoparticle suspension in an ice-water bath, then set the ultrasonic power to 500W, sonicate for 2s, pause for 3s, and process the magnetic nanoparticle suspension in the ice-water bath for 10min to obtain 2.1kg of activated magnetic nanoparticle suspension. 4: Transfer 1 kg of composite bacterial algae mud and 2 kg of activated magnetic nanoparticle suspension to a vacuum homogenizer, then add 200 g of activated zeolite powder, 150 g of hydroxypropyl starch and 20 g of potassium dihydrogen phosphate. Set the vacuum homogenizer temperature to 4℃ and the rotation speed to 200 rpm. Homogenize and mix for 30 min under a vacuum of -0.08 MPa. After homogenization, transfer to a 4℃ refrigerator and let stand for 30 min to obtain mature slurry. 5: The matured slurry is extruded into thin strips through an extrusion granulator with a screen size of 0.8mm. Then it is transferred to a high-speed rotary granulator and rolled into spherical wet particles with a particle size of 0.5mm. The spherical wet particles are spread evenly in a material tray and the material tray is placed in a -45℃ ultra-low temperature freezer for 4 hours to obtain pre-frozen symbiotic particles. 6: Transfer the pre-frozen symbiont particles to a freeze-drying chamber, set the freeze-drying chamber temperature to -25℃ and the pressure to 10Pa, and perform the first drying for 24 hours. After the first drying is completed, set the freeze-drying chamber temperature to 25℃ and the pressure to 10Pa, and perform the second drying for 6 hours to obtain dried symbiont particles. 7: The dried symbiotic particles were crushed in a turbine pulverizer and then sieved using a vibrating screen. The dried symbiotic particles that passed through the 80-mesh screen but remained in the 120-mesh screen were collected to obtain the magnetically targeted bacterial and algal symbiotic prepared in Example 1. The magnetically targeted bacterial and algal symbiotic was vacuum-packed and stored at a low temperature of 4°C. Example 2 1: High-density expansion culture of Bacillus subtilis, Bacillus amyloliquefaciens, Bacillus thuringiensis, nitrifying bacteria, denitrifying bacteria, Rhodopseudomonas palustris, Saccharomyces cerevisiae, Chlorella vulgaris, and Scenedesmus was carried out in a fermenter, and then centrifuged at 4°C and 10,000 rpm to obtain Bacillus subtilis bacterial sludge, denitrifying bacteria bacterial sludge, Rhodopseudomonas palustris bacterial sludge, nitrifying bacteria bacterial sludge, Saccharomyces cerevisiae bacterial sludge, Chlorella vulgaris algae sludge, and Scenedesmus algae sludge. 2: Transfer 240g of Bacillus subtilis sludge, 120g of Bacillus amyloliquefaciens sludge, 60g of Bacillus thuringiensis sludge, 600g of denitrifying bacteria sludge, 360g of Rhodopseudomonas palustris sludge, 240g of nitrifying bacteria sludge, 180g of Saccharomyces cerevisiae sludge, 720g of Chlorella vulgaris sludge, and 480g of Scenedesmus spp. sludge into a reaction vessel. Set the stirrer speed to 500 rpm and the temperature to 4℃, and stir for 5 minutes to obtain primary bacterial and algal sludge. Then add trehalose and glycerol to the primary bacterial and algal sludge, keeping the stirrer temperature and speed constant, and repeat stirring for 60 minutes to obtain 3kg of composite bacterial and algal sludge. 3: Accurately weigh 300g of aminated superparamagnetic iron oxide nanoparticles using an analytical balance. Then, add the aminated superparamagnetic iron oxide nanoparticles to 6kg of sterile phosphate buffer to obtain a magnetic nanoparticle suspension. Place the magnetic nanoparticle suspension in an ice-water bath, then set the ultrasonic power to 500W, sonicate for 2s, pause for 3s, and process the magnetic nanoparticle suspension in the ice-water bath for 15min to obtain an activated magnetic nanoparticle suspension. 4: Transfer 3 kg of composite bacterial algae mud and 6 kg of activated magnetic nanoparticle suspension to a vacuum homogenizer, then add 600 g of activated zeolite powder, 450 g of hydroxypropyl starch and 60 g of potassium dihydrogen phosphate. Set the vacuum homogenizer temperature to 4℃ and the rotation speed to 200 rpm. Homogenize and mix for 30 min under a vacuum of -0.05 MPa. After homogenization, transfer to a 4℃ refrigerator and let stand for 45 min to obtain mature slurry. 5: The matured slurry is extruded into thin strips through an extrusion granulator with a screen size of 0.8mm. Then it is transferred to a high-speed rotary granulator and rolled into spherical wet particles with a particle size of 1mm. The spherical wet particles are spread evenly in a material tray and the material tray is placed in a -45℃ ultra-low temperature freezer for 4 hours to obtain pre-frozen symbiotic particles. 6: Transfer the pre-frozen symbiont particles to a freeze-drying chamber, set the freeze-drying chamber temperature to -25℃ and the pressure to 10Pa, and perform the first drying for 24 hours. After the first drying is completed, set the freeze-drying chamber temperature to 25℃ and the pressure to 10Pa, and perform the second drying for 6 hours to obtain dried symbiont particles. 7: The dried symbiotic particles were crushed in a turbine pulverizer and then sieved using a vibrating screen. The dried symbiotic particles that passed through the 80-mesh screen but remained on the 120-mesh screen were collected to obtain the magnetically targeted bacterial and algal symbiotic prepared in Example 2. The magnetically targeted bacterial and algal symbiotic was vacuum-packed and stored at 4°C. Example 3 1. Weigh 400g of sodium alginate and add it to 5kg of deionized water. Then transfer it to a 45℃ water bath, set the stirrer speed to 500rpm, stir and dissolve for 15min, and let it stand for 10min to obtain sodium alginate gel. Weigh 120g of polyvinyl alcohol and add it to 1.2kg of 85℃ hot water, set the stirrer speed to 2000rpm, stir and dissolve for 15min to obtain polyvinyl alcohol solution. 2: Weigh 30g of chitosan and add it to 1.5kg of 1% acetic acid solution. Set the stirrer speed to 500rpm and stir for 10min to dissolve the chitosan and obtain a chitosan acetic acid solution. Weigh 40g of biochar nanoparticles and add them to 100ml of deionized water. Disperse the biochar using ultrasonication at 400W for 10min to obtain a biochar dispersion. 3: 220g of the magnetically targeted bacterial-algal symbiont prepared in Example 1, 60g of sodium carboxymethyl cellulose, biochar dispersion, 60g of trehalose, 40g of glycerol and 30g of calcium carbonate were put into a vacuum homogenizer. The temperature of the vacuum homogenizer was set to 25℃, the rotation speed to 800rpm and the vacuum degree to -0.07MPa. The mixture was vacuum mixed for 15min to obtain an active mixture. 4: Add sodium alginate solution, polyvinyl alcohol solution and chitosan acetic acid solution to the active mixture in sequence, set the speed of the vacuum homogenizer to 1200 rpm and the vacuum degree to -0.09 MPa, and stir for 30 min to obtain the composite gel slurry; 5: Transfer the composite gel slurry to a pressure pelletizing machine. Set the pressure of the pressure pelletizing machine to 0.3 MPa and the nozzle orifice diameter to 0.8 mm. Drop the composite gel slurry into a curing tank containing 4% calcium chloride solution. The composite gel slurry drops into the curing tank to form solid gel microspheres. Let the solid gel microspheres stand and cure in the curing tank for 30 minutes to obtain wet-process formulation microspheres. 6: The wet-process formulation microspheres were retrieved, rinsed three times with deionized water, and then transferred to a fluidized bed dryer. The inlet air temperature was set to 35°C, and the drying time was 45 minutes. After drying, the microspheres were transferred to a vibrating sieve to collect uniform particles that passed through 20 mesh but remained at 40 mesh, thus obtaining the water pollution treatment composite microbial preparation prepared in Example 3. The water pollution treatment composite microbial preparation was vacuum-packed and sealed in an aluminum foil composite bag and stored at 4°C.

