Preparation method of bacteria-algae immobilized polyurethane carrier capable of being stored for long time and rapidly activated

Abstract

The invention discloses a preparation method of a bacteria-algae immobilized polyurethane carrier capable of being quickly activated and preserved. The method comprises the following steps: drying and dehydrating bacteria / algae at low temperature, and preparing composite powder particles from the dehydrated bacteria / algae, sodium alginate, a nutritive salt compound, inorganic skeleton powder and the like; then adding the mixture into a polyether polyol system, and uniformly distributing the bacteria and algae in the polyurethane three-dimensional porous skeleton through an in-situ foaming process, so as to realize in-situ synthesis of the stable and high-activity bacteria and algae; the carrier can be stored in a dark and sealed condition after being added into a protective solution; in a use stage, natural light is utilized for activating and putting into a polluted water body, so that the denitrification function can be quickly recovered. According to the method, a technical path integrating in-situ synthesis, sealed storage and quick start of the bacteria and algae is constructed, and the method has the advantages of retaining the activity of the bacteria and algae, realizing long-term storage and quick field application and the like.

Description

Technical Field

[0001] This invention belongs to the field of water treatment, specifically relating to a method for preparing a polyurethane carrier for immobilizing bacteria and algae that can be preserved for a long time and activated rapidly. Background Technology

[0002] Nitrogen pollution in water bodies has become one of the major challenges in the fields of global environmental science and ecological engineering. Excessive accumulation of nitrogen in water bodies can easily lead to eutrophication, which in turn causes a series of environmental problems such as the death of aquatic organisms and ecosystem imbalance.

[0003] Traditional biological nitrogen removal processes typically employ aeration to supply oxygen to the system, first oxidizing ammonia nitrogen into nitrite and nitrate, and then further reducing it back to nitrogen gas. However, the aeration process requires continuous power input, increasing system energy consumption and potentially disturbing functional bacterial communities, leading to bacterial loss and impacting nitrogen removal efficiency. Algae are widespread autotrophic organisms in natural water bodies, releasing oxygen through photosynthesis under light conditions. Nitrifying bacteria can oxidize ammonia nitrogen into nitrite and nitrate under aerobic conditions, but as chemoautotrophic microorganisms, they have long generation cycles and slow growth and reproduction. Constructing a symbiotic system of algae and nitrifying bacteria allows algae to produce oxygen through photosynthesis, which the nitrifying bacteria then use to oxidize ammonia nitrogen, thus achieving ammonia nitrogen oxidation without external aeration. Therefore, constructing a system where algae and nitrifying bacteria coexist is crucial for achieving the synergistic removal of ammonia nitrogen by algae and nitrifying bacteria.

[0004] Microbial immobilization technology, by embedding bacteria or microalgae into porous materials, can increase local microbial biomass, stabilize microbial community structure, and reduce microbial loss, making it an important strategy for maintaining microbial concentration and activity. Existing immobilization carriers mainly include natural organic carriers, inorganic carriers, and polymer carriers. Natural organic carriers, such as chitosan, alginate, and carrageenan, are widely used in microbial immobilization due to their good biocompatibility and relatively mild immobilization process. However, these materials typically suffer from low mechanical strength, susceptibility to swelling or degradation, and insufficient long-term stability, making them unsuitable for long-term operation in actual wastewater treatment. In contrast, inorganic carriers, represented by activated carbon, zeolite, and bentonite, can compensate for the structural strength deficiencies of natural materials to some extent due to their high specific surface area, stable physicochemical properties, and strong physical adsorption capacity. However, inorganic carriers also suffer from high material rigidity and low plasticity, which are not conducive to achieving efficient encapsulation and uniform dispersion of bacterial and algal cells. Polymer carriers, such as polyvinyl alcohol and polyacrylamide, possess both good mechanical strength and high durability, thus combining the advantages of natural and inorganic carriers to some extent. However, in practical applications, these carriers have long biofilm formation cycles (15-30 days), generally making it difficult to rapidly construct highly active bacterial and algal systems. Furthermore, the immobilized bacteria and algae are prone to activity decay under water-free conditions, with long recovery periods. Therefore, existing carriers cannot simultaneously meet the requirements of "efficient bacterial and algal immobilization" and "long-term stable maintenance of activity."

