Immobilized bacteria-algae microspheres as well as preparation method and application thereof

Abstract

The invention discloses immobilized bacteria-algae microspheres, a preparation method thereof and application of the immobilized bacteria-algae microspheres in removal of ammonia nitrogen and total phosphorus in aquaculture water. The preparation method comprises the following steps: adding polyvinyl alcohol and sodium alginate into deionized water, heating and sterilizing while fully dissolving polyvinyl alcohol and sodium alginate to obtain an embedding medium; adding the iron modified biochar powder into a mixed solution of chlorella and photosynthetic bacteria for adsorption to obtain a bacteria-algae-biochar mixture; adding the bacteria-algae-biochar mixture into an embedding medium to form a gel solution; adding the gel solution into a cross-linking agent for cross-linking, and after cross-linking, separating a solid and cleaning to obtain immobilized bacteria-algae microspheres; the preparation method of the iron-modified biochar powder comprises the following steps: adjusting the pH value of a mixed solution containing biochar powder, Fe < 2 + > and Fe < 3 + > to 9.5-10.5, carrying out heat preservation stirring at 75-85 DEG C for 1-5 hours, carrying out solid-liquid separation, taking a solid, washing, and drying to obtain the iron-modified biochar powder.

Description

Technical Field

[0001] The present invention relates to the technical field of wastewater biological treatment, and in particular to an immobilized bacteria-algae microsphere and a preparation method and application thereof. Background Art

[0002] In recent decades, the scale of aquaculture has grown significantly, and the breeding model has rapidly shifted from extensive and low-density to intensive and high-density. The introduction of large amounts of feed has caused excessive accumulation of nitrogen in the aquaculture water, which not only causes aquatic animal diseases, but also the discharge of tail water without effective treatment can easily destroy the ecological balance of the natural water environment. Therefore, how to use biological treatment technology to effectively decompose and treat nitrogen in aquaculture water and improve the aquaculture environment is a key technical requirement for maintaining the sustainable development of the aquaculture industry.

[0003] The bacteria-algae symbiosis utilizes the synergistic effect between bacteria and microalgae to promote the assimilation and absorption of ammonia nitrogen in water bodies. It does not require the addition of additional carbon sources or aeration, and is considered to be a sustainable biological treatment technology and has received increasing attention. The immobilized bacteria-algae symbiosis combines bacteria and algae in a stable spatial structure through physical adsorption and embedding, which reduces the loss of bacteria and algae. It has the advantages of high biological density, fast reaction rate, strong impact resistance, and high denitrification efficiency, and solves the defects of the free bacteria-algae system.

[0004] The patent specification with publication number CN108034624A discloses a biological preparation for treating high-concentration ammonia nitrogen wastewater, and its preparation raw materials are composed of the following components in parts by weight: 10-15 parts of Chlorella DH2 algae liquid, 20-30 parts of Pseudomonas aeruginosa DT1 fermentation liquid, 15-20 parts of Bacillus megaterium NCT-2 fermentation liquid, 10-15 parts of Acinetobacter baumannii AL-6 fermentation liquid, 5-10 parts of photosynthetic bacterial fermentation liquid, 1-3 parts of agar, and 3-5 parts of polyvinyl alcohol.

[0005] The patent specification with publication number CN115304169A discloses a bacteria-algae symbiotic system and a method and device for treating domestic sewage. The bacteria-algae symbiotic system includes immobilized algae balls and aerobic granular sludge. The immobilized algae balls are made by embedding microalgae with sodium alginate and polyvinyl alcohol as carriers and calcium chloride as a fixing agent. The immobilized algae balls are mixed with aerobic granular sludge and then cultured in a photobioreactor to construct a bacteria-algae symbiotic system.

[0006] The study found that the traditional sodium alginate and polyvinyl alcohol encapsulation and the formed gel particles have poor porosity and mass transfer performance. The growth of microorganisms embedded in the gel particles is restricted and it is difficult for them to fully contact with pollutants in the water, which greatly reduces the decomposition of pollutants in the water by microorganisms and affects the water purification effect. Summary of the invention

[0007] In view of the above-mentioned technical problems and the shortcomings in the art, the present invention provides an immobilized bacteria-algae microsphere and a preparation method and application thereof. The immobilized bacteria-algae microsphere can be used to remove ammonia nitrogen and total phosphorus in aquaculture water.