[0011] Example 4 1. Weigh 350g of sodium alginate and add it to 4.375kg of deionized water. Then transfer it to a 50℃ water bath, set the stirrer speed to 500rpm, stir and dissolve for 15min, and let it stand for 10min to obtain sodium alginate gel. Weigh 100g of polyvinyl alcohol and add it to 1kg of 90℃ hot water, set the stirrer speed to 2000rpm, stir and dissolve for 15min to obtain polyvinyl alcohol solution. 2: Weigh 20g of chitosan and add it to 1kg of 1% acetic acid solution. Set the stirrer speed to 500rpm and stir for 10min to dissolve the chitosan and obtain a chitosan acetic acid solution. Weigh 30g of biochar nanoparticles and add them to 100ml of deionized water. Disperse the biochar dispersion using ultrasonication at 400W for 10min. 3: Add 350g of the magnetically targeted bacterial-algae symbiotic prepared in Example 2, 50g of sodium carboxymethyl cellulose, biochar dispersion, 50g of trehalose, 30g of glycerol and 20g of calcium carbonate into a vacuum homogenizer. Set the temperature of the vacuum homogenizer to 25℃, the rotation speed to 1000rpm and the vacuum degree to -0.07MPa. Vacuum mix for 20min to obtain an active mixture. 4: Add sodium alginate solution, polyvinyl alcohol solution and chitosan acetic acid solution to the active mixture in sequence, set the speed of the vacuum homogenizer to 1500 rpm and the vacuum degree to -0.09 MPa, and stir for 45 min to obtain the composite gel slurry; 5: Transfer the composite gel slurry to a pressure pelletizing machine. Set the pressure of the pressure pelletizing machine to 0.3 MPa and the nozzle orifice diameter to 0.8 mm. Drop the composite gel slurry into a curing tank containing 4% calcium chloride solution. The composite gel slurry drops into the curing tank to form solid gel microspheres. Let the solid gel microspheres stand and cure in the curing tank for 30 minutes to obtain wet-process formulation microspheres. 6: The wet-process formulation microspheres were retrieved, rinsed three times with deionized water, and then transferred to a fluidized bed dryer. The inlet air temperature was set to 35°C, and the drying time was 60 minutes. After drying, the microspheres were transferred to a vibrating sieve to collect uniform particles that passed through 20 mesh but remained at 40 mesh, thus obtaining the water pollution treatment composite microbial preparation prepared in Example 4. The water pollution treatment composite microbial preparation was vacuum-packed and sealed in an aluminum foil composite bag and stored at 4°C.

