Modified graphene oxide immobilized microorganism composite material and preparation method and application thereof

Abstract

The application discloses a modified graphene oxide immobilized microorganism composite material and a preparation method and application thereof, and belongs to the technical field of sewage treatment. The modified graphene oxide is obtained by specific chemical modification treatment on the graphene oxide, and is modified into a material with good biocompatibility. The modified graphene oxide is used for immobilizing functional microorganisms, the activity inhibition of the functional microorganisms is overcome, the biomass of denitrifying and dephosphorizing bacteria in a treatment reactor is greatly increased, and efficient simultaneous denitrification and dephosphorization treatment of landfill leachate is realized.

Description

Technical Field

[0001] This invention belongs to the field of wastewater treatment technology, and more specifically, relates to a modified graphene oxide immobilized microbial composite material, its preparation method, and its application. Background Technology

[0002] Landfill leachate refers to a high-concentration organic wastewater formed from the moisture contained in the landfill itself, rainwater and snowmelt entering the landfill, and other water sources, after deducting the saturated water-holding capacity of the landfill and cover layer, and passing through the landfill and cover layer. Landfill leachate is characterized by high levels of organic pollutants, high nitrogen and phosphorus content, and complex composition. If discharged directly into the environment without treatment, it will cause serious environmental pollution. Traditional activated sludge processes have relatively low efficiency in treating nitrogen and phosphorus pollutants in landfill leachate due to the limited biomass of microorganisms in the reactor.

[0003] Graphene oxide is a novel carbon material with a high specific surface area and abundant oxygen-containing functional groups such as hydroxyl, epoxy, carboxyl, and carbonyl groups, making it an ideal adsorbent. Graphene oxide can be used as an adsorbent in wastewater treatment. For example, Chinese invention patent application No. 201610476174.4, filed on June 27, 2016, discloses an adsorbent composed of attapulgite and graphene oxide and its preparation method. Graphene oxide has a typical two-dimensional planar structure and a large number of oxygen-containing functional groups such as hydroxyl, carboxyl, and epoxy groups. Hydrophilic molecules can be intercalated onto the surface of graphene oxide through interlayer hydrogen bonds, ionic bonds, and covalent bonds to form a complex. This complex has advantages such as good hydrophilicity, strong adsorption capacity, low cost, easy separation from wastewater, and high material recycling efficiency, making it practically valuable in the field of water treatment.

[0004] For example, Chinese invention patent application No. 201811277203.X, filed on October 30, 2018, discloses a microbial composite preparation immobilized with graphene oxide nanocomposite material, its preparation method, and its application in coking wastewater. First, graphene oxide nanocomposite material is prepared; second, the immobilized biological composite preparation is prepared. Immobilizing Citrobacter and Pseudomonas aeruginosa using graphene oxide nanocomposite material can better remove COD from coking wastewater effluent. However, graphene oxide also has a certain inhibitory effect on the enzymatic activity of some microorganisms, such as urease and ammonia monooxygenase, thus inhibiting the activity of microorganisms and reducing their biocompatibility. Summary of the Invention

[0005] 1. The problem to be solved

[0006] To address the problems of poor treatment efficiency and the inhibitory effect of graphene oxide on microbial activity in existing wastewater treatment methods, this invention provides a modified graphene oxide-immobilized microbial composite material, its preparation method, and its application. By subjecting graphene oxide to specific chemical modification and immobilizing functional microorganisms with the modified graphene oxide, the inhibitory effect on the activity of functional microorganisms is eliminated, achieving efficient and simultaneous denitrification and phosphorus removal of landfill leachate.

[0007] 2. Technical Solution

[0008] To solve the above problems, the technical solution adopted by the present invention is as follows:

[0009] The present invention discloses a method for preparing a modified graphene oxide immobilized microbial composite material, comprising the following steps:

[0010] S10, Graphene Oxide Pretreatment: Graphene oxide, dispersant, biocompatibility modifier, and enzyme cofactor are added to water and ultrasonically treated to obtain a graphene oxide suspension; then the graphene oxide suspension is dried to obtain pretreated graphene oxide solid, denoted as GO1.

[0011] S20, Graphene Oxide Modification: The pretreated graphene oxide solid GO1 is dispersed in an organic solvent, and then methacrylic acid, butyl methacrylate and azobisisobutyronitrile are added. Polymerization and grafting are carried out under nitrogen protection to modify the graphene oxide solid GO1, and the modified graphene oxide solid is denoted as GO2.

[0012] S30. Modified graphene oxide immobilized microorganisms: Microorganisms are activated and cultured to obtain bacterial solution. Then, the bacterial solution is mixed evenly with modified graphene oxide GO2, cultured by shaking, and dried to obtain modified graphene oxide immobilized microorganism composite material.

[0013] Preferably, the dispersant is tetrasodium 1,3,6,8-pyrenetetrasulfonate, the biocompatibility modifier is a mixture of polylactic acid and polyethylene glycol, and the enzyme cofactor is magnesium ions.

[0014] Preferably, in step S10, 1–1.2 g / L of graphene oxide, 2–2.5 g / L of tetrasodium 1,3,6,8-pyrenetetrasulfonic acid, 0.5–0.7 g / L of polylactic acid, 0.1–0.15 g / L of polyethylene glycol, and 0.1–0.2 g / L of magnesium chloride are added to water and ultrasonically treated to obtain a graphene oxide suspension.

[0015] Preferably, in step S20, 200-250 mg of pretreated graphene oxide solid GO1 is dispersed in 100-150 mL of N,N-dimethylformamide, and then 140-150 μL of methacrylic acid, 770-790 μL of butyl methacrylate, and 15-18 mg of azobisisobutyronitrile are added to carry out a modification reaction.

[0016] Preferably, in step S20, the modification reaction conditions are as follows: under nitrogen protection, the reaction is carried out by stirring at 70°C for 10 hours; after the reaction is completed, the mixture is cooled to room temperature, washed with acetone, and dried at 60°C to obtain modified graphene oxide solid GO2.

[0017] Preferably, in step S30, the microorganism is a combination of *Gastrodia elata* and *Pseudomonas kunmingense*, and the volume ratio between *Gastrodia elata* and *Pseudomonas kunmingense* in the bacterial solution is 1:3 to 1:3.5.

[0018] Preferably, in step S30, the mass ratio between the modified graphene oxide GO2 and the bacterial solution is 1:8 to 1:10.

[0019] Preferably, in step S30, the viable count of *Microcystis aeruginosa* in the bacterial solution is controlled to be 10. 9 CFU / mL, viable count of Pseudomonas kunmingense was 10. 10 The bacterial culture was mixed with modified graphene oxide (GO2) at CFU / mL and cultured with shaking at 30°C for 24–36 h. After centrifugation at 4°C and 5000 rpm for 10 min, the mixture was dried to obtain the composite material.

[0020] Preferably, in step S30, the drying is freeze-drying. The freeze-drying method is as follows: pre-freezing at -45 to -50°C for 2.7 to 3.0 hours, then evacuating to a vacuum. Once the vacuum level reaches below 0.15 mbar, the next drying cycle begins, with the vacuum level set at 0.28 mbar and the temperature set at 20°C for 3.0 to 3.5 hours. After reaching this temperature, the temperature is further increased to 30°C for 7.0 to 7.5 hours. After reaching this temperature, the temperature is slowly increased to 32°C for 13 to 15 hours. The final drying temperature is 32°C, and the drying time is 20 to 22 hours, resulting in the dried modified graphene oxide immobilized microbial composite material.

[0021] The present invention discloses a modified graphene oxide immobilized microbial composite material, which is prepared by the above preparation method. The modified graphene oxide immobilized microbial composite material contains *Gnaphalium affine* and *Pseudomonas kunmingense*.

[0022] The present invention discloses the application of a modified graphene oxide immobilized microbial composite material in the simultaneous denitrification and phosphorus removal of landfill leachate. The method includes inoculating the modified graphene oxide immobilized microbial composite material into an SBR reactor at a mass ratio of 20%, filling the reactor with landfill leachate for aeration and activation; after activation, controlling the SBR reactor to cycle through aerobic and anoxic stages, repeatedly performing aerobic and anoxic treatments, controlling the pH in the SBR reactor during the reaction, and sampling to measure the changes in ammonia nitrogen, total nitrogen, and total phosphorus content in the landfill leachate before and after treatment.

[0023] Preferably, the aerobic phase is maintained for 12 hours with dissolved oxygen controlled at 3 mg / L, the hypoxic phase is maintained for 6 hours with dissolved oxygen controlled at 0.2 mg / L, and the aerobic and hypoxic treatments are repeated for 5 days.

[0024] Preferably, the pH in the SBR reactor is controlled at 7.5 to 8.2 during the reaction process.

[0025] 3. Beneficial effects

[0026] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0027] (1) A modified graphene oxide immobilized microbial composite material of the present invention, which immobilizes functional microorganisms such as Phosphorus-accumulating bacteria and Pseudomonas kunmingense, effectively achieves efficient and simultaneous denitrification and phosphorus removal of landfill leachate by utilizing the large specific surface area of ​​modified graphene oxide and the good performance of functional microorganisms.

[0028] (2) A method for preparing a modified graphene oxide immobilized microbial composite material of the present invention involves selecting a suitable compound and modifying graphene oxide to make it a material with good biocompatibility, which can be used as an immobilization carrier for functional microorganisms such as Phosphorus spp. and Pseudomonas kunmingii.

[0029] (3) The modified graphene oxide immobilized microbial composite material of the present invention can be applied to the simultaneous denitrification and phosphorus removal of landfill leachate, which can significantly increase the biomass of denitrification and phosphorus removal bacteria in the treatment reactor and improve the treatment effect. Attached Figure Description

[0030] Figure 1 This is a schematic diagram of the process flow for the application of a modified graphene oxide immobilized microbial composite material in landfill leachate treatment according to the present invention. Detailed Implementation

[0031] The present invention will be further described below with reference to specific embodiments.

[0032] like Figure 1As shown, a method for preparing a modified graphene oxide immobilized microbial composite material according to the present invention includes the following steps:

[0033] S10. 1–1.2 g / L graphene oxide, 2–2.5 g / L tetrasodium 1,3,6,8-pyrenetetrasulfonic acid, 0.5–0.7 g / L polylactic acid, 0.1–0.15 g / L polyethylene glycol, and 0.1–0.2 g / L magnesium chloride were added to water and ultrasonically treated at room temperature for 1 hour to obtain a graphene oxide suspension. The graphene oxide suspension was then dried at 55–65 °C to obtain pretreated graphene oxide solid, denoted as GO1. The copolymer formed by polylactic acid and polyethylene glycol can improve the biocompatibility of graphene oxide, and magnesium ions, as a cofactor for enzymes, can enhance the activity of subsequently immobilized microorganisms.

[0034] S20. 200-250 mg of pretreated graphene oxide solid GO1 was dispersed in 100-150 mL of N,N-dimethylformamide, and then 140-150 μL of methacrylic acid, 770-790 μL of butyl methacrylate and 15-18 mg of azobisisobutyronitrile were added. The modification reaction was carried out at 70 °C for 10 h under nitrogen protection. After the reaction was completed, the mixture was cooled to room temperature, washed with acetone, and dried at 60 °C to obtain modified graphene oxide solid, denoted as GO2.

[0035] S30. Select *Gnaphalium affine* (strain number DSM NM-1, commercially available) and activate it on DSMZ Medium 776 medium. Select *Pseudomonas kunmingense* (strain number DSM 25974, commercially available) and activate it on DSMZ Medium 1a medium. After culturing to the logarithmic growth phase, mix the two bacterial cultures at a volume ratio of 1:3 to 1:3.5, controlling the viable count of *Gnaphalium affine* to be 10⁻⁶. 9 CFU / mL, viable count of Pseudomonas kunmingense was 10. 10 CFU / mL was used to obtain a mixed bacterial solution; then, modified graphene oxide GO2 was mixed with the bacterial solution at a mass ratio of 1:8 to 1:10, and cultured with shaking at 30℃ for 24 to 36 h. After centrifugation at 4℃ and 5000 rpm for 10 min, the mixture was dried to obtain the modified graphene oxide immobilized microbial composite material.

[0036] It should be noted that in step S30, the drying is freeze-drying. The freeze-drying method is as follows: pre-freezing at -45 to -50°C for 2.7 to 3.0 hours, followed by vacuuming. Once the vacuum level reaches below 0.15 mbar, the next drying cycle begins, with the vacuum level set at 0.28 mbar and the temperature set at 20°C for 3.0 to 3.5 hours. After reaching this temperature, the temperature is further increased to 30°C for 7.0 to 7.5 hours. Then, the temperature is slowly increased to 32°C for 13 to 15 hours. The final drying temperature is 32°C, and the drying time is 20 to 22 hours. After drying, the modified graphene oxide immobilized microbial composite material is obtained. The freeze-drying method used in this invention is more conducive to maintaining the high activity of microorganisms and extending the storage time.

[0037] A modified graphene oxide immobilized microbial composite material of the present invention was inoculated into an SBR reactor at a mass ratio of 20%, and landfill leachate was filled and aerated for 12 hours, with dissolved oxygen controlled at 2 mg / L. After activation, the SBR reactor was cyclically subjected to aerobic and anoxic phases. The aerobic phase was maintained for 12 hours with dissolved oxygen controlled at 3 mg / L, and the anoxic phase was maintained for 6 hours with dissolved oxygen controlled at 0.2 mg / L. The aerobic and anoxic treatments were repeated for 5 days, and the pH in the SBR reactor was controlled at 7.5–8.2 during the reaction. Samples were taken to measure the changes in ammonia nitrogen, total nitrogen, and total phosphorus content in the landfill leachate before and after treatment.

[0038] Example 1

[0039] The preparation method of the modified graphene oxide immobilized microbial composite material of this embodiment includes the following steps:

[0040] S10. 1 g / L commercially available graphene oxide, 2 g / L tetrasodium 1,3,6,8-pyrenetetrasulfonic acid, 0.5 g / L polylactic acid, 0.1 g / L polyethylene glycol, and 0.1 g / L magnesium chloride were added to water and ultrasonically treated at room temperature for 1 h to obtain a graphene oxide suspension. The graphene oxide suspension was then dried at 55 °C to obtain pretreated graphene oxide solid GO1.

[0041] S20. 200 mg of pretreated graphene oxide solid GO1 was dispersed in 100 mL of N,N-dimethylformamide. Then, 140 μL of methacrylic acid, 770 μL of butyl methacrylate and 15 mg of azobisisobutyronitrile were added to the reaction vessel. Under nitrogen protection, the mixture was stirred at 70 °C for 10 h. After the reaction was completed, the mixture was cooled to room temperature and washed with acetone. Then, it was dried at 60 °C to obtain modified graphene oxide solid GO2.

[0042] S30. *Pseudomonas kunmingense* was activated on DSMZ Medium 776 medium, and *Pseudomonas kunmingense* was activated on DSMZ Medium 1a medium. After culturing to the logarithmic growth phase, the two bacterial cultures were mixed at a volume ratio of 1:3, controlling the viable count of *Pseudomonas kunmingense* to be 10⁻⁶. 9 CFU / mL, viable count of Pseudomonas kunmingense was 10. 10 CFU / mL was used to obtain a mixed bacterial culture; then the modified graphene oxide GO2 was mixed with the mixed bacterial culture at a mass ratio of 1:8, and cultured at 30℃ with shaking for 24h. Then, the mixture was centrifuged at 4℃ and 5000rpm for 10min to obtain the composite material.

[0043] The composite material was placed in a freeze dryer for freeze drying. The freeze drying method was as follows: pre-freezing at -45℃ for 2.7h, then evacuating the chamber until the vacuum degree reached below 0.15mbar, and then starting the next drying cycle. The chamber vacuum degree was set to 0.28mbar, the temperature was set to 20.0℃, and the heating time was 3.0h. After reaching this point, the temperature was further increased to 30℃ for 7.0h. After reaching this point, the temperature was slowly increased to 32℃ for 13h. The desorption drying temperature was 32℃, and the drying time was 20h, resulting in the modified graphene oxide immobilized microbial composite material.

[0044] The modified graphene oxide immobilized microbial composite material prepared in this embodiment was divided into two parts. One part was used for stability testing, with an initial viable count of 10⁻⁶. 8 After storage at 25°C for 3 months, the number of viable bacteria on the functional microorganisms immobilized by the modified graphene oxide was determined to be 10 CFU / g. 7 CFU / g.

[0045] Another study involved inoculating a modified graphene oxide-immobilized microbial composite material into an SBR reactor at a mass ratio of 20%, filling it with landfill leachate, and then activating it with aeration for 12 hours, controlling the dissolved oxygen at 2 mg / L. After activation, the SBR reactor was cyclically subjected to aerobic and anoxic phases. The aerobic phase was maintained for 12 hours, controlling the dissolved oxygen at 3 mg / L; the anoxic phase was maintained for 6 hours, during which nitrogen was used to purge residual oxygen in the water, controlling the dissolved oxygen at 0.2 mg / L. This aerobic and anoxic cycle was repeated for 5 consecutive days. During the reaction, the pH in the SBR reactor was maintained at 7.5, and samples were taken to measure changes in ammonia nitrogen, total nitrogen, and total phosphorus content in the water.

[0046] The influent to the landfill leachate contained 2560 mg / L of ammonia nitrogen, 3180 mg / L of total nitrogen, and 88 mg / L of total phosphorus. After treatment with the modified graphene oxide immobilized microbial composite material of this embodiment for 5 days, the ammonia nitrogen in the wastewater was 142 mg / L, the total nitrogen was 310 mg / L, and the total phosphorus was 17 mg / L. The removal rates of ammonia nitrogen, total nitrogen, and total phosphorus were 94.5%, 90.3%, and 80.7%, respectively.

[0047] Example 2

[0048] The basic content of this embodiment is the same as that of Embodiment 1, except that: the preparation method of the modified graphene oxide immobilized microbial composite material in this embodiment includes the following steps:

[0049] S10. 1.2 g / L commercially available graphene oxide, 2.5 g / L tetrasodium 1,3,6,8-pyrenetetrasulfonic acid, 0.7 g / L polylactic acid, 0.15 g / L polyethylene glycol, and 0.2 g / L magnesium chloride were added to water and ultrasonically treated at room temperature for 1 hour to obtain a graphene oxide suspension. The graphene oxide suspension was then dried at 65°C to obtain pretreated graphene oxide solid GO1.

[0050] S20. 250 mg of pretreated graphene oxide solid GO1 was dispersed in 150 mL of N,N-dimethylformamide. Then, 150 μL of methacrylic acid, 790 μL of butyl methacrylate and 18 mg of azobisisobutyronitrile were added to the reaction vessel. Under nitrogen protection, the mixture was stirred at 70 °C for 10 h. After the reaction was completed, the mixture was cooled to room temperature and washed with acetone. Then it was dried at 60 °C to obtain modified graphene oxide solid GO2.

[0051] S30. *Pseudomonas kunmingense* was activated on DSMZ Medium 776 medium, and *Pseudomonas kunmingense* was activated on DSMZ Medium 1a medium. After culturing to the logarithmic growth phase, the two bacterial cultures were mixed at a volume ratio of 1:3.5, controlling the viable count of *Pseudomonas kunmingense* to be 10⁻⁶. 9 CFU / mL, viable count of Pseudomonas kunmingense was 10. 10 CFU / mL was used to obtain a mixed bacterial culture; then the modified graphene oxide GO2 was mixed with the mixed bacterial culture at a mass ratio of 1:10, and cultured at 30℃ with shaking for 36h. Then, the mixture was centrifuged at 4℃ and 5000rpm for 10min to obtain the composite material.

[0052] The composite material was placed in a freeze dryer for freeze drying. The freeze drying method was as follows: pre-freezing at -50℃ for 3.0h, then evacuating the chamber until the vacuum degree reached below 0.15mbar, and then starting the next drying cycle. The chamber vacuum degree was set to 0.28mbar, the temperature was set to 20.0℃, and the heating time was 3.5h. After reaching this point, the temperature was further increased to 30℃ for 7.5h. After reaching this point, the temperature was slowly increased to 32℃ for 15h. The desorption drying temperature was 32℃, and the drying time was 22h, resulting in the modified graphene oxide immobilized microbial composite material.

[0053] The modified graphene oxide immobilized microbial composite material prepared in this embodiment was inoculated into an SBR reactor at a mass ratio of 20%, filled with landfill leachate, and then activated by aeration for 12 hours, with dissolved oxygen controlled at 2 mg / L. After activation, the SBR reactor was cyclically subjected to aerobic and anoxic phases. The aerobic phase was maintained for 12 hours, with dissolved oxygen controlled at 3 mg / L; the anoxic phase was maintained for 6 hours, during which residual oxygen in the water was purged with nitrogen, and dissolved oxygen was controlled at 0.2 mg / L. The aerobic and anoxic cycles were repeated for 5 consecutive days. During the reaction, the pH in the SBR reactor was controlled at 7.5, and samples were taken to measure the changes in ammonia nitrogen, total nitrogen, and total phosphorus content in the water.

[0054] The influent to the landfill leachate contained 2560 mg / L of ammonia nitrogen, 3180 mg / L of total nitrogen, and 88 mg / L of total phosphorus. After treatment with the modified graphene oxide immobilized microbial composite material of this embodiment for 5 days, the ammonia nitrogen in the wastewater was 195 mg / L, the total nitrogen was 372 mg / L, and the total phosphorus was 14 mg / L. The removal rates of ammonia nitrogen, total nitrogen, and total phosphorus were 92.4%, 88.3%, and 84.1%, respectively.

[0055] Comparative Example 1

[0056] The basic content of this comparative example is the same as that of Example 1, except that the graphene oxide was not pretreated and was not modified in the composite material preparation method of this comparative example.

[0057] This comparative example describes a method for preparing a graphene oxide-immobilized microbial composite material, comprising activating *Gnaphalium affine* on DSMZ Medium 776 medium and activating *Pseudomonas kunmingense* on DSMZ Medium 1a medium, culturing them separately to the logarithmic growth phase, and then mixing the two bacterial cultures at a volume ratio of 1:3, controlling the viable count of *Gnaphalium affine* to be 10⁻⁶. 9 CFU / mL, viable count of Pseudomonas kunmingense was 10. 10CFU / mL was used to obtain a mixed bacterial culture; then commercially available graphene oxide was mixed with the mixed bacterial culture at a mass ratio of 1:10, and cultured at 30℃ with shaking for 24h. Then, the mixture was centrifuged at 4℃ and 5000rpm for 10min to obtain the composite material.

[0058] The composite material was freeze-dried in a freeze dryer. The freeze-drying method involved pre-freezing at -45℃ for 2.7 hours, followed by evacuation of the chamber until the vacuum level reached below 0.15 mbar. The next drying cycle was then initiated with a vacuum level of 0.28 mbar and a temperature of 20.0℃ for 3.0 hours. After reaching this temperature, the temperature was further increased to 30℃ for 7.0 hours, and then slowly increased to 32℃ for 13 hours. The final drying temperature was 32℃, and the drying time was 20 hours, yielding the graphene oxide-immobilized microbial composite material.

[0059] The graphene oxide-immobilized microbial composite material prepared in this comparative example was divided into two parts. One part was used for stability testing, with an initial viable count of 10⁻⁶ bacteria. 8 After being stored at 25°C for 3 months, the number of viable bacteria on the graphene-immobilized functional microorganisms was determined to be 10 CFU / g. 4 CFU / g. Experiments showed that directly immobilizing functional microorganisms with graphene oxide was less effective than immobilizing functional microorganisms with modified graphene oxide, which was not conducive to maintaining microbial vitality.

[0060] Another method involved inoculating the graphene oxide-immobilized microbial composite material into the SBR reactor at a 20% mass ratio, filling it with landfill leachate, and then activating it with aeration for 12 hours, controlling the dissolved oxygen at 2 mg / L. After activation, the SBR reactor was cyclically subjected to aerobic and anoxic phases. The aerobic phase was maintained for 12 hours, controlling the dissolved oxygen at 3 mg / L; the anoxic phase was maintained for 6 hours, during which nitrogen was used to purge residual oxygen in the water, controlling the dissolved oxygen at 0.2 mg / L. This aerobic and anoxic cycle was repeated for 5 consecutive days. During the reaction, the pH in the SBR reactor was maintained at 7.5, and samples were taken to measure changes in ammonia nitrogen, total nitrogen, and total phosphorus content in the water.

[0061] The influent to the landfill leachate contained 2560 mg / L of ammonia nitrogen, 3180 mg / L of total nitrogen, and 88 mg / L of total phosphorus. After 5 days of treatment with the graphene oxide-immobilized microbial composite material used in this comparative example, the ammonia nitrogen, total nitrogen, and total phosphorus in the wastewater decreased to 1530 mg / L, 2538 mg / L, and 54 mg / L, with removal rates of 40.2%, 20.2%, and 38.6%, respectively. The results indicate that directly immobilizing functional microorganisms with graphene oxide negatively impacts their activity, hindering efficient nitrogen and phosphorus removal from landfill leachate.

[0062] Comparative Example 2

[0063] The basic content of this comparative example is the same as that of Example 1, except that the graphene oxide was not pretreated in the composite material preparation method of this comparative example.

[0064] The preparation method of the modified graphene oxide immobilized microbial composite material in this comparative example includes the following steps:

[0065] S10. 250 mg of commercially available graphene oxide solid was dispersed in 150 mL of N,N-dimethylformamide. Then, 150 μL of methacrylic acid, 790 μL of butyl methacrylate and 18 mg of azobisisobutyronitrile were added to the reaction vessel. Under nitrogen protection, the mixture was stirred at 70 °C for 10 h. After the reaction was completed, the mixture was cooled to room temperature and washed with acetone. Then, it was dried at 60 °C to obtain modified graphene oxide solid.

[0066] S20. *Pseudomonas kunmingense* was activated on DSMZ Medium 776 medium, and *Pseudomonas kunmingense* was activated on DSMZ Medium 1a medium. After culturing to the logarithmic growth phase, the two bacterial cultures were mixed at a volume ratio of 1:3.5, controlling the viable count of *Pseudomonas kunmingense* to be 10⁻⁶. 9 CFU / mL, viable count of Pseudomonas kunmingense was 10. 10 CFU / mL was used to obtain a mixed bacterial culture; then the modified graphene oxide was mixed with the mixed bacterial culture at a mass ratio of 1:10, and cultured at 30℃ with shaking for 36h. Then, the mixture was centrifuged at 4℃ and 5000rpm for 10min to obtain the composite material.

[0067] The composite material was placed in a freeze dryer for freeze drying. The freeze drying method was as follows: pre-freezing at -50℃ for 3.0h, then evacuating the chamber until the vacuum degree reached below 0.15mbar, and then starting the next drying cycle. The chamber vacuum degree was set to 0.28mbar, the temperature was set to 20.0℃, and the heating time was 3.5h. After reaching this point, the temperature was further increased to 30℃ for 7.5h. After reaching this point, the temperature was slowly increased to 32℃ for 15h. The desorption drying temperature was 32℃, and the drying time was 22h, resulting in a composite material with modified graphene oxide immobilized functional microorganisms.

[0068] The modified graphene oxide-immobilized functional microorganism composite material prepared in this comparative example was inoculated into an SBR reactor at a mass ratio of 20%, filled with landfill leachate, and then aerated for 12 hours, with dissolved oxygen controlled at 2 mg / L. After activation, the SBR reactor was cyclically subjected to aerobic and anoxic phases. The aerobic phase was maintained for 12 hours, with dissolved oxygen controlled at 3 mg / L; the anoxic phase was maintained for 6 hours, with nitrogen used to purge residual oxygen in the water, and dissolved oxygen controlled at 0.2 mg / L. This aerobic and anoxic cycle was repeated for 5 consecutive days. During the reaction, the pH in the SBR reactor was controlled at 7.5, and samples were taken to measure changes in ammonia nitrogen, total nitrogen, and total phosphorus content in the water.

[0069] The influent of landfill leachate contained 2560 mg / L of ammonia nitrogen, 3180 mg / L of total nitrogen, and 88 mg / L of total phosphorus. After 5 days of treatment with the modified graphene oxide immobilized microbial composite material used in this comparative example, the wastewater contained 739 mg / L of ammonia nitrogen, 1096 mg / L of total nitrogen, and 39 mg / L of total phosphorus. The removal rates of ammonia nitrogen, total nitrogen, and total phosphorus were 71.1%, 65.5%, and 55.7%, respectively.

[0070] Comparative Example 3

[0071] The basic content of this comparative example is the same as that of Example 1, except that the ratio of bacterial agent, the ratio of mixed bacterial solution to modified graphene oxide immobilized microbial composite material is different in this comparative example.

[0072] The preparation method of the modified graphene oxide immobilized microbial composite material in this comparative example includes the following steps:

[0073] S10. 1.2 g / L commercially available graphene oxide, 2.5 g / L tetrasodium 1,3,6,8-pyrenetetrasulfonic acid, 0.7 g / L polylactic acid, 0.15 g / L polyethylene glycol, and 0.2 g / L magnesium chloride were added to water and ultrasonically treated at room temperature for 1 hour to obtain a graphene oxide suspension. The graphene oxide suspension was then dried at 65°C to obtain pretreated graphene oxide solid GO1.

[0074] S20. 250 mg of pretreated graphene oxide solid GO1 was dispersed in 150 mL of N,N-dimethylformamide. Then, 150 μL of methacrylic acid, 790 μL of butyl methacrylate and 18 mg of azobisisobutyronitrile were added to the reaction vessel. Under nitrogen protection, the mixture was stirred at 70 °C for 10 h. After the reaction was completed, the mixture was cooled to room temperature and washed with acetone. Then it was dried at 60 °C to obtain modified graphene oxide solid GO2.

[0075] S30. *Pseudomonas kunmingense* was activated on DSMZ Medium 776 medium, and *Pseudomonas kunmingense* was activated on DSMZ Medium 1a medium. After culturing to the logarithmic growth phase, the two bacterial cultures were mixed at a 1:1 volume ratio, controlling the viable count of *Pseudomonas kunmingense* to be 10⁻⁶. 9 CFU / mL, viable count of Pseudomonas kunmingense was 10. 9 CFU / mL was used to obtain a mixed bacterial culture; then the modified graphene oxide GO2 was mixed with the mixed bacterial culture at a mass ratio of 1:4, and cultured at 30℃ with shaking for 36h. Then, the mixture was centrifuged at 4℃ and 5000rpm for 10min to obtain the composite material.

[0076] The composite material was placed in a freeze dryer for freeze drying. The freeze drying method was as follows: pre-freezing at -50℃ for 3.0h, then evacuating the chamber until the vacuum degree reached below 0.15mbar, and then starting the next drying cycle. The chamber vacuum degree was set to 0.28mbar, the temperature was set to 20.0℃, and the heating time was 3.5h. After reaching this point, the temperature was further increased to 30℃ for 7.5h. After reaching this point, the temperature was slowly increased to 32℃ for 15h. The desorption drying temperature was 32℃, and the drying time was 22h, resulting in the modified graphene oxide immobilized microbial composite material.

[0077] The modified graphene oxide immobilized microbial composite material prepared in this comparative example was inoculated into an SBR reactor at a mass ratio of 20%, filled with landfill leachate, and then activated by aeration for 12 hours, with dissolved oxygen controlled at 2 mg / L. After activation, the SBR reactor was cyclically subjected to aerobic and anoxic phases. The aerobic phase was maintained for 12 hours, with dissolved oxygen controlled at 3 mg / L; the anoxic phase was maintained for 6 hours, with nitrogen used to purge residual oxygen in the water, and dissolved oxygen controlled at 0.2 mg / L. The aerobic and anoxic cycles were repeated for 5 consecutive days. During the reaction, the pH in the SBR reactor was controlled at 7.5, and samples were taken to measure the changes in ammonia nitrogen, total nitrogen, and total phosphorus content in the water.

[0078] The influent of landfill leachate contained 2560 mg / L of ammonia nitrogen, 3180 mg / L of total nitrogen, and 88 mg / L of total phosphorus. After treatment with the modified graphene oxide immobilized microbial composite material of this comparative example for 5 days, the ammonia nitrogen, total nitrogen, and total phosphorus in the wastewater were reduced to 844 mg / L, 1228 mg / L, and 35 mg / L, with removal rates of 67.0%, 61.4%, and 60.2%, respectively.

[0079] Comparative Example 4

[0080] The basic content of this comparative example is the same as that of Example 1, except that the composite material in this comparative example is not freeze-dried.

[0081] The preparation method of the modified graphene oxide immobilized microbial composite material in this comparative example includes the following steps:

[0082] S10. 1 g / L commercially available graphene oxide, 2 g / L tetrasodium 1,3,6,8-pyrenetetrasulfonic acid, 0.5 g / L polylactic acid, 0.1 g / L polyethylene glycol, and 0.1 g / L magnesium chloride were added to water and ultrasonically treated at room temperature for 1 h to obtain a graphene oxide suspension. The graphene oxide suspension was then dried at 55 °C to obtain pretreated graphene oxide solid GO1.

[0083] S20. 200 mg of pretreated graphene oxide solid GO1 was dispersed in 100 mL of N,N-dimethylformamide. Then, 140 μL of methacrylic acid, 770 μL of butyl methacrylate and 15 mg of azobisisobutyronitrile were added to the reaction vessel. Under nitrogen protection, the mixture was stirred at 70 °C for 10 h. After the reaction was completed, the mixture was cooled to room temperature and washed with acetone. Then, it was dried at 60 °C to obtain modified graphene oxide solid GO2.

[0084] S30. *Pseudomonas kunmingense* was activated on DSMZ Medium 776 medium, and *Pseudomonas kunmingense* was activated on DSMZ Medium 1a medium. After culturing to the logarithmic growth phase, the two bacterial cultures were mixed at a volume ratio of 1:3, controlling the viable count of *Pseudomonas kunmingense* to be 10⁻⁶. 9 CFU / mL, viable count of Pseudomonas kunmingense was 10. 10 CFU / mL was used to obtain a mixed bacterial culture; then the modified graphene oxide GO2 was mixed with the mixed bacterial culture at a mass ratio of 1:8, and cultured at 30℃ with shaking for 24h. Then, the mixture was centrifuged at 4℃ and 5000rpm for 10min to obtain the composite material.

[0085] The composite material was subjected to stability testing, with an initial sample having a viable bacterial count of 10. 9 After storage at 25°C for 3 months, the viable bacterial count on the functional microorganisms immobilized by the modified graphene oxide was determined to be 10 CFU / mL. 3 The concentration of CFU / g was found, and visible microbial growth was observed, indicating spoilage and an off-odor. This demonstrates that without freeze-drying, the composite material is difficult to preserve stably for extended periods.

[0086] Comparative Example 5

[0087] The basic content of this comparative example is the same as that of Example 1, except that: in the composite material preparation method of this comparative example, no bacterial agent is added for immobilized microorganism treatment.

[0088] The preparation method of the modified graphene oxide in this comparative example includes the following steps:

[0089] S10. 1.2 g / L commercially available graphene oxide, 2.5 g / L tetrasodium 1,3,6,8-pyrenetetrasulfonic acid, 0.7 g / L polylactic acid, 0.15 g / L polyethylene glycol, and 0.2 g / L magnesium chloride were added to water and ultrasonically treated at room temperature for 1 hour to obtain a graphene oxide suspension. The graphene oxide suspension was then dried at 65°C to obtain pretreated graphene oxide solid GO1.

[0090] S20. 250 mg of pretreated graphene oxide solid GO1 was dispersed in 150 mL of N,N-dimethylformamide. Then, 150 μL of methacrylic acid, 790 μL of butyl methacrylate and 18 mg of azobisisobutyronitrile were added to the reaction vessel. Under nitrogen protection, the mixture was stirred at 70 °C for 10 h. After the reaction was completed, the mixture was cooled to room temperature and washed with acetone. Then, it was dried at 60 °C to obtain modified graphene oxide solid GO2.

[0091] Modified graphene oxide (GO2) was inoculated into the SBR reactor at a mass ratio of 20%, and the reactor was filled with landfill leachate. Aeration and activation were then performed for 12 hours, with dissolved oxygen controlled at 2 mg / L. After activation, the SBR reactor was cyclically subjected to aerobic and anoxic phases. The aerobic phase was maintained for 12 hours, with dissolved oxygen controlled at 3 mg / L; the anoxic phase was maintained for 6 hours, during which residual oxygen in the water was purged with nitrogen, and dissolved oxygen was controlled at 0.2 mg / L. This aerobic and anoxic cycle was repeated for 5 consecutive days. During the reaction, the pH in the SBR reactor was maintained at 7.5, and samples were taken to measure changes in ammonia nitrogen, total nitrogen, and total phosphorus content in the water.

[0092] The influent of landfill leachate contained 2560 mg / L of ammonia nitrogen, 3180 mg / L of total nitrogen, and 88 mg / L of total phosphorus. After 5 days of treatment with modified graphene oxide in this comparative example, the ammonia nitrogen, total nitrogen, and total phosphorus in the wastewater were 2212 mg / L, 2457 mg / L, and 72 mg / L, with removal rates of 13.6%, 22.7%, and 18.2%, respectively.

[0093] Comparative Example 6

[0094] The basic content of this comparative example is the same as that of Example 1, except that the application method of the composite material prepared in this comparative example in the simultaneous denitrification and phosphorus removal of landfill leachate is different.

[0095] The preparation method of the modified graphene oxide immobilized microbial composite material in this comparative example includes the following steps:

[0096] S10. 1.2 g / L commercially available graphene oxide, 2.5 g / L tetrasodium 1,3,6,8-pyrenetetrasulfonic acid, 0.7 g / L polylactic acid, 0.15 g / L polyethylene glycol, and 0.2 g / L magnesium chloride were added to water and ultrasonically treated at room temperature for 1 hour to obtain a graphene oxide suspension. The graphene oxide suspension was then dried at 65°C to obtain pretreated graphene oxide solid GO1.

[0097] S20. 250 mg of pretreated graphene oxide solid GO1 was dispersed in 150 mL of N,N-dimethylformamide. Then, 150 μL of methacrylic acid, 790 μL of butyl methacrylate and 18 mg of azobisisobutyronitrile were added to the reaction vessel. Under nitrogen protection, the mixture was stirred at 70 °C for 10 h. After the reaction was completed, the mixture was cooled to room temperature and washed with acetone. Then it was dried at 60 °C to obtain modified graphene oxide solid GO2.

[0098] S30. *Pseudomonas kunmingense* was activated on DSMZ Medium 776 medium, and *Pseudomonas kunmingense* was activated on DSMZ Medium 1a medium. After culturing to the logarithmic growth phase, the two bacterial cultures were mixed at a volume ratio of 1:3.5, controlling the viable count of *Pseudomonas kunmingense* to be 10⁻⁶. 9 CFU / mL, viable count of Pseudomonas kunmingense was 10. 10 CFU / mL was used to obtain a mixed bacterial culture; then the modified graphene oxide GO2 was mixed with the mixed bacterial culture at a mass ratio of 1:10, and cultured at 30℃ with shaking for 36h. Then, the mixture was centrifuged at 4℃ and 5000rpm for 10min to obtain the composite material.

[0099] The composite material was placed in a freeze dryer for freeze drying. The freeze drying method was as follows: pre-freezing at -50℃ for 3.0h, then evacuating the chamber until the vacuum degree reached below 0.15mbar, and then starting the next drying cycle. The chamber vacuum degree was set to 0.28mbar, the temperature was set to 20.0℃, and the heating time was 3.5h. After reaching this point, the temperature was further increased to 30℃ for 7.5h. After reaching this point, the temperature was slowly increased to 32℃ for 15h. The desorption drying temperature was 32℃, and the drying time was 22h, resulting in the modified graphene oxide immobilized microbial composite material.

[0100] The modified graphene oxide immobilized microbial composite material of this comparative example was inoculated into an SBR reactor at a mass ratio of 10%, and the reactor was filled with landfill leachate. The SBR reactor was cyclically subjected to aerobic and anoxic phases. The aerobic phase was maintained for 4 hours, with dissolved oxygen controlled at 2 mg / L; the anoxic phase was maintained for 2 hours, with nitrogen used to purge residual oxygen in the water, and dissolved oxygen controlled at 0.1 mg / L. The aerobic and anoxic cycles were repeated for 5 consecutive days. During the reaction, the pH in the SBR reactor was controlled at 7.0, and the changes in ammonia nitrogen, total nitrogen, and total phosphorus content in the water were measured.

[0101] The influent of landfill leachate contained 2560 mg / L of ammonia nitrogen, 3180 mg / L of total nitrogen, and 88 mg / L of total phosphorus. After treatment with the modified graphene oxide immobilized microbial composite material of this comparative example for 5 days, the ammonia nitrogen, total nitrogen, and total phosphorus in the wastewater were 617 mg / L, 981 mg / L, and 48 mg / L, with removal rates of 75.9%, 69.2%, and 45.5%, respectively.

[0102] The present invention and its embodiments have been described above illustratively. This description is not restrictive, and the data used is only one embodiment of the present invention. The actual combination of data is not limited to this. Therefore, if those skilled in the art are inspired by this description and, without departing from the spirit of the present invention, devise similar embodiments and examples of the technical solution without creative design, all such embodiments and examples should fall within the protection scope of the present invention.

Claims

1. A method for preparing a modified graphene oxide immobilized microbial composite material, characterized in that: Includes the following steps: S10. Graphene oxide pretreatment: Graphene oxide, dispersant, biocompatibility modifier, and enzyme cofactor are added to water and ultrasonically treated to obtain a graphene oxide suspension; then the graphene oxide suspension is dried to obtain pretreated graphene oxide solid, denoted as GO1, wherein the dispersant is tetrasodium 1,3,6,8-pyrenetetrasulfonate, the biocompatibility modifier is a mixture of polylactic acid and polyethylene glycol, and the enzyme cofactor is magnesium ions; S20, Graphene Oxide Modification: The pretreated graphene oxide solid GO1 is dispersed in an organic solvent, and then methacrylic acid, butyl methacrylate and azobisisobutyronitrile are added. Polymerization and grafting are carried out under nitrogen protection to modify the graphene oxide solid GO1, and the modified graphene oxide solid is denoted as GO2. S30. Modified graphene oxide immobilized microorganisms: Microorganisms are activated and cultured to obtain bacterial solution. Then, the bacterial solution is mixed evenly with modified graphene oxide GO2, cultured by shaking, and dried to obtain modified graphene oxide immobilized microorganism composite material.

2. The method for preparing a modified graphene oxide immobilized microbial composite material according to claim 1, characterized in that: In step S10, 1–1.2 g / L of graphene oxide, 2–2.5 g / L of tetrasodium 1,3,6,8-pyrenetetrasulfonic acid, 0.5–0.7 g / L of polylactic acid, 0.1–0.15 g / L of polyethylene glycol, and 0.1–0.2 g / L of magnesium chloride are added to water and ultrasonically treated to obtain a graphene oxide suspension.

3. The method for preparing a modified graphene oxide immobilized microbial composite material according to claim 1, characterized in that: In step S20, 200-250 mg of pretreated graphene oxide solid GO1 is dispersed in 100-150 mL of N,N-dimethylformamide, and then 140-150 μL of methacrylic acid, 770-790 μL of butyl methacrylate and 15-18 mg of azobisisobutyronitrile are added to carry out the modification reaction. or In step S20, the modification reaction conditions are as follows: under nitrogen protection, the reaction is carried out by stirring at 70°C for 10 hours; after the reaction is completed, the mixture is cooled to room temperature, washed with acetone, and dried at 60°C to obtain modified graphene oxide solid GO2.

4. The method for preparing a modified graphene oxide immobilized microbial composite material according to claim 1, characterized in that: In step S30, the microorganism is a combination of *Gastrodia elata* and *Pseudomonas kunmingense*, and the volume ratio between *Gastrodia elata* and *Pseudomonas kunmingense* in the bