Composite algae preparation for removing nitrogen and phosphorus pollutants and preparation method thereof

Abstract

The invention relates to the field of river pollution treatment, and discloses a composite algae preparation for removing nitrogen and phosphorus pollutants and a preparation method thereof, and the composite algae preparation is prepared by compounding phosphorus-accumulating algae species, nitrogen-fixing algae species and phosphate-solubilizing bacteria which are subjected to function screening according to a specific cell number proportion. The key step of the preparation method is as follows: co-culturing the screened three microorganisms under specific pH and environmental conditions to form phycomycete symbionts with complementary functions; and then carrying out phosphorus deficiency hunger stress treatment on the obtained symbiont to induce and strengthen the excessive phosphorus uptake physiological activity of the phosphorus-accumulating algae species. By constructing a microbial system with a synergistic effect and combining a key physiological stress means, the obtained preparation can efficiently and synchronously remove nitrogen and phosphorus pollutants in a water body. The preparation prepared by the method has the advantages that the phosphorus removal rate and capability are obviously improved, the preparation process is clear, the product performance is stable, and the preparation can be prepared into wet paste or powder which is easy to store and use.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of river pollution treatment, in particular to a composite algal preparation for removing nitrogen and phosphorus pollutants and a preparation method thereof. BACKGROUND

[0002] Water eutrophication is one of the serious environmental problems currently faced, and its main cause is the discharge of excessive nutrients such as nitrogen and phosphorus in wastewater. Using microbial technology, especially using microalgae to remove nitrogen and phosphorus in water, has become a research hotspot because it is environmentally friendly, relatively low in cost, and can achieve resource utilization. Microalgae can assimilate inorganic nitrogen and phosphate in water into their own cell components during growth and reproduction, thereby achieving the purpose of purifying water quality.

[0003] However, in the prior art, there are still some technical bottlenecks in using microalgae for water remediation. Conventional algal water treatment technology mostly uses a single algal species, but different algal species have different preferences and efficiencies for nitrogen and phosphorus absorption. A single algal species often cannot achieve efficient simultaneous removal of the two pollutants, resulting in an imbalance in the proportion of nitrogen and phosphorus in the treated water. In addition, some industrial wastewater or domestic sewage contains complex organic phosphorus, which cannot be directly utilized by most microalgae, limiting their total phosphorus removal capacity in actual wastewater treatment.

[0004] To solve the above problems, researchers have tried to compound multiple microorganisms in order to take advantage of the functional complementarity between different species. For example, bacteria with phosphorus solubilizing ability are coexisting with microalgae. In theory, bacteria can decompose organic phosphorus into orthophosphate that can be utilized by algae. However, simple microbial mixing often fails to form a stable and efficient synergistic system. There may be problems such as mismatched growth rates, nutrient competition, or mutual inhibition between different species, resulting in unstable actual treatment effect of the composite microbial agent and large batch-to-batch performance difference. At the same time, the preparation methods of existing preparations often fail to fully stimulate the metabolic potential of microorganisms, resulting in limited pollutant removal rate and removal capacity of the final product, which is difficult to meet the engineering application requirements of rapid and deep treatment. Therefore, developing a composite microbial preparation with controllable preparation process, stable performance and efficient nitrogen and phosphorus simultaneous removal capacity is a technical problem to be solved in the field. SUMMARY

[0005] The technical problem to be solved by the present application is that in the prior art, using a single microalgae or an algal-bacterial composite system without specific physiological regulation to treat wastewater has the problems of unstable nitrogen and phosphorus pollutant removal efficiency and low removal rate.

[0006] To solve the above technical problems, the present application aims to provide a composite algal preparation that can synergistically and efficiently remove nitrogen and phosphorus pollutants in water, and a preparation method that can significantly improve the pollutant removal activity of the preparation.

[0007] To achieve the above objectives, the present invention adopts the following technical solution:

[0008] The first aspect of this invention provides a compound algae preparation for removing nitrogen and phosphorus pollutants. This compound algae preparation is a complex composed of microorganisms screened for specific functions, specifically comprising:

[0009] Screened polyphosphate-accumulating algae species;

[0010] Nitrogen-fixing algae strains that have been screened;

[0011] And screened phosphate-solubilizing bacteria.

[0012] In this compound algae preparation, the components are configured in specific proportions to form a synergistic system.

[0013] In one specific embodiment, the cell ratio of the polyphosphate-accumulating algae species to the nitrogen-fixing algae species is 1:3 to 1:5; simultaneously, the ratio of the total cell number of the polyphosphate-accumulating algae species and the nitrogen-fixing algae species to the cell number of the phosphate-solubilizing bacteria is 100:1 to 500:1. This ratio facilitates the effective exchange of matter and energy among the various microbial components during the pollutant removal process.

[0014] As a preferred embodiment, the polyphosphate-accumulating algae are one or more species of the genus *Chlorella*, the nitrogen-fixing algae are one or more species of the genus *Anabaena*, and the phosphate-solubilizing bacteria are *Bacillus megaterium*. These algae and bacteria species are characterized by rapid growth rate, strong environmental adaptability, and well-defined functions in nitrogen and phosphorus removal.

[0015] To facilitate storage, transportation, and application, the compound algae preparation can be formulated into a specific physical form. For example, the preparation can be a paste with a water content of 75% to 85%, or a powder with a water content of less than 10% prepared by processes such as freeze-drying.

[0016] A second aspect of this invention provides a method for preparing the aforementioned compound algae preparation for removing nitrogen and phosphorus pollutants. This method, through a series of precisely controlled steps, ensures that the final compound algae preparation possesses high biological activity. Specifically, the method includes the following steps:

[0017] Screening steps: Based on their core functions, polyphosphate-accumulating algae, nitrogen-fixing algae, and phosphate-solubilizing bacteria used to prepare compound formulations are quantitatively screened to obtain highly efficient functional strains.

[0018] In one embodiment, the screening criteria are:

[0019] Algal strains with an instantaneous phosphate uptake rate (as phosphorus) of not less than 1.5 mg / (gDW·h) were selected as polyphosphate-accumulating algae species.

[0020] Algal strains with nitrogenase activity of not less than 500 nmol C2H4 / (mgPro·h) were selected as nitrogen-fixing algal species;

[0021] Phosphate-solubilizing bacteria were selected from strains that could increase the concentration of soluble phosphorus in the culture medium by no less than 150 mg / L after 72 hours of incubation.

[0022] Co-cultivation steps: The selected polyphosphate-accumulating algae, nitrogen-fixing algae, and phosphate-solubilizing bacteria are mixed and co-cultivated according to a preset ratio until a structurally stable algal-bacterial symbiosis is formed.

[0023] In one embodiment, the co-culture conditions include maintaining the pH of the culture medium in the range of 7.0 to 8.5 and co-culturing for 5 to 7 days.

[0024] Starvation stress treatment step: The algae-bacterial symbionts obtained in the co-culture step are subjected to specific physiological stress treatments to induce and enhance their ability to subsequently absorb pollutants.

[0025] In one embodiment, the hunger stress treatment specifically includes:

[0026] First, the algae-bacteria symbiotic organisms were collected by centrifugation or filtration and washed with a phosphorus-free solution until the phosphate concentration in the supernatant after washing was less than 0.05 mg / L.

[0027] Then, the cleaned algal-bacterial symbionts were transferred to a phosphorus-free medium at an initial density of 1.0 to 1.5 g DW / L (dry weight) for further cultivation.

[0028] As a preferred option, the culture time in phosphorus-free medium is 24 to 48 hours.

[0029] Finished product preparation steps:

[0030] In a preferred embodiment, after the starvation stress treatment step, the method further includes preparing the obtained compound algae preparation into a paste or powder finished product by means of concentration or freeze-drying.