[0012] Comparative Example 1 Example 7 of Chinese Patent Publication No. CN119797610A was selected as Comparative Example 1.

[0013] Comparative Example 2 The specific implementation of Chinese Patent No. CN117204443A was selected as Comparative Example 2.

[0014] Core colony count test Accurately weigh 1.0 g of the formulations prepared in Examples 1, 2, and 1 Comparative Example, place them in an Erlenmeyer flask containing 100 ml of sterile phosphate buffer, add sterile glass beads, and shake on a shaker at 200 rpm for 30 min to ensure that the particles are fully broken and the bacteria are completely eluted to obtain bacterial suspensions of Example 1, 2, and 3 Comparative Example; take 1 ml of the formulation prepared in Comparative Example 2 directly as a bacterial suspension sample and label it as Comparative Example 2 bacterial suspension. The bacterial suspensions from Example 1, Example 2, and Comparative Example 3 were diluted to a concentration of 10 μL using sterile physiological saline. -7 10 -8 and 10 -9 The test samples were diluted with sterile physiological saline to a concentration of 10 μL for the control sample. -5 10 -6 and 10 -7 For each test sample, take 0.1 ml of all the above test samples and spread them on a nutrient agar plate. Perform three replicates for each dilution. After coating, the plates were inverted and incubated at 37°C for 72 hours. Plates with colony counts between 30 and 300 were selected for counting. The entire operation was performed under aseptic conditions.

