Algal-bacterial symbiotic system capable of degrading microplastics, and construction method and application thereof

Abstract

This invention provides a biodegradable microplastic algae-bacterial symbiotic system, its construction method, and its application, belonging to the field of wastewater biological treatment technology. The invention involves enriching and culturing a mixture of algae, aerobic activated sludge, and microplastics to obtain an enriched culture. A portion of the enriched culture is screened and separated to obtain at least two pure algal strains, and another portion is extracted to obtain a bacterial filtrate. The pure algal strains, bacterial filtrate, and microplastics are then mixed and co-cultured to obtain a biodegradable microplastic algae-bacterial symbiotic system. Using the method of this invention to construct a biodegradable microplastic algae-bacterial symbiotic system significantly reduces the time and economic costs of cultivating and obtaining germplasm resources. Furthermore, the algae-bacterial symbiotic system can degrade microplastics in situ under ambient temperature conditions, is simple to operate, and is easy to promote and use.

Description

Technical Field

[0001] This invention relates to the field of wastewater biological treatment technology, and in particular to an algae-bacteria symbiotic system for biodegradable microplastics, its construction method, and its application. Background Technology

[0002] Microplastics, as an important category of emerging pollutants, have the potential to act as carriers, exacerbating the accumulation of various new pollutants in organisms and threatening human health through the food chain. More than 95% of microplastics enter wastewater treatment plants through industrial wastewater and municipal sewage. However, large amounts of microplastics, and even nanoplastics, remain in the effluent from traditional water treatment processes. Therefore, wastewater treatment plants are considered an important source and sink for microplastics.

[0003] Biodegradation is a low-carbon and green sustainable technology for removing plastic polymers. It utilizes enzymatic depolymerization to convert plastic polymers into intermediates that can be bioassimilated and metabolized. However, existing biodegradable germplasm resources for plastic polymers (especially microplastics) have poor performance and minimal conversion efficiency. In addition, most biodegradable microplastic processes require high-temperature conditions, which is not conducive to widespread application. Summary of the Invention

[0004] The purpose of this invention is to provide an algae-bacterial symbiotic system for degradable microplastics, its construction method, and its application. By using the method of this invention to construct an algae-bacterial symbiotic system for degradable microplastics, the time and economic costs of cultivating and obtaining germplasm resources are greatly reduced. Moreover, the algae-bacterial symbiotic system can degrade microplastics in situ under normal temperature conditions, and the operation is simple and easy to promote and use.

[0005] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0006] This invention provides a method for constructing an algae-bacterial symbiotic system with biodegradable microplastics, comprising the following steps:

[0007] Algae mixture, aerobic activated sludge and microplastics were mixed and enriched to obtain enriched culture;

[0008] At least two pure algal species were obtained by screening and separating a portion of the enriched culture, and bacterial filtrate was obtained by extracting another portion of the enriched culture.

[0009] The pure algae strain, bacterial filtrate, and microplastics were mixed and co-cultured to obtain the algae-bacteria symbiotic system of the biodegradable microplastics.

[0010] Preferably, the aerobic activated sludge is collected from the secondary sedimentation tank of the wastewater treatment plant, and the algae mixture is collected from the tank wall of the secondary sedimentation tank of the same wastewater treatment plant.

[0011] Preferably, the microplastic is made of one or more of polyvinyl chloride, polyethylene terephthalate, polyethylene, and polystyrene; the microplastic is sterilized before use.

[0012] Preferably, the culture medium used for enrichment culture includes BG-11 medium; the volume ratio of the BG-11 medium to the mass ratio of the microplastics used for enrichment culture is 25 mL: 50-80 mg; and the volume ratio of the algae mixture, the volume of the aerobic activated sludge, to the mass ratio of the microplastics used for enrichment culture is 0.5 mL: 0.5 mL: 50-80 mg.

[0013] Preferably, the enrichment culture conditions include: a culture temperature of 26-27°C; a culture method of periodic alternation of light and darkness, with each cycle consisting of 12 hours of light and 12 hours of darkness, and a light intensity of 10,000-13,000 Lux; and oscillation during the enrichment culture process, with an oscillation rate of 130 rpm.

[0014] Preferably, the method for preparing the pure algal strain includes the following steps:

[0015] A portion of the enriched culture was filtered through a microporous membrane with a pore size of 0.2 μm. The filtrate was collected and centrifuged. The precipitate obtained by centrifugation was coated onto the surface of an agar plate, and then the pure algae strain was obtained by streak plating. The centrifugation force was 2500 g, and the centrifugation time was 10 min. The agar plate was prepared using BG-11 medium, microplastics, and agar. The microplastic content on the agar plate was 2-3 mg / mL, and the agar content was 1.5 wt%.

[0016] The method for preparing the bacterial filtrate includes the following steps:

[0017] Another portion of the enriched culture was filtered using microporous membranes with pore sizes of 0.6 μm and 1 μm, respectively, and the liquid with a size of 0.6–1 μm was collected as the bacterial filtrate.

[0018] Preferably, the culture medium used for the algae-bacteria co-culture includes BG-11 medium; the volume ratio of the BG-11 medium to the mass ratio of the microplastics used in the algae-bacteria co-culture is 25 mL: 50-80 mg; the volume ratio of the pure algae species, the total volume of the bacterial filtrate, to the mass ratio of the microplastics used in the algae-bacteria co-culture is 0.5 mL: 0.5 mL: 50-80 mg.

[0019] Preferably, the conditions for the co-culture of algae and bacteria include: a culture temperature of 26-27°C; a culture method of alternating light and dark, with each cycle consisting of 12 hours of light and 12 hours of darkness, and a light intensity of 10,000-13,000 Lux; and oscillation during the co-culture of algae and bacteria at a rate of 130 rpm.

[0020] The present invention provides a biodegradable microplastic algae-bacteria symbiotic system constructed by the construction method described above.

[0021] This invention provides the application of the algae-bacteria symbiotic system for degrading microplastics described in the above technical solution in the degradation of microplastics in wastewater.

[0022] This invention provides a method for constructing an algae-bacterial symbiotic system for degrading microplastics, comprising the following steps: mixing algae, aerobic activated sludge, and microplastics for enrichment culture to obtain an enriched culture; screening and separating a portion of the enriched culture to obtain at least two pure algal strains; extracting a bacterial filtrate from another portion of the enriched culture; and mixing the pure algal strains, bacterial filtrate, and microplastics for algae-bacterial co-culture to obtain the algae-bacterial symbiotic system for degrading microplastics. This invention employs a cultivation strategy of enriching algal bacteria with pure algal strains, ensuring that the algal bacteria recruited during the enrichment process are sustained by algae-derived organic matter, thus significantly reducing the time and economic costs of cultivating and acquiring germplasm resources. Simultaneously, the algae-bacterial symbiotic system constructed by this invention possesses advantages such as strong environmental adaptability and high stress resistance, and can efficiently degrade microplastics in wastewater. Specifically, the algae-bacterial symbiotic system of this invention can degrade microplastics in situ under ambient temperature conditions, is simple to operate, and is easy to promote and use. Attached Figure Description

[0023] Figure 1 The flowchart illustrates the construction of the algae-bacteria symbiotic system for biodegradable microplastics according to this invention.

[0024] Figure 2 This is a diagram showing the microbial composition of bacteria in the algae-bacterial symbiotic system of biodegradable microplastics using Desmodesmus_abundans and Chlorella_sorokiniana as algae species in Example 1.

[0025] Figure 3 The images show the FTIR spectra of PVC microplastics before and after degradation by the algae-bacteria symbiotic system in Example 1.

[0026] Figure 4 SEM image of the original PVC microplastics;

[0027] Figure 5 SEM images of PVC microplastics in the culture medium;

[0028] Figure 6 This is a SEM image of PVC microplastics after being treated with the algae-bacteria symbiotic system in Example 1. Detailed Implementation

[0029] This invention provides a method for constructing an algae-bacterial symbiotic system with biodegradable microplastics, comprising the following steps:

[0030] Algae mixture, aerobic activated sludge and microplastics were mixed and enriched to obtain enriched culture;

[0031] At least two pure algal species were obtained by screening and separating a portion of the enriched culture, and bacterial filtrate was obtained by extracting another portion of the enriched culture.

[0032] The pure algae strain, bacterial filtrate, and microplastics were mixed and co-cultured to obtain the algae-bacteria symbiotic system of the biodegradable microplastics.

[0033] In related technologies, the biodegradation of microplastics is usually an ex-situ degradation process, requiring high temperatures (above 40°C). The algae-bacterial symbiotic system constructed using the method of this invention can degrade microplastics in situ at room temperature, is simple to operate, and is easy to promote and use. Specifically, this invention employs a cultivation strategy of enriching algal bacteria with pure algal strains, ensuring that the algal bacteria recruited during the enrichment process are sustained by algal-derived organic matter, significantly reducing the time and economic costs of cultivating and acquiring germplasm resources. Meanwhile, most existing studies on functional strains for degrading plastic polymers are conducted in pure culture systems, resulting in difficulties in bacterial colonization, lack of functional synergy, and minimal actual microplastic degradation effects. The algae-bacterial symbiotic system constructed by the method of this invention effectively solves the problems of insufficient germplasm resources and high cultivation difficulty in biodegradable microplastics, and possesses advantages such as strong environmental adaptability and high stress resistance, enabling efficient degradation of microplastics in wastewater. The method of this invention will be described in detail below.

[0034] In this invention, unless otherwise specified, all raw materials used are commercially available products well known to those skilled in the art or prepared using methods well known to those skilled in the art.

[0035] This invention involves enriching and cultivating a mixture of algae, aerobic activated sludge, and microplastics to obtain an enriched culture. In one embodiment, the aerobic activated sludge can be collected from the secondary sedimentation tank of a wastewater treatment plant, and the algae mixture can be collected from the tank wall of the same secondary sedimentation tank. In another embodiment, the microplastics can be made of one or more of polyvinyl chloride (PVC), polyethylene terephthalate (PET), polyethylene, and polystyrene, specifically polyvinyl chloride; the average particle size of the microplastics can be 10–100 μm, specifically 75 μm. In yet another embodiment, the microplastics are preferably pre-treated for sterilization before use, specifically by washing them with an ethanol-water solution (75% by volume); the washing can be performed 3–5 times, specifically 3 times; and the microplastics can be dried in a sterile operating table after washing.

[0036] In one embodiment of the present invention, the culture medium used for enrichment culture may include BG-11 medium; the volume ratio of the BG-11 medium to the mass ratio of the microplastics used for enrichment culture may be 25 mL: 50-80 mg, or more preferably 25 mL: 75-80 mg; the volume ratio of the algal mixture, the volume of the aerobic activated sludge, to the mass ratio of the microplastics used for enrichment culture may be 0.5 mL: 0.5 mL: 50-80 mg, or more preferably 0.5 mL: 0.5 mL: 75-80 mg. In another embodiment of the present invention, the enrichment culture conditions include: a culture temperature of 26-27°C; culture using a periodic alternation of light and dark, with each period consisting of 12 hours of light and 12 hours of darkness, and a light intensity of 10,000-13,000 Lux; and oscillation occurring during the enrichment culture process at a rate of 130 rpm. In one embodiment of the present invention, the enrichment culture can be performed 2 to 5 times, specifically 2 times; the duration of each enrichment culture can be 2 to 8 weeks, specifically 4 weeks. In this embodiment, the culture obtained from the first enrichment culture can be re-inoculated into a tissue culture flask containing fresh culture medium for the next enrichment culture. In this embodiment, the tissue culture flask used for the enrichment culture can be 25 cm². 2 The enrichment culture can be carried out in a light-illuminated, temperature-controlled shaker.

[0037] After obtaining the enriched culture, the present invention screens and separates a portion of the enriched culture to obtain at least two pure algal species, and extracts another portion of the enriched culture to obtain bacterial filtrate. These will be described separately below.

[0038] In one embodiment of the present invention, the method for preparing the pure algal strain includes the following steps: filtering a portion of the enriched culture using a microporous membrane with a pore size of 0.2 μm, collecting the filtrate (i.e., the liquid with a particle size less than 0.2 μm) and centrifuging it, collecting the precipitate obtained by centrifugation and coating it onto the surface of an agar plate, and then obtaining the pure algal strain by streak plating. In one embodiment of the present invention, the microporous membrane can specifically be a nylon microporous membrane, which will not be described further. In one embodiment of the present invention, the centrifugal force can be 2500 g, and the centrifugation time can be 10 min; the agar plate is specifically prepared using BG-11 medium, microplastics, and agar, and the content of microplastics on the agar plate can be 2-3 mg / mL, and the content of agar can be 1.5 wt%. In this embodiment of the invention, the precipitate is uniformly spread onto an agar plate using a sterile pipette in a sterile workbench. The spread agar plate is then inverted in a 27°C light-controlled shaking incubator and cultured for 3 weeks under alternating 12-hour light and 12-hour dark conditions. During the culture, the shaking speed of the incubator is 130 rpm, and the light intensity during the light phase is 13000 Lux. After the culture is completed, the agar plate is streak-strike separation method is used for repeated separation and microscopic purification until a pure algal strain that can grow on the agar plate is obtained. This pure algal strain is a biodegradable microplastic algal strain and serves as the algal host for the algal-bacterial symbiotic system. In this embodiment of the invention, the algal strain is identified as belonging to *Desmodesmus_abundans* and *Chlorella_sorokiniana* by comparison with the NCBI-NR database. The purified algal strain is then placed in a 27°C light-controlled shaking incubator and cultured under alternating 12-hour light and 12-hour dark conditions for a transfer period of 60 days. As one embodiment of the present invention, when the composition and structure of the original algal mixture and aerobic activated sludge are relatively complex, the precipitate can be molecularly sequenced, and the key algal species can be identified by collinear network analysis. Based on the analysis results, the isolated algal species are repeatedly separated, examined under a microscope and purified until a pure algal species that can grow on the agar plate is obtained.

[0039] As one embodiment of the present invention, the method for preparing the bacterial filtrate may include the following steps: filtering another portion of the enriched culture using microporous membranes with pore sizes of 0.6 μm and 1 μm, respectively, and collecting the liquid with a size of 0.6 to 1 μm as the bacterial filtrate.

[0040] After obtaining pure algal strains and bacterial filtrates, the present invention mixes the pure algal strains, bacterial filtrates, and microplastics for algal-bacterial co-culture to obtain the algal-bacterial symbiotic system with degradable microplastics. As one embodiment of the present invention, the culture medium used for the algal-bacterial co-culture may include BG-11 medium; the volume ratio of the BG-11 medium to the mass ratio of the microplastics used in the algal-bacterial co-culture may be 25 mL: 50-80 mg, specifically 25 mL: 75-80 mg; the volume ratio of the pure algal strain, the total volume of the bacterial filtrate, and the mass ratio of the microplastics used in the algal-bacterial co-culture may be 0.5 mL: 0.5 mL: 50-80 mg, further specifically 0.5 mL: 0.5 mL: 75-80 mg. As one embodiment of the present invention, taking the pure algal strains as Desmodesmus abundans and Chlorella sorokinina as examples, the volume ratio of Desmodesmus abundans and Chlorella sorokinina may be 1:1.

[0041] In one embodiment of the present invention, the conditions for the co-culture of algae and bacteria include: a culture temperature of 26–27°C; a periodic alternation of light and dark, with each cycle consisting of 12 hours of light and 12 hours of darkness, and a light intensity of 10,000–13,000 Lux; and oscillation occurring during the co-culture process at a rate of 130 rpm. In another embodiment of the present invention, the co-culture of algae and bacteria can be performed 2–5 times; specifically, in this embodiment, the co-culture is repeated 5 times every 2 weeks.

[0042] In one embodiment of the present invention, after completing the algae-bacterial co-culture, the enriched material obtained after algae-bacterial co-culture is preferably centrifuged. The precipitate obtained by centrifugation is collected and mixed with microplastics for continuous cultivation to ensure that the algal bacteria recruited during the enrichment process have sufficient time to adapt and survive on algae-derived organic matter, ultimately obtaining the algae-bacterial symbiotic system with biodegradable microplastics. In one embodiment of the present invention, the number of centrifugation separations can be 3 to 5 times; the conditions for each centrifugation separation include: a centrifugal force of 2500g and a centrifugation time of 10min. In one embodiment of the present invention, the specific conditions for continuous cultivation can refer to the aforementioned conditions for algae-bacterial co-culture, and will not be repeated here.

[0043] This invention provides an algae-bacterial symbiotic system for biodegradable microplastics constructed using the method described in the above technical solution. As one embodiment of this invention, the algae species in the algae-bacterial symbiotic system may specifically include *Desmodesmus abundans* and *Chlorella sorokinina*. As another embodiment of this invention, the bacteria in the algae-bacterial symbiotic system may specifically include *Porphyrobacter*, *Rhizobiaceae*, *Brevundimonas*, and *Rhodobacteraceae*, wherein the relative abundance of *Porphyrobacter* is 40.38%, *Rhizobiaceae* is 23.66%, *Brevundimonas* is 6.63%, and *Rhodobacteraceae* is 6.91%.

[0044] This invention provides the application of the algae-bacterial symbiotic system for degrading microplastics described above in the degradation of microplastics in wastewater. The algae-bacterial symbiotic system for degrading microplastics described in this invention can degrade microplastics in situ under ambient temperature conditions. As one embodiment of this invention, the wastewater may include domestic sewage or industrial wastewater.

[0045] Figure 1 The flowchart below shows the process of constructing an algae-bacterial symbiotic system for biodegradable microplastics according to the present invention. The present invention constructs an algae-bacterial symbiotic system for biodegradable microplastics through a series of steps, including collecting algal mixtures and aerobic activated sludge from wastewater treatment plants, screening and separating algal species that can degrade microplastics, and enriching algal bacteria.

[0046] The technical solutions of this invention will be clearly and completely described below with reference to the embodiments thereof. Obviously, the described embodiments are only a part of the embodiments of this invention, and not all of them. All other embodiments obtained by those skilled in the art based on the embodiments of this invention without creative effort are within the scope of protection of this invention.

[0047] Example 1

[0048] In this embodiment, the algae-bacteria symbiotic system for biodegradable microplastics is constructed according to the following steps:

[0049] Step 1: Inoculation and Microbial Collection

[0050] Aerobic activated sludge was collected from the secondary sedimentation tank of a sewage treatment plant in Hangzhou, Zhejiang Province. Algae mixture was collected from the side wall of the secondary sedimentation tank of the same sewage treatment plant. The collected aerobic activated sludge and algae mixture were transported to the laboratory and stored at 4°C for later use.

[0051] Step 2: Enrichment Culture

[0052] Take a tissue culture flask (25cm) containing 25mL of BG-11 medium. 2 1 mL of a mixed microbial sample (obtained by mixing algae and aerobic activated sludge collected in step 1 at a volume ratio of 1:1) was inoculated with 80 mg of PVC microplastics (the average particle size of the PVC microplastics was 75 μm, and the microplastics were pre-treated by washing three times with a 75% ethanol aqueous solution before addition and then dried in a sterile operating table). The tissue culture flask was then placed in a 27°C light-controlled shaking incubator and cultured in alternating 12-hour light and 12-hour dark cycles. The shaking speed of the incubator was 130 rpm, and the light intensity during the light phase was 13000 Lux. After 4 weeks of culture, 1 mL of the resulting mixed culture was re-inoculated into a 25 cm tissue culture flask containing 25 mL of fresh BG11 medium. 2 In the process, the above steps are repeated to perform secondary enrichment, resulting in secondary enriched products.

[0053] Step 3: Algal strain screening and separation

[0054] Agar plates were prepared using BG-11 medium, PVC microplastics, and agar. The agar plates contained 1.5 wt% agar and 3 mg / mL uniformly dispersed PVC microplastics.

[0055] The secondary enrichment obtained in step 2 was filtered through a 0.2 μm nylon microporous membrane to separate and remove prokaryotes. The filtrate (i.e., the liquid with a particle size of less than 0.2 μm) was centrifuged at 2500g for 10 min. The supernatant obtained by centrifugation (which is free bacteria) was discarded, and the precipitate obtained by centrifugation was collected as algae and bacteria.

[0056] In a sterile workbench, 100 μL of the precipitate was evenly spread onto an agar plate using a sterile pipette. The agar plate was then inverted in a 27°C light-controlled shaking incubator and cultured for 3 weeks with alternating 12-hour light and 12-hour dark cycles. During the culture, the shaking speed of the incubator was 130 rpm, and the light intensity during the light phase was 13000 Lux. After the culture, the agar plate was streak-strike separation method was used for repeated separation and microscopic purification until a pure algal species that could grow on the agar plate was obtained. This pure algal species was biodegradable and served as the algal host for the algal-bacterial symbiotic system. By comparing with the NCBI-NR database, the algal species was identified as belonging to the genus *Desmodesmus abundans* and *Chlorella sorokinina*. The purified algal species was then placed in a 27°C light-controlled shaking incubator and cultured with alternating 12-hour light and 12-hour dark cycles for 60 days.

[0057] Step 4: Bacterial filtrate extraction

[0058] The secondary enrichment obtained in step 2 was filtered through a 0.6 μm pore size nylon microporous membrane and a 1 μm pore size nylon microporous membrane to remove larger algal cells and culture medium impurities, resulting in a bacterial filtrate (bacterial size in the range of 0.6 to 1 μm).

[0059] Step 5: Algae-bacteria co-culture

[0060] Take a tissue culture flask (25cm) containing 25mL of BG-11 medium. 2 The tissue culture flask was inoculated with 1 mL of algae-bacteria mixture (obtained by mixing the bacteria-free Desmodesmus_abundans and Chlorella_sorokiniana cultures obtained in step 3 with the bacterial filtrate obtained in step 4 at a volume ratio of 1:1:1) and 80 mg of PVC microplastics. The flask was then placed in a 27°C light-controlled shaking incubator and co-cultured with algae and bacteria for 4 weeks in alternating 12-hour light and 12-hour dark cycles. During the culture, the shaking speed of the incubator was 130 rpm to prevent algae from adhering to the wall and growing. The light intensity during the light phase was 13000 Lux.

[0061] Step 6: Construction of the Algae-Fungi Symbiotic System

[0062] After the co-culture of algae and bacteria in step 5, the material was centrifuged at 2500g for 10 min. The precipitate was collected and filtered through 0.6μm and 1μm nylon microporous membranes to obtain bacterial filtrate (bacterial size in the range of 0.6-1μm). Then, the bacterial filtrate (0.5mL) was inoculated into a tissue culture flask (25cm) containing 25mL of fresh BG-11 medium, 80mg of PVC microplastics, and 0.5mL of the bacteria-free Desmodesmus abundans and Chlorella sorokiniana cultures obtained in step 3. 2 In the process, the algae-bacteria co-culture is carried out under the conditions in step 5. The above algae-bacteria co-culture operation is repeated 5 times every 2 weeks (after each algae-bacteria co-culture, the algae are centrifuged at 2500g for 10 minutes and the precipitate is collected for the next algae-bacteria co-culture) to ensure that the algal bacteria recruited during the enrichment process can adapt and live on algae-derived organic matter in a sufficient period of time.

[0063] The enriched product obtained after the 5th co-culture of algae and bacteria was collected and centrifuged three times at 2500g for 10 min each time. The precipitate (1 mL) obtained after centrifugation was inoculated into a tissue culture flask (25 cm²) containing 25 mL of fresh BG-11 medium and 80 mg of PVC microplastics. 2The tissue culture flasks were then placed in a 27°C light-controlled shaker and continuously cultured in a 12-hour light-12-hour dark cycle. During the culture, the shaking rate of the shaker was 130 rpm, and the light intensity during the light phase was 13000 Lux. After continuous culture, a biodegradable microplastic algae-bacteria symbiotic system was obtained using Desmodesmus abundans and Chlorella sorokinia as algal species.

[0064] Test Example 1

[0065] Figure 2 The diagram shows the microbial composition of bacteria in the algae-bacterial symbiotic system for biodegradable microplastics using Desmodesmus abundans and Chlorella sorokinina as algae species in Example 1 (the relative abundance of each microorganism was measured three times). The results show that the bacteria in the algae-bacterial symbiotic system are mainly composed of Porphyrobacter, Rhizobium, Brevundimonas, and Rhodobacteraceae. Among these bacteria, the relative abundance of Porphyrobacter was 40.38%, that of Rhizobium was 23.66%, that of Brevundimonas was 6.63%, and that of Rhodobacteraceae was 6.91%.

[0066] Test Example 2

[0067] Collect PVC microplastics from the system obtained after continuous culture in step 6 of Example 1; and follow the continuous culture method in step 6 of Example 1, except that only the PVC microplastics are cultured in BG-11 medium, which serves as control group 1; in addition, use the original PVC microplastics (i.e., PVC microplastics without any treatment) as control group 2.

[0068] Figure 3 The images show the FTIR spectra of PVC microplastics before and after degradation by the algae-bacterial symbiotic system in Example 1. "PVC after treatment with the algae-bacterial symbiotic system" refers to the PVC microplastics in the system obtained after continuous cultivation in step 6 of Example 1; "PVC in the culture medium" refers to the PVC microplastics in control group 1; and "original PVC" refers to the PVC microplastics in control group 2. Figure 3 As shown, in the degraded PVC microplastics, the OH absorption peak (3300–3500 cm⁻¹) is present. -1 The intensity increased significantly, and the CH2 absorption peak (~966 cm⁻¹) was observed. -1 The strength decreased significantly, and COC stretching bonds (1270–1010 cm⁻¹) appeared. -1This indicates that hydrolysis products were produced after the degradation of PVC microplastics, and traditional biological treatment methods (such as activated sludge process, biofilm process, etc.) are almost unable to degrade PVC. Based on the amount of OH groups produced, the degradation efficiency of PVC can be calculated to be 83.8%.

[0069] Figures 4-6 These are SEM images of PVC microplastics before and after degradation by the algae-bacteria symbiotic system in Example 1, wherein... Figure 4 This is a SEM image of the original PVC microplastics. Figure 5 SEM images of PVC microplastics in culture medium. Figure 6 This is a SEM image of PVC microplastics after treatment in an algae-bacterial symbiotic system. The surface morphology of the PVC microplastics observed by SEM can also visually demonstrate the erosion and weathering processes they underwent in the algae-bacterial symbiotic system, such as... Figures 4-6 As shown, the original PVC particles were rough and irregular in shape. However, after being treated with culture medium and algae-bacteria symbiotic system, smaller polymer particles were generated on their surface. Compared with the original PVC microplastic particles, they had a smoother spherical shape. Larger voids and pores appeared on the surface and on the spheres. There was even a phenomenon of sheet-like polymer peeling off from the surface, indicating that the algae-bacteria symbiotic system aggravated the biological degradation and biological fragmentation of PVC microplastics.

[0070] The results above show that the present invention employs a cultivation strategy of enriching algal bacteria with multiple pure algal species, ensuring that the algal bacteria recruited during the enrichment process sustain themselves on algal-derived organic matter, thus significantly reducing the time and economic costs of cultivating and acquiring germplasm resources. Meanwhile, most existing studies on functional strains for degrading plastic polymers are conducted in pure culture systems, resulting in difficulties in bacterial colonization, lack of functional synergy, and minimal actual microplastic degradation effects. The algal-bacterial symbiotic system constructed using the method of this invention can effectively solve the problems of insufficient germplasm resources and high cultivation difficulty for biodegradable microplastics. Compared to algal-bacterial symbiotic systems with a single algal species, it possesses advantages such as strong environmental adaptability and high stress resistance, making it a highly efficient and feasible method for degrading various types of microplastics from wastewater treatment plants.

[0071] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A method for constructing an algae-bacterial symbiotic system with biodegradable microplastics, comprising the following steps: Algae mixture, aerobic activated sludge and microplastics were mixed and enriched to obtain enriched culture; At least two pure algal species were obtained by screening and separating a portion of the enriched culture, and bacterial filtrate was obtained by extracting another portion of the enriched culture. The pure algae strain, bacterial filtrate, and microplastics were mixed and co-cultured to obtain the algae-bacteria symbiotic system with biodegradable microplastics. The aerobic activated sludge was collected from the secondary sedimentation tank of the wastewater treatment plant, and the algae mixture was collected from the tank wall of the secondary sedimentation tank of the same wastewater treatment plant. The at least two pure algae species are Desmodesmus_abundans and Chlorella_sorokiniana ; the preparation method of the pure algae species comprises the following steps: filtering part of the enrichment culture by using a microporous filter membrane with a pore size of 0.2 μm, collecting a filtrate obtained by filtering, performing centrifugal separation on the filtrate, collecting a precipitate obtained by centrifugal separation, coating the precipitate on the surface of an agar plate, and then obtaining the pure algae species by using a streak plate method; the centrifugal force of the centrifugal separation is 2500 g, and the centrifugal time is 10 min; the agar plate is prepared by using a BG-11 culture medium, microplastics and agar, the content of the microplastics on the agar plate is 2-3 mg / mL, and the content of the agar is 1.5 wt%. The bacteria in the bacterial filtrate include Porphyromonas, Rhizobium, Shortwave Monoclonal bacteria, and Rhodopsin. The preparation method of the bacterial filtrate includes the following steps: filtering another portion of the enriched culture using microporous membranes with pore sizes of 0.6 μm and 1 μm, respectively, and collecting the liquid with a size of 0.6~1 μm as the bacterial filtrate.

2. The construction method according to claim 1, characterized in that, The microplastics are made of one or more of polyvinyl chloride, polyethylene terephthalate, polyethylene, and polystyrene; the microplastics are sterilized before use.

3. The construction method according to claim 1 or 2, characterized in that, The enrichment culture medium includes BG-11 medium; the volume ratio of the BG-11 medium to the mass ratio of the microplastics used in the enrichment culture is 25 mL: 50~80 mg; the volume ratio of the algae mixture, the volume of the aerobic activated sludge, to the mass ratio of the microplastics used in the enrichment culture is 0.5 mL: 0.5 mL: 50~80 mg.

4. The construction method according to claim 3, characterized in that, The enrichment culture conditions include: a culture temperature of 26-27℃; a culture method of alternating light and dark, with each cycle consisting of 12 hours of light and 12 hours of darkness, and a light intensity of 10,000-13,000 Lux; and oscillation during the enrichment culture process at a rate of 130 rpm.

5. The construction method according to claim 1 or 2, characterized in that, The culture medium used for the algae-bacteria co-culture includes BG-11 medium; the volume ratio of BG-11 medium to the mass ratio of microplastics used in the algae-bacteria co-culture is 25 mL: 50~80 mg; the volume ratio of the pure algae species, the total volume of the bacterial filtrate, to the mass ratio of microplastics used in the algae-bacteria co-culture is 0.5 mL: 0.5 mL: 50~80 mg.

6. The construction method according to claim 5, characterized in that, The conditions for the co-culture of algae and bacteria include: a culture temperature of 26-27℃; a periodic alternation of light and dark, with each period consisting of 12 hours of light and 12 hours of darkness, and a light intensity of 10,000-13,000 Lux; and oscillation during the co-culture of algae and bacteria at a rate of 130 rpm.

7. The algae-bacterial symbiotic system of biodegradable microplastics constructed by the construction method according to any one of claims 1 to 6.

8. The application of the algae-bacteria symbiotic system for degrading microplastics as described in claim 7 in the degradation of microplastics in wastewater.

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