[0005] In summary, existing bioimmobilization methods struggle to construct and maintain highly active bacterial and algal systems over the long term, hindering their widespread application in practical engineering. Therefore, there is an urgent need to develop a novel method for preparing bacterial and algal immobilization carriers that enables efficient immobilization, long-term preservation, and rapid activation. Summary of the Invention

[0006] To address the aforementioned problems in existing technologies, this invention provides a method for preparing a polyurethane carrier for immobilized bacteria and algae that can be preserved long-term and activated rapidly. This invention constructs a polyurethane carrier that combines a highly active bacteria-algae system with long-term stable preservation capabilities, providing a stable attachment substrate and a favorable metabolic microenvironment for the bacteria-algae symbiotic system. Simultaneously, this invention uses a foaming process to immobilize bacteria and algae biomass in situ onto the polyurethane carrier. After adding a cell preservation solution and placing it in a sealed, light-protected environment, the carrier can be preserved long-term, and rapid reactivation and activation can be achieved after addition, thereby enabling the rapid start-up of an integrated bacteria-algae wastewater treatment system.

[0007] The present invention discloses a method for preparing a polyurethane carrier for immobilizing bacteria and algae that can be stored for a long time and activated rapidly, comprising the following steps:

[0008] Step 1: Cultivation of nitrifying bacteria and microalgae

[0009] Nitrifying bacteria were inoculated into a sequencing batch reactor (SBR) using ammonia nitrogen as the sole nitrogen source and enriched under conditions of 25-30℃, pH 7.5-8.5, and dissolved oxygen concentration ≥2 mg / L. The nitrifying bacteria culture was completed when the ammonia nitrogen removal rate in the system consistently reached over 90% and the nitrite accumulation rate was between 0-0.5 mg / L, with MLSS typically stable at 3000-4000 mg / L. Simultaneously, microalgae were inoculated into BG-11 medium and photoautotrophically cultured in a light incubator under the following conditions: temperature 25±1℃, light-dark cycle 12 h:12 h, and light intensity 5000 lx. The culture continued until the microalgae entered the logarithmic growth phase, reaching a concentration of (1.0-5.0)×10⁻⁶. 7 cells / mL.

[0010] Step 2: Algae and Bacteria Concentration

[0011] Nitrifying bacteria were removed from the SBR reactor and centrifuged at 4000-6000 rpm for 10-15 min. The supernatant was discarded to obtain nitrifying bacteria with a water content of 90-95%. The obtained nitrifying bacteria were then evenly spread in a thin layer of 3-5 mm thickness and placed in a freeze dryer at 0-4℃ for low-temperature pre-drying to an overall water content of 70-80%, forming nitrifying bacteria biomass that is easy to process later.

[0012] The algal solution was taken out from the microalgae light incubator and concentrated to a water content of 90-95% by dissolved air flotation to obtain concentrated microalgae. The obtained concentrated microalgae was evenly spread into a thin layer with a thickness of 3-5 mm and then placed in a freeze dryer at 0-4℃ for low-temperature pre-drying to an overall water content of 70-80%, forming concentrated microalgae biomass that is easy to process later.

[0013] Step 3: Pretreatment for the protection of the biomass activity of bacteria and algae

[0014] The nitrifying bacteria biomass and concentrated microalgae biomass obtained in step 2 are placed in a mixing container at a mass ratio of 1:1. First, 2-10 wt% sodium alginate is added to the resulting mixture as a structural support agent. A low-shear ribbon mixer is used to premix the mixture at a speed of 40-60 rpm for 5-10 minutes, controlling the temperature of the mixture to not exceed 35°C, so that the sodium alginate forms a uniform coating layer on the surface of the bacterial and algal biomass. After the above premixing is completed, 0.5-8 wt% of the nutrient salt compound is slowly added to the mixture while maintaining the same speed, so that it is evenly dispersed. Sodium alginate is used to coat the structure; then 1-15 wt% of inorganic framework powder is added, and mixing continues until a wet compound material with preliminary shaping properties is formed; finally, an ionic crosslinking precursor is added to the wet compound material to form an enhanced micro-crosslinking structure, improve the breakage resistance of the obtained powder, and adjust the overall moisture content of the mixture to 55%-65% to ensure compressibility and flowability during powder processing; the wet bacterial-algae compound formed by the above steps is granulated by passing it through a 60-200 mesh shaping sieve to obtain uniform bacterial-algae compound powder particles.

[0015] In step 3, the nutrient salt compound includes a nitrogen source, a phosphorus source, and trace metal ions, wherein the nitrogen source is NO3. - and / or NH4 + The phosphorus source is H2PO4. - and / or HPO4 2- Trace metal ions include Fe 3+ Mg 2+ Co 2+ and MoO4 2- It is used to promote the reactivation of the subsequent algae removal complex system. The molar ratio of nitrogen source (calculated as N) to phosphorus source (calculated as P) is 10:1; the mass content of all trace metal ions in the nutrient salt complex is ≤1%.

[0016] In step 3, the inorganic framework powder is bentonite, diatomite, or zeolite powder; the ionic crosslinking precursor can be 0.05-0.2wt% CaCl2 solid.

[0017] Step 4: Preparation of polyurethane carrier for bacterial and algal immobilization

[0018] The algae-microbe composite powder obtained in step 3 was added to the polyether polyol system. The algae-microbe composite particles were uniformly dispersed in the polyol system by mechanical stirring at 500-800 rpm to form an algae-microbe polyol premix with good flowability. Then, a foaming agent was added to the algae-microbe polyol premix, and stirring was continued until the system was homogeneous. The mixture was then degassed under a negative pressure of 50-80 kPa for 5-10 min to remove the mixed air and promote the uniformity of closed cells in the subsequent foaming structure. Under the condition of maintaining negative pressure, isocyanate was added to the degassed premix in a slow and continuous dripping manner to allow the isocyanate to undergo an in-situ foaming polymerization reaction with the polyol system. After the dripping was completed, the reaction system was allowed to stand and solidify at room temperature. Then, it was cured at 25-35℃ for 24-48 h to obtain the algae-microbe immobilized polyurethane carrier.

[0019] In step 4, the polyether polyol system includes polyether polyol, triethanolamine, dimethylolpropionic acid, and stabilizer.

[0020] Furthermore, the polyether polyol is selected from one or more of polyether 330, polyether 330N, and polyether 320, and is used to provide flexible soft segments of the polyurethane skeleton and adjust the mechanical properties and pore structure of the carrier. The stabilizer is silicone oil, which is used to improve the stability of the foam structure and prevent foam collapse.

[0021] In step 4, the foaming agent is a mixture of triethylamine and dichloromethane, prepared at a mass ratio of 1:1, used to adjust the foaming rate and cell uniformity of the polyurethane system. The isocyanate is selected from one or more of toluene-2,4-diisocyanate (TDI-2,4), toluene-2,6-diisocyanate (TDI-2,6), and naphthalene-1,5-diisocyanate (NDI-1,5), used to react with polyether polyol and auxiliary crosslinking agent to generate a polyurethane network.

[0022] In step 4, the components are proportioned by mass as follows:

[0023] The mixture consists of 5 parts of algal-bacterial complex powder particles, 80 parts of polyether polyol, 5 parts of triethanolamine, 2 parts of dimethylolpropionic acid, 2 parts of stabilizer, 5 parts of isocyanate, and 1 part of foaming agent.

[0024] In step 4, the curing process means that the surface has a certain strength, the internal pore structure is formed, and the residual isocyanate content is less than 1%.

[0025] Step 5: Media Preservation

[0026] Spray a cell protection solution of 6%-8% (w / v) trehalose and 0.2 mmol / L ascorbic acid onto the surface of the polyurethane carrier for immobilized bacteria and algae obtained in step 4. Let it stand for 20-30 minutes to allow the protectant to fully penetrate into the pores of the carrier and the microenvironment of bacteria and algae, forming a uniform permeable protective layer. Then, dry it in a ventilated environment at 25±5℃ and 50-70% relative humidity. After drying, store it under nitrogen-filled, sealed, and light-protected conditions. It can maintain its activity for about 12 months at room temperature.

[0027] Step 6: Carrier Activation and Use

[0028] After taking out the carrier stored in step 5, activate it under natural light conditions and place it in a water body with an ammonia nitrogen concentration of 1-15 mg / L, a pH of 7.0-8.0, and a temperature of 10-30℃. The amount of carrier added should cover the surface of the water body. Run the system under light conditions. When the ammonia nitrogen removal rate is ≥85% for 3 consecutive days and the nitrite accumulation rate in the water body is 0-0.5 mg / L, it is determined that the carrier's activity has been restored and it can be used in natural slightly polluted water bodies.

[0029] The polyurethane carrier for immobilized bacteria and algae constructed in this invention achieves long-term preservation and rapid activation of bacteria and algae cells through the in-situ foaming interface coupling of the pretreated bacteria and algae with the polyurethane system, while also possessing bioactivity protection, mechanical stability, and ease of engineering application.

[0030] The beneficial effects of this invention are reflected in the following aspects:

[0031] (1) This invention uses sodium alginate and nutrient salt compound as a pretreatment for bacteria and algae to achieve full-process biological activity protection. Sodium alginate molecules contain abundant carboxyl and hydroxyl functional groups, which can form a flexible coating layer on the cell surface through hydrogen bonding and electrostatic interaction; the inorganic salts and small molecule protectants in the nutrient salt compound can regulate local osmotic pressure and maintain microenvironmental stability; the inorganic framework powder can improve the specific surface area and water retention capacity of the compound; the ionic crosslinking precursor constructs micro-crosslinked stable junctions to improve the powder's resistance to breakage under foaming stress.

[0032] (2) This invention provides a coupling of bacterial and algal powder with polyurethane in situ foaming to achieve synchronous embedding and strong interfacial bonding. In the three-dimensional porous network formed by the foaming reaction, the bacterial and algal powder is uniformly embedded in the polyurethane matrix during the foam formation process, resulting in a stable, uniform and high-density distribution of microorganisms; the hydrophilic functional groups formed by the polyurethane chain segments during the curing process can generate hydrogen bonds, electrostatic adsorption or weak complexation with the polysaccharides and protein layers on the bacterial and algal surface, which helps to form a stronger interfacial bonding structure and improve the resistance to shearing and shearing.

[0033] (3) This invention utilizes the pressure difference introduced during the preparation of foamed polyurethane material to control bubble formation. By adjusting the vacuum level and component ratio, a polyurethane carrier structure suitable for algal and bacterial loading is constructed. This porous system forms interconnected gas-liquid channels at the microscale, thereby creating a regional structure with both aerobic and hypoxic microenvironments within the same carrier. This provides ideal conditions for the symbiosis of algal photosynthetic oxygen production and bacterial heterotrophic denitrification, achieving active regulation of oxygen distribution within the system and improving reaction efficiency.

[0034] (4) The present invention adopts a composite protection system of trehalose and ascorbic acid, which achieves long-term preservation of bacteria and algae (target 12 months) through osmotic protection, membrane / protein stabilization and antioxidant synergistic effect, thereby solving the bottleneck of storage and transportation of biological carriers and rapid on-site deployment.

[0035] (5) This invention constructs a comprehensive technical path that integrates bacterial and algal fixation, long-term preservation, rapid activation and application. The preparation process is simple, the carrier structure is stable, the preservation period is long, and the start-up period is short. It has good engineering operability and promotion and application value. Attached Figure Description

[0036] Figure 1 This is a schematic diagram of the polyurethane carrier structure for immobilizing bacteria and algae according to the present invention.

[0037] Figure 2 This is a flowchart illustrating the preparation process of the bacteria-algae immobilized polyurethane carrier in Example 1 of the present invention. (a) Microscopic morphology of the concentrated nitrifying bacteria solution and nitrifying bacteria powder; (b) Microscopic morphology of the concentrated algal solution and dehydrated microalgae powder; (c) Surface morphology of the integrated bacteria-algae polyurethane carrier; (d) Surface morphology and SEM image of the integrated bacteria-algae polyurethane carrier.

[0038] Figure 3 This refers to the ammonia nitrogen removal efficiency of the bacteria and algae immobilized polyurethane carriers with different dehydration ratios in Example 2.

[0039] Figure 4 This refers to the removal efficiency of ammonia nitrogen by the polyurethane carrier immobilized with bacteria and algae for different storage times in Example 3.

[0040] Figure 5 This refers to the changes in ammonia nitrogen and nitrate nitrogen during the startup process of the polyurethane carrier immobilized with bacteria and algae in Example 3. Detailed Implementation

[0041] The present invention will be further described below with reference to the accompanying drawings and specific embodiments.

[0042] This invention provides a method for preparing a rapidly activated and preserved polyurethane carrier for immobilizing bacteria and algae, the specific method of which is as follows:

[0043] (1) Cultivation of nitrifying bacteria and microalgae. Nitrifying bacteria were inoculated into a sequencing batch reactor (SBR) using ammonia nitrogen as the sole nitrogen source and enriched under conditions of 25-30℃, pH 7.5-8.5, and dissolved oxygen concentration not less than 2.0 mg / L. The cultivation of nitrifying bacteria was completed when the ammonia nitrogen removal rate in the system reached more than 90% and the nitrite accumulation rate was 0-0.5 mg / L. The MLSS was usually stable at 3000-4000 mg / L. At the same time, microalgae were inoculated into BG-11 medium and photoautotrophically cultured in a light incubator under the following conditions: temperature 25±1℃, light-dark cycle of 12 h:12 h, and light intensity of 5000 lx. The microalgae were cultured until they entered the logarithmic growth phase and the concentration reached (1.0-5.0)×10 7 cells / mL.

[0044] (2) Algal and bacterial concentration. Stable nitrifying bacteria were taken from the SBR reactor and centrifuged at 4000-6000 rpm for 10-15 min. The supernatant was discarded to obtain nitrifying bacteria with a water content of 90-95% for later use. At the same time, the microalgae suspension in the light incubator was concentrated by dissolved air flotation to achieve a water content of 90-95% for the algae. The obtained nitrifying bacteria and concentrated microalgae were evenly spread in thin layers of 3-5 mm thickness and placed in a freeze dryer at 0-4℃ for low-temperature pre-drying treatment. Free water was removed by slow sublimation to reduce the overall water content to 70-80%, forming algal and bacterial biomass suitable for subsequent processing.

[0045] (3) Pretreatment for the protection of the activity of bacterial and algal biomass. The bacterial and algal biomass obtained in step 2 is placed in a mixing container at a mass ratio of 1:1. First, 2-10 wt% sodium alginate is added. The mixture is premixed for 5-10 min at a speed of 40-60 rpm using a low-shear ribbon mixer, with the temperature not exceeding 35℃. While maintaining the same speed, 0.5-8 wt% of nutrient salt compound is slowly added to the mixing system. Then, 1-15 wt% of inorganic framework powder is added, and the mixture is continued until a wet compound material with preliminary shaping properties is formed. Finally, an ionic crosslinking precursor is added to the mixture to form an enhanced micro-crosslinked structure. The overall moisture content of the mixture is adjusted to 55%-65%. The wet bacterial and algal compound formed by the above steps is granulated by passing it through a 60-200 mesh shaping sieve to obtain uniform bacterial and algal compound powder particles.

[0046] (4) Preparation of polyurethane carrier for bacterial and algal immobilization. The bacterial and algal composite powder particles, polyether polyol, triethanolamine, dimethylolpropionic acid and stabilizer prepared in step 3 are mixed evenly in the following proportions (5 parts of bacterial and algal composite powder particles, 100 parts of polyether polyol, 6 parts of triethanolamine, 3 parts of dimethylolpropionic acid, 2 parts of stabilizer, 6 parts of foaming agent, 1 part of isocyanate; the polyether polyol can be selected from polyether 330 and polyether 330N). The foaming agent is added and stirred thoroughly at 500~800 rpm. After mixing, the system is placed under a negative pressure of 50-80 kPa for degassing for 5-10 min. Subsequently, isocyanate (one or more of toluene-2,4-diisocyanate (TDI-2,4), toluene-2,6-diisocyanate (TDI-2,6), and naphthalene-1,5-diisocyanate (NDI-1,5)) is slowly added dropwise; during the foaming process, the algae and bacteria powder particles are uniformly distributed in the polyurethane pore wall structure during carrier formation; after addition, the system is kept static and the reaction continues until complete curing; the cured carrier is then aged at 25-35℃ for 24-48 hours. After aging, an algae and bacteria immobilized polyurethane carrier with a porous structure, good mechanical strength, and biocompatibility is obtained.

[0047] (5) Carrier preservation. Spray the surface of the polyurethane carrier for immobilized bacteria and algae obtained in step 4 with a cell protection solution of 6%-8% (w / v) trehalose and 0.2 mmol / L ascorbic acid, and let it stand for 20-30 minutes to allow the protectant to fully penetrate into the carrier pores and the microenvironment of bacteria and algae, forming a uniform permeable protective layer; then place it in a ventilated environment at 25±1℃ and a relative humidity of about 50-70% to dry; after drying, store it under nitrogen-filled, sealed and light-proof conditions, and it can maintain its activity for about 12 months at room temperature.

[0048] (6) Carrier activation and use. After taking out the carrier stored in step 5, activate it under natural light and put it into a water body with an ammonia nitrogen concentration of 1-15 mg / L, a pH of 7.0-8.0, and a temperature of 10-30℃. The amount of carrier added should cover the surface of the water body. Run it under light conditions. When the ammonia nitrogen removal rate is not less than 85% for 3 consecutive days and no obvious accumulation of nitrite in the water body is observed, it is determined that the activity of the carrier has been restored and it can be put into use in natural slightly polluted water bodies.

[0049] Example 1:

[0050] The detailed implementation steps of the bacteria-algae polyurethane carrier constructed in this invention are as follows, and a schematic diagram of the integrated bacteria-algae polyurethane carrier is shown below. Figure 2 As shown:

[0051] 1. Pretreatment of bacteria and algae

[0052] Nitrifying bacteria were removed from the SBR reactor and centrifuged at 4000-6000 rpm for 10-15 min to remove the supernatant, yielding nitrifying bacteria with a water content of 90-95%. Figure 2 a) Take the microalgae suspension and concentrate it to a water content of 90-95% by dissolved air flotation. Spread the nitrifying bacteria and concentrated microalgae separately in thin layers on aluminum foil, and place them in a freeze dryer at 0-4℃ for low-temperature pre-drying to reduce the overall water content to 70-80%, obtaining bacterial and algal biomass for later use. Figure 2 (b) The obtained bacterial and algal biomass is placed in a mixing container. First, 2-10 wt% sodium alginate is added. A low-shear ribbon mixer is used to premix the mixture at 40-60 rpm for 5-10 minutes, with the temperature not exceeding 35°C. While maintaining the same speed, 0.5-8 wt% nutrient salt compound is slowly added to the mixing system. Then, 1-15 wt% inorganic framework powder is added, and mixing continues until a wet compound material with preliminary shaping properties is formed. Finally, an ionic crosslinking precursor is added to the mixture to form an enhanced micro-crosslinked structure. The overall moisture content of the mixture is adjusted to 55%-65%. The wet bacterial and algal compound formed through the above steps is granulated through a 60-200 mesh shaping sieve to obtain uniform bacterial and algal composite powder particles.

[0053] 2. Preparation of Algae-Immobilized Polyurethane: Algae-bacterial composite powder particles are added to a pre-proportioned polyether polyol system, which includes polyether polyol, triethanolamine, dimethylolpropionic acid, and a stabilizer. The algae-bacterial composite particles are uniformly dispersed in the polyol system by mechanical stirring at 500-800 rpm to form a well-flowing algae-polyol premix. A foaming agent is then added to the premix, and stirring continues until the system is homogeneous. The mixture is then degassed under a negative pressure of 50-80 kPa for 5-10 minutes to remove air and promote the uniformity of closed-cell structures in subsequent foaming. While maintaining the negative pressure, isocyanate is slowly and continuously added dropwise to the degassed premix, causing in-situ foaming polymerization of the isocyanate with the polyol system. After the addition is complete, the reaction system is allowed to stand and solidify at room temperature. A curing treatment is then performed at 25-35℃ for 24-48 hours to obtain the algae-immobilized polyurethane carrier. Figure 2 c and d).

[0054] Example 2:

[0055] This embodiment systematically explores the optimal water content of the algae-immobilized polyurethane carrier system in the proposed denitrification method:

[0056] After dissolved air flotation treatment, the algae-bacterial system had a water content of 95%. To determine the optimal water content, five groups with different water contents (100%, 75%, 50%, 25%, and 0%) were set up for the experiment. Algae-bacterial immobilized polyurethane carriers were prepared in corresponding proportions, and their denitrification performance was evaluated. The experiment was conducted in 100 mL beakers, with 50 mL of NH4Cl solution (initial ammonia nitrogen concentration of 10.0 ± 0.2 mg / L) added to each beaker, and the initial pH adjusted to 8.0 ± 0.2. The five algae-bacterial immobilized polyurethane carriers prepared with different dehydration ratios were added to the reaction system at a biomass concentration of 5 g / L, and then placed in a constant temperature and light incubator (temperature 30℃, light intensity 20 μmol·m²). -2 ·s -1 The denitrification performance was tested in the reaction mixture. During the reaction, 1 mL water samples were collected at 0 h, 6 h, 12 h, 18 h, and 24 h. After filtration through a 0.45 μm microporous membrane, the ammonia nitrogen concentration in the filtrate was determined spectrophotometrically. The denitrification rate was calculated based on this, and its trend over time was analyzed. The experimental results are as follows: Figure 3 As shown in the figure. Experimental results indicate that the denitrification efficiency of the system is highest when the moisture content is 75%. When the moisture content is 50%, 25%, and 0%, the denitrification efficiency decreases, mainly because excessive dehydration leads to the loss of bound water from bacteria and algae, resulting in decreased activity or even death. Therefore, the optimal moisture content determined by this method is 75%, which is the moisture content for low-temperature pre-drying.

[0057] Example 3:

[0058] This embodiment is used to compare and analyze the effect of polyurethane carriers immobilized with bacteria and algae on their activity under different storage time conditions.

[0059] The experiment used polyurethane immobilized bacterial and algal carriers stored for 0, 3, 6, 9, and 12 months. The experiment was conducted in a light-controlled incubator. Immobilized carriers with different storage times were placed in 250 mL beakers, and 200 mL of ammonium chloride (NH4Cl) solution was added to each beaker. The initial ammonia nitrogen concentration in the solution was set at 10 mg / L, the pH was adjusted to 7.5, and the incubation temperature was controlled at 25 ℃. The experimental light intensity was 20 μmol·m⁻². -2 ·s -1 The light-dark cycle was 12 h:12 h. Ammonia nitrogen concentration was continuously monitored during the culture process, and the denitrification activity of the immobilized carrier was considered to have been restored when the ammonia nitrogen removal rate reached or exceeded 80%.

[0060] Experimental results are as follows Figure 4As shown, storage time has a significant impact on the recovery of activity of the bacterial and algal immobilized carrier. Freshly prepared carriers (stored for 0 months) only require 4 days to recover denitrification activity; as the storage time increases, the activation time of the carrier gradually increases. For immobilized carriers stored for 12 months, their denitrification activity can be recovered within 6 days, still meeting the usage requirements.

[0061] Example 4:

[0062] The denitrification performance of the bacteria-algae immobilized polyurethane carrier of this invention in slightly polluted wastewater was investigated.

[0063] The experiment used 1000 mL beakers as reactors, with 500 mL of simulated wastewater containing NH4Cl (initial ammonia nitrogen concentration 30.0 ± 2.0 mg / L) added to each beaker, and the initial pH adjusted to 8.0. The activated polyurethane carrier immobilized with bacteria and algae was added to the reaction system and placed in a constant temperature and light incubator (30℃, light intensity 20 μmol·m²). -2 ·s -1 The culture was carried out under a light-dark ratio of 8h:8h. During the reaction, samples were taken at 0h, 8h, 16h, 24h, 32h, 40h, and 48h. After filtration through a 0.45μm microporous membrane, the concentrations of ammonia nitrogen, nitrite nitrogen, and nitrate nitrogen were determined spectrophotometrically, and the denitrification rate was calculated accordingly. The results showed that the algae-bacterial polyurethane composite carrier had a significant removal effect on ammonia nitrogen under weakly alkaline conditions (see...). Figure 5 After 48 hours of reaction, the ammonia nitrogen removal rate reached 78.11%, indicating that the bacteria and algae maintained high biological activity even after long-term storage. During the reaction, ammonia nitrogen was mainly oxidized into harmless nitrogen gas, with a small portion converted into nitrite and nitrate. The microorganisms within the carrier system further promoted the reaction of nitrite and ammonia nitrogen to generate nitrogen gas, thus achieving a synergistic denitrification process of simultaneous nitrification and denitrification. This demonstrates the high efficiency and stability of the carrier in nitrogen removal from slightly polluted wastewater.

Claims

1. A method for preparing a polyurethane carrier for immobilizing bacteria and algae that can be stored for a long time and activated rapidly, characterized in that... Includes the following steps: Step 1: Cultivation of nitrifying bacteria and microalgae 1a. Inoculate nitrifying bacteria into the SBR reactor, using ammonia nitrogen as the sole nitrogen source, and carry out enrichment culture under the conditions of temperature 25-30℃, pH 7.5-8.5, and dissolved oxygen concentration ≥2 mg / L; when the ammonia nitrogen removal rate in the system stably reaches more than 90% and the nitrite accumulation rate is 0-0.5 mg / L, the culture of nitrifying bacteria is completed. 1b. Inoculate the microalgae into BG-11 medium and culture them in a photoautotrophic incubator until they enter the logarithmic growth phase. The concentration is (1.0-5.0)×10⁻⁶. 7 cells / mL; Step 2: Algae and Bacteria Concentration 2a. Remove nitrifying bacteria from the SBR reactor, centrifuge and discard the supernatant to obtain nitrifying bacteria with a water content of 90-95%; then spread the obtained nitrifying bacteria evenly with a thickness of 3-5 mm, and place them in a freeze dryer at 0-4℃ for low-temperature pre-drying to an overall water content of 70-80%, forming nitrifying bacteria biomass that is easy to process later. 2b. Take out the algal solution from the microalgae light incubator and concentrate it to a water content of 90-95% through dissolved air flotation treatment to obtain concentrated microalgae; spread the obtained concentrated microalgae evenly with a thickness of 3-5mm and place it in a freeze dryer at 0-4℃ for low-temperature pre-drying to an overall water content of 70-80% to form concentrated microalgae biomass that is easy to process later. Step 3: Pretreatment for the protection of the biomass activity of bacteria and algae The nitrifying bacteria biomass and concentrated microalgae biomass obtained in step 2 were placed in a mixing container. Sodium alginate was first added to the mixing system as a structural support agent. A low-shear ribbon mixer was used to premix the mixture at a speed of 40-60 rpm for 5-10 minutes, controlling the temperature of the mixture to ≤35℃, so that sodium alginate formed a uniform coating layer on the surface of the bacteria and algae biomass. After the above premixing was completed, the nutrient salt compound was slowly added to the mixing system at the same speed to make it uniformly dispersed in the sodium alginate coating structure. Then, inorganic framework powder was added and mixing continued until a wet compound material with preliminary shaping properties was formed. Finally, an ionic crosslinking precursor was added to the wet compound material to form an enhanced micro-crosslinking structure, improve the breakage resistance of the obtained powder, and adjust the overall moisture content of the mixture to 55%-65% to ensure compressibility and flowability during powder processing. The wet bacteria and algae compound formed in the above steps was granulated by passing it through a 60-200 mesh shaping sieve to obtain uniform bacteria and algae compound powder particles. Step 4: Preparation of polyurethane carrier for bacterial and algal immobilization The algae-bacterial composite powder obtained in step 3 was added to the polyether polyol system. Mechanical stirring was used to uniformly disperse the algae-bacterial composite particles in the polyol system, forming an algae-bacterial polyol premix with good flowability. A foaming agent was then added to the algae-bacterial polyol premix, and stirring continued until the system was homogeneous. The mixture was then degassed under a negative pressure of 50-80 kPa to remove air and promote the uniformity of closed-cell structures in subsequent foaming. While maintaining the negative pressure, isocyanate was continuously added dropwise to the degassed premix, allowing the isocyanate to undergo an in-situ foaming polymerization reaction with the polyol system. After the addition was complete, the reaction system was allowed to stand and solidify at room temperature. Finally, a curing treatment was performed at 25-35°C to obtain an algae-immobilized polyurethane carrier. Step 5: Media Preservation Spray a cell protection solution containing trehalose and ascorbic acid onto the surface of the polyurethane carrier for immobilized bacteria and algae obtained in step 4. Allow it to stand to allow the protectant to fully penetrate into the carrier pores and the microenvironment of bacteria and algae, forming a uniform permeable protective layer. Then, place it in a ventilated environment at 25±5℃ and 50-70% relative humidity to dry. After drying, store it under nitrogen-filled, sealed, and light-protected conditions. Step 6: Carrier Activation and Use After taking out the carrier stored in step 5, activate it under natural light conditions and place it in a water body with an ammonia nitrogen concentration of 1-15 mg / L, a pH of 7.0-8.0, and a temperature of 10-30℃. The amount of carrier added should cover the surface of the water body. Run the system under light conditions. When the ammonia nitrogen removal rate is ≥85% for 3 consecutive days and the nitrite accumulation rate in the water body is 0-0.5 mg / L, it is determined that the carrier's activity has been restored and it can be used in natural slightly polluted water bodies.

2. The preparation method according to claim 1, characterized in that: In step 1b, the culture conditions are: temperature 25±1℃, light-dark cycle of 12 h:12 h, and light intensity of 5000 lx.

3. The preparation method according to claim 1, characterized in that: In step 3, when the nitrifying bacteria biomass and concentrated microalgae biomass obtained in step 2 are placed in a mixing container, the mass ratio of the two is 1:

1.

4. The preparation method according to claim 1, characterized in that: In step 3, the amount of sodium alginate added is 2-10 wt% of the total mass of the mixed system, the amount of nutrient salt compound added is 0.5-8 wt% of the total mass of the mixed system, the amount of inorganic framework powder added is 1-15 wt% of the total mass of the mixed system, and the amount of ion crosslinking precursor added is 0.05-0.2 wt% of the total mass of the mixed system.

5. The preparation method according to claim 4, characterized in that: The nutrient salt compound includes a nitrogen source, a phosphorus source, and trace metal ions, wherein the nitrogen source is NO3. - and / or NH4 + The phosphorus source is H2PO4. - and / or HPO4 2- Trace metal ions include Fe 3+ Mg 2+ Co 2+ and MoO4 2- The inorganic framework powder is bentonite, diatomite, or zeolite powder; the ionic crosslinking precursor is CaCl2.

6. The preparation method according to claim 1, characterized in that: In step 4, the polyether polyol system includes polyether polyol, triethanolamine, dimethylolpropionic acid, and stabilizer.

7. The preparation method according to claim 1, characterized in that: In step 4, the foaming agent is a mixture of triethylamine and dichloromethane, prepared in a mass ratio of 1:

1.

8. The preparation method according to claim 1, characterized in that: In step 4, the isocyanate is selected from one or more of toluene-2,4-diisocyanate, toluene-2,6-diisocyanate, and naphthalene-1,5-diisocyanate.

9. The preparation method according to claim 1, 6, 7 or 8, characterized in that: In step 4, the components are proportioned by mass as follows: The mixture consists of 5 parts of algal-bacterial complex powder particles, 80 parts of polyether polyol, 5 parts of triethanolamine, 2 parts of dimethylolpropionic acid, 2 parts of stabilizer, 5 parts of isocyanate, and 1 part of foaming agent.

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