[0015] Table 1. Results of core colony count test

[0016] Performance testing of removal rates of key pollutants in eutrophic water bodies Take tap water and add glucose, ammonium chloride, potassium dihydrogen phosphate, yeast extract and trace element solution to prepare synthetic wastewater. The initial concentrations of key pollutants in the synthetic wastewater are: ammonia nitrogen 15 mg / L, chemical oxygen demand (COD) 200 mg / L, total nitrogen 20 mg / L and total phosphorus 3 mg / L. Take 1.5L of synthetic wastewater and place it in a 2L clean beaker as the test water. Add the preparations prepared in Examples 3, 4, Comparative Example 1 and Comparative Example 2 to the test water at a uniform dosage of 1g / L. Set up a blank group and add an equal amount of deionized water. Each test sample is repeated 3 times and the blank group is repeated 2 times. Place the test beaker in a 25°C environment, protect it from light and let it stand. Stir it manually 3 times a day to simulate a slight water flow. Then, take samples on the 3rd, 5th and 7th day after addition, detect the concentration of key pollutants in the supernatant and calculate the removal rate. Table 2. Performance test results of removal rates of key pollutants in eutrophic water bodies

[0017] Analysis of Table 2 shows that, based on all the data, the pollution control capabilities of the formulations prepared in Examples 3 and 4 are continuous, efficient, and comprehensive. As time goes on, the advantages continue to expand and eventually reach a high level of efficient removal. In contrast, Comparative Example 1 only performs well in the removal of total phosphorus, while Comparative Example 2, as an algae inhibitor, performs poorly in the treatment of complex water bodies.

[0018] Eco-acute toxicity test Following OECD guidelines, the formulations prepared in Examples 3, 4, Comparative Examples 1, and 2 were diluted with purified water to prepare test solutions of 10, 50, 100, 300, and 500 mg / L. For each concentration group, 20 healthy daphnia juveniles less than 24 hours old were placed in 50 ml of the test solution. A blank control group and a positive control group were also set up. The blank control was purified water, and the positive control was potassium dichromate solution at concentrations of 10, 50, 100, 300, and 500 mg / L. The juvenile daphnia were statically cultured at 20°C under a 16:8 hour light-dark cycle for 48 hours without feeding. At 48 hours, the number of immobile daphnia in each container was recorded, and the half-maximal effect concentration (EC50) was calculated. Referring to ISO 11348, the formulations prepared in Examples 3, 4, Comparative Example 1, and Comparative Example 2 were diluted with 2% NaCl solution to prepare test solutions of 10, 50, 100, 300, and 500 mg / L. 10 ml of the test solution and 10 ml of the revived luminescent bacteria culture were added to a test tube, and the mixture was reacted in a water bath at 15°C for 15 min. Blank control and quality control were set up at the same time. The luminescence intensity of each tube was measured using a luminometer, and the luminescence inhibition rate at each concentration was calculated and the half-maximal inhibitory concentration (IC50) was calculated. Table 3. Results of Eco-acute toxicity tests

[0019] As can be seen from the analysis of Table 3, the formulations prepared in Examples 3 and 4 showed the highest environmental safety among all tested samples. Their EC50 and IC50 values ​​were much higher than the threshold of 100 mg / L for conventional toxic substances, indicating that under normal use conditions that achieve effective purification concentrations, the formulations prepared in Examples 3 and 4 are safe for aquatic ecosystems and will not cause acute harm to non-target organisms. Comparative Example 1 showed low toxicity, indicating that in the actual application of Comparative Example 1, the nanoparticles can penetrate the biomembrane and cause oxidative stress. At the same time, the slow release of metal ions in the material can also produce some toxicity. Comparative Example 2, as an algae inhibitor, works by killing and inhibiting microorganisms through toxicity. In practical applications, while killing harmful algae, Comparative Example 2 also causes serious damage to non-target aquatic organisms such as Daphnia macrocarpa and bioluminescent bacteria.

[0020] The foregoing has shown and described the basic principles, main features, and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited to the above embodiments. The embodiments and descriptions in the specification are merely illustrative of the principles of the invention. Various changes and modifications can be made to the invention without departing from its spirit and scope, and all such changes and modifications fall within the scope of the present invention as claimed. The scope of protection of this invention is defined by the appended claims and their equivalents.