[0031] To ensure the physiological activity of the bacteria and algae in the above-mentioned cultivation steps, the cultivation processes in the co-cultivation step and the starvation stress treatment step can be carried out under specific environmental conditions. These conditions include: maintaining a temperature of 23 to 27°C, maintaining a light intensity of 3000 to 4000 lux, and using a light-dark cycle of 12 hours of light and 12 hours of darkness.

[0032] In summary, the present invention has at least one of the following beneficial technical effects:

[0033] 1. This invention constructs a complementary and synergistic microecological system by combining polyphosphate-accumulating algae, nitrogen-fixing algae, and phosphate-solubilizing bacteria in a specific ratio. In this system, nitrogen-fixing algae remove nitrogen from the water while providing a nitrogen source for other microorganisms; phosphate-solubilizing bacteria not only decompose complex organic phosphorus into orthophosphates that can be directly utilized by algae, but also exert a significant synergistic effect with polyphosphate-accumulating algae by secreting bioactive substances or improving the microenvironment, jointly promoting the efficient absorption and storage of phosphates in the water; while polyphosphate-accumulating algae perform the final efficient absorption of phosphates. This multi-level synergistic mechanism achieves the efficient and simultaneous removal of both nitrogen and phosphorus pollutants from the water.

[0034] 2. The preparation method of the present invention significantly improves the pollutant removal efficiency of the compound preparation by introducing two key steps: screening and starvation stress. First, the bacterial and algal species are screened by quantitative indicators to ensure that each biological unit constituting the preparation has a high level of initial functional activity. Second, by applying phosphorus-deficient starvation stress to the formed algal-bacterial symbiosis, the polyphosphate-accumulating algae species are induced to produce a physiological effect of excessive phosphorus uptake, so that the preparation can absorb phosphate instantaneously at a rate much higher than that under normal conditions after being put into sewage, thereby greatly improving the phosphorus removal rate and capacity.

[0035] 3. This invention provides clear numerical ranges for key technical aspects of the preparation method, including the ratio of bacteria and algae, co-culture conditions, stress treatment parameters, and environmental control parameters. This precise control of process parameters throughout the entire process ensures the stability and repeatability of the preparation process, resulting in a final compound algae preparation product with uniform quality and reliable performance. It solves the technical problem of large batch-to-batch differences in the effects of existing biological agents and has good prospects for industrial application. Detailed Implementation

[0036] The present invention will be further described in detail below with reference to embodiments, comparative examples and test examples.

[0037] Experimental materials:

[0038] The main raw materials and reagents used in the following examples, comparative examples and test cases are sourced and specified as follows. Reagents not specifically mentioned are all commercially available analytical grade or higher grade products.

[0039] Microbial strains:

[0040] Chlorella, accession number: FACHB-8.

[0041] Anabaena, accession number: FACHB-83.

[0042] Bacillus megaterium, accession number: CCTCAB207465.

[0043] Main chemical reagents for culture medium and simulated wastewater:

[0044] The main chemical reagents used in the analysis and testing are:

[0045] Potassium persulfate, CAS No.: 7727-21-1.

[0046] Examples 1-3:

[0047] Example 1:

[0048] This embodiment provides a method for preparing a compound algae preparation, the specific steps of which are as follows:

[0049] S1. Screening and preparation of bacterial and algal strains:

[0050] The original strains of Chlorella sp., Anabaenasp., and Bacillus megaterium were obtained from the Freshwater Algae Bank (FACHB) of the Institute of Hydrobiology, Chinese Academy of Sciences, and the China Center for Type Culture Collection (CCTCC), respectively.

[0051] The original bacterial strains were screened using conventional enrichment culture, isolation and purification, and functional detection methods in this field, and the following functional bacteria and algae were finally obtained:

[0052] An polyphosphate-accumulating algae species has an instantaneous phosphate uptake rate of 1.5 mg / (gDW·h) based on phosphorus.

[0053] A nitrogen-fixing algal species has a nitrogenase activity of 500 nmol C2H4 / (mgPro·h);

[0054] A phosphate-solubilizing bacterium can increase the concentration of soluble phosphorus in the culture medium by 150 mg / L after 72 hours of cultivation.

