Phycomycete symbiotic system and application thereof in field of aquaculture wastewater treatment

Abstract

The invention particularly relates to a phycomycete symbiotic system and application thereof in the field of aquaculture wastewater treatment. Aiming at the characteristics of high ammonia nitrogen element content and antibiotic inhibition in wastewater in the breeding industry, the invention provides a phycomycete symbiotic system suitable for degrading organic wastewater, which comprises composite bacteria of bacillus proteolyticus, bacillus tropicalis and pseudomonas stutzeri, and inoculating the mixed algae of chlorella vulgaris, chlorella pyrenoidosa and scenedesmus quadricauda to the biofilm carrier. Through verification, when the phycomycete symbiotic system is applied to treatment of pig-raising biogas slurry wastewater, the content of ammonia nitrogen elements in the wastewater can be effectively reduced, degradation of antibiotic components is promoted, and emission standards are met.

Description

Technical Field

[0001] This invention relates to the field of algae-bacterial symbiotic biofilm technology, and in particular to an algae-bacterial symbiotic system and its application in the field of aquaculture wastewater treatment. Background Technology

[0002] The information disclosed in this background section is intended only to enhance understanding of the overall background of the invention and is not necessarily to be construed as an admission or in any way implying that such information constitutes prior art known to those skilled in the art.

[0003] Currently, the commonly used treatment processes for pig farm biogas slurry wastewater mainly include three categories: biological treatment, physicochemical treatment, and natural treatment. Biological treatment is based on anaerobic digestion (such as UASB, CSTR), which decomposes organic matter and recovers biogas through methanogenic bacteria. However, the effluent still has high levels of ammonia nitrogen and phosphorus, requiring further nitrogen and phosphorus removal through subsequent processes (such as activated sludge process, biofilm process). Physicochemical treatment efficiently removes pollutants through chemical precipitation (aluminum salts / iron salts) or advanced oxidation (such as Fenton), but it suffers from problems such as high reagent costs and increased sludge production. Natural treatment relies on constructed wetlands or stabilization ponds, which have low operating costs but require a large area and are significantly affected by seasonality. While current process combinations (such as "anaerobic + aerobic + wetland") can meet emission standards, they still face challenges such as carbon-nitrogen ratio imbalance, high energy consumption, and antibiotic residues. Emerging technologies such as microalgae-microbe symbiotic systems and short-cut nitrification-anaerobic ammonia oxidation (PN / A) have become research hotspots due to their combination of low carbon and high efficiency and resource utilization potential (algae powder production and carbon source saving), driving the transformation of treatment modes towards energy conservation and resource recovery.

[0004] Microbial-microalgae symbiotic biofilm technology has developed rapidly in the field of wastewater treatment. It achieves efficient nitrogen and phosphorus removal through the synergistic effect of bacteria and algae (microalgae produce oxygen to promote the degradation of organic matter by aerobic bacteria, and bacteria release CO2 to support algal growth). At the same time, algal powder can be used to produce oils, realizing resource utilization. Current technologies focus on issues such as bacterial-algae regulation strategies, ammonia nitrogen emission compliance, and reactor optimization. However, large-scale application is still limited by bottlenecks such as antibiotic inhibition, low ammonia nitrogen removal rate, and high harvesting costs.

[0005] In view of the above-mentioned research status, this invention believes that existing algae-bacteria symbiotic systems are difficult to meet the emission standards for aquaculture pollutants, especially in terms of SM2 residue and nitrogen and phosphorus removal, and are unable to meet the needs of aquaculture wastewater treatment. Summary of the Invention

[0006] To address the shortcomings of the existing technologies, this invention provides a more effective algae-bacteria symbiotic preparation. By optimizing the types and ratios of microorganisms, it achieves better treatment results for organic wastewater from aquaculture.

[0007] In a first aspect, the present invention provides an algae-bacterial symbiotic system, the symbiotic system comprising a carrier, a composite bacteria and a mixed algae, wherein the composite bacteria is a combination of Bacillus proteolyticus, Bacillus tropicalis and Pseudomonas schrenckii, and the mixed algae is a combination of Chlorella vulgaris, Chlorella proteoglycinae and Scenedesmus tetracaulis.

[0008] The above-mentioned compound bacteria have the following preferred embodiments: The *Bacillus proteolyticus* was collected from the sediment of a eutrophic water body in Jinan. Genome sequencing comparison showed a high degree of identity with known *Bacillus proteolyticus*, thus identifying it as *Bacillus proteolyticus*, and naming it *Bacillus proteolyticus*. Bacillus proteolyticus B1, whose 16S rDNA sequence is shown in SEQ ID NO:1, was deposited on May 23, 2025 at the China Center for Type Culture Collection (CCTCC), located at Wuhan University, Wuhan, China, with accession number CCTCC M 20251164.

[0009] The tropical spore-forming bacteria specifically refers to tropical spore-forming bacteria (Bacillus tropicalis). Bacillus tropicus This strain, B8, is tolerant of enriched pig farm wastewater and can survive in simulated wastewater with high levels of antibiotics and ammonia nitrogen. Its 16S rDNA sequence is shown in SEQ ID NO:2. This invention names it *Bacillus tropicalis* (B8). Bacillus tropicus )B8, or B8.

[0010] The *Pseudomonas schrenckii* strain was purchased from the China General Microbiological Culture Collection Center (CGMCC), with accession number CGMCC:1.10279. This invention names it *Pseudomonas schrenckii*. Pseudomonas stutzeri B3, abbreviated as "Pseudomonas stearothermii B3" or "B3".

[0011] The above-mentioned mixed algae also have the following preferred embodiments: The Chlorella, further, is common Chlorella ( Chlorella vulgaris )FACHB-32.

[0012] The *Chlorella proteoglycans*, further, is *Chlorella proteoglycans* (…). Chlorella pyrenoidosa) FACHB-1216.

[0013] The aforementioned *Scenedesmus tetracaudus*, further, is *Scenedesmus tetracaudus* ( Scenedesmus quadricauda ) FACHB-1297.

[0014] All three algal strains were purchased from the Freshwater Algae Culture Bank of the Wuhan Institute of Hydrobiology, Chinese Academy of Sciences.

[0015] The carrier is used for the attachment and growth of fungi and algae, and is preferably a material with good biocompatibility, high specific surface area, porous structure, and chemical stability. Feasible materials include organic polymers (polyethylene, polyurethane, or polyester fibers), inorganic porous materials (activated carbon, zeolite, or ceramic materials), and cellulose-based materials (such as plants, straw, and coconut shells). In one embodiment of this invention, the biofilm is made of long-fiber polyester material, i.e., an algae-bacterial symbiotic biofilm. The preparation method of the biofilm is as follows: Mixed algae solution and compound bacterial solution are mixed at an inoculation ratio of 1:1 to 1:4 to obtain mixed algae and bacteria. The total inoculation amount of compound bacteria and mixed algae is 1~1.5g / L. The algae and bacteria symbiotic biofilm is obtained by attaching the biofilm using a peristaltic pump.

[0016] Further, the compound bacterial solution is prepared as follows: *Bacillus proteolyticus* B1, *Bacillus tropicalis* B8, and *Pseudomonas schrenckii* B3 are cultured to the logarithmic growth phase, and the bacterial cells are collected and inoculated into the aquaculture wastewater to be treated for resuspension. The inoculation ratio of *Bacillus proteolyticus* B1, *Bacillus tropicalis* B8, and *Pseudomonas schrenckii* B3 is 0.8~2.2:0.8~2.2:0.8~2.2; further, the inoculation ratio is 1~2:1~2:1~2. In some embodiments with better results, the inoculation ratio is 0.8~1.2:0.8~1.2:1.5~2.5, specifically, such as 1:1:2.

[0017] Further, the mixed algal solution is prepared as follows: *Chlorella vulgaris* FACHB-32, *Chlorella proteoglycans* FACHB-1216, and *Scenedesmus tetracoralis* FACHB-1297 are cultured to the logarithmic growth phase, centrifuged to obtain algal sludge, and inoculated into the wastewater to be treated to obtain the algal solution. The total inoculation amount of the three algae is 0.2~0.8 g / L; wherein, the inoculation amount ratio of *Chlorella vulgaris* FACHB-32, *Chlorella proteoglycans* FACHB-1216, and *Scenedesmus tetracoralis* FACHB-1297 is 0.8~2.2:0.8~1.2:0~1.2; further, the inoculation amount ratio of the three algae is 1~2:0.8~1.2:0.8~1.2, and in some embodiments with better results, the above inoculation amount ratio is 1~2:1:1.

[0018] Furthermore, the above-mentioned algae-bacteria symbiotic biofilm is applied to the treatment of aquaculture wastewater in the following ways: The algae-bacteria symbiotic biofilm was placed on an inclined plane, and the aquaculture wastewater to be treated was continuously dripped from above the inclined plane at a flow rate of 28-32 mL / min. During the biofilm formation process, the ambient temperature was maintained at 22-26℃, and continuous lighting (36 W, 60 μmol / m² / s) was provided.

[0019] In a second aspect, the present invention provides the application of the algae-bacteria symbiotic system described in the first aspect in the field of aquaculture wastewater treatment.

[0020] The livestock and poultry breeding wastewater mentioned in the second aspect above further refers to livestock and poultry breeding wastewater, including but not limited to wastewater from pigs, cattle, sheep, rabbits, and chickens. In some embodiments verified by this invention, the aforementioned livestock and poultry breeding wastewater is biogas slurry wastewater, that is, liquid byproducts after anaerobic fermentation.

[0021] The purposes of the above applications include, but are not limited to, any of the following: 1) Reduce COD and ammonia nitrogen (NH4+) in aquaculture wastewater + -N), total phosphorus (TP) and / or antibiotic content; 2) Improve aquaculture wastewater to meet discharge standards into the environment; 3) Improve aquaculture wastewater to meet the standards for use as liquid fertilizer.

[0022] Compared with the prior art, the beneficial effects of the present invention are: This invention compared the degradation effects of three different systems (a control group composite bacterial suspension system, a control group mixed algal film system, and a dominant algae-bacterial combined biofilm system) on actual pig farm biogas slurry wastewater. The biomass accumulation and pollutant removal efficiency of different systems in actual wastewater were examined. Furthermore, the content of biochemical components in the biofilm was measured, revealing their resource utilization value. The results are as follows: The algae-bacteria symbiotic system provided by this invention can effectively control TN and NH4 in actual wastewater. + The removal rates of -N, COD, TP, and SM2 were 86.21%, 91.01%, 96.42%, 97.92%, and 100%, respectively. Compared with the control group (algae-mixed or bacteria-mixed systems), the NH4+ removal rate of the algae-bacteria symbiotic system was significantly higher. + The residual concentrations of NH4+, COD, and TP were 41.49 mg / L, 349.81 mg / L, and 0.15 mg / L, respectively, all meeting the Chinese standards for livestock and poultry wastewater effluent (NH4+). + -N≤ 80 mg / L, COD ≤ 400 mg / L, TP ≤ 8 mg / L). Attached Figure Description

[0023] The accompanying drawings, which form part of this invention, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an improper limitation of the invention.

[0024] Figure 1 It is an inclined plate biofilm photoreactor; Figure 1 In the middle, 'a' is the main view. Figure 1 In the middle, b is the top view; Figure 2 for Bacillus tropicus Identification phylogenetic tree of strain B8; MCC 1A01406 in the figure is... Bacillus tropicus B8; Figure 3 for Bacillus proteolyticus Identification phylogenetic tree of strain B1; the number MCCC1A00365 in the figure is... Bacillus proteolyticus B1; Figure 4 A graph showing the pH changes in wastewater; The microbial preservation information is as follows: Bacillus protein hydrolysate ( Bacillus proteolyticus B1, this strain was deposited on May 23, 2025, at the China Center for Type Culture Collection (CCTCC), located at Wuhan University, Wuhan, China, with the accession number CCTCCCM 20251164. Detailed Implementation

[0025] It should be noted that the following detailed description is illustrative and intended to provide further explanation of the invention. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0026] It should be noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of exemplary embodiments according to the invention. As used herein, the singular form is intended to include the plural form as well, unless the context clearly indicates otherwise. Furthermore, it should be understood that when the terms "comprising" and / or "including" are used in this specification, they indicate the presence of features, steps, operations, devices, components, and / or combinations thereof.

[0027] In the following description, "B1" indicates "Bacillus proteolyticus (B1)". Bacillus proteolyticus "B1" and "B8" indicate "Tropical Bacillus ( Bacillus tropicus "B8" and "B3" indicate "Pseudomonas stearothermiae" (B3). Pseudomonas stutzeri B3”; "C.v” Represents common Chlorella ( Chlorella vulgaris FACHB-32 or common Chlorella FACHB-32, C.p Representative Chlorella proteoglycans ( Chlorellapyrenoidosa FACHB-1216 or Chlorella proteoglycans FACHB-1216, S.q Represents Scenedesmus tetra-tailed ( Scenedesmus quadricauda FACHB-1297 or Scenedesmus tetracoccinea FACHB-1297.

[0028] The aforementioned Bacillus tropicalis B8 has been published in a paper, the information of which is as follows: Lu Tianxiang. Research on the construction of dominant algal biofilms and their effect on the treatment of pig biogas slurry [D]. Qilu University of Technology, 2024. This strain is currently deposited in the applicant's laboratory, and the applicant promises to release this biological material to the public within 20 years from the date of this application. As described in the background section, existing technologies struggle to meet the emission standards for SM2, nitrogen, and phosphorus residues in aquaculture pollutants. To address these technical issues, this invention designs and provides an algae-bacterial symbiotic system. To achieve this technical objective, this invention explores composite bacteria, microalgae biofilms, and algae-bacterial symbiotic biofilms: I. Experimental Preparation 1. Water used in the experiment In the early stages of the experiment, simulated pig farm biogas slurry wastewater (composition shown in Table 1) was selected. In the final stage of the experiment, actual biogas slurry wastewater from a pig farm in Jinan was used for treatment research. The pollutant indicators and contents of the simulated and actual pig farm biogas slurry wastewater are shown in Table 2. After the actual pig farm biogas slurry wastewater was collected, it was refrigerated at 4 °C and allowed to stand for 24 hours. Then, it was filtered through double-layered gauze, and the filtered wastewater was centrifuged at 5000 r / min for 10 min. The supernatant was kept for later use (stored at 4 °C).

[0029] Table 1. Formula for simulated pig farming biogas slurry wastewater Table 2. Components and content of simulated and actual pig farming biogas slurry wastewater. 2. Biomembrane experimental setup The reactor used in the experiment was an inclined plate biofilm photoreactor developed in previous laboratory research, used for algal and bacterial biofilm cultivation and wastewater treatment. The reactor is as follows... Figure 1 As shown, the outer shell is made of polymethyl methacrylate (PMMA) and is 0.25 m long, 0.05 m wide, and 0.1 m high. Each reactor includes an inlet tank for uniform wastewater distribution and an inclined 0.01 m tank for biofilm growth. 2 The system includes a biofilm attachment area and an outlet tank for wastewater outflow. Both the inlet and outlet tanks have 10 mm diameter inlet and outlet holes on the PMMA plate. The carrier for biofilm growth is placed at a 15° angle to ensure smooth flow of the medium across the material to the outlet tank. This design exposes the biofilm to air, resulting in higher gas transfer efficiency compared to submerged biofilms.

[0030] 3. Membrane carrier materials This study selected long-pile polyester fabric as the biofilm carrier material.

[0031] II. Construction of the Compound Bacteria This invention isolated a strain of Bacillus proteolyticus from the sediment of a eutrophic water body in Jinan. Bacillus proteolyticus B1, the above strains were resuspended in sterile physiological saline to OD. 600 The seed culture concentration was approximately 0.4%. One mL of the seed culture was inoculated into 100 mL of denitrification performance testing medium and cultured at 30°C and 180 r / min for 24 h. This achieved a TN removal rate of 92.8% and NO3- removal... - -N removal rate 98.99%, NO2 - -N accumulation is 0.036 mg / L, indicating good denitrification effect.

[0032] Because pig farming wastewater has a high nitrogen content, this invention designs a compound microbial preparation for purifying pig farming wastewater, specifically a compound microbial agent containing the aforementioned strain B1. In the inventors' prior research, a tropical Bacillus (…) Bacillus tropicus B8, the strain described, was collected from pig farm biogas slurry wastewater and showed degradation effects on multiple pollutants in the wastewater. The following compound microbial agent also includes a strain of *Pseudomonas stearothermiae*, purchased from the China General Microbiological Culture Collection Center (CGMCC), with accession number CGMCC:1.10279, designated as B3.

[0033] In this embodiment, the above-mentioned strains were selected and combined in pairs or in combination of three strains at different dosage ratios. The three strains cultured to the logarithmic phase were centrifuged at 4 ℃ and 4000 r / min for 10 min, the supernatant was discarded, and the bacterial cells were washed three times with the prepared simulated biogas slurry wastewater. Then, they were inoculated into conical flasks containing simulated biogas slurry wastewater at 3% of the total inoculum amount. The total volume of the culture was 800 mL.

[0034] Furthermore, in this embodiment, an algae-bacterial co-culture system is also provided. Chlorella vulgaris cultured to the logarithmic growth phase and a compound bacterial agent (B1:B8:B3=1:1:1) are centrifuged separately for 10 min (4 ℃, 4000 r / min). The supernatant is discarded, and the algal cells and bacterial bodies are washed three times with prepared simulated biogas slurry wastewater. The algae and bacterial inoculation ratio is 2:1 (dry weight ratio, g / L) into an Erlenmeyer flask containing 800 mL of simulated pig-farming biogas slurry wastewater. The total concentration of inoculated algae and bacteria is approximately 0.1 g / L. During the culture process, the environment is maintained at 25±1 °C, and a white fluorescent lamp (Philips, 36 W) with 60 μmol / m³ is used. 2 Continuous illumination at / s. The results are shown in Table 3 below: Table 3. Pollutant degradation effect of compound bacterial agent and algae-bacterial co-culture system According to Table 3, the combined use of B1 with B8 or B3 still results in insufficient TP or COD degradation rates. However, the combined use of B1, B8, and B3 has a good degradation effect on all pollutants in pig farm biogas slurry wastewater. By adjusting the ratio of the three strains, the B1:B8:B3=2:1:1 dosage group has the best degradation effect.

[0035] Compare B1:B8:B3=2:1:1 with C.v + B1 + B8 + B3 The results show that introducing Chlorella and bacterial strains together can further improve the degradation effect on various pollutants, especially antibiotic components, with an effect of nearly double that of compound bacterial agents.

[0036] III. Establishment of a Mixed Algal Biofilm System Based on the research results in Part II, this embodiment designs and provides a composite algal biofilm system with better performance.

[0037] 1. Experimental algal strains Based on previous literature review, this study selected three algal strains with good tolerance to pig farm biogas slurry wastewater: *Chlorella vulgaris* FACHB-32, *Chlorella proteoglycans* FACHB-1216, and *Scenedesmus tetracauda* FACHB-1297, all purchased from the Freshwater Algae Culture Bank of the Wuhan Institute of Hydrobiology, Chinese Academy of Sciences. The algal strains were first cultured in conical flasks containing BG11 culture medium (composition and dosage shown in Table 1) in a constant temperature and light incubator (light: 5000 lux; light: dark: 12 h: 12 h; temperature: 25 ± 1 ℃) for regeneration, and then transferred to a photoreactor for further cultivation and expansion under the same light and temperature conditions.

[0038] Table 4. Composition and dosage of BG11 culture medium 2. Mixed algal biofilm ① Microalgae culture Place the algal culture in an inclined plate photoreactor (e.g.) Figure 1 Propagation was carried out in ) until it grew in the logarithmic growth phase (OD). 680 Centrifuge the algal solution at a concentration of approximately 2.0 g / L. Centrifuge the seed culture to achieve an initial biomass concentration of approximately 1.2 g / L. Centrifuge the algal solution at 4000 r / min for 10 min and discard the supernatant. The concentrated algal sludge is then resuspended for later use.

[0039] ② Microalgae biofilm The simulated wastewater was sterilized at high temperature and then placed into Erlenmeyer flasks and sealed for later use. Long-fiber polyester was dried at 105 °C to constant weight (approximately 24 h). The reactor, silicone tubing, and adapters used in the experiment were sterilized by immersion in dilute hydrochloric acid for 24 h. The dried long-fiber polyester was laid on the inclined plate of the reactor (0.01 m²), and the reactor was connected to the Erlenmeyer flasks and a peristaltic pump. Each reactor was connected to a 500 mL Erlenmeyer flask, and the peristaltic pump was started (20 mL / min) for 24 h of biofilm formation treatment.

[0040] ③ Experimental Design This experiment investigated the effects of six mixed algal formulations (Table 2) on the growth of microalgal biofilms and their pollutant removal efficiency. After the mixed algae formed biofilms, each system was connected to simulated biogas slurry wastewater. The reactor was connected to a 1 L Erlenmeyer flask (containing 800 mL of wastewater), and the wastewater was pumped to the membrane carrier channel via a peristaltic pump to ensure continuous contact and circulation between the algal biofilm and the wastewater. Each experiment was conducted in triplicate, with an experimental period of 8 days. The apparatus was maintained at 25±1 ℃, and continuous illumination was provided by white fluorescent tubes.

[0041] Water samples were taken every 48 hours. After measuring biomass and pH, the samples were filtered through 0.45 µm filter paper and then used for NH4 testing. + The analysis of N, COD, TN, TP and SM2 was conducted, and the growth of six mixed algal biofilms and the removal of pollutants in wastewater were analyzed based on the experimental data.

[0042] Table 5 Mixed algae group formulation design and inoculation concentration 3. Analytical Methods ① Detection of wastewater quality indicators (1) Routine water quality index determination Table 6. Methods for Testing Conventional Water Quality Indicators (2) SM2 measurement The concentration of SM2 in wastewater was determined by high-performance liquid chromatography (HPLC). First, the sample was passed through a microporous membrane with a pore size of 0.22 μm to remove impurities such as particles and bacteria. Methanol and water were selected as the mobile phases, with a final flow ratio of organic phase (methanol) to inorganic phase (water) of 3:7. The detection method was as follows: flow rate controlled at 1 mL / min, column temperature at 35 ℃, column pressure at 11–17 Pa, detection wavelength at 268 nm, and retention time at 10–15 min.

[0043] 4. Analysis Results - Effects of Different Mixed Algal Biofilm Ratios on the Treatment Efficacy of Pig Farm Wastewater The wastewater was introduced into the biofilm of the above-mentioned mixed algae, and the removal of each pollutant is shown in Table 7 below: Table 7. Removal amount and removal rate of pollutants in wastewater by different mixed algal biofilm groups According to Table 7, among the six different proportions of mixed microalgae in the biofilm... C.v : C.p : S.q The 2:1:1 ratio group had a biomass yield of 176.34 g / m³. 2 ), yield (22.04 g / m 2 / d) and TN (71.05%) and NH4 in wastewater + The best results were achieved in the removal of -N (71.51%) and SM2 (60.80%). Taking all factors into account, [the following was chosen]. C.v : C.p : S.q The 2:1:1 ratio was used as a mixed algal biofilm group for constructing an algal-bacterial symbiotic biofilm system.

[0044] IV. Constructing an Algae-Fungi Symbiotic Biofilm System The experimental design employed the optimal compound bacterial formulation selected in Parts II and III (Bacillus tropicalis: Bacillus proteolyticus: Pseudomonas stearothermia = 1:1:2) and mixed algae (Chlorella vulgaris: Chlorella proteoglycinae: Scenedesmus tetracaulis = 2:1:1) to construct a dominant algae-bacterial combination. The effects of five algae-bacterial ratios (1:1, 1:2, 2:1, 1:3, 3:1) on the growth of the algae-bacterial biofilm and the pollutant removal efficiency were investigated. The total inoculum concentration of algae and bacteria in each system was 1.2 g / L. After centrifugation, concentration, resuspending, and sealing, the algae and bacteria solutions were stored in Erlenmeyer flasks for later use. The microalgae biofilm formation process and experimental design were the same as in Part III.

[0045] The analysis results showed that biofilms with different algae-bacteria inoculation ratios affected TN and NH4 in simulated wastewater. + The removal of -N, TP, COD, and SM2 is shown in Table 8. When the algae-bacterial inoculation ratio was 1:3, the algae-bacterial combined biofilm system showed the best removal effect on pollutants in the simulated wastewater, particularly for NH4+. + The removal amounts of -N, TN, TP, COD, and SM2 were 407.68 mg / L, 413.08 mg / L, 45.00 mg / L, 927.90 mg / L, and 8.20 mg / L, respectively, with removal rates of 90.60%, 89.80%, 99.99%, 93.73%, and 82.00%. Among the five algae-to-bacteria ratios, the 1:3 ratio showed the highest removal rate for pollutants in the simulated wastewater. Therefore, a dominant algae-to-bacteria biofilm system was constructed based on an algae-to-bacteria ratio of 1:3.

[0046] Table 8. Removal amount and removal rate of pollutants in simulated wastewater by different algae-to-bacteria ratio systems. V. The effect of algae-bacteria symbiotic biofilm on the degradation of actual pig farm biogas slurry wastewater 1. Water used in the experiment The actual pig farm biogas slurry wastewater was taken from the effluent of the anaerobic fermentation tank of a pig farm in Jinan. After the wastewater was brought back to the laboratory, it was first refrigerated in a refrigerator at 4 ℃ for 24 h, and then impurities and particulate matter were filtered out with double-layer gauze. The filtered wastewater was then centrifuged at 5000 r / min for 10 min, and the supernatant was kept for later use (stored at 4 ℃).

[0047] 2. Culture medium preparation The preparation and formulation of LB medium are shown in Table 9.

[0048] Table 9 LB Culture Medium Formulation 3. Control group treated with suspended compound bacteria system for actual pig biogas slurry Take 800 mL of the pretreated wastewater and place it in a 1 L Erlenmeyer flask. Add the concentrated compound bacteria (B1:B8:B3=1:1:2) after centrifugation (4000 rpm, 10 min) to each Erlenmeyer flask, keeping the total inoculum at 3%. Place the inoculated Erlenmeyer flasks in a constant temperature incubator at 25±1 ℃ for continuous culture. Set up 3 parallel experiments with an experimental period of 8 days. Shake the Erlenmeyer flasks 1-2 times each in the morning, noon and evening to prevent bacterial sedimentation.

[0049] 4. Control group mixed algae film system for treating actual pig biogas slurry The pretreated wastewater was poured into a sealed Erlenmeyer flask and kept for later use. The algae solution was then mixed... C.v : C.p : S.q The inoculum concentration was 2:1:1 (maintaining a total inoculum concentration of 1.2 g / L). After centrifugation, concentration, and resuspending, the inoculum was also placed in Erlenmeyer flasks and sealed for later use. The biofilm formation process and experimental design were the same as in Part III.

[0050] 5. Treatment of actual pig-farming biofilm slurry using a combination of dominant algae and bacteria The pretreated actual pig farm biogas slurry wastewater was placed into Erlenmeyer flasks, sealed, and kept for later use. The compound bacteria and mixed algae solution were mixed at an inoculation ratio of 3:1, centrifuged and concentrated, then resuspended in the actual pig farm biogas slurry wastewater and placed into Erlenmeyer flasks, sealed, and kept for later use. The total inoculation concentration of algae and bacteria was kept at 1.2 g / L. The biofilm formation process and experimental design were the same as above.

[0051] 6. Results and Discussion - Removal of Pollutants by the Three Systems Table 7 shows the results of three systems in treating pollutants in actual pig farm biogas slurry wastewater. As can be seen from Table 7, the treatment of TN and NH4+... +Regarding the removal of -N, the dominant algae-bacteria biofilm system demonstrated high efficiency and stability throughout the process. The removal effects of the three systems on COD and TP are shown in Table 7. From day 2 to day 8, the TP removal rate of the dominant algae-bacteria biofilm system increased from 84.72% to 97.92% (removal amount increased from 6.10 mg / L to 7.05 mg / L), significantly exceeding the removal efficiency of the suspended composite bacterial system (TP: 78.61%) and the mixed algae biofilm system (TP: 87.51%). The COD removal rate of the dominant algae-bacteria biofilm system jumped from 48.16% to 96.42% (removal amount increased from 4704.95 mg / L to 9420.09 mg / L), also significantly higher than the suspended composite bacterial system (COD: 58.40%) and the mixed algae biofilm system (COD: 81.61%). All three systems achieved complete removal of SM2 within the first two days. In summary, the dominant algae-bacteria combined biofilm system demonstrates high efficiency, stability, and sustainability in treating high-concentration wastewater from actual pig farming. Its TN and NH4 content are significantly reduced. + The removal rates of nitrogen, COD and TP were significantly improved compared with the compound bacteria and mixed algae system, and no chemical additives or external carbon sources were required. The ammonia nitrogen, COD, TP and SM2 in the wastewater all met the "Emission Standard for Livestock and Poultry Farming (GB18956-2001)".

[0052] Table 10. Removal amount and removal rate of pollutants in wastewater by different systems To enable those skilled in the art to better understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below with reference to specific embodiments.

[0053] Example 1 In this embodiment, an algae-bacterial symbiotic system is provided, comprising composite bacteria and mixed algal biofilm, and the preparation and application methods are as follows: 1. Preparation of compound bacteria (1) Inoculate B1, Bacillus tropicalis B8, and Pseudomonas schrenckii B3 into test tubes containing 5 mL of LB medium and incubate at 25±1 ℃ for 24 h. Take 5 mL of the culture and inoculate it into a conical flask containing 250 mL of LB medium, place it on a constant temperature shaker at 25±1 ℃ and a shaking rate of 180 r / min and incubate for 16 h.

[0054] (2) The protein-hydrolyzed Bacillus B1, Bacillus tropicalis B8, and Pseudomonas schrenckii B3 from step (1) were cultured to the logarithmic phase, centrifuged at 4℃ and 4000 r / min for 10 min, and the supernatant was discarded. The bacterial cells were inoculated into the wastewater to be treated at a total inoculum of 3% and resuspended. The three bacterial solutions were mixed at an inoculum ratio of B1:B8:B3=1:1:2 to obtain a composite bacterial solution, which was then placed in an Erlenmeyer flask and sealed for later use.

[0055] 2. Preparation of mixed algae Chlorella vulgaris FACHB-32, Chlorella proteoglycans FACHB-1216, and Scenedesmus tetrapoda FACHB-1297 were revived in BG11 medium under the following conditions: light: 5000 lux; light: dark: 12 h: 12 h; temperature: 25 ± 1 °C. They were then transferred to a photoreactor and cultured and expanded under the same light and temperature conditions. The revival was achieved when the phylogenetic cycle reached the logarithmic growth phase (OD2). 680 When the algal concentration is around 2.0, centrifuge the algal solution at 4000 r / min for 10 min and discard the supernatant to obtain the centrifuged and concentrated algal sludge. Inoculate the aquaculture wastewater to be treated (inoculation amount 1.2 g / L), mix the algal solution at an inoculation ratio of 2:1:1 to obtain a mixed algal solution, and put it into an Erlenmeyer flask and seal it for later use.

[0056] 3. Preparation of mixed algae and bacteria biofilm (1) The above compound bacterial solution and mixed algal solution were mixed at an inoculation ratio of 3:1 to obtain mixed algae and bacteria, with a total inoculation concentration of 1.2 g / L. The mixture was placed in an Erlenmeyer flask for subsequent biofilm treatment. (2) The long-fiber polyester was dried at 105°C to constant weight (approximately 24 h) and laid on the inclined plate of the reactor (0.01 m). 2 The reactor was connected to a 500 mL Erlenmeyer flask and a peristaltic pump. Each reactor was connected to a 500 mL Erlenmeyer flask. The peristaltic pump (20 mL / min) was started for 24 h of biofilm formation treatment to obtain an algae-bacterial symbiotic biofilm. (3) After the biofilm formation is completed, the pig biogas slurry wastewater to be treated is introduced. The wastewater is transported to the mixed algae biofilm formation via a peristaltic pump at a flow rate of 30 mL / min. During the mixed algae biofilm formation treatment, the ambient temperature is maintained at 25±1 ℃, and continuous lighting (36 W, 60 μmol / m² / s) is provided.

[0057] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. An algae-bacteria symbiotic system, characterized in that, The symbiotic system includes a carrier, a compound bacteria and a mixed algae. The compound bacteria is a combination of Bacillus proteolyticus, Bacillus tropicalis and Pseudomonas schrenckii, and the mixed algae is a combination of Chlorella vulgaris, Chlorella proteoglycinae and Scenedesmus tetracaulis.

2. The algae-bacteria symbiotic system as described in claim 1, characterized in that, The protein-hydrolyzing Bacillus is a protein-hydrolyzing Bacillus ( Bacillus proteolyticus B1 was deposited on May 23, 2025, at the China Center for Type Culture Collection (CCTCC), located at Wuhan University, Wuhan, China, with accession number CCTCC M 20251164. Its 16S rDNA sequence is shown in SEQ ID NO:

1. The tropical spores are tropical spores ( Bacillus tropicus B8, whose 16S rDNA sequence is shown in SEQ ID NO:2; The *Pseudomonas schrenckii* strain was purchased from the China General Microbiological Culture Collection Center (CGMCC), and its accession number is CGMCC:1.10279.

3. The algae-bacteria symbiotic system as described in claim 2, characterized in that, The Chlorella mentioned is common Chlorella ( Chlorella vulgaris )FACHB-32; The *Chlorella proteoglycans* is *Chlorella proteoglycans* (…). Chlorella pyrenoidosa )FACHB-1216; The four-tailed Scenedesmus is *Scenedesmus four-tailed* ( Scenedesmus quadricauda ) FACHB-1297.

4. The algae-bacteria symbiotic system as described in claim 1, characterized in that, The carrier material is selected from polyethylene, polyurethane, polyester fiber, activated carbon, zeolite, ceramic materials, plants, straw or coconut shell.

5. The algae-bacteria symbiotic system as described in claim 4, characterized in that, The carrier is a long-pile polyester material.

6. The algae-bacteria symbiotic system as described in claim 5, characterized in that, The carrier is made of long-fiber polyester fiber material, and the algae-bacterial symbiotic system is an algae-bacterial symbiotic biofilm. The preparation method of the biofilm is as follows: Mixed algae solution and compound bacterial solution are mixed at an inoculation ratio of 1:1 to 1:4 to obtain mixed algae and bacteria. The total inoculation amount of compound bacteria and mixed algae is 1~1.5g / L. The algae and bacteria symbiotic biofilm is obtained by attaching the biofilm using a peristaltic pump.

7. The algae-bacteria symbiotic system as described in claim 6, characterized in that, The compound bacterial solution was prepared as follows: Bacillus protein hydrolysate B1, Bacillus tropicalis B8, and Pseudomonas schrenckii B3 were cultured to the logarithmic growth phase, and the bacterial cells were collected and inoculated into the aquaculture wastewater to be treated and resuspended. The inoculation ratio of Bacillus protein hydrolysate B1, Bacillus tropicalis B8, and Pseudomonas schrenckii B3 was 0.8~2.2:0.8~2.2:0.8~2.

2.

8. The algae-bacteria symbiotic system as described in claim 6, characterized in that, The mixed algal solution was prepared as follows: Chlorella vulgaris FACHB-32, Chlorella proteoglycans FACHB-1216, and Scenedesmus tetracoralis FACHB-1297 were cultured to the logarithmic growth phase, centrifuged to obtain algal sludge, and inoculated into the wastewater to be treated to obtain the algal solution. The total inoculation amount of the three algae was 0.2~0.8 g / L; wherein, the inoculation amount ratio of Chlorella vulgaris FACHB-32, Chlorella proteoglycans FACHB-1216, and Scenedesmus tetracoralis FACHB-1297 was 0.8~2.2:0.8~1.2:0~1.

2.

9. The application of the algae-bacteria symbiotic system according to any one of claims 1-8 in the field of aquaculture wastewater treatment.

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