Claims

1. A compound microbial preparation for water pollution treatment, characterized in that, It is composed of the following components by weight percentage: 22-35% magnetically targeted bacterial and algal symbiotic, 5-6% sodium carboxymethyl cellulose, 3-4% biochar nanoparticles, 35-40% sodium alginate, 10-12% polyvinyl alcohol, 2-3% chitosan, 5-6% trehalose, 3-4% glycerol and 2-3% calcium carbonate.

2. The compound microbial preparation for water pollution treatment according to claim 1, characterized in that, The method for preparing the magnetically targeted bacterial-algal symbiotic includes the following steps: A1: High-density expansion culture of Bacillus subtilis, Bacillus amyloliquefaciens, Bacillus thuringiensis, nitrifying bacteria, denitrifying bacteria, Rhodopseudomonas palustris, Saccharomyces cerevisiae, Chlorella vulgaris, and Scenedesmus was carried out in a fermenter. Subsequently, the cultures were centrifuged at 4°C and 10,000 rpm to obtain Bacillus subtilis bacterial sludge, denitrifying bacteria bacterial sludge, Rhodopseudomonas palustris bacterial sludge, nitrifying bacteria bacterial sludge, Saccharomyces cerevisiae bacterial sludge, Chlorella vulgaris algae sludge, and Scenedesmus algae sludge. A2: Transfer the Bacillus subtilis, Bacillus amyloliquefaciens, Bacillus thuringiensis, denitrifying bacteria, Rhodopseudomonas palustris, nitrifying bacteria, Saccharomyces cerevisiae, Chlorella, and Scenedesmus to a reaction vessel. Set the stirrer speed to 500 rpm and the temperature to 4°C, and stir for 5 minutes to obtain the primary bacterial and algal slurry. Then, add trehalose and glycerol to the primary bacterial and algal slurry, and repeat stirring for 60 minutes while keeping the stirrer temperature and speed constant to obtain the composite bacterial and algal slurry. A3: Accurately weigh the aminated superparamagnetic iron oxide nanoparticles using an analytical balance, then add the aminated superparamagnetic iron oxide nanoparticles to sterile phosphate buffer to obtain a magnetic nanoparticle suspension. Place the magnetic nanoparticle suspension in an ice-water bath, then set the ultrasonic power to 500W, sonicate for 2 seconds, pause for 3 seconds, and treat the magnetic nanoparticle suspension in the ice-water bath for 10-15 minutes to obtain an activated magnetic nanoparticle suspension. A4: Transfer the composite bacterial algae mud and activated magnetic nanoparticle suspension to a vacuum homogenizer, then add activated zeolite powder, hydroxypropyl starch and potassium dihydrogen phosphate. Set the vacuum homogenizer temperature to 4℃ and the rotation speed to 200rpm. Homogenize and mix for 30min under a vacuum condition of -0.05MPa to -0.08MPa. After homogenization and mixing, transfer to a 4℃ refrigerator and let stand for 30-45min to obtain a mature slurry. A5: The matured slurry is extruded into thin strips through an extrusion granulator with a screen size of 0.8mm. Then it is transferred to a high-speed rotary granulator and rolled into spherical wet granules with a particle size of 0.5-1mm. The spherical wet granules are spread evenly in a material tray and the material tray is placed in a -45℃ ultra-low temperature freezer for 4 hours to obtain pre-frozen symbiotic granules. A6: Transfer the pre-frozen symbiont particles to a freeze-drying chamber, set the freeze-drying chamber temperature to -25℃ and the pressure to 10Pa, and perform the first drying for 24 hours. After the first drying is completed, set the freeze-drying chamber temperature to 25℃ and the pressure to 10Pa, and perform the second drying for 6 hours to obtain dried symbiont particles. A7: Place the dried symbiotic particles in a turbine pulverizer to crush them, and then use a vibrating screener to screen them. Collect the dried symbiotic particles that pass through the 80-mesh screen but remain on the 120-mesh screen to obtain the magnetic-targeted bacterial-algae symbiotic. Vacuum pack the magnetic-targeted bacterial-algae symbiotic and store it at a low temperature of 4°C.