[0008] The bacteria-algae symbiotic system utilizes the synergistic effect between bacteria and algae to remove pollutants such as nitrogen and phosphorus in sewage. Different ratios of bacteria and algae will affect the interaction between algae and bacteria. The appropriate ratio of bacteria and algae is one of the key factors to improve the performance of the bacteria-algae symbiotic system.

[0009] Biochar is a carbon-containing solid substance formed by high-temperature cracking of biomass raw materials. It has a loose porous structure and a high specific surface area. The adsorption capacity of biochar is related to the number and properties of functional groups on its surface. It is expected that the adsorption capacity of biochar will be significantly increased by chemical or physical modification of it.

[0010] To achieve the above object, the technical solution of the present invention is as follows:

[0011] A method for preparing immobilized bacteria-algae microspheres, comprising:

[0012] Adding polyvinyl alcohol and sodium alginate into deionized water and heating to sterilize the water while fully dissolving the polyvinyl alcohol and sodium alginate to obtain an embedding agent;

[0013] The iron-modified biochar powder is added to a mixture of Chlorella and photosynthetic bacteria for adsorption to obtain a bacteria-algae-biochar mixture;

[0014] Adding the bacteria-algae-biochar mixture into the embedding agent to form a gel solution;

[0015] The gel solution is added to a cross-linking agent for cross-linking, and after cross-linking, the solid is separated and washed to obtain immobilized bacteria and algae microspheres;

[0016] The preparation method of the iron-modified biochar powder comprises: 2+ and Fe 3+ The pH value of the mixed solution is adjusted to 9.5-10.5 (e.g., 10, etc.), and the mixture is kept stirred at 75-85° C. (e.g., 80° C., etc.) for 1-5 hours (e.g., 4 hours, etc.), the solid-liquid separation is performed, the solid is washed, and dried to obtain the iron-modified biochar powder.

[0017] In the method for preparing the iron-modified biochar powder, the biochar powder may include at least one of bamboo biochar powder, straw biochar powder, and rice husk biochar powder, and preferably includes (corn) straw biochar powder.

[0018] In the method for preparing the iron-modified biochar powder, the particle size of the biochar powder is preferably less than 50 meshes.

[0019] In the method for preparing the iron-modified biochar powder, the biochar powder can be obtained by oxygen-free sintering of biomass raw materials at 400-600° C. (preferably 500° C.). Further, the biomass raw materials can include at least one of bamboo, straw, and rice husk, preferably (corn) straw. The oxygen-free sintering time can be 3 hours.

[0020] In the method for preparing the iron-modified biochar powder, the biochar powder is preferably soaked in deionized water to release phosphorus, washed and dried before use.

[0021] In the method for preparing the iron-modified biochar powder, the biochar powder, Fe 2+ and Fe 3+ In the mixed solution, Fe 2+ and Fe 3+ The molar ratio of Fe is preferably 1:2. 3+ The molar ratio of to the mass of the biochar powder is preferably 6-7 mmol:2g.

[0022] In the method for preparing the iron-modified biochar powder, the biochar powder, Fe 2+ and Fe 3+ The pH of the mixed solution was adjusted to 9.5-10.5.

[0023] In the mixed solution of Chlorella and photosynthetic bacteria, the ratio of the number of cells of Chlorella to that of photosynthetic bacteria may be 3:1 to 9, preferably 2:1.

[0024] In the mixed solution of Chlorella and photosynthetic bacteria, the number of cells of Chlorella can be 10 7 ~10 9 / mL (e.g. 1×10 8 / mL, etc.), the number of cells of photosynthetic bacteria can be 10 7 ~10 8 CFU / mL (e.g. 5×10 7 CFU / mL, etc.).

[0025] The mixed solution of Chlorella and photosynthetic bacteria can be obtained by centrifuging the Chlorella solution and the photosynthetic bacteria solution, washing with deionized water, and resuspending with saline. The number of living Chlorella cells in the Chlorella solution can be 10 5 ~10 6 The number of living cells of photosynthetic bacteria in the photosynthetic bacteria liquid may be 10 7 ~10 8 CFU / mL.

[0026] The photosynthetic bacteria may include Rhodopseudomonas sp. Further, the Rhodopseudomonas sp. may include R-7 with a preservation number of CCTCC NO: M 20241789. The Rhodopseudomonas sp. R-7 is deposited in the China Center for Type Culture Collection, the preservation date is August 14, 2024, and the preservation address is China Center for Type Culture Collection, Wuhan University, Wuhan, Hubei Province (No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province, China).

[0027] In the method for preparing the immobilized bacteria-algae microspheres, the adsorption temperature can be room temperature to 35° C., and the adsorption time can be 8 to 14 hours.

[0028] The cross-linking agent may be a calcium chloride solution.

[0029] In the preparation method of the immobilized bacteria-algae microspheres, the ratio of the polyvinyl alcohol, the sodium alginate, the iron-modified biochar powder, the mixed solution of the chlorella and photosynthetic bacteria, and the deionized water in the embedding agent can be: 4-12g: 1-3g: 0.3-0.7g (for example, 0.5g, etc.): 5-15mL: 100mL, preferably 4g: 3g: 0.5g: 5mL: 100mL. Under this condition, the molding effect and ammonia nitrogen removal rate of the immobilized bacteria-algae microspheres obtained are the best.

[0030] The invention further provides immobilized bacteria and algae microspheres prepared by the preparation method.

[0031] The present invention also provides the use of the immobilized bacteria-algae microspheres in removing ammonia nitrogen and total phosphorus in aquaculture water.

[0032] As a general inventive concept, the present invention also provides a strain of Rhodopseudomonas sp., strain number R-7, and the preservation number is CCTCC NO: M 20241789. The Rhodopseudomonas sp. R-7 is deposited in the China Center for Type Culture Collection, the preservation date is August 14, 2024, and the preservation address is China Center for Type Culture Collection, Wuhan University, Wuhan City, Hubei Province (No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province, China).

[0033] Compared with the prior art, the present invention has the following beneficial effects:

[0034] 1. The present invention is applied to aquaculture, and the bacteria and algae used are photosynthetic bacteria and Chlorella, respectively. The photosynthetic bacteria can quickly degrade ammonia nitrogen in aquaculture wastewater, and can also be used as bait to increase the yield. Chlorella is an algae that aquatic animals such as fish and shrimp like to eat, and can also play a role in improving water quality without affecting the economic benefits of aquaculture.

[0035] 2. The adsorbent biochar powder used in the present invention is treated with iron modification. Ordinary biochar has a general adsorption effect on ammonia nitrogen. After the biochar is modified with iron, the oxygen functional groups increase and the adsorption effect is enhanced, thereby obtaining a better ammonia nitrogen adsorption effect.

[0036] 3. Ordinary biochar will release phosphorus pollutants in the water body, which is easy to cause secondary pollution. The present invention performs iron modification and soaking treatment on the biochar to inhibit the release of phosphorus.

[0037] 4. The conventional gel beads formed by embedding materials such as sodium alginate and polyvinyl alcohol have poor porosity and poor mass transfer performance. The immobilized bacteria-algae microspheres provided by the present invention utilize the loose porous structure of biochar to fully adsorb photosynthetic bacteria and microalgae before embedding. On the one hand, the loss of bacteria and algae is reduced to a greater extent; on the other hand, the addition of biochar increases the specific surface area of ​​the gel beads, improves the mass transfer performance of the immobilized bacteria-algae microspheres, promotes the contact between bacteria and algae and pollutants in the water, and improves the removal efficiency of pollutants. In addition, the immobilized bacteria-algae microspheres are easy to recycle and reuse, and the use of biochar also realizes the resource utilization of agricultural waste to a certain extent, reducing the treatment cost. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1 This is a diagram showing the formation of immobilized bacteria and algae microspheres in each group of experiments in Example 5.

[0039] Figure 2 This is a graph showing the removal efficiency of ammonia nitrogen and total phosphorus (TP) for 72 hours using three types of immobilized bacteria and algae microspheres in Example 7. DETAILED DESCRIPTION

[0040] The present invention will be further described below in conjunction with the accompanying drawings and specific embodiments. It should be understood that these embodiments are only used to illustrate the present invention and are not used to limit the scope of the present invention.

[0041] Example 1: Isolation and screening of photosynthetic bacteria

[0042] 1.1 Sample processing

[0043] An environmental sample of tail water from an aquaculture enterprise was collected. The water sample was filtered through a 0.22 μm filter membrane in the laboratory, the filtrate was discarded, and the filter membrane was retained for later use.

[0044] 1.2 Enrichment culture

[0045] The enrichment medium is a photosynthetic bacteria medium purchased from Qingdao Haibo Biotechnology Co., Ltd. Prepare 100 mL of enrichment medium in a 250 mL conical flask and sterilize it under high pressure at 121 ° C for 15 minutes. After the culture medium is cooled, the filter membrane described in 1.1 is connected to the enrichment medium, and 5 mL of sterilized liquid paraffin is covered on the surface of the culture medium to ensure an anaerobic environment; the enrichment culture solution is placed in a light incubator with a light intensity of 3000 lux, a temperature of 30 ° C, and a light-dark ratio of 24h:0h for enrichment culture. After the enrichment culture solution turns dark red, take 1 mL and transfer it to a new enrichment medium. Repeat the enrichment 3 times until the culture solution turns brown-red and the enrichment is complete.

[0046] 1.3 Separation and purification

[0047] Add 20 g / L agar powder to the enrichment medium to prepare a solid separation medium, sterilize under high pressure at 121°C for 15 min, pour 15 mL of the plate under sterile conditions, cool and set aside; dilute the enrichment solution in a sterile environment to 10 -4 , 10 -5 , 10 -6 , 10 -7 , 10 -8 , take 50 μL of the diluted enrichment solution and evenly spread it on the photosynthetic bacteria isolation medium plate, place the coated solid plate in an anaerobic bag and culture it under the culture conditions described in 1.2 until obvious red colonies grow on the plate; use a burned inoculation loop to pick up a typical red single colony on the plate and repeatedly streak it on a new isolation medium plate for purification; put the streaked plate into an anaerobic bag and place it in a light incubator under the same conditions as above, and culture it until pure colonies with uniform colony morphology grow on the plate; inoculate the obtained pure colonies into a bacterial preservation tube filled with 1 mL of a mixture of 5% α-lactose and 25% glycerol, and store it in a 4°C refrigerator as a strain, and transfer it to a -80°C refrigerator for storage one day later.

[0048] 1.4 Identification of bacterial species

[0049] 16S rDNA gene extraction: After activating the target strain, use a DNA extraction kit to extract genomic DNA.

[0050] 16S rDNA gene amplification: PCR amplification was performed using genomic DNA or bacterial solution as template, using bacterial 16SrDNA universal primers 27F (SEQ ID NO: 1) and 1492R (SEQ ID NO: 2). The 25μL PCR system was: premix 12.5μL, double distilled water 9.5μL, DNA template 1μL, and forward and reverse primers 1μL each. The amplified product was sent to Beijing Qingke Biotechnology Co., Ltd. for sequencing after being detected by 1.5% agarose gel electrophoresis, and the sequencing results were submitted to the NCBI official website for BLAST comparison and retrieval of bacterial gene sequences with a sequence similarity of more than 97% with the fragment. The 16S rDNA sequence (SEQ ID NO: 3) was analyzed and compared to confirm that it was Rhodopseudomonas sp., with a sequence similarity of 99.63%.

[0051] The isolated strain was deposited in the China Type Culture Collection on August 14, 2024. The deposit address is China Type Culture Collection of Wuhan University, Wuhan City, Hubei Province (No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province, China). The strain deposit number is CCTCC NO: M 20241789, named R-7, and all photosynthetic bacteria used subsequently are this strain. The phenotypic characteristics of the above-mentioned Rhodopseudomonas sp. R-7 are as follows: R-7 is a Gram-negative bacterium with rod-shaped cells; after culturing at 30°C on photosynthetic bacterial solid culture medium for 5 to 7 days, the colony changes from white to red, with smooth edges, convex surfaces, and opaque; the pH growth range is 6.8 to 7.2.

[0052] Example 2: Biochar Optimization Experiment

[0053] 2.1 Biochar phosphorus release experiment

[0054] In order to ensure that the introduction of biochar would not cause secondary pollution, the phosphorus-releasing capacity of biochar itself in water bodies was studied.

[0055] Preparation of biochar powder: The biomass raw materials (bamboo, corn stalks) are preliminarily crushed (rice husks do not need to be crushed), washed with deionized water 2-3 times, and dried in an 80°C oven for use; the dried biomass raw materials are placed in a muffle furnace at 500°C for 3 hours in an oxygen-free environment, and cooled at room temperature to obtain biochar; the biochar is further ground and crushed to pass through a 50-mesh sieve for collection. Unless otherwise specified, biochar powder is prepared in this way.

[0056] Preparation of phosphorus-containing solution: Prepare a solution with a total phosphorus (TP) of 2 mg / L and sterilize at 121°C for 15 min.

[0057] Phosphorus release detection: Bamboo, corn straw and rice husk biochar were added to the phosphorus-containing solution at a dosage of 0.5% (0.5 g / 100 mL), and the total phosphorus content in the solution was measured at 0, 3, 6, 24 and 48 h. The determination method referred to the ammonium molybdate spectrophotometry method. The results are shown in Table 1.

[0058] Table 1 Phosphorus release capacity of various types of biochar

[0059]

[0060] The results showed that the phosphorus release rate of bamboo biochar was faster within 3 hours, while that of corn straw biochar was relatively slower. The phosphorus release trends of the three types of biochar were similar within 24 hours. After 24 hours, bamboo biochar and corn straw biochar basically stopped releasing phosphorus, but the total amount of phosphorus released by bamboo biochar was higher than that of corn straw biochar.

[0061] 2.2 Biochar ammonia nitrogen adsorption experiment

[0062] Preparation of simulated wastewater: simulated wastewater with an ammonia nitrogen content of 5 mg / L was prepared. The composition and content of the simulated wastewater are shown in Table 2. Unless otherwise specified, the simulated wastewater will be prepared in this way.

[0063] Table 2 Simulated wastewater formulation

[0064]

[0065] Ammonia nitrogen adsorption capacity test: 3 types of biochar were added to the simulated wastewater described in Table 2 at a dosage of 0.5% (0.5 g / 100 mL), and the ammonia nitrogen content in the solution was measured at 0, 2, 4, 24, 48, and 72 h. The determination method referred to the Nessler reagent spectrophotometry method. The results are shown in Table 3.

[0066] Table 3 Ammonia nitrogen adsorption capacity of various biochars

[0067]

[0068] The results showed that rice husk biochar had no significant adsorption effect on ammonia nitrogen in water within 72 hours, while bamboo and corn straw biochar had a more obvious adsorption effect on ammonia nitrogen in water. The adsorption capacity of corn straw biochar on ammonia nitrogen was stronger than that of bamboo charcoal within 72 hours.

[0069] Considering the phosphorus release capacity and ammonia nitrogen adsorption capacity of the three types of biochar, corn straw biochar has a better application effect in sewage treatment. Therefore, the corn straw biochar powder prepared according to this embodiment is used as an adsorbent to prepare immobilized bacteria and algae microspheres.

[0070] Example 3: Cultivation of living cell fluid of Chlorella and photosynthetic bacteria

[0071] Chlorella and photosynthetic bacteria seed liquid were inoculated into BG11 medium and photosynthetic bacteria medium at an inoculation rate of 10%, respectively, and cultured in a constant temperature illumination culture with a light intensity of 3000 lux and a temperature of 30° C. BG11 medium and photosynthetic bacteria medium were purchased from Qingdao Haibo Biotechnology Co., Ltd.

[0072] After culturing for a period of time, the light absorption values ​​of Chlorella and photosynthetic bacteria were measured at 660nm and 680nm respectively. According to the formula y = 2.53x-0.15 (10 7 / mL) to calculate the algae density of Chlorella culture solution, according to the formula y = 9.41x-0.37 (10 7 / mL) to calculate the bacterial density of photosynthetic bacteria culture solution, where x is the light absorption value and y is the bacterial or algae density.

[0073] According to the density calculation formula of Chlorella and photosynthetic bacteria, when the number of living cells in the algae liquid reaches 10 5 -10 6 / mL, the number of photosynthetic bacterial cells reached 10 7 -10 8 CFU / mL, collected for later use.

[0074] Example 4: Optimization of the ratio of bacterial and algal cells

[0075] The bacterial and Chlorella culture solutions were prepared to have a viable cell count of 1×10 7 The free bacteria-algae mixture was prepared according to the algae-bacteria cell number ratio of 1:1, 1:2, 1:3, 2:1, and 3:1, and was added into 100 mL of simulated wastewater containing 5 mg / L ammonia nitrogen at a dosage of 5% (i.e., 5 mL of the free bacteria-algae mixture relative to 100 mL of simulated wastewater). The removal amount of ammonia nitrogen in the simulated wastewater was measured every 24 h for 72 h to determine the optimal ratio of the number of Chlorella and photosynthetic bacteria cells. The measurement method was according to the Nessler spectrophotometry. The results are shown in Table 4.

[0076] Table 4 Removal rate of ammonia nitrogen by different ratios of bacteria and algae

[0077]

[0078] The results showed that when the cell number ratio of Chlorella to photosynthetic bacteria was 2:1, the free bacteria-algae system had the highest removal rate for ammonia nitrogen in simulated wastewater.

[0079] Example 5. Optimization of preparation conditions for immobilized bacteria and algae microspheres

[0080] 5.1 Biochar pretreatment

[0081] Soak the corn straw biochar powder in deionized water for 48 hours to release phosphorus in the raw materials and avoid secondary pollution. Wash the soaked corn straw biochar powder with deionized water for 3 times and dry it in an oven at 80°C. Unless otherwise specified, all subsequent biochar is pretreated.

[0082] 5.2 Preparation of bacteria-algae mixture

[0083] According to the cell number ratio of Chlorella: photosynthetic bacteria of 2:1 and the calculated density of bacteria and algae in the culture medium, take the corresponding volume of Chlorella solution and photosynthetic bacteria solution, centrifuge the Chlorella solution and photosynthetic bacteria solution at 4000rpm and 6500rpm for 5min respectively, discard the supernatant, and wash repeatedly with sterile deionized water for 3 times to completely remove the nutrients in the culture medium.

[0084] The washed bacteria and algae were resuspended in physiological saline and mixed to form a photosynthetic bacterial density of 5×10 7 CFU / mL, Chlorella density is 1×10 8 The bacteria-algae mixture with a concentration of 100 / mL was collected for later use.

[0085] 5.3 Optimization of the optimal preparation conditions for immobilized bacterial and algal microspheres

[0086] The embedding agent is obtained by adding polyvinyl alcohol and sodium alginate into deionized water and heating and sterilizing at 121° C. to fully dissolve the polyvinyl alcohol and sodium alginate.

[0087] Select L9(3 4 ) was used to design an orthogonal experiment with three factors and three levels. The removal rate of ammonia nitrogen was used as the main indicator, and the formation of microspheres was observed. The orthogonal test results were analyzed and the optimal preparation conditions of immobilized bacteria and algae microspheres were determined. The orthogonal experimental factor levels are shown in Table 5.

[0088] Table 5 Orthogonal experimental factor levels

[0089]

[0090] According to the selected factors and levels, L9(3 4 ) orthogonal table, and the experimental scheme is determined as shown in Table 6 below.

[0091] Table 6 Orthogonal experimental scheme

[0092]

[0093] In Tables 5 and 6: the amount of polyvinyl alcohol (x%, x=4, 8, 12) refers to the amount of polyvinyl alcohol x g relative to 100 mL of deionized water in the embedding agent; the amount of sodium alginate (y%, y=4, 8, 12) refers to the amount of sodium alginate y g relative to 100 mL of deionized water in the embedding agent; the amount of bacteria-algae mixed solution (z mL) refers to the amount of bacteria-algae mixed solution z mL relative to 100 mL of deionized water in the embedding agent.

[0094] Different embedding agents were prepared according to the experimental scheme to embed different volumes of bacteria-algae mixed solutions. The amount of corn straw biochar was 0.5% (i.e., the amount of corn straw biochar was 0.5g relative to the deionized water in 100mL embedding agent). The immobilized bacteria-algae microspheres prepared in each experimental group were added to the simulated wastewater containing 5mg / L ammonia nitrogen at a dosage of 10% of the wet weight. The amount of ammonia nitrogen removed from the wastewater was measured every 24h, and the measurement was continued for 72h to calculate the ammonia nitrogen removal rate. The orthogonal experimental results are shown in Table 7 and Figure 1 shown.

[0095] Table 7 Orthogonal experiment results

[0096]

[0097] The intuitive analysis of the orthogonal experimental results is shown in Table 8 below.

[0098] Table 8 Orthogonal experiment results analysis

[0099]

[0100] The results showed that the best conditions for the preparation of immobilized bacteria-algae microspheres were A1B3C1, that is, the amount of polyvinyl alcohol was 4%, the amount of sodium alginate was 3%, and the amount of bacteria-algae embedding was 5 mL. Under the best conditions, the microspheres were well formed.

[0101] Example 6. Preparation of iron-modified biochar

[0102] Weigh 6.8 g of ferrous chloride and 21.4 g of ferrous sulfate and dissolve them in 500 mL of deionized water to form Fe 2+ :Fe 3+A mixed solution with a molar ratio of 1:2; 20g of straw biochar powder was dispersed in 300mL of deionized water, and then 300mL of the above mixed solution was added, and the pH was adjusted to 10 with 4.5mol / L sodium hydroxide solution, and heated and stirred on a magnetic stirrer at 80°C for 4h, and washed with deionized water until neutral when cooled to room temperature, and dried in an oven at 80°C to obtain iron-modified biochar powder. Corn straw biochar powder before and after modification was added to the above simulated wastewater at a dosage of 0.5% (0.5g / 100mL), and the ammonia nitrogen content in the solution at 0, 2, 4, 24, 48, and 72h was determined. The determination method referred to the Nessler reagent spectrophotometry method, and the results are shown in Table 9.

[0103] Table 9 Comparison of ammonia nitrogen adsorption capacity before and after modification

[0104]

[0105] The results showed that the adsorption rate of ammonia nitrogen by the modified corn straw biochar powder was about 74.95% within 72 hours, which was about 13.50% higher than that before modification.

[0106] Example 7. Preparation of immobilized bacteria and algae microspheres

[0107] The immobilized bacteria-algae microspheres were prepared according to the optimal parameter conditions optimized in Example 5. In order to further illustrate the advantages of the immobilized bacteria-algae microspheres of the present invention, i.e., the bacteria-algae microspheres prepared by adding iron-modified biochar adsorbents in removing ammonia nitrogen from aquaculture water bodies, three types of immobilized bacteria-algae microspheres were prepared without adding biochar, adding unmodified ordinary corn straw biochar, and adding iron-modified corn straw biochar. The amount of biochar added was 0.5%, that is, 0.5g of biochar was added to every 100mL of deionized water in the embedding agent. The three types of immobilized bacteria-algae microspheres were added to the simulated wastewater at a dosage of 10% of the wet weight, and the ammonia nitrogen content in the wastewater was measured for 72 hours. The measurement method referred to the Nessler reagent spectrophotometry; at the same time, the total phosphorus content in the wastewater was measured, indicating that the iron modification of biochar and its co-encapsulation with bacteria and algae as an adsorbent reduced the amount of phosphorus released by biochar in the water body and synergized with bacteria and algae to show a certain removal effect on phosphorus. For results, see Figure 2 .

[0108] In addition, it should be understood that after reading the above description of the present invention, those skilled in the art may make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the claims attached to this application.

Claims

1. A method for preparing immobilized bacteria-algae microspheres, characterized in that: include: Adding polyvinyl alcohol and sodium alginate into deionized water and heating to sterilize the water while fully dissolving the polyvinyl alcohol and sodium alginate to obtain an embedding agent; The iron-modified biochar powder is added to a mixture of Chlorella and photosynthetic bacteria for adsorption to obtain a bacteria-algae-biochar mixture; Adding the bacteria-algae-biochar mixture into the embedding agent to form a gel solution; The gel solution is added to a cross-linking agent for cross-linking, and after cross-linking, the solid is separated and washed to obtain immobilized bacteria and algae microspheres; The preparation method of the iron-modified biochar powder comprises: 2+ and Fe 3+ The pH value of the mixed solution is adjusted to 9.5-10.5, and the mixture is kept warm and stirred at 75-85° C. for 1-5 hours. The solid-liquid separation is performed, and the solid is washed and dried to obtain the iron-modified biochar powder.

2. The method for preparing immobilized bacteria-algae microspheres according to claim 1, characterized in that: In the method for preparing the iron-modified biochar powder: The biochar powder includes at least one of bamboo biochar powder, straw biochar powder, and rice husk biochar powder; The particle size of the biochar powder is less than 50 mesh; The biochar powder is obtained by sintering biomass raw materials at 400°C to 600°C (preferably 500°C) without oxygen; the biomass raw materials include at least one of bamboo, straw, and rice husk; The biochar powder is soaked in deionized water to release phosphorus, washed and dried before use; The biochar powder, Fe 2+ and Fe 3+ In the mixed solution, Fe 2+ and Fe 3+ The molar ratio of Fe is 1:

2. 3+ The molar ratio of the biochar powder to the biochar powder is 6-7 mmol:2 g; Sodium hydroxide solution was used to 2+ and Fe 3+ The pH of the mixed solution was adjusted to 9.5-10.

5.

3. The method for preparing immobilized bacteria-algae microspheres according to claim 1, characterized in that: In the mixed solution of Chlorella and photosynthetic bacteria, the ratio of the number of Chlorella to the number of photosynthetic bacteria is 3:1 to 9, preferably 2:1; In the mixed solution of Chlorella and photosynthetic bacteria, the number of cells of Chlorella is 10 7 ~10 9 / mL, the number of cells of photosynthetic bacteria is 10 7 ~10 8 CFU / mL.

4. The method for preparing immobilized bacteria-algae microspheres according to claim 1, characterized in that: The mixed solution of Chlorella and photosynthetic bacteria is obtained by centrifuging the Chlorella solution and the photosynthetic bacteria solution respectively, washing with deionized water, and resuspending with physiological saline; The number of living cells of Chlorella in the Chlorella liquid is 10 5 ~10 6 Pieces / mL; The number of living cells of photosynthetic bacteria in the photosynthetic bacteria liquid is 10 7 ~10 8 CFU / mL.

5. The method for preparing immobilized bacteria-algae microspheres according to claim 1, characterized in that: The photosynthetic bacteria include Rhodopseudomonas sp.; The Rhodopseudomonas sp. includes R-7 with a deposit number of CCTCC NO: M 20241789.

6. The method for preparing immobilized bacteria-algae microspheres according to claim 1, characterized in that: The adsorption temperature is from room temperature to 35° C., and the adsorption time is from 8 to 14 hours.

7. The method for preparing immobilized bacteria-algae microspheres according to claim 1, characterized in that: The cross-linking agent is a calcium chloride solution; The ratio of the polyvinyl alcohol, the sodium alginate, the iron-modified biochar powder, the mixed solution of Chlorella and photosynthetic bacteria, and the deionized water in the embedding agent is: 4-12g:1-3g:0.3-0.7g:5-15mL:100mL.

8. The immobilized bacteria-algae microspheres prepared according to the preparation method according to any one of claims 1 to 7.

9. Use of the immobilized bacteria-algae microspheres according to claim 8 in removing ammonia nitrogen and total phosphorus from aquaculture water.

10. A strain of Rhodopseudomonas sp., characterized in that: The strain number is R-7, and the deposit number is CCTCCNO: M 20241789.

Images