[0055] The polyphosphate-accumulating algae and nitrogen-fixing algae obtained from the screening were propagated in BG11 medium, while the phosphate-solubilizing bacteria were propagated in LB medium. All were cultured to the late logarithmic growth phase for later use.

[0056] S2. Co-culture to form an algal-bacterial symbiosis:

[0057] Take the algal and bacterial solutions prepared in step S1, mix them according to the ratio of polyphosphate-accumulating algae to nitrogen-fixing algae cells of 1:3 and the ratio of total algal cell number to phosphate-solubilizing bacteria cell number of 100:1, and inoculate them into a new BG11 medium.

[0058] Co-culturing was performed under the following conditions:

[0059] Incubation temperature: 23℃;

[0060] Illumination intensity: 3000 lux;

[0061] Light-dark cycle: 12 hours of light, 12 hours of darkness;

[0062] pH value: The pH value of the culture medium is maintained at 7.0 by adding a buffer;

[0063] Total incubation time: 5 days.

[0064] After the culture is completed, a culture medium containing algal-bacterial symbiosis is obtained.

[0065] S3, Management of starvation stress:

[0066] Centrifuge the culture medium obtained in step S2 at 4000 rpm for 10 minutes, discard the supernatant to collect the algal-bacterial symbiont. Use phosphorus-free BG11 medium as the washing solution to resuspend and centrifuge the collected algal-bacterial symbiont, repeating this operation until the phosphate concentration in the supernatant is detected to be below 0.05 mg / L.

[0067] The cleaned algal-bacterial symbiont was transferred to a new phosphorus-free BG11 medium, and the inoculation density was adjusted so that the initial concentration of algal-bacterial symbiont in the culture medium was 1.0 g / L (on a dry weight basis).

[0068] Starvation stress culture was carried out under the same environmental conditions as step S2 (temperature 23℃, light intensity 3000 lux, light-dark cycle 12h:12h) for 24 hours.

[0069] After cultivation, the compound algae preparation of Example 1 is obtained by centrifugation. This preparation can be used directly in wet paste form or subjected to subsequent freeze-drying.

[0070] Example 2:

[0071] This embodiment provides a method for preparing a compound algae preparation, which differs from Example 1 mainly in the different process parameters used. The specific steps are as follows:

[0072] S1. Screening and preparation of bacterial and algal strains:

[0073] Using the same methods and sources as in Example 1, bacterial and algal strains were obtained and screened, ultimately yielding the following functional bacterial and algal strains:

[0074] An polyphosphate-accumulating algae species has an instantaneous phosphate uptake rate of 2.2 mg / (gDW·h) based on phosphorus.

[0075] A nitrogen-fixing algal species has a nitrogenase activity of 750 nmol C2H4 / (mgPro·h);

[0076] A phosphate-solubilizing bacterium can increase the concentration of soluble phosphorus in the culture medium by 225 mg / L after 72 hours of cultivation.

[0077] The selected bacterial and algal strains were propagated and cultured to the late logarithmic growth phase for later use.

[0078] S2. Co-culture to form an algal-bacterial symbiosis:

[0079] Take the algal and bacterial solutions prepared in step S1, mix them according to the ratio of polyphosphate-accumulating algae to nitrogen-fixing algae cells of 1:4 and the ratio of total algal cell number to phosphate-solubilizing bacteria cell number of 300:1, and inoculate them into a new BG11 medium.

[0080] Co-culturing was performed under the following conditions:

[0081] Incubation temperature: 25℃;

[0082] Illumination intensity: 3500 lux;

[0083] Light-dark cycle: 12 hours of light, 12 hours of darkness;

[0084] pH value: The pH value of the culture medium was maintained at 7.7 by adding a buffer;

[0085] Total incubation time: 6 days.

[0086] After the culture is completed, a culture medium containing algal-bacterial symbiosis is obtained.

[0087] S3, Management of starvation stress:

[0088] The algae-bacterial symbiont obtained in step S2 was collected and cleaned using the same centrifugation and washing methods as in Example 1.

