A method for synchronous recovery of sewage treatment and poly-beta-hydroxybutyric acid based on a bacteria-algae symbiotic system

Abstract

The present application belongs to the field of environmental engineering and biological resource recycling technology, and particularly relates to a wastewater treatment and poly-beta-hydroxybutyric acid synchronous recovery method based on a bacteria-algae symbiotic system. The present application independently screens a dominant algal group and bacterial group capable of generating PHB (poly-beta-hydroxybutyric acid) from a local water environment, constructs a bacteria-algae symbiotic system for degrading wastewater, and can degrade main pollutants (COD, ammonia nitrogen and total phosphorus) in the wastewater within 48 hours, greatly improves the ability of the system to generate PHB synchronously during the wastewater treatment process, and realizes high-yield recovery. The present application uses an orthogonal test method to systematically optimize five key operating parameters, provides a complete and clear process scheme for engineering applications of different scales and different water qualities, ensures the replicability and amplification potential of the technology, and realizes the concept of "waste control by waste and waste into treasure". The whole process is green and low-carbon, and the final product can be completely biodegraded, which conforms to the concept of circular economy and sustainable development.

Description

Technical Field

[0001] This invention belongs to the field of environmental engineering and biological resource recycling technology, specifically relating to a method for simultaneous wastewater treatment and polyβ-hydroxybutyric acid recovery based on a bacterial-algae symbiotic system. Background Technology

[0002] Poly(β-hydroxybutyric acid) (PHB), a natural polyester synthesized by microorganisms, possesses complete biodegradability and excellent biocompatibility, and is considered one of the ideal materials to replace traditional petroleum-based plastics and alleviate the problem of "white pollution." However, current mainstream PHB production processes mainly rely on pure microorganisms (such as...) Copper-hungry killer Fermentation in expensive culture media results in high production costs, which severely restricts the large-scale commercial application of PHB.

[0003] In the field of wastewater treatment, traditional activated sludge processes and their derivatives have long faced challenges such as high energy consumption, large amounts of residual sludge, significant greenhouse gas emissions, and difficulties in resource recovery. In recent years, algae-microbe symbiotic systems have attracted widespread attention as a green and sustainable technology that couples water treatment with resource recovery. This system utilizes the photosynthesis of microalgae to provide dissolved oxygen and absorb carbon dioxide for symbiotic bacteria, while the respiration of the bacteria provides carbon sources (such as carbon dioxide) for the microalgae. The two form a mutually beneficial synergistic relationship, thereby achieving efficient removal of nitrogen, phosphorus, and organic matter from wastewater, and possessing the potential to synthesize high-value-added bio-based products.

[0004] With in-depth research, significant progress has been made in the process design and operational control of algae-bacterial symbiotic systems. For example, patent application number 2024101676528 discloses a low-energy-consumption, high-concentration self-circulating algae-bacterial symbiotic wastewater treatment system and method. This system achieves low-energy operation and efficient utilization of light energy by incorporating a reflective concentrating photovoltaic system, a self-circulating aeration device, and a PLC control system. A key feature of this system is the use of a carrier to fix microalgae balls and form an algae-bacterial symbiotic biofilm with aerobic granular sludge, enhancing system stability. However, this technology primarily focuses on achieving wastewater treatment standards and reducing energy consumption; the treatment process is relatively simple and does not involve the recovery of value-added products.

[0005] On the other hand, to improve the pollutant removal performance of the system, existing technologies have begun to focus on optimizing the function of the microbial community through external regulation. For example, patent application number 202510358647X reveals the application of the quorum sensing signaling molecule C8-HSL in promoting the denitrification and phosphorus removal capabilities of algal-microbe symbiotic systems, by regulating specific functional genes (such as... narGHI、nirKS、phnX The expression of [various microorganisms] significantly improved nitrogen and phosphorus removal efficiency. Such studies provide new insights into optimizing system performance through the regulation of microbial community behavior. However, they still do not address promoting the biogeneration of high-value products (such as PHB).

[0006] Despite the progress made in the aforementioned aspects, the integrated bacterial-algae symbiotic technology for wastewater treatment and resource recovery still has the following prominent limitations: (1) Single functional objective: Most existing systems focus on the efficient removal of pollutants or the reduction of energy consumption, and fail to systematically couple the wastewater treatment process with the targeted generation and efficient recycling of specific high-value products such as PHB, resulting in limited resource recovery benefits.

[0007] (2) Lack of product generation regulation: Existing process parameter optimization and external regulation strategies (such as quorum sensing regulation) are mainly aimed at removal functions such as nitrogen and phosphorus removal, and lack targeted design and regulation of microbial biosynthetic pathways (such as PHB generation pathway), resulting in low product yield and poor economic efficiency.

[0008] (3) Insufficient system efficiency and stability: Although some technologies (such as self-circulation and immobilization) have improved system stability or treatment efficiency, for complex wastewater, the system generally has problems such as long start-up and degradation cycles and weak resistance to shock loads. In addition, it relies on exogenous microorganisms, and its stability in actual engineering applications still needs to be improved.

[0009] (4) Limited process integration and replicability: Existing technologies often focus on improvements in a single aspect (such as energy utilization and specific function regulation), lacking integrated design and clear, scalable process guidance for the entire process of microbial screening, symbiotic system construction, synergistic optimization of operating parameters and product recovery.

[0010] Based on the above analysis, existing technologies have failed to construct an efficient, stable, and economical integrated system to simultaneously achieve rapid and advanced wastewater treatment and high-yield targeted recovery of PHB. Summary of the Invention

[0011] The technical problem to be solved by this invention is to provide a method for simultaneous wastewater treatment and poly-β-hydroxybutyric acid (PHB) recovery based on a bacterial-algae symbiotic system. Using high PHB production as the core screening tool, dominant algal groups capable of generating PHB are autonomously screened from the local aquatic environment. These algae and high PHB-producing bacteria are then used to construct a bacterial-algae symbiotic system that can degrade major pollutants (COD, ammonia nitrogen, and total phosphorus) in wastewater in a very short time. Through multi-parameter synergistic optimization, the system's ability to simultaneously generate PHB during wastewater treatment is significantly improved, achieving high-yield recovery.

[0012] The technical solution adopted is as follows: A method for simultaneous wastewater treatment and polyβ-hydroxybutyric acid (PHA) recovery based on a bacterial-algae symbiotic system includes the following steps: Step S1. Autonomous screening of dominant algal communities capable of generating PHB: Collect algae-rich water samples from the wastewater treatment environment, enrich and culture them in a culture medium to obtain dominant algal communities; Step S2. Acclimation and enrichment of PHB microbial community: The activated sludge is acclimated, enriched and functionally screened in stages to obtain microbial community that has both pollutant degradation ability and PHB generation ability. Step S3. Construction and domestication of the bacterial-algal symbiotic system: The dominant algal community obtained in step S1 is mixed with the bacterial community obtained in step S2 and inoculated into wastewater. Co-culture and directional domestication are carried out under light and intermittent aeration conditions to obtain a domesticated bacterial-algal symbiotic system. Step S4. Operation of the integrated treatment process: The matured bacterial-algae symbiotic culture from step S3 is added to the wastewater to be treated at an inoculum volume of 8%–12%, simultaneously achieving wastewater purification and PHB accumulation in the bacterial-algae symbiotic culture; and: Step S5. Recovery and Quantitative Analysis of PHB: Plot the PHB standard curve; recover and quantify PHB from the bacterial-algal symbiont treated in Step S4.

[0013] Preferably, step S1 includes obtaining the following method: S11. Acclimation and enrichment: Place the algae-rich water sample in a liquid culture medium for PHB production, and culture it continuously for 10 to 15 generations under continuous light conditions at 25-28°C to gradually improve the ability to produce PHB. Screen out single colonies with PHB production ≥20% to obtain high-producing PHB algal communities. The liquid culture medium for producing PHB comprises NaNO3 80 mg / L, K2HPO4 80 mg / L, MgSO4·7H2O 75 mg / L, CaCl2·2H2O 36 mg / L, Na2CO3 20 mg / L, citric acid 6 mg / L, ferric ammonium citrate 6 mg / L, EDTA-Na2 1 mg / L, and A5 trace element solution 1 ml / L. S12. Molecular identification: Metagenomic sequencing analysis confirmed that more than 90% of the algae in the algal community carried key genes capable of generating PHB. phbA、phbB、phbC Furthermore, through specific PCR and high-throughput sequencing of the 18S rRNA gene, its dominant algae were identified as... Chlorella sorokiniana、Chlorella vulgaris、Scenedesmus oblique and Chlamydomonas reinhardtii One or more of them.

[0014] Preferably, in step S2, the method for domestication and enrichment of high-PHB-producing bacterial communities includes: S21. Source collection: Collect activated sludge from the aeration tank of a wastewater treatment plant; S22. Construction of the specific culture medium: A selective culture medium with a high carbon-to-nitrogen ratio of 20–60:1 was used. The specific formula included 20–30 g / L glucose, 0.5–1.0 g / L NaNO3, 1.5 g / L K2HPO4, 0.2 g / L MgSO4·7H2O, 0.05 g / L CaCl2·2H2O, 0.01 g / L FeSO4·7H2O, 0.005 g / L MnSO4·H2O, and 0.5 g / L yeast extract. S23. Phased acclimatization: In the sequencing batch reactor, the process goes through three phases in sequence: adaptation, enhancement and stabilization. By gradually increasing the carbon-nitrogen ratio to 60:1 and adopting an anaerobic-aerobic alternating operation mode, low-yielding bacteria are eliminated and high-yielding PHB bacteria with a stable PHB content of more than 25% and a stable COD removal rate of more than 90% are enriched. S24. Identification and Verification: The core genera of the bacterial community were identified using high-throughput 16S rRNA gene sequencing. Cupriavidus sp., Bacillus sp. .and Pseudomonas sp. And verify the key gene that enables it to generate PHB. phbA、phbB、phbC All were positive.

[0015] Preferably, in step S23, the temperature is controlled at 30±1℃ throughout the sequencing batch reactor, dissolved oxygen is adjusted according to the acclimatization stage, and the hydraulic retention time (HRT) is 24h; the three-stage cultivation process is as follows: Adaptation period: Inoculate activated sludge into an SBR reactor containing the above-mentioned liquid culture medium at an inoculation rate of 10% (v / v), aerate and culture, with dissolved oxygen concentration of 3-4 mg / L, and replace 50% of the culture medium with fresh medium every day to allow the microorganisms in the sludge to adapt to the high carbon and low nitrogen culture medium environment and initially enrich the bacterial community that can generate PHB; the time is from day 1 to day 5. Enhancement phase: Gradually increase the carbon source concentration in the culture medium, raising glucose from 20 g / L to 30 g / L; decrease the nitrogen source concentration (NaNO3) from 1.0 g / L to 0.5 g / L, increasing the C / N ratio to 60:1; adopt an alternating anaerobic-aerobic aeration method, alternating between 4 hours of anaerobic and 4 hours of aerobic aeration, repeating the cycle. Utilize the carbon source storage during the anaerobic phase and the nitrogen and phosphorus limitation during the aerobic phase to induce microbial PHB production. Measure the PHB content of the sludge in the reactor daily, eliminating bacteria with PHB production below 10%; this phase lasts from day 6 to 15. Stabilization period: Maintain a C / N ratio of 60:1 in the culture medium and alternate between anaerobic and aerobic cycles of 4h:4h. Replace the culture medium with simulated domestic sewage to allow the bacteria to adapt to the sewage substrate while retaining their ability to degrade pollutants and generate PHB. When the PHB content in the sludge stabilizes above 25% and the COD removal rate in the sewage stabilizes above 90%, the acclimation is complete, and a high-yield PHB-producing bacterial community is obtained. This period is from day 16 to 20.

[0016] Preferably, in step S24, the screening of high-yielding PHB bacterial communities requires primary screening and secondary screening, specifically as follows: Initial screening: Take the acclimated sludge culture medium and perform screening for 10⁻¹~10⁻ 6 The bacterial suspensions were serially diluted and spread onto solid culture plates capable of generating PHB. They were then incubated at 30°C for 24–48 h. Single colonies with regular morphology, full colonies, and clear edges were selected and inoculated into liquid culture medium in shake flasks. The culture was carried out at 150 r / min and 30°C for 24 h. The PHB content was determined by the chloroform-sodium hypochlorite method. Single colonies with PHB production ≥10% were screened out and mixed to obtain a complex bacterial community. Secondary screening: The complex bacterial groups obtained from the initial screening were inoculated into simulated domestic sewage and cultured continuously in an SBR reactor for 7 days. The removal rates of pollutants COD, NH4⁺-N, TP and PHB production were measured daily. Complex bacterial groups with pollutant removal rates ≥90% and PHB production ≥25% were screened as high-producing PHB candidate bacterial groups and then subjected to molecular biological identification.

[0017] Preferably, in step S3, the construction and directional acclimatization of the algae-bacterial symbiotic system includes: mixing the dominant PHB algae and bacteria in the logarithmic growth phase at a biomass dry weight ratio of 1:1 and inoculating them into simulated domestic sewage in an SBR reactor; culturing them under conditions of 25-30°C, a light-dark cycle of 12h:12h, and intermittent aeration, and acclimatizing them by gradually increasing the influent pollution load for 10-12 days until the system's pollutant removal efficiency stabilizes at over 95%.

[0018] Preferably, in step S4, the operating conditions are: light-dark time ratio of (24-x):x, where x ranges from 0 to 24, hydraulic retention time of 12 to 48 hours, anaerobic to aerobic time ratio of 1 to 11:1, carbon-nitrogen ratio of 8 to 20:1, and nitrogen-phosphorus ratio of 2 to 10:1.

[0019] Preferably, in step (5), the standard curve is plotted as follows: weigh the PHB standard sample, prepare a stock solution with chloroform, and perform gradient dilution to prepare a series of working solutions of 0.0 to 5.0 mg / L. Measure its absorbance at a wavelength of 235 nm using a UV-Vis spectrophotometer. Plot the concentration on the x-axis and the absorbance on the y-axis, and fit the curve using the least squares method, with R² ≥ 0.999.

[0020] Preferably, in step (5), after the treatment is completed, all the mixture in the reactor is centrifuged at 4°C and 8000-10000 r / min for 8-15 minutes to collect the bacterial-algal symbiotic mud. The bacterial-algal symbiotic mud is freeze-dried to constant weight and then pulverized into dry powder. The PHB extraction method is chloroform-sodium hypochlorite method, specifically: the freeze-dried bacterial-algal symbiotic powder is extracted with chloroform in a 60°C water bath for 4-6 hours, the organic phase is taken after centrifugation, and the chloroform is removed by rotary evaporation to obtain crude polyβ-hydroxybutyric acid extract. The crude extract of poly-β-hydroxybutyric acid was dissolved in 98% concentrated sulfuric acid and heated in a 100°C water bath for 5–15 minutes to hydrolyze it into crotonic acid. After cooling, the absorbance was measured at a wavelength of 235 nm, and the content of poly-β-hydroxybutyric acid was calculated according to the standard curve.

[0021] The present invention also provides a fungal-algae symbiotic system for implementing the method, comprising: The dominant algal community capable of generating PHB, wherein the dominant algae of the algal community are Chlorella sorokiniana, Chlorella vulgaris、Scenedesmus obliquus and Chlamydomonas reinhardtii One or more of these, and carrying key genes capable of generating PHB. phbA、phbB、phbC ;as well as: High-yield PHB bacterial communities, the core genera of which include Cupriavidus sp.、Bacillus sp. and Pseudomonas sp. And possesses the key gene capable of generating PHB. phbA、phbB、phbC All were positive. The specific formula for the A5 trace element solution is as follows: 2.86 g of H3BO3, 1.81 g of MnCl2·4H2O, 0.222 g of ZnSO4·7H2O, 0.39 g of Na2MoO4·2H2O, 0.079 g of CuSO4·5H2O, and 49.4 mg (0.0494 g) of Co(NO3)2·6H2O. The solution is diluted to 1 L with deionized water, filtered to remove bacteria (0.22 μm), and stored at 4°C in the dark.

[0022] Compared with the prior art, the beneficial effects of the present invention are as follows: (1) Excellent wastewater treatment efficiency: This invention achieves complete removal of COD, ammonia nitrogen and total phosphorus in wastewater (removal rate of 100%) through the optimized symbiosis of specific algae strains and domesticated bacterial communities, and systematically controls key operating parameters. This significantly surpasses the treatment cycle of traditional bacterial and algal systems, which usually requires more than 5 days, greatly improving treatment efficiency and reactor utilization, and is closer to the actual engineering needs for rapid treatment.

[0023] (2) Significant resource recovery value: This invention pioneered a model that simultaneously drives high PHB production during efficient wastewater treatment. Algal photosynthesis provides oxygen to the system and fixes CO2, while bacteria efficiently convert pollutants to generate PHB. Under optimized conditions, algae themselves can also accumulate PHB, forming a "dual synthesis" effect, which enables the PHB yield to reach an industry-leading level (>45% dry weight), greatly improving the resource recovery benefits of wastewater treatment.

[0024] (3) Innovation and Adaptability of Algal Community Source: This invention adopts the method of independently screening and identifying local algal communities from the environment, rather than purchasing and preserving bacterial strains. The algal community has natural adaptability to local water quality and environment, and the anti-interference ability and shock load resistance of the multi-algal community are significantly better than those of a single algal strain. At the same time, the algal community is detected and quality controlled in real time through specific primer PCR technology, which ensures the stability of the algal community composition during system operation. The system starts up quickly and has low long-term operating costs, which solves the problems of traditional technology relying on exogenous single strain bacteria / algae and poor stability, and reduces long-term operating costs.

[0025] (4) Scientific and reliable process with strong reproducibility: This invention abandons empirical operation and uses orthogonal experimental design to systematically optimize five key operating parameters, thus obtaining a scientifically optimal process window. This provides a complete, clear, and repeatable process scheme for engineering applications of different scales and water qualities, ensuring the reproducibility and scale-up potential of the technology.

[0026] (5) Win-win for both environment and economy: This invention successfully integrates the deep purification of wastewater with the production of high-value-added biomaterial PHB in the same system and process, breaking through the limitations of the separation of "treatment" and "recycling" in traditional technologies, and truly realizing "treating waste with waste and turning waste into treasure". Using wastewater as a free culture medium to produce high-value PHB significantly reduces the production cost of PHB. The entire process is green and low-carbon, and the final product is completely biodegradable, which is in line with the concept of circular economy and sustainable development.

[0027] (6) Low operating cost and stable system: This invention uses sewage as a nutrient source directly, without the need to add expensive exogenous culture medium; the bacteria-algae symbiotic system produces oxygen itself, which can greatly reduce or replace the energy consumption of mechanical aeration; the system has carbon sequestration capacity, low overall energy consumption, stronger competitiveness and survival ability in actual sewage environment, high system robustness, and can be widely promoted. Attached Figure Description

[0028] Figure 1 This is a standard curve diagram of PHB production in this invention.

[0029] Figure 2 The curves show the changes in COD, NH4⁺-N, and TP concentrations in wastewater with treatment time (0-48 h) under optimal process parameters.

[0030] Figure 3 The bar charts show the effects of a five-factor, three-level orthogonal experiment on PHB production. (a) shows the effects of different light-dark time ratios, (b) shows different hydraulic retention time ratios, (c) shows different C / N ratios, (d) shows different N / P ratios, and (e) shows the effects of different anaerobic and aerobic time ratios on PHB production. Detailed Implementation

[0031] The accompanying drawings are for illustrative purposes only; the technical solutions of the present invention will be described in detail below with reference to the embodiments and the accompanying drawings. It should be understood that prior art and common knowledge may be omitted.

[0032] Unless otherwise specified, the chemicals or instruments used in this invention are all available through conventional commercial channels.

[0033] like Figure 1 As shown, the process of plotting the standard curve is as follows: a. Experimental materials and reagents Standards: Poly-β-hydroxybutyrate (PHB) standard (purity ≥98%, Sigma-Aldrich P3649); chloroform (analytical grade, used to dissolve PHB); concentrated sulfuric acid (98%, analytical grade, used for digestion); deionized water (used for dilution); UV-Vis spectrophotometer (with 1 cm quartz cuvette); constant temperature water bath (or digester, temperature controlled at 100℃); analytical balance (accuracy 0.0001 g); digestion tubes, volumetric flasks, pipettes, cuvettes, etc., select the size according to your needs.

[0034] b. Preparation of standard solutions: 1) Stock solution: Accurately weigh 10.00 mg PHB standard, dilute to 100 mL with chloroform to obtain a 100 mg / L stock solution, and store at 4℃ protected from light; 2) Gradient working solutions: Serially dilute the stock solution to working solutions of 0.0, 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, and 5.0 mg / L, with 3 replicates for each concentration.

[0035] Digestion and determination: Take 2.0 mL of working solution, evaporate chloroform to dryness at 100 °C, add 5.0 mL of concentrated sulfuric acid and digest at 100 °C for 10 min, dilute 10 times, and measure the absorbance at 235 nm. Blank control: Take 2.0 mL of chloroform and process it according to the above digestion steps to serve as a reagent blank.

[0036] Absorbance measurement: Turn on the UV spectrophotometer, preheat for 30 min, and set the measurement wavelength to 235 nm.

[0037] Zero the instrument with a blank control solution and measure the absorbance (A) of each concentration of standard solution in sequence.

[0038] Each sample was measured three times, and the average value was taken as the absorbance at that concentration point.

[0039] Calculation of PHB content in samples: Process the samples according to the same digestion-determination procedure to obtain the absorbance. .

[0040] Substitute into the regression equation to calculate the sample concentration: C sample = (A 样品 - 0.0079) / 0.2109; Calculate the PHB content (%, w / w) by combining parameters such as sample dilution factor and dry weight: PHB content = (C 样品 ×V 消解液 × Dilution factor / m 样品干重 )×100; Standard curve plotting: PHB concentration was plotted on the x-axis and absorbance on the y-axis. The regression equation obtained was: y = 0.2109x + 0.0079 (R² = 0.9998), which meets the quantitative requirements.

[0041] Example 1 A method for simultaneous wastewater treatment and polyβ-hydroxybutyric acid (PHA) recovery based on a bacterial-algae symbiotic system includes the following steps: (1) Domestication, enrichment and molecular identification of dominant algal communities that can generate PHB.

[0042] Sample source and acclimatization enrichment: Algae-rich water samples were collected from the aeration tank and receiving water of a wastewater treatment plant. A specific high-efficiency PHB production liquid culture medium (components: NaNO3 80 mg / L, K2HPO4 80 mg / L, MgSO4・7H2O 75 mg / L, CaCl2・2H2O 36 mg / L, Na2CO3 20 mg / L, citric acid 6 mg / L, ferric ammonium citrate 6 mg / L, EDTA-Na 21 mg / L, A5 trace element solution 1 ml / L, sterilized at 121℃ for 20 min) was used for acclimatization and enhancement. The algae were continuously acclimatized and enhanced under conditions of 25-28°C and continuous light (approximately 50 μmol photons m⁻² s⁻¹). By gradually increasing the PHB production selection pressure and adjusting the nutrient ratio of the culture medium, the algae were continuously subcultured for 10 generations, ultimately obtaining a high-PHB-producing algal community.

[0043] Molecular biological identification: Genomic DNA extraction: Take 10 mL of the domesticated algal culture medium, centrifuge at 8000 r / min for 10 min to collect the algal sludge, and extract total genomic DNA from the algal community using a plant genomic DNA extraction kit (centrifuge column type). The DNA integrity is detected by 1% agarose gel electrophoresis, and the DNA concentration and purity are detected by a nucleic acid protein analyzer. The required OD260 / OD280 is 1.8~2.0, and OD260 / OD230 is ≥2.0. Store at -20℃ for later use.

[0044] Metagenomic analysis: Total DNA from qualified algal communities was sent for metagenomic sequencing to construct a PE150 library. Sequencing was performed using the Illumina NovaSeq platform. The sequencing data underwent quality control, assembly, gene prediction, and functional annotation, based on the ability to generate PHB-related functional genes. phbA、phbB、phbC The abundance and species annotation results of the algae were analyzed to determine the proportion of high PHB-producing algae in the algal community. The results showed that more than 90% of the algae were high PHB-producing algae carrying key genes for PHB production.

[0045] Specific PCR screening: Primer design and modification: Specific primer pairs were used (upstream: 3NDF, 5'-CGCGGATCCGGCAAGTCTGGTGCCAG-3'; downstream: V4-euk-R1R, 5'-CCGGAATTCGACTACGACGGTATCTRATCRTCTTCG-3'). The primers were synthesized by a professional biotechnology company and purified by PAGE.

[0046] PCR reaction system: A 25 μL reaction system was used, including 2.5 μL of 10×PCR Buffer (containing Mg2+), 2 μL of dNTP Mix (2.5 mmol / L), 1 μL each of forward and reverse primers (10 μmol / L), 0.2 μL of Taq DNA polymerase (5 U / μL), 1 μL of algal DNA template (50 ng / μL), and ddH2O to a final volume of 25 μL.

[0047] PCR amplification program: 94℃ pre-denaturation for 5 min; 94℃ denaturation for 30 s, 58℃ annealing for 30 s, 72℃ extension for 40 s, for a total of 35 cycles; 72℃ final extension for 10 min; store at 4℃.

[0048] Product detection: Take 5 μL of PCR amplification product and detect it by 1.5% agarose gel electrophoresis (1×TAE buffer, 120V, 30min). Observe and take pictures with a gel imaging system to screen out algal samples that can amplify specific bands of about 500bp.

[0049] High-throughput sequencing and species identification: A 16S / 18S rRNA gene library was constructed from PCR-positive algal DNA samples, and high-throughput sequencing was performed using the Illumina MiSeq platform. The sequencing results were quality-controlled filtered, OTU clustered (97% similarity), and species were annotated in the NCBI database. Combined with microscopic morphological observation (single-celled, spherical, 3-5 μm in diameter, with a distinct protein nucleus), the dominant algae of this high-PHB-producing algal community were identified as... Chlorella sorokiniana, Chlorella vulgaris, Scenedesmus obliquus, Chlamydomonas reinhardtii All Chlorella sorokiniana The similarity is over 99.0%.

[0050] (2) Domestication, enrichment and identification of high-producing PHB bacteria.

[0051] This invention screens bacterial sources from activated sludge in aeration tanks of local urban wastewater treatment plants, enriches and acclimates them in stages using a dedicated, highly efficient PHB-producing selective culture medium, and combines functional screening and molecular identification to obtain a high-PHB-producing bacterial community that possesses both high PHB-generating capacity and wastewater pollutant degradation capacity. The specific steps are as follows: Source of bacteria collection: 1000 mL of activated sludge from the aeration tank of a local urban wastewater treatment plant was collected, placed in a sterile brown sampling bottle, and transported at room temperature away from light. The acclimatization process was completed within 24 hours.

[0052] A selective culture medium formulation with exclusive, highly efficient PHB generation capability: Liquid culture medium: glucose 20 g / L, NaNO3 1.0 g / L, K2HPO4 1.5 g / L, MgSO4·7H2O 0.2 g / L, CaCl2·2H2O 0.05 g / L, FeSO4·7H2O 0.01 g / L, MnSO4·H2O 0.005 g / L, yeast extract 0.5 g / L, pH adjusted to 7.0~7.2, sterilized at 121℃ for 20 min; this culture medium is high carbon and low nitrogen type, with a C / N ratio of 20:1, suitable for the nutritional requirements of bacteria capable of generating PHB, and trace elements are added to ensure the growth of the bacterial community.

[0053] Solid culture medium: Add 20 g / L of agar powder to the above liquid culture medium, sterilize at 121℃ for 20 min, cool to 50~60℃ and pour into plates for later use.

[0054] A phased acclimatization and enrichment strategy was employed: a sequencing batch reactor (SBR) was used for phased acclimatization, with the temperature controlled at 30±1℃ throughout the process. Dissolved oxygen was adjusted according to the acclimatization stage, and the hydraulic retention time (HRT) was 24 hours, divided into three stages. Adaptation period (5 days): Inoculate activated sludge into an SBR reactor containing the above liquid culture medium at an inoculation rate of 10% (v / v), aerate and culture (dissolved oxygen 3~4 mg / L), and replace 50% of the culture medium with fresh medium every day to allow the microorganisms in the sludge to adapt to the high carbon and low nitrogen culture medium environment and initially enrich high PHB-producing bacteria.

[0055] Enhancement period (10 days): Gradually increase the carbon source concentration in the culture medium (glucose from 20 g / L to 30 g / L) and decrease the nitrogen source concentration (NaNO3 from 1.0 g / L to 0.5 g / L), increasing the C / N ratio to 60:1; adopt an alternating anaerobic-aerobic aeration method (anaerobic 4h, aerobic 4h, repeated in cycles), utilizing the carbon source storage in the anaerobic stage and the nitrogen and phosphorus limitation in the aerobic stage to induce microbial PHB production. Measure the PHB content of the sludge in the reactor daily and eliminate bacterial groups with PHB production below 10%.

[0056] Stabilization period (5 days): Maintain the C / N ratio of the culture medium at 60:1 and alternate between anaerobic and aerobic cycles of 4h:4h. Replace the culture medium with simulated domestic sewage (COD≈300 mg / L, NH4⁺-N≈40 mg / L, TP≈8 mg / L) to allow the bacteria to adapt to the sewage substrate and retain the ability to degrade pollutants and generate PHB. When the PHB content in the sludge stabilizes above 25% and the COD removal rate in the sewage stabilizes above 90%, the acclimatization is complete, and a high-PHB-producing bacterial community is obtained.

[0057] Functional screening of gut microbiota: Initial screening: Take the acclimated sludge culture medium and perform serial dilution (10⁻¹~10⁻¹). 6 ), take 100 μL of bacterial suspension at different dilutions and spread it on high-efficiency PHB-producing solid medium plates, and incubate at 30℃ for 24~48h; pick single colonies with regular morphology, full colonies and clear edges, inoculate them in liquid medium and shake flask culture (150r / min, 30℃, 24h), determine the PHB content by chloroform-sodium hypochlorite method, screen out single colonies with high PHB production, and mix them to obtain a complex bacterial community.

[0058] Secondary screening: The composite bacterial groups obtained from the initial screening were inoculated into simulated domestic sewage and cultured continuously in an SBR reactor for 7 days. The removal rates of pollutants (COD, NH4⁺-N, TP) and the production of PHB were measured daily. Composite bacterial groups with a pollutant removal rate ≥90% and a PHB production ≥25% were selected as high-producing PHB candidate bacterial groups.

[0059] Molecular biological identification of the bacterial community: Total DNA extraction: Take 10 mL of candidate bacterial culture medium, centrifuge at 8000 r / min for 10 min to collect bacterial sludge, extract total DNA of bacterial community using bacterial genomic DNA extraction kit, and test its integrity, concentration and purity. After passing the test, store at -20℃.

[0060] PCR amplification of 16S rRNA gene: PCR amplification was performed using the universal bacterial primer pairs 27F (5'-AGAGTTTGATCCTGGCTCAG-3') and 1492R (5'-GGTTACCTTGTTACGACTT-3'). The reaction volume was 25 μL, and the amplification program was as follows: pre-denaturation at 94℃ for 5 min, 30 cycles (94℃ for 30 s, 55℃ for 30 s, 72℃ for 1 min), and final extension at 72℃ for 10 min.

[0061] High-throughput sequencing and bioinformatics analysis: PCR products were used to construct libraries and high-throughput sequencing was performed using the Illumina NovaSeq platform; quality control of sequencing data was performed (removal of low-quality sequences and chimeras), OTU clustering (97% similarity), and species annotation was performed in RDP and NCBI databases; the species composition and relative abundance of the bacterial community were analyzed to identify the core genera of the high PHB-producing bacterial community.

[0062] Results Verification: Molecular identification results showed that the high-PHB-producing bacterial community obtained in this invention mainly includes Cupriavidus sp. (Relative abundance 35%~40%) Bacillus sp. (25%~30%) and Pseudomonas sp.(20%~25%), the rest being small amounts. Acinetobacter sp. and Paracoccus sp. All of these are previously reported high-producing PHB bacteria; at the same time, the key genes of this bacterial community capable of producing PHB were analyzed. phbA、phbB、phbC The tests all showed positive results, proving that it has the molecular basis for high PHB production.

[0063] Microbial community performance verification: The identified high-PHB-producing microbial community was inoculated into simulated domestic sewage and cultured at 30℃ under anaerobic-aerobic conditions for 48 hours. The results showed that the COD removal rate was 92.5%, the NH4⁺-N removal rate was 88.3%, the TP removal rate was 85.7%, and the PHB yield reached 28.5% (dry weight), proving that the microbial community has both efficient pollutant degradation and PHB generation capabilities.

[0064] (3) Construction and targeted domestication of bacterial-algal symbiotic systems.

[0065] Algal community: The dominant algal community that has been domesticated, enriched, and molecularly identified in step (1) above (dominant algae include...) Chlorella sorokiniana, Chlorella vulgaris, Scenedesmus obliquus and Chlamydomonas Reinhardt ); Source of bacteria: The high-PHB-producing bacterial groups (mainly including those that have undergone phased domestication, enrichment, functional screening, and molecular identification in step (2) above) Cupriavidus sp.、Bacillus sp. and Pseudomonas sp. ); Symbiotic system construction and acclimatization: High-PHB-producing algal solution in the logarithmic growth phase was mixed with the aforementioned high-PHB-producing bacterial community at a biomass dry weight ratio of 1:1 and inoculated into an SBR reactor simulating domestic sewage (COD≈300 mg / L, NH4⁺-N≈40 mg / L, TP≈8 mg / L, pH 7.0±0.2). Reactor operating parameters: temperature 25-30°C, light-dark cycle 12h:12h (light intensity 3000 lux), intermittent mild aeration (dissolved oxygen maintained at 4-6 mg / L). Every 24 hours, the influent pollution load was gradually increased (COD increased by 50 mg / L each time, NH4⁺-N increased by 5 mg / L each time, and TP increased by 1 mg / L each time), and the acclimatization process lasted for 12 days. During this period, the stability of the algal community composition was detected by specific PCR and the bacterial community composition was detected by 16S rRNA high-throughput sequencing until the system’s removal efficiency of pollutants stabilized at over 95%, thus obtaining a mature acclimatized algal-bacterial symbiotic system.

[0066] (4) Optimization of key operating parameters of integrated processing technology.

[0067] System startup: Add the domesticated and mature bacterial-algal symbiotic organism to the actual municipal / industrial wastewater at an inoculation rate of 8%-12% (v / v) to start the integrated treatment system.

[0068] Orthogonal experimental design optimization: In order to maximize the pollutant removal rate and PHB generation capacity of the system, this invention designs a five-factor, four-level orthogonal experiment with the following optimization indices: light-dark time ratio, hydraulic retention time (HRT), anaerobic-aerobic time ratio, carbon-nitrogen ratio (C / N), and nitrogen-phosphorus ratio (N / P).

[0069] Optimal process combination: Through orthogonal experimental range analysis (see Table 1, setting five factors: light-dark ratio, HRT, anaerobic / aerobic ratio, C / N ratio, and N / P ratio, each with four levels, for a total of 16 experimental groups; each group ran for 48 hours, and the pollutant removal rate and PHB production results were detected), the optimal parameters were determined to be: light-dark time ratio 12:12, HRT 2d, anaerobic / aerobic time ratio 4:4, carbon-nitrogen ratio 80:5, and nitrogen-phosphorus ratio 5:1. Under these conditions, the system achieved 100% removal rates of COD, NH4⁺-N, and TP in the wastewater, and the PHB production reached 42.3% (dry weight).

[0070] (5) Extraction and quantitative analysis methods of PHB.

[0071] Biomass harvesting: After the treatment is completed, all the mixed liquid in the reactor is centrifuged at 4°C and 8000 r / min for 10 minutes to collect the bacterial-algal symbiotic mud.

[0072] PHB extraction (chloroform-sodium hypochlorite method): The bacterial and algal mud is freeze-dried to constant weight; a certain amount of dry powder is weighed, chloroform is added, and the mixture is stirred and extracted in a 60°C water bath for 4-6 hours; after centrifugation, the lower organic phase is taken out, and the chloroform is removed by rotary evaporation to obtain a light white crude PHB extract.

[0073] PHB quantification (concentrated sulfuric acid hydrolysis method): Accurately weigh an appropriate amount of crude PHB extract (or standard), dissolve it in 98% concentrated sulfuric acid, and heat it in a 100°C water bath for 10 minutes to completely hydrolyze PHB into crotonic acid; after cooling to room temperature, measure its absorbance at a wavelength of 235 nm using a UV-Vis spectrophotometer; calculate the PHB content in the sample according to the pre-plotted PHB standard curve.

[0074] Table 1. Orthogonal experimental design table and corresponding PHB yield results comparison By comparing the results of 16 sets of orthogonal experiments, range analysis was performed using the Spass orthogonal experiment analysis software to generate Table 2, and the optimal parameter combination was determined. The results are shown in Table 2.

[0075] Table 2 Comparison of range analysis results using SPASS orthogonal experimental design software After optimization through orthogonal experiments, the optimal process combination was determined as follows: the optimal parameters were determined through orthogonal experimental range analysis: light-dark time ratio of 12:12, hydraulic retention time of 48h, anaerobic-aerobic time ratio of 4:4, carbon-nitrogen ratio of 80:5, and nitrogen-phosphorus ratio of 5:1. Under these conditions, the pollutant removal rate reached 100%, and the PHB production reached 42.3g / L.

[0076] like Figure 2 As shown, under optimal process parameters, the concentrations of COD, NH4⁺-N, and TP in wastewater decreased continuously with treatment time, intuitively demonstrating complete degradation within 48 hours.

[0077] like Figure 3 As shown, this demonstrates the magnitude of the impact of different levels of various factors on PHB production.

[0078] Example 2 The complete acclimatized algal-microbe symbiotic system from Example 1 was inoculated and set to optimal parameters, with a total operating time of 48 hours. The degradation results of COD, NH4⁺-N, and TP in the water were measured, along with the content of PHB produced by the symbiotic system.

[0079] (1) Domestication, enrichment and identification of dominant algal communities that generate PHB.

[0080] Sample collection: 2L of mixed algae-rich water sample was collected from the aeration tank and lake of a sewage treatment plant near a school and preserved aseptically; Acclimation and enrichment: Take 50 mL of water sample and inoculate it into 1 L of high-efficiency PHB-producing liquid medium (BG11 basic upper limit nitrogen and phosphorus enrichment). Acclimate it for 30 days at 26℃, 3000 lux light, and 12h:12h light-dark ratio. Replace 50% of the medium with fresh medium every 24 hours to gradually improve the ability to produce PHB. Metagenomic analysis: Total DNA was extracted from the algal community for metagenomic sequencing, and the results showed that 90% of the algae were carriers of the virus. phbA、phbB、phbC Genetically advanced PHB-producing algae; Specific PCR and High-Throughput Identification: PCR amplification was performed using specific primers with 5' restriction enzyme sites and protective bases, yielding a 500bp specific band; high-throughput sequencing combined with microscopic observation confirmed that the dominant algae in the algal community were... Chlorella sorokiniana, Chlorella vulgaris, Scenedesmus obliquus, Chlamydomonas ReinhardtThe homology with the model strain was ≥99.0%; Performance verification: After culturing the algal community in simulated wastewater for 48 hours, the PHB yield reached 15.3% (dry weight), which was significantly higher than that of the unacclimated algal community.

[0081] (2) Domestication and enrichment of high-producing PHB bacteria and construction of bacterial-algal symbiotic system.

[0082] Source of bacteria collection: 1000 mL of activated sludge from the aeration tank of a local wastewater treatment plant was collected; Phased acclimatization: The culture medium was acclimatized in a specific high-efficiency PHB-producing selective medium through an adaptation period (5 days), an enhancement period (10 days), and a stabilization period (5 days). The C / N ratio was increased from 20:1 to 60:1, and anaerobic-aerobic alternating aeration was adopted. Functional screening: After initial and secondary screening, the obtained compound microbial community achieved a PHB production of 28.5% (dry weight) and a COD removal rate of 92.5% in simulated wastewater; Molecular identification: 16S rRNA high-throughput sequencing showed that the core bacteria of the bacterial community were... Cupriavidus sp. (38%) Bacillus sp. (29%) Pseudomonas sp. (twenty four%), phbA、phbB、phbC All genes were positive.

[0083] Construction and domestication of fungal-algal symbiotic systems: The superior algal solution with the ability to generate a large amount of PHB was mixed with the bacterial community at a dry weight ratio of 1:1 and inoculated into an SBR reactor simulating domestic sewage. It was acclimatized for 11 days under the conditions of 28℃, 12h:12h light and dark, and 5mg / L dissolved oxygen. The pollution load was gradually increased, and the final pollutant removal rate of the system was stabilized at over 96%.

[0084] With the optimal parameters of light-dark ratio 12:12, HRT 2d, anaerobic / aerobic ratio 4:4, C / N ratio 80:5, and N / P ratio 5:1, the PHB yield reached 42.3% (dry weight).

[0085] Influent water quality: Prepare simulated wastewater with COD=275±10 mg / L, NH4⁺-N=36±2 mg / L, TP=7.0±0.5 mg / L, and pH=7.1.

[0086] Results: After 48 hours, the effluent COD was less than 15 mg / L, NH4⁺-N was less than 0.5 mg / L, and TP was less than 0.3 mg / L, with removal rates approaching 100%.

[0087] PHB recovery: The collected biomass dry weight was 2.15 g / L. Extraction and analysis revealed a PHB content of 1.01 g / L, representing 47.0% of the dry weight of the algal-bacterial symbiosis.

[0088] Other areas not mentioned are the same as in Example 1.

[0089] Comparative Example 1 Pure dominant algal community control: Only the dominant algal community was inoculated, and the degradation results of COD, NH4⁺-N, and TP in the water were measured, along with the PHB content produced by the pure algal system. Other aspects not mentioned are the same as in Example 2.

[0090] Comparative Example 2 High-yield PHB mixed bacterial colony control: Only the screened bacterial colony capable of producing PHB was inoculated, and the degradation results of COD, NH4⁺-N, and TP in the water were measured, along with the PHB production content of the mixed bacterial colony. Other aspects not mentioned are the same as in Example 2.

[0091] The containers used in Example 2, Comparative Examples 1 and 2 are conventional laboratory containers and meet the processing requirements of this invention.

[0092] Example 2 was compared with Comparative Examples 1 and 2, and the results are shown in Table 3.

[0093] Table 3. Comparison of pollutant removal and PHB production between Example 2 and Comparative Examples 1-2 As shown in Table 3, the method of this invention has significant advantages in the removal speed and thoroughness of ammonia nitrogen and total phosphorus in terms of pollutant removal. Regarding PHB production, the PHB production of Example 2 (47.0% dry weight) was significantly higher than that of Comparative Example 1 (15.3% dry weight) and Comparative Example 2 (28.5% dry weight), demonstrating the synergistic effect of algal-microbe symbiosis.

[0094] Of course, the above description is not intended to limit the present invention, and the present invention is not limited to the examples given above. Any changes, modifications, additions or substitutions made by those skilled in the art within the scope of the present invention should also fall within the protection scope of the present invention.

Claims

1. A method for simultaneous wastewater treatment and polyβ-hydroxybutyric acid (PHA) recovery based on a bacterial-algae symbiotic system, characterized in that, Includes the following steps: Step S1. Autonomous screening of dominant algal communities capable of generating PHB: Collect algae-rich water samples from the wastewater treatment environment, enrich and culture them in a culture medium to obtain dominant algal communities; Step S2. Acclimation and enrichment of PHB microbial community: The activated sludge is acclimated, enriched and functionally screened in stages to obtain microbial community with both pollutant degradation ability and PHB generation ability. Step S3. Construction and domestication of the bacterial-algal symbiotic system: The dominant algal community obtained in step S1 is mixed with the bacterial community obtained in step S2 and inoculated into wastewater. Co-culture and directional domestication are carried out under light and intermittent aeration conditions to obtain a domesticated bacterial-algal symbiotic system. Step S4. Operation of integrated treatment process: The matured bacteria-algae symbiotic culture from step S3 is added to the wastewater to be treated at an inoculation rate of 8% to 12% by volume, and the wastewater is purified and PHB is accumulated in the bacteria-algae symbiotic culture simultaneously. as well as: Step S5. Recovery and Quantitative Analysis of PHB: Plot the PHB standard curve; recover and quantify PHB from the bacterial-algal symbiont treated in Step S4.

2. The method for simultaneous wastewater treatment and polyβ-hydroxybutyric acid recovery based on a bacterial-algae symbiotic system according to claim 1, characterized in that, Step S1 includes obtaining the following methods: S11. Acclimation and enrichment: Place the algae-rich water sample in a liquid culture medium for PHB production, and culture it continuously for 10 to 15 generations under continuous light conditions at 25-28°C to gradually improve the ability to produce PHB. Screen out single colonies with PHB production ≥20% to obtain high-producing PHB algal communities. The liquid culture medium for producing PHB comprises NaNO3 80 mg / L, K2HPO4 80 mg / L, MgSO4·7H2O 75 mg / L, CaCl2·2H2O 36 mg / L, Na2CO3 20 mg / L, citric acid 6 mg / L, ferric ammonium citrate 6 mg / L, EDTA-Na2 1 mg / L, and A5 trace element solution 1 ml / L. S12. Molecular identification: Metagenomic sequencing analysis confirmed that more than 90% of the algae in the algal community carried key genes capable of generating PHB. phbA, phbB, phbC Furthermore, through specific PCR and high-throughput sequencing of the 18S rRNA gene, its dominant algae were identified as... Chlorella sorokiniana, Chlorella vulgaris, Scenedesmus obliquus and Chlamydomonas reinhardtii One or more of them.

3. The method for simultaneous wastewater treatment and polyβ-hydroxybutyric acid recovery based on a bacterial-algae symbiotic system according to claim 1, characterized in that, In step S2, the methods for domestication and enrichment of PHB microbiota include: S21. Source collection: Collect activated sludge from the aeration tank of a wastewater treatment plant; S22. Construction of the specific culture medium: A selective culture medium with a high carbon-to-nitrogen ratio of 20–60:1 was used. The specific formula included 20–30 g / L glucose, 0.5–1.0 g / L NaNO3, 1.5 g / L K2HPO4, 0.2 g / L MgSO4·7H2O, 0.05 g / L CaCl2·2H2O, 0.01 g / L FeSO4·7H2O, 0.005 g / L MnSO4·H2O, and 0.5 g / L yeast extract. S23. Phased acclimatization: In the sequencing batch reactor, the process goes through three phases in sequence: adaptation, enhancement and stabilization. By gradually increasing the carbon-nitrogen ratio to 60:1 and adopting an anaerobic-aerobic alternating operation mode, low-yield bacteria are eliminated and PHB bacteria with a stable PHB content of more than 25% and a stable COD removal rate of more than 90% are enriched. S24. Identification and Verification: High-throughput 16S rRNA gene sequencing was used to identify the core genera of the bacterial community, including... Cupriavidus sp., Bacillus sp. .and Pseudomonas sp. And verify the key gene that enables it to generate PHB. phbA, phbB, phbC All were positive.

4. The method for simultaneous wastewater treatment and polyβ-hydroxybutyric acid recovery based on a bacterial-algae symbiotic system according to claim 3, characterized in that, In step S23, the temperature is controlled at 30±1℃ throughout the sequencing batch reactor, dissolved oxygen is adjusted according to the acclimatization stage, and the hydraulic retention time (HRT) is 24h; the three-stage cultivation process is as follows: Adaptation period: Inoculate activated sludge into an SBR reactor containing the above-mentioned liquid culture medium at an inoculation rate of 10% (v / v), aerate and culture, with dissolved oxygen concentration of 3-4 mg / L, and replace 50% of the culture medium with fresh medium every day to allow the microorganisms in the sludge to adapt to the high carbon and low nitrogen culture medium environment and initially enrich the bacterial community that can generate PHB; the time is from day 1 to day 5. Enhancement phase: Gradually increase the carbon source concentration in the culture medium, raising glucose from 20 g / L to 30 g / L; decrease the nitrogen source concentration (NaNO3) from 1.0 g / L to 0.5 g / L, increasing the C / N ratio to 60:1; adopt an alternating anaerobic-aerobic aeration method, alternating between 4 hours of anaerobic and 4 hours of aerobic aeration, repeating the cycle. Utilize the carbon source storage during the anaerobic phase and the nitrogen and phosphorus limitation during the aerobic phase to induce microbial PHB production. Measure the PHB content of the sludge in the reactor daily, eliminating bacteria with PHB production below 10%; this phase lasts from day 6 to 15. Stabilization period: Maintain a C / N ratio of 60:1 in the culture medium and alternate between anaerobic and aerobic cycles of 4h:4h. Replace the culture medium with simulated domestic sewage to allow the bacteria to adapt to the sewage substrate while retaining their ability to degrade pollutants and generate PHB. When the PHB content in the sludge stabilizes above 25% and the COD removal rate in the sewage stabilizes above 90%, the acclimation is complete, and a high-yield PHB-producing bacterial community is obtained. This period lasts from day 16 to 20.

5. The method for simultaneous wastewater treatment and polyβ-hydroxybutyric acid recovery based on a bacterial-algae symbiotic system according to claim 3, characterized in that, In step S24, the screening of PHB bacteria requires primary screening and secondary screening, specifically as follows: Initial screening: Take the acclimated sludge culture medium and perform screening for 10⁻¹~10⁻ 6 The bacterial suspensions were serially diluted and spread onto solid culture plates capable of generating PHB. They were then incubated at 30°C for 24–48 h. Single colonies with regular morphology, full colonies, and clear edges were selected and inoculated into liquid culture medium in shake flasks. The culture was carried out at 150 r / min and 30°C for 24 h. The PHB content was determined by the chloroform-sodium hypochlorite method. Single colonies with PHB production ≥10% were screened out and mixed to obtain a complex bacterial community. Secondary screening: The complex bacterial groups obtained from the initial screening were inoculated into simulated domestic sewage and cultured continuously in an SBR reactor for 7 days. The removal rates of pollutants COD, NH4⁺-N, TP and PHB production were measured daily. Complex bacterial groups with pollutant removal rates ≥90% and PHB production ≥25% were screened as high-producing PHB candidate bacterial groups and then subjected to molecular biological identification.

6. The method for simultaneous wastewater treatment and polyβ-hydroxybutyric acid recovery based on a bacterial-algae symbiotic system according to claim 1, characterized in that, In step S3, the construction and directional acclimatization of the algae-bacterial symbiotic system includes: mixing the dominant PHB algae and bacteria in the logarithmic growth phase at a biomass dry weight ratio of 1:1 and inoculating them into simulated domestic sewage in the SBR reactor; culturing them under conditions of temperature 25-30°C, light-dark cycle of 12h:12h, and intermittent aeration, and acclimatizing them by gradually increasing the influent pollution load for 10-12 days until the system's pollutant removal efficiency stabilizes at over 95%.

7. The method for simultaneous wastewater treatment and polyβ-hydroxybutyric acid recovery based on a bacterial-algae symbiotic system according to claim 1, characterized in that, In step S4, the operating conditions are as follows: the light-dark time ratio is (24-x):x, where x ranges from 0 to 24; the hydraulic retention time is 12 to 48 hours; the anaerobic to aerobic time ratio is 1 to 11:1; the carbon-nitrogen ratio is 8 to 20:1; and the nitrogen-phosphorus ratio is 2 to 10:

1.

8. The method for simultaneous wastewater treatment and polyβ-hydroxybutyric acid recovery based on a bacterial-algae symbiotic system according to claim 1, characterized in that, In step (5), the standard curve is plotted as follows: weigh the PHB standard sample, prepare a stock solution with chloroform, and perform gradient dilution to prepare a series of working solutions of 0.0 to 5.0 mg / L. Measure the absorbance at a wavelength of 235 nm using a UV-Vis spectrophotometer. Plot the concentration on the x-axis and the absorbance on the y-axis, and fit the curve using the least squares method. R² ≥ 0.

999.

9. The method for simultaneous wastewater treatment and polyβ-hydroxybutyric acid recovery based on a bacterial-algae symbiotic system according to claim 1, characterized in that, In step (5), after the treatment is completed, all the mixture in the reactor is centrifuged at 4°C and 8000-10000 r / min for 8-15 minutes to collect the bacterial-algal symbiotic mud. The bacterial-algal symbiotic mud is freeze-dried to constant weight and then pulverized into dry powder. The PHB extraction method is chloroform-sodium hypochlorite method, specifically: the freeze-dried bacterial-algal symbiotic powder is extracted with chloroform in a 60°C water bath for 4-6 hours, the organic phase is taken after centrifugation, and the chloroform is removed by rotary evaporation to obtain crude polyβ-hydroxybutyric acid extract. The crude extract of poly-β-hydroxybutyric acid was dissolved in 98% concentrated sulfuric acid and heated in a 100°C water bath for 5–15 minutes to hydrolyze it into crotonic acid. After cooling, the absorbance was measured at a wavelength of 235 nm, and the content of poly-β-hydroxybutyric acid was calculated according to the standard curve.

10. A symbiotic system for implementing the method according to any one of claims 1-9, characterized in that, include: The dominant algal community capable of generating PHB, wherein the dominant algae of the algal community are Chlorella sorokiniana,Chlorella vulgaris, Scenedesmus obliquus and Chlamydomonas reinhardtii One or more of these, and carrying key genes that can generate PHB. phbA, phbB, phbC ; as well as: High-yield PHB bacterial communities, the core genera of which include Cupriavidus sp., Bacillus sp. and Pseudomonas sp. And the key gene that can generate PHB phbA, phbB, phbC All were positive.

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