3. The compound microbial preparation for water pollution treatment according to claim 2, characterized in that, The mass ratio of Bacillus subtilis sludge, Bacillus amyloliquefaciens sludge, Bacillus thuringiensis sludge, denitrifying bacteria sludge, Rhodopseudomonas palustris spp. spp. nitrifying bacteria sludge, Saccharomyces cerevisiae sludge, Chlorella pulveratum sludge and Scenedesmus spp. ...

4. The compound microbial preparation for water pollution treatment according to claim 2, characterized in that, The mass ratio of the primary bacterial algae mud, trehalose, and glycerol mentioned in step A2 is 100:10:

5.

5. The compound microbial preparation for water pollution treatment according to claim 2, characterized in that, The mass ratio of the aminated superparamagnetic iron oxide nanoparticles to the sterile phosphate buffer solution in step A3 is 1:

20.

6. The compound microbial preparation for water pollution treatment according to claim 2, characterized in that, The mass ratio of the composite bacterial-algae mud, activated magnetic nanoparticle suspension, activated zeolite powder, hydroxypropyl starch and potassium dihydrogen phosphate mentioned in step A4 is 100:200:20:15:

2.

7. A method for preparing the composite microbial agent for water pollution treatment according to claim 1, characterized in that, Includes the following steps: B1: Weigh sodium alginate and add it to deionized water, then transfer it to a 45-50℃ water bath. Set the stirrer speed to 500 rpm and stir to dissolve for 15 minutes. Let it stand for 10 minutes to obtain sodium alginate gel. Weigh polyvinyl alcohol and add it to hot water at 85-90℃. Set the stirrer speed to 2000 rpm and stir to dissolve for 15 minutes to obtain polyvinyl alcohol solution. B2: Weigh chitosan and add it to a 1% acetic acid solution. Set the stirrer speed to 500 rpm and stir for 10 minutes to dissolve the chitosan in acetic acid solution. Weigh biochar nanopowder and add it to 100 ml of deionized water. Disperse the biochar using ultrasonication at 400 W for 10 minutes to obtain a biochar dispersion. B3: Add the magnetically targeted algal symbiont, sodium carboxymethyl cellulose, biochar dispersion, trehalose, glycerol and calcium carbonate into a vacuum homogenizer. Set the vacuum homogenizer temperature to 25℃, the rotation speed to 800-1000 rpm, and the vacuum degree to -0.07 MPa. Vacuum mix for 15-20 min to obtain the active mixture. B4: Add sodium alginate solution, polyvinyl alcohol solution and chitosan acetic acid solution to the active mixture in sequence, set the speed of the vacuum homogenizer to 1200-1500 rpm and the vacuum degree to -0.09 MPa, and stir for 30-45 min to obtain the composite gel slurry; B5: Transfer the composite gel slurry to a pressure dripping pellet mill. Set the pressure of the pressure dripping pellet mill to 0.3 MPa and the nozzle orifice diameter to 0.8 mm. Drip the composite gel slurry into a curing tank containing 4% calcium chloride solution. The composite gel slurry drips into the curing tank to form solid gel microspheres. Let the solid gel microspheres stand and cure in the curing tank for 30 minutes to obtain wet-process formulation microspheres. B6: The wet-process formulation microspheres are retrieved, rinsed three times with deionized water, and then transferred to a fluidized bed dryer. The inlet air temperature is set to 35℃, and the drying time is 45-60 minutes. After drying, the microspheres are transferred to a vibrating sieve to collect uniform particles that pass through 20 mesh but remain at 40 mesh to obtain the water pollution treatment compound microbial preparation. The water pollution treatment compound microbial preparation is vacuum-packed and sealed in an aluminum foil composite bag and stored at 4℃.

8. The method for preparing a compound microbial agent for water pollution treatment according to claim 7, characterized in that, The mass ratio of sodium alginate to deionized water in step B1 is 4:

50.

9. The method for preparing a compound microbial agent for water pollution treatment according to claim 7, characterized in that, The mass ratio of polyvinyl alcohol to hot water in step B1 is 1:

10.

10. The method for preparing a compound microbial agent for water pollution treatment according to claim 7, characterized in that, The mass ratio of chitosan to 1% acetic acid solution in step B2 is 1:50.

Images