[0089] The cleaned algal-bacterial symbiont was transferred to a new phosphorus-free BG11 medium, and the inoculation density was adjusted to make the initial concentration of algal-bacterial symbiont in the culture medium 1.25 g / L (on a dry weight basis).

[0090] Starvation stress culture was carried out under the same environmental conditions as step S2 (temperature 25℃, light intensity 3500 lux, light-dark cycle 12h:12h) for 36 hours.

[0091] After cultivation, the compound algae preparation of Example 2 is obtained by centrifugation.

[0092] Example 3:

[0093] This embodiment provides a method for preparing a compound algae preparation, which differs from Embodiments 1 and 2 mainly in the different process parameters used. The specific steps are as follows:

[0094] S1. Screening and preparation of bacterial and algal strains:

[0095] Using the same methods and sources as in Example 1, bacterial and algal strains were obtained and screened, ultimately yielding the following functional bacterial and algal strains:

[0096] An polyphosphate-accumulating algae species has an instantaneous phosphate uptake rate of 3.1 mg / (gDW·h) based on phosphorus.

[0097] A nitrogen-fixing algal species has a nitrogenase activity of 980 nmol C2H4 / (mgPro·h);

[0098] A phosphate-solubilizing bacterium can increase the concentration of soluble phosphorus in the culture medium by 280 mg / L after 72 hours of cultivation.

[0099] The selected bacterial and algal strains were propagated and cultured to the late logarithmic growth phase for later use.

[0100] S2. Co-culture to form an algal-bacterial symbiosis:

[0101] Take the algal and bacterial solutions prepared in step S1, mix them according to the ratio of polyphosphate-accumulating algae to nitrogen-fixing algae cells of 1:5 and the ratio of total algal cell number to phosphate-solubilizing bacteria cell number of 500:1, and inoculate them into a new BG11 medium.

[0102] Co-culturing was performed under the following conditions:

[0103] Incubation temperature: 27℃;

[0104] Illumination intensity: 4000 lux;

[0105] Light-dark cycle: 12 hours of light, 12 hours of darkness;

[0106] pH value: The pH value of the culture medium is maintained at 8.5 by adding a buffer;

[0107] Total incubation time: 7 days.

[0108] After the culture is completed, a culture medium containing algal-bacterial symbiosis is obtained.

[0109] S3, Management of starvation stress:

[0110] The algae-bacterial symbiont obtained in step S2 was collected and cleaned using the same centrifugation and washing methods as in Example 1.

[0111] The cleaned algae-bacterial symbiont was transferred to a new phosphorus-free BG11 medium, and the inoculation density was adjusted so that the initial concentration of algae-bacterial symbiont in the culture medium was 1.5 g / L (on a dry weight basis).

[0112] Starvation stress culture was carried out under the same environmental conditions as step S2 (temperature 27℃, light intensity 4000 lux, light-dark cycle 12h:12h) for 48 hours.

[0113] After cultivation, the compound algae preparation of Example 3 was obtained by centrifugation.

[0114] Comparative Examples 1-3:

[0115] Comparative Example 1:

[0116] The difference from Example 2 is that, in the co-culture step, only the selected polyphosphate-accumulating algae and nitrogen-fixing algae are mixed and co-cultured, without the addition of phosphate-solubilizing bacteria. All other steps and conditions are exactly the same as in Example 2.

[0117] Comparative Example 2:

[0118] The difference from Example 2 is that, after the co-cultivation step, the obtained algal-bacterial symbiont was directly collected by centrifugation as the final preparation, omitting the entire starvation stress treatment step. All other steps and conditions were exactly the same as in Example 2.

[0119] Comparative Example 3

[0120] The difference between Example 2 and Example 3 is that the polyphosphate-accumulating algae, nitrogen-fixing algae, and phosphate-solubilizing bacteria used were all original strains obtained from the bacterial culture center and had not undergone functional screening. All other steps and conditions were exactly the same as in Example 2.

[0121] Test Example 1-2:

[0122] Test Example 1: Nitrogen and Phosphorus Pollutant Removal Efficiency Test

[0123] To verify the removal effect of the compound algae preparation prepared in the embodiments of the present invention on nitrogen and phosphorus pollutants, this test was designed and conducted.

[0124] Experimental methods:

[0125] Simulated wastewater preparation: KH₂PO₄ and KNO₃ were used as phosphorus and nitrogen sources, respectively, and dissolved in sterile water to prepare simulated wastewater. After adjustment, the initial total phosphorus (TP) concentration of the wastewater was 5.0 mg / L, and the initial total nitrogen (TN) concentration was 50.0 mg / L.

[0126] Experimental grouping and inoculation: Prepare six identical conical flasks, each containing 500 mL of the simulated wastewater described above. Take the compound algae preparations (all in wet paste form) prepared in Examples 1, 2, and 3, and Comparative Examples 1, 2, and 3, respectively, and inoculate them into the six conical flasks at the same dry weight dosage (0.2 g DW / L), labeling them as Example 1 group, Example 2 group, Example 3 group, Comparative Example 1 group, Comparative Example 2 group, and Comparative Example 3 group.

[0127] Processing procedure: All conical flasks were placed in a light incubator and cultured with shaking under the conditions of 25℃ temperature, 3500 lux light intensity, and 12h:12h light-dark cycle.

[0128] Sample Testing: Water samples were taken from each conical flask 24 hours after the start of incubation. After filtration through a 0.45 μm filter membrane, the total nitrogen and total phosphorus concentrations in the water samples were determined according to the methods specified in "Determination of Total Nitrogen in Water - Alkaline Potassium Persulfate Digestion Ultraviolet Spectrophotometric Method" (GB11894-89) and "Determination of Total Phosphorus in Water - Ammonium Molybdate Spectrophotometric Method" (GB11893-89). The removal rate was calculated based on the initial and 24-hour concentrations.

[0129] Experimental results:

[0130] The data on the removal efficiency of total nitrogen and total phosphorus in the simulated wastewater for each experimental group are recorded in the table below.

[0131] Experimental group 24-hour total nitrogen removal rate (%) 24-hour total phosphorus removal rate (%) Example 1 91.3 92.5 Example 2 94.7 95.8 Example 3 96.2 97.1 Comparative Example 1 93.1 45.3 Comparative Example 2 92.8 68.7 Comparative Example 3 61.5 55.4

[0132] Results analysis:

[0133] As shown in Table 1, the compound algae preparations prepared in Examples 1, 2, and 3 all exhibited high removal rates for both total nitrogen and total phosphorus. Comparison with the comparative examples confirms that the specific combination and preparation steps in this technical solution are the foundation for achieving the desired technical effect. A comparison of the results of Example 2 with Comparative Example 1 reveals the core advantages of the composite system of this invention. It is worth noting that the phosphorus source of the simulated wastewater used in this test example is readily available inorganic phosphate (KH2PO4), theoretically requiring no decomposition by phosphate-solubilizing bacteria. However, the experimental results show that even under these conditions, the total phosphorus removal rate (45.3%) of Comparative Example 1, lacking phosphate-solubilizing bacteria, still showed a precipitous decrease compared to Example 2 (95.8%), which contained phosphate-solubilizing bacteria. **This strongly demonstrates that the role of the phosphate-solubilizing bacteria (Bacillus megaterium) in this invention is multifaceted, and its importance is not limited to its phosphorus-solubilizing function in treating wastewater containing complex organic phosphorus.** More importantly, by coexisting with two types of algae, it can form a stable micro-ecology. Through secreting growth-promoting substances, optimizing the microenvironment around algal cells, or utilizing its own phosphorus storage capacity, it produces a strong synergistic effect with polyphosphate-accumulating algae, thereby greatly enhancing the overall absorption capacity of the composite preparation for inorganic phosphorus. Therefore, the ternary composite system composed of polyphosphate-accumulating algae, nitrogen-fixing algae, and phosphate-solubilizing bacteria is a key technological guarantee for achieving efficient purification of various phosphorus-containing wastewaters (whether inorganic or organic phosphorus).

[0134] The comparison of the results of Example 2 and Comparative Example 2 shows that the formulation treated with starvation stress (Example 2) has a significantly higher total phosphorus removal rate than the formulation that did not undergo this step (Comparative Example 2). This confirms that starvation stress treatment for a specific period of time in a phosphorus-free environment can effectively induce the physiological activity of polyphosphate-accumulating algae to absorb excess phosphorus, and this step is a key technical step in improving the phosphorus removal capacity of the formulation.

[0135] A comparison of the results of Example 2 and Comparative Example 3 shows that the formulation prepared using bacteria and algae that have undergone functional quantitative screening (Example 2) has significantly higher removal rates of total nitrogen and total phosphorus than the formulation prepared using unscreened original strains (Comparative Example 3). This result indicates that screening the starting bacteria and algae species based on their nitrogen fixation, phosphorus solubilization, and phosphorus uptake capabilities is a necessary prerequisite to ensure that the final composite formulation has high pollutant removal performance.

[0136] Test Example 2: Formulation and Stability Testing

[0137] To verify the effectiveness of the compound algae preparation of the present invention in different physical forms and its ability to maintain activity under storage conditions, this test was designed and conducted.

[0138] Experimental methods:

[0139] Sample preparation: The wet paste-like compound algae preparation (with a water content of about 80%) prepared in Example 2 was used as the base material and divided into three groups for treatment.

[0140] Group A (wet ointment preparations): Freshly prepared wet ointment preparations are used directly.

[0141] Group B (Fresh Powdered Formulation): Take a portion of the freshly prepared wet paste-like formulation, place it in a freeze dryer for processing until its moisture content is less than 10%, and use it immediately after it is made into a powdered formulation.

[0142] Group C (Storage of Powdered Formulations): The powdered formulations prepared in Group B should be sealed and stored at room temperature (approximately 25°C) in a dry, light-protected environment for 90 days before use.

[0143] Activity Test: Three experimental groups, A, B, and C, were set up, corresponding to the three formulation samples mentioned above. The pollutant removal efficiency was tested using the same experimental method as in Test Example 1. Specifically, the three formulations were added to simulated wastewater with an initial total nitrogen concentration of 50.0 mg / L and a total phosphorus concentration of 5.0 mg / L, respectively, at a dosage of 0.2 g DW / L (dry weight). After treatment under the same culture conditions for 24 hours, samples were taken to measure the final concentrations of total nitrogen and total phosphorus in the water, and the removal rate was calculated.

[0144] Experimental results:

[0145] The data on the removal effects of formulations in different forms and under different storage conditions on total nitrogen and total phosphorus in simulated wastewater are recorded in the table below.

[0146] Experimental group 24-hour total nitrogen removal rate (%) 24-hour total phosphorus removal rate (%) Group A: wet paste preparation (fresh) 94.7 95.8 Group B: powdered preparation (fresh) 93.9 95.1 Group C: powdered preparation (stored for 90 days) 90.5 92.3

[0147] Results analysis:

[0148] Table 2 shows that the nitrogen and phosphorus removal capacity of the wet paste formulation was not significantly reduced when converted into a low-moisture powder formulation via freeze-drying. This indicates that the composite microbial system obtained by this preparation method has good processing tolerance, and its intrinsic functional structure and physiological activity are effectively maintained during dehydration. These results confirm the technical feasibility of preparing solid formulations with low moisture content, a form that is beneficial for product packaging, transportation, and application.

[0149] A comparison of fresh powdered formulations with those stored for 90 days showed that the latter maintained a high removal rate of total nitrogen and total phosphorus. This indicates that inducing microorganisms into a dormant state through dehydration effectively preserves their biological activity before application. The synergistic structure between the pre-constructed polyphosphate-accumulating algae, nitrogen-fixing algae, and phosphate-solubilizing bacteria within the formulation, as well as the pre-induced excessive phosphorus uptake physiological state through starvation stress, were retained even after long-term storage.

[0150] The above results confirm that the preparation method provided by this technical solution can not only produce biological agents with highly efficient pollutant removal capabilities, but also process these agents into a stable solid form. Even after long-term storage, this solid form can still effectively exert its technical effects based on multi-species synergy and specific physiological stress effects, providing technical support for the practical application of this agent.

[0151] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

Claims

1. A compound algae preparation for removing nitrogen and phosphorus pollutants, characterized in that, The compound algae preparation includes: Screened polyphosphate-accumulating algae species; Screened nitrogen-fixing algae; and Screened phosphate-solubilizing bacteria; The cell ratio of the polyphosphate-accumulating algae species to the nitrogen-fixing algae species is 1:3 to 1:

5. The ratio of the total number of cells of the polyphosphate-accumulating algae and nitrogen-fixing algae to the number of cells of the phosphate-solubilizing bacteria is 100:1 to 500:

1.

2. The compound algae preparation for removing nitrogen and phosphorus pollutants according to claim 1, characterized in that, The polyphosphate-accumulating algae is Chlorella vulgaris, the nitrogen-fixing algae is Anabaena, and the phosphate-solubilizing bacteria is Bacillus megaterium.

3. The compound algae preparation for removing nitrogen and phosphorus pollutants according to claim 2, characterized in that, The preparation is a paste with a water content of 75%-85% or a powder with a water content of less than 10%.

4. A method for preparing a compound algae preparation for removing nitrogen and phosphorus pollutants as described in any one of claims 1-3, characterized in that, The method specifically includes: The polyphosphate-accumulating algae, nitrogen-fixing algae, and phosphate-solubilizing bacteria were screened. A symbiotic organism is formed by co-culturing polyphosphate-accumulating algae, nitrogen-fixing algae, and phosphate-solubilizing bacteria. The algae-bacterial symbiotic obtained in the above steps is subjected to starvation stress treatment to prepare a compound algae preparation.

5. The method for preparing a compound algae preparation for removing nitrogen and phosphorus pollutants according to claim 4, characterized in that, The screening criteria are as follows: The instantaneous phosphate absorption rate of the polyphosphate-accumulating algae species, calculated as phosphorus, is not less than 1.5 mg / (gDW·h); The nitrogenase activity of the nitrogen-fixing algae species is not less than 500 nmol C2H4 / (mgPro·h); The phosphate-solubilizing bacteria increased the concentration of soluble phosphorus in the culture medium by no less than 150 mg / L after 72 hours of cultivation.

6. The method for preparing a compound algae preparation for removing nitrogen and phosphorus pollutants according to claim 4, characterized in that, The co-culture conditions include maintaining the pH of the culture medium between 7.0 and 8.5, and co-culturing for 5 to 7 days.

7. The method for preparing a compound algae preparation for removing nitrogen and phosphorus pollutants according to claim 4, characterized in that, The hunger stress treatment specifically includes: First, the algae-bacterial symbiont was washed until the phosphate concentration in the supernatant was below 0.05 mg / L; The cleaned algal-bacterial symbionts were then transferred to phosphorus-free medium at an initial density of 1.0 to 1.5 g DW / L for culture.

8. The method for preparing a compound algae preparation for removing nitrogen and phosphorus pollutants according to claim 7, characterized in that, The culture time in the phosphorus-free medium is 24 to 48 hours.

9. The method for preparing a compound algae preparation for removing nitrogen and phosphorus pollutants according to claim 4, characterized in that, Following the starvation stress treatment step, the method further includes a step of preparing the compound algae preparation into a paste or powder formulation by concentration or freeze-drying.

10. The method for preparing a compound algae preparation for removing nitrogen and phosphorus pollutants according to claim 5, characterized in that, Both the co-culture and starvation stress treatments were carried out under the following conditions: temperature of 23 to 27°C, light intensity of 3000 to 4000 lux, and light-dark cycle of 12h:12h.