Alcomycete symbiotic system capable of degrading microplastics and construction method and application of phycomycete symbiotic system

Abstract

The invention provides a phycomycete symbiotic system capable of degrading microplastics as well as a construction method and application of the phycomycete symbiotic system, and belongs to the technical field of biological treatment of wastewater. The method comprises the following steps: mixing an algae mixture, aerobic activated sludge and microplastics, and carrying out enrichment culture to obtain an enrichment culture; screening and separating a part of the enrichment culture to obtain at least two pure algae species, and extracting the other part of the enrichment culture to obtain a bacterial filtrate; and mixing the pure algae species, the bacterial filtrate and the microplastics for algae-bacteria co-culture to obtain the algae-bacteria symbiotic system capable of degrading the microplastics. The phycomycete symbiotic system capable of degrading the microplastics is constructed by adopting the method disclosed by the invention, so that the time cost and the economic cost for culturing and obtaining germplasm resources are greatly reduced; and the phycomycete symbiotic system can perform in-situ degradation on the micro-plastics at normal temperature, and is simple to operate and convenient to popularize and use.

Description

Technical Field

[0001] The present invention relates to the technical field of wastewater biological treatment, and particularly to an algae-bacteria symbiotic system capable of degrading microplastics, a construction method thereof, and an application thereof. Background Art

[0002] As an important category of emerging pollutants, the potential carrier role of microplastics exacerbates the enrichment of various emerging pollutants in organisms and threatens human health through the food chain. More than 95% of microplastics enter wastewater treatment plants through industrial wastewater and urban sewage, but a large amount of microplastics and even nanoplastics still remain in the effluent of traditional water treatment processes. Therefore, wastewater treatment plants are considered important sources and sinks of microplastics.

[0003] Biodegradation is a low-carbon and green sustainable plastic polymer removal technology, which can convert plastic polymers into intermediates that can be biologically assimilated and metabolically transformed through enzymatic depolymerization. However, the performance of existing plastic polymer (especially microplastic) degradation functional germplasm resources is poor and the transformation effect is very small. In addition, the general process of biodegradating microplastics needs to be carried out under high temperature conditions, which is not conducive to popularization and use. Summary of the Invention

[0004] The purpose of the present invention is to provide an algae-bacteria symbiotic system capable of degrading microplastics, a construction method thereof, and an application thereof. By using the method of the present invention to construct an algae-bacteria symbiotic system capable of degrading microplastics, the time cost and economic cost of cultivating and obtaining germplasm resources are greatly reduced; moreover, the algae-bacteria symbiotic system can in-situ degrade microplastics at room temperature, with simple operation and convenient for popularization and use.

[0005] In order to achieve the above-mentioned invention purpose, the present invention provides the following technical solutions:

[0006] The present invention provides a construction method of an algae-bacteria symbiotic system capable of degrading microplastics, comprising the following steps:

[0007] Mix an algae mixture, aerobic activated sludge and microplastics for enrichment culture to obtain an enrichment culture;

[0008] Screen and separate at least two pure algal species from a part of the enrichment culture, and extract a bacterial filtrate from another part of the enrichment culture;

[0009] Mix the pure algal species, the bacterial filtrate and microplastics for algae-bacteria co-culture to obtain the algae-bacteria symbiotic system capable of degrading microplastics.

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

[0011] Preferably, the material of the microplastics includes one or more of polyvinyl chloride, polyethylene terephthalate, polyethylene, and polystyrene; the microplastics are subjected to sterilization pretreatment before use.

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

[0013] Preferably, the conditions for enrichment culture include: the culture temperature is 26 - 27 °C; it is cultured in a periodic alternating manner of light and darkness, and the light time and darkness time in each cycle are 12 h respectively, and the light intensity is 10000 - 13000 Lux; the enrichment culture process is accompanied by oscillation, and the oscillation rate is 130 rpm.

[0014] Preferably, the preparation method of the pure algal species includes the following steps:

[0015] Filter a part of the enrichment culture with a microporous filter membrane with a pore size of 0.2 μm, collect the filtrate obtained by filtration for centrifugation separation, collect the precipitate obtained by centrifugation separation and coat it on the surface of an agar plate, and then use the streak plate method to obtain the pure algal species; the centrifugal force for centrifugation separation is 2500 g, and the centrifugation time is 10 min; the agar plate is prepared with BG-11 medium, microplastics and agar, and the content of microplastics on the agar plate is 2 - 3 mg / mL, and the content of agar is 1.5 wt%.

[0016] The preparation method of the bacterial filtrate includes the following steps:

[0017] Filter another part of the enrichment culture with a microporous filter membrane with a pore size of 0.6 μm and a microporous filter membrane with a pore size of 1 μm respectively, and collect the liquid with a size of 0.6 - 1 μm as the bacterial filtrate.

[0018] Preferably, the medium used for algal-bacterial co-culture includes BG-11 medium; the volume ratio of the BG-11 medium to the mass of the microplastics used for algal-bacterial co-culture is 25 mL: 50 - 80 mg; the volume ratio of a single type of the pure algal species, the total volume of the bacterial filtrate to the mass of the microplastics used for algal-bacterial 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: the culture temperature is 26-27°C; the culture is carried out in a periodic alternating manner of light and darkness, with the light time and darkness time being 12 h each in each cycle, and the light intensity is 10,000-13,000 Lux; during the co-culture of algae and bacteria, oscillation is accompanied, and the oscillation rate is 130 rpm.

[0020] The present invention provides an algae-bacteria symbiotic system capable of degrading microplastics constructed by the construction method described in the above technical solution.

[0021] The present invention provides the application of the algae-bacteria symbiotic system capable of degrading microplastics described in the above technical solution in degrading microplastics in sewage.

[0022] The present invention provides a construction method for an algae-bacteria symbiotic system capable of degrading microplastics, comprising the following steps: mixing an algae mixture, aerobic activated sludge and microplastics for enrichment culture to obtain an enrichment culture; screening and separating part of the enrichment culture to obtain at least two pure algal species, and extracting another part of the enrichment culture to obtain a bacterial filtrate; mixing the pure algal species, the bacterial filtrate and microplastics for co-culture of algae and bacteria to obtain the algae-bacteria symbiotic system capable of degrading microplastics. The present invention adopts a culture strategy of enriching algal bacteria with pure algal species, ensuring that the algal bacteria recruited during the enrichment of algae rely on algae-derived organic matter for survival, greatly reducing the time cost and economic cost of culturing and obtaining germplasm resources. At the same time, the algae-bacteria symbiotic system constructed by the method of the present invention has advantages such as strong environmental adaptability and high stress resistance, and can efficiently degrade microplastics in sewage. Specifically, the algae-bacteria symbiotic system in the present invention can in-situ degrade microplastics under normal temperature conditions, with simple operation and easy promotion and use. Description of the Drawings

[0023] Figure 1 It is a flow chart for constructing an algae-bacteria symbiotic system capable of degrading microplastics according to the present invention;

[0024] Figure 2 It is a microbial composition diagram of bacteria in an algae-bacteria symbiotic system capable of degrading microplastics with Desmodesmus abundans and Chlorella sorokiniana as algal species in Example 1;

[0025] Figure 3 It is an FTIR spectrum of PVC microplastics before and after degradation by the algae-bacteria symbiotic system in Example 1;

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

[0027] Figure 5 It is an SEM image of PVC microplastics in the culture medium;

[0028] Figure 6 It is the SEM image of PVC microplastics after being treated by the algal-bacterial symbiotic system in Example 1. Specific implementation manners

[0029] The present invention provides a method for constructing an algal-bacterial symbiotic system for degrading microplastics, comprising the following steps:

[0030] Mix an algal mixture, aerobic activated sludge and microplastics for enrichment culture to obtain an enrichment culture;

[0031] Screen and separate part of the enrichment culture to obtain at least two pure algal species, and extract another part of the enrichment culture to obtain a bacterial filtrate;

[0032] Mix the pure algal species, the bacterial filtrate and microplastics for algal-bacterial co-culture to obtain the algal-bacterial symbiotic system for degrading microplastics.

[0033] In the related technologies, the process of biodegradable microplastics is usually ex-situ degradation and needs to be carried out under the condition of a relatively high temperature (above 40 °C). The algal-bacterial symbiotic system constructed by the method of the present invention can degrade microplastics by in-situ degradation and can be carried out at room temperature, with simple operation and easy popularization and use. Specifically, the present invention adopts a culture strategy of enriching algal bacteria with pure algal species to ensure that the algal bacteria recruited during the enrichment process of algae live on the organic matter derived from algae, greatly reducing the time cost and economic cost of culturing and obtaining germplasm resources. At the same time, most of the existing studies on plastic polymer degrading functional strains are carried out in pure culture systems, with difficult colonization of the bacterial community and lack of functional syntrophy, and the actual microplastic degradation effect is very small. The algal-bacterial symbiotic system constructed by the method of the present invention can effectively solve the problems of lack of germplasm resources for biodegradable microplastics and high culture difficulty at present, and has advantages such as strong environmental adaptability and high stress resistance, and can efficiently degrade microplastics in sewage. The method of the present invention will be described in detail below.

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

[0035] The present invention enriches and cultures a mixture of algae, aerobic activated sludge and microplastics to obtain an enriched culture. As an embodiment of the present invention, the aerobic activated sludge can be collected from the secondary sedimentation tank of a sewage treatment plant, and the algae mixture can be collected from the wall of the secondary sedimentation tank of the same sewage treatment plant. As an embodiment of the present invention, the material of the microplastics can include one or more of polyvinyl chloride (PVC), polyethylene terephthalate, polyethylene and polystyrene, specifically polyvinyl chloride; the average particle size of the microplastics can be 10-100 μm, specifically 75 μm. As an embodiment of the present invention, the microplastics are preferably pretreated by sterilization before use. Specifically, the microplastics can be washed with an ethanol aqueous solution to achieve sterilization. The volume fraction of the ethanol aqueous solution can be 75%; the number of washing times can be 3-5 times, specifically 3 times; after washing, it can also include drying in a sterile workbench.

[0036] As an embodiment of the present invention, the culture medium used for the enrichment culture can include BG-11 medium; the mass ratio of the volume of the BG-11 medium to the mass of the microplastics used for the enrichment culture can be 25 mL: 50-80 mg, further 25 mL: 75-80 mg; the mass ratio of the volume of the algae mixture, the volume of the aerobic activated sludge to the mass of the microplastics used for the enrichment culture can be 0.5 mL: 0.5 mL: 50-80 mg, further 0.5 mL: 0.5 mL: 75-80 mg. As an embodiment of the present invention, the conditions for the enrichment culture include: the culture temperature can be 26-27 °C; it is cultured in a periodic alternating manner of light and darkness. In each cycle, the light time and the darkness time can be 12 h respectively, and the light intensity can be 10000-13000 Lux; during the enrichment culture process, oscillation is accompanied, and the oscillation rate can be 130 rpm. As an embodiment of the present invention, the number of times of the enrichment culture can be 2-5 times, specifically 2 times; the time for each enrichment culture can be 2-8 weeks, specifically 4 weeks. In the examples of the present invention, specifically, 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 the examples of the present invention, the specification of the tissue culture flask used for the enrichment culture can be 25 cm 2 , and the enrichment culture can be specifically carried out in a light incubator shaker.

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

[0038] As an embodiment of the present invention, the method for preparing the pure algal species comprises the following steps: filtering a part of the enrichment culture with a microporous filter membrane with a pore size of 0.2 μm, collecting the filtrate obtained by filtration (i.e., the liquid material with a particle size less than 0.2 μm) for centrifugal separation, collecting the precipitate obtained by centrifugal separation and coating it on the surface of an agar plate, and then obtaining the pure algal species by the streak plate method. As an embodiment of the present invention, the microporous filter membrane of the present invention can specifically be a nylon microporous filter membrane, which will not be elaborated hereinafter. As an embodiment of the present invention, the centrifugal force for the centrifugal separation can be 2500 g, and the centrifugation time can be 10 min; the agar plate is specifically prepared from a BG-11 medium, microplastics and agar, 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 the embodiments of the present invention, specifically, in a sterile workbench, a sterile pipette is used to evenly coat the precipitate on the agar plate, and the coated agar plate is inverted and placed in a 27°C light constant temperature shaker, and cultured for 3 weeks under the condition of periodic alternation of 12 h of light and 12 h of darkness. During the culture process, the shaking rate of the shaker is 130 rpm, and the light intensity during the light stage is 13,000 Lux; after the culture is completed, the streak plate method is adopted on the agar plate, and repeated separation and microscopic examination are carried out for purification until a pure algal species that can grow on the agar plate is obtained. It is an algal species that can degrade microplastics and serves as the algal host of the algal-bacterial symbiotic system. In the embodiments of the present invention, through comparison with the NCBI-NR database, it is identified that the algal species belongs to Desmodesmus_abundans and Chlorella_sorokiniana; the algal species obtained after the above separation and purification is placed in a 27°C light constant temperature shaker and cultured under the condition of periodic alternation of 12 h of light and 12 h of darkness, and the transfer period is 60 days. As an embodiment of the present invention, when the composition structure of the original algal mixture and aerobic activated sludge is relatively complex, the precipitate can be subjected to molecular sequencing, and the key algal species therein can be determined by the co-linear network analysis method. Based on the analysis results, the isolated algal species are repeatedly separated and microscopically examined for purification until a pure algal species that can grow on the agar plate is obtained.

[0039] As an embodiment of the present invention, the method for preparing the bacterial filtrate may comprise the following steps: filtering another part of the enrichment culture with a microporous filter membrane with a pore size of 0.6 μm and a microporous filter membrane with a pore size of 1 μm respectively, and collecting the liquid material with a size of 0.6 - 1 μm as the bacterial filtrate.

[0040] After obtaining the pure algal strain and the bacterial filtrate, the present invention mixes the pure algal strain, the bacterial filtrate and microplastics for co-cultivation of algae and bacteria to obtain the algae-bacteria symbiotic system capable of degrading microplastics. As an embodiment of the present invention, the culture medium used for the co-cultivation of algae and bacteria may include BG-11 medium; the volume ratio of the BG-11 medium to the mass of microplastics used for the co-cultivation of algae and bacteria may be 25 mL: 50-80 mg, specifically 25 mL: 75-80 mg; the volume of a single type of the pure algal strain, the total volume of the bacterial filtrate and the mass of microplastics used for the co-cultivation of algae and bacteria may be 0.5 mL: 0.5 mL: 50-80 mg, further 0.5 mL: 0.5 mL: 75-80 mg. As an embodiment of the present invention, taking the two algal strains of Desmodesmus abundans and Chlorella sorokiniana as the pure algal strain, the volume ratio of Desmodesmus abundans and Chlorella sorokiniana may be 1:1.

[0041] As an embodiment of the present invention, the conditions for the co-cultivation of algae and bacteria include: the culture temperature may be 26-27 °C; it is cultured in a periodic alternating manner of light and darkness, and the light time and darkness time in each cycle may be 12 h respectively, and the light intensity may be 10000-13000 Lux; oscillation is accompanied during the co-cultivation of algae and bacteria, and the oscillation rate may be 130 rpm. As an embodiment of the present invention, the number of times of the co-cultivation of algae and bacteria may be 2-5 times; specifically, in the examples of the present invention, the co-cultivation of algae and bacteria is repeated 5 times every 2 weeks.

[0042] As an embodiment of the present invention, after the co-cultivation of algae and bacteria is completed, it is preferably to perform centrifugal separation on the enriched product obtained after the co-cultivation of algae and bacteria, collect the precipitate obtained by centrifugal separation and mix it with microplastics for continuous culture to ensure that the algal bacteria recruited during the enrichment process of the algae can adapt and survive on the algae-derived organic matter within sufficient time, and finally obtain the algae-bacteria symbiotic system capable of degrading microplastics. As an embodiment of the present invention, the number of times of the centrifugal separation may be 3-5 times; the conditions for each centrifugal separation include: the centrifugal force may be 2500 g and the centrifugal time may be 10 min. As an embodiment of the present invention, the conditions for the continuous culture may specifically refer to the conditions for the co-cultivation of algae and bacteria described above, and will not be elaborated here.

[0043] The present invention provides an algal-bacterial symbiotic system capable of degrading microplastics constructed by the construction method described in the above technical solution. As an embodiment of the present invention, the algal species in the algal-bacterial symbiotic system capable of degrading microplastics of the present invention may specifically include Desmodesmus abundans and Chlorella sorokiniana. As an embodiment of the present invention, the bacteria in the algal-bacterial symbiotic system may specifically include Porphyrobacter, Rhizobiaceae, Brevundimonas, and Rhodobacteraceae. The relative abundance of Porphyrobacter in the bacteria is 40.38%, the relative abundance of Rhizobiaceae is 23.66%, the relative abundance of Brevundimonas is 6.63%, and the relative abundance of Rhodobacteraceae is 6.91%.

[0044] The present invention provides an application of the algal-bacterial symbiotic system capable of degrading microplastics described in the above technical solution in degrading microplastics in sewage. The algal-bacterial symbiotic system capable of degrading microplastics of the present invention can in-situ degrade microplastics under normal temperature conditions. As an embodiment of the present invention, the sewage may include domestic sewage or industrial sewage.

[0045] Figure 1 It is a flow chart for constructing an algal-bacterial symbiotic system capable of degrading microplastics in the present invention. The present invention finally constructs an algal-bacterial symbiotic system capable of degrading microplastics through a series of steps such as collecting an algal mixture and aerobic activated sludge from a sewage treatment plant, screening and separating algal species capable of degrading microplastics, and enriching algal bacteria.

[0046] Next, the technical solutions in the present invention will be clearly and completely described in conjunction with the embodiments in the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments in the present invention without making creative efforts belong to the scope protected by the present invention.

[0047] Example 1

[0048] In this example, the algal-bacterial symbiotic system capable of degrading microplastics is constructed specifically according to the following steps:

[0049] Step 1: Inoculation and microorganism collection

[0050] Collect aerobic activated sludge from the secondary sedimentation tank of a certain urban sewage treatment plant in Hangzhou, Zhejiang Province, and collect an algal mixture from the side wall of the secondary sedimentation tank of the same sewage treatment plant. Transport the collected aerobic activated sludge and algal mixture to the laboratory and store them at 4°C for standby;

[0051] Step 2: Enrichment culture

[0052] Take a tissue culture flask containing 25 mL of BG-11 medium (with a specification of 25 cm 2 ), inoculate 1 mL of the mixed microbial sample (obtained by mixing the algal mixture collected in Step 1 and aerobic activated sludge at a volume ratio of 1:1) and 80 mg of PVC microplastics (the average particle size of the PVC microplastics is 75 μm, and before dosing, it is sterilized and pretreated by washing 3 times with an ethanol aqueous solution with a volume fraction of 75% and dried in a sterile operating table). Then, place the tissue culture flask in a 27°C light constant temperature shaker and culture it in a cycle of 12 h of light and 12 h of darkness. During the culture process, the shaking rate of the shaker is 130 rpm, and the light intensity during the light stage is 13000 Lux. After 4 weeks of culture, take 1 mL of the obtained mixed culture and re-inoculate it into a tissue culture flask containing 25 mL of fresh BG11 medium (with a specification of 25 cm 2 ), and repeat the above steps for secondary enrichment to obtain a secondary enrichment product;

[0053] Step 3: Algal species screening and isolation

[0054] Prepare an agar plate using BG-11 medium, PVC microplastics, and agar. The agar plate contains 1.5 wt% agar and 3 mg / mL of uniformly dispersed PVC microplastics;

[0055] Filter the secondary enrichment product obtained in Step 2 using a nylon microporous filter membrane with a pore size of 0.2 μm to separate and remove prokaryotes. Centrifuge the filtered filtrate (i.e., the liquid with a particle size less than 0.2 μm) at 2500 g for 10 min, discard the centrifuged supernatant (free bacteria), and collect the centrifuged precipitate as algae and bacteria;

[0056] In a sterile workbench, use a sterile pipette to take 100 μL of the precipitate and evenly coat it on the agar plate. The coated agar plate is inverted and placed in a 27°C light constant temperature shaker and cultured for 3 weeks in a cycle of 12 h of light and 12 h of darkness. During the culture process, the shaking rate of the shaker is 130 rpm, and the light intensity during the light stage is 13000 Lux. After the culture is completed, use the streak plate method on the agar plate, and repeatedly separate and microscopically examine and purify until a pure algal species that can grow on the agar plate is obtained. It is an algal species that can degrade microplastics and serves as the algal host of the algal-bacterial symbiotic system. Through comparison with the NCBI-NR database, it is identified that this algal species belongs to the Scenedesmus genus, namely Desmodesmus_abundans and Chlorella_sorokiniana. Place the above-obtained algal species after separation and purification in a 27°C light constant temperature shaker and culture it in a cycle of 12 h of light and 12 h of darkness, and the transfer cycle is 60 days;

[0057] Step 4: Bacterial filtrate extraction

[0058] Filter the secondary enriched product obtained in step 2 using a 0.6 μm pore size nylon microporous filter membrane and a 1 μm pore size nylon microporous filter membrane to remove larger-sized algal cells and culture medium impurities, obtaining a bacterial filtrate (the size of bacteria is in the range of 0.6 - 1 μm).

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

[0060] Take a tissue culture flask containing 25 mL of BG-11 medium (specification: 25 cm 2 ), inoculate 1 mL of an algae-bacteria mixture (obtained by mixing the bacteria-free Desmodesmus abundans and Chlorella sorokiniana cultures obtained in step 3 and the bacterial filtrate obtained in step 4 in a volume ratio of 1:1:1) and 80 mg of PVC microplastics. Then place the tissue culture flask in a 27 °C light incubator shaker and conduct algae-bacteria co-culture for 4 weeks in a cycle of 12 h light and 12 h darkness. During the culture process, the shaking rate of the shaker is 130 rpm to avoid algal adhesion growth, and the light intensity during the light stage is 13000 Lux.

[0061] Step 6: Construction of an algae-bacteria symbiotic system

[0062] Centrifuge the material obtained after the algae-bacteria co-culture in step 5 at 2500 g for 10 min, collect the precipitate and filter it using a 0.6 μm pore size nylon microporous filter membrane and a 1 μm pore size nylon microporous filter membrane to obtain a bacterial filtrate (the size of bacteria is in the range of 0.6 - 1 μm). Then inoculate the bacterial filtrate (0.5 mL) into a tissue culture flask (specification: 25 cm 2 ) containing 25 mL of fresh BG-11 medium, 80 mg of PVC microplastics and the bacteria-free Desmodesmus abundans and Chlorella sorokiniana cultures (0.5 mL) obtained in step 3, and conduct algae-bacteria co-culture according to the conditions in step 5. Repeat the above algae-bacteria co-culture operation 5 times every 2 weeks (each time after algae-bacteria co-culture, centrifuge at 2500 g for 10 min and collect the precipitate for the next algae-bacteria co-culture) to ensure that the phycosphere bacteria recruited by the algae during the enrichment process can adapt and survive on the algae-derived organic matter within sufficient time.

[0063] Collect the enriched product obtained after the 5th algae-bacteria co-culture, centrifuge it 3 times at 2500 g, with each centrifugation time being 10 min. Inoculate the centrifuged precipitate (1 mL) into a tissue culture flask (specification: 25 cm 2) After that, place the tissue culture flask in a constant temperature shaker with light at 27°C and continuously culture it in a cycle of 12 hours of light and 12 hours of darkness. During the culture process, the shaking rate of the shaker is 130 rpm, and the light intensity during the light stage is 13,000 Lux; after continuous culture, an algal-bacterial symbiotic system capable of degrading microplastics with Desmodesmus_abundans and Chlorella_sorokiniana as algal species is obtained.

[0064] Test Example 1

[0065] Figure 2 It is the microbial composition map of bacteria in the algal-bacterial symbiotic system capable of degrading microplastics with Desmodesmus_abundans and Chlorella_sorokiniana as algal species in Example 1 (the relative abundances of various microorganisms were measured three times). The results show that the bacteria in the algal-bacterial symbiotic system are mainly composed of Porphyrobacter, Rhizobiaceae, Brevundimonas, and Rhodobacteraceae. The relative abundance of Porphyrobacter in the bacteria is 40.38%, the relative abundance of Rhizobiaceae is 23.66%, the relative abundance of Brevundimonas is 6.63%, and the relative abundance of Rhodobacteraceae is 6.91%.

[0066] Test Example 2

[0067] Collect the PVC microplastics in the system obtained after continuous culture in Step 6 of Example 1; at the same time, operate according to the method of continuous culture in Step 6 of Example 1, with the difference that only the PVC microplastics are cultured in BG-11 medium, and this is used as Control Group 1; in addition, the original PVC microplastics (i.e., PVC microplastics without any treatment) are used as Control Group 2.

[0068] Figure 3 It is the FTIR spectra of PVC microplastics before and after degradation by the algal-bacterial symbiotic system in Example 1, where "PVC after being treated by the algal-bacterial symbiotic system" refers to the PVC microplastics in the system obtained after continuous culture in Step 6 of Example 1; "PVC in the medium" refers to the PVC microplastics of Control Group 1; "original PVC" refers to the PVC microplastics of Control Group 2. As Figure 3 shown, in the degraded PVC microplastics, the intensity of the OH absorption peak (3300 - 3500 cm -1 ) increased significantly, the intensity of the CH2 absorption peak (~966 cm -1 ) decreased significantly, and a C-O-C stretching bond (1270 - 1010 cm -1) This indicates that hydrolysis products have appeared in the degraded PVC microplastics, while traditional biological treatment methods (such as the activated sludge method, biofilm method, etc.) can hardly degrade PVC. The degradation efficiency of PVC can be calculated as 83.8% based on the output of OH groups.

[0069] Figures 4 to 6 SEM images of PVC microplastics before and after degradation by the algal-bacterial symbiotic system in Example 1, where Figure 4 is the SEM image of the original PVC microplastics, Figure 5 is the SEM image of the PVC microplastics in the culture medium, Figure 6 is the SEM image of the PVC microplastics after treatment by the algal-bacterial symbiotic system. The surface morphology of the PVC microplastics observed by SEM can also intuitively reflect the erosion and weathering processes they have experienced in the algal-bacterial symbiotic system. For example, Figures 4 to 6 as shown, the shape of the original PVC is rough and irregular, while after treatment with the culture medium and the algal-bacterial symbiotic system, smaller-sized polymer particles have appeared on its surface, and they have a more rounded spherical shape compared to the original PVC microplastic particles. There are large holes and pores on the surface and the spheres, and even the phenomenon of sheet polymers peeling off from the surface has occurred, indicating that the algal-bacterial symbiotic system has intensified the biological deterioration and biological fragmentation of PVC microplastics.

[0070] From the above results, it can be seen that the present invention adopts a culture strategy of enriching algal bacteria with multiple pure algal species, ensuring that the algal bacteria recruited during the enrichment process rely on the organic matter derived from the algae for survival, greatly reducing the time cost and economic cost of culturing and obtaining germplasm resources. At the same time, most of the existing research on plastic polymer-degrading functional strains is carried out in pure culture systems, with difficulties in bacterial colonization and lack of functional syntrophy, and the actual microplastic degradation effect is very small. The algal-bacterial symbiotic system constructed by the method of the present invention can effectively solve the problems such as the lack of germplasm resources and high cultivation difficulty of biodegradable microplastics at present. Moreover, compared with the algal-bacterial symbiotic system of a single algal species, it has advantages such as strong environmental adaptability and high stress resistance, and is an efficient and feasible method for degrading various types of microplastics in sewage treatment plants.

[0071] The above description is only the preferred embodiment of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and refinements can be made, and these improvements and refinements should also be regarded as the protection scope of the present invention.

Claims

1. A method for constructing an algae-bacteria symbiotic system that can degrade microplastics, comprising the following steps: The algae mixture, aerobic activated sludge and microplastics are mixed for enrichment culture to obtain an enrichment culture; Screening and separating a portion of the enriched culture to obtain at least two pure algae species, and extracting another portion of the enriched culture to obtain a bacterial filtrate; The pure algae species, bacterial filtrate and microplastics are mixed for algae-bacteria co-cultivation to obtain the algae-bacteria symbiotic system capable of degrading microplastics.

2. The construction method according to claim 1, characterized in that: The aerobic activated sludge is collected from the secondary sedimentation tank of a sewage treatment plant, and the algae mixture is collected from the wall of the secondary sedimentation tank of the same sewage treatment plant.

3. The construction method according to claim 1, characterized in that: The material of the microplastic includes one or more of polyvinyl chloride, polyethylene terephthalate, polyethylene and polystyrene; the microplastic is sterilized before use.

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

5. The construction method according to claim 4, characterized in that: The conditions of the enrichment culture include: a culture temperature of 26-27° C.; culture in a periodic alternation of light and darkness, with the light time and dark time in each cycle being 12 hours respectively, and the light intensity being 10,000-13,000 Lux; and the enrichment culture process is accompanied by oscillation, with the oscillation rate being 130 rpm.

6. The construction method according to claim 1, characterized in that: The preparation method of the pure algae species comprises the following steps: Part of the enriched culture is filtered through a microporous filter membrane with a pore size of 0.2 μm, the filtrate obtained by filtration is collected and centrifuged, the precipitate obtained by centrifugation is collected and coated on the surface of an agar plate, and then the pure algae species is obtained by streaking separation; the centrifugal force of the centrifugal separation is 2500g, and the centrifugation time is 10min; the agar plate is prepared using BG-11 culture medium, microplastics and agar, the content of microplastics on the agar plate is 2-3 mg / mL, and the content of agar is 1.5wt%; The preparation method of the bacterial filtrate comprises the following steps: Another part of the enriched culture is filtered through a microporous filter membrane with a pore size of 0.6 μm and a microporous filter membrane with a pore size of 1 μm, and the feed liquid with a size of 0.6 to 1 μm is collected as the bacterial filtrate.

7. The construction method according to claim 1, 3 or 6, characterized in that: The culture medium used for algae-bacteria co-cultivation includes BG-11 culture medium; the mass ratio of the volume of the BG-11 culture medium to the microplastics used for algae-bacteria co-cultivation is 25 mL: 50-80 mg; the mass ratio of the volume of the pure algae species of a single species, the total volume of the bacterial filtrate and the microplastics used for algae-bacteria co-cultivation is 0.5 mL: 0.5 mL: 50-80 mg.

8. The construction method according to claim 7, characterized in that: The conditions for the algae-bacteria co-culture include: a culture temperature of 26-27° C.; culture in a manner of periodic alternation of light and darkness, with the light time and dark time in each cycle being 12 hours respectively, and the light intensity being 10,000-13,000 Lux; and oscillation during the algae-bacteria co-culture process, with the oscillation rate being 130 rpm.

9. The algae-bacteria symbiotic system for degradable microplastics constructed by the construction method described in any one of claims 1 to 8.

10. Application of the algae-bacteria symbiotic system capable of degrading microplastics as described in claim 9 in degrading microplastics in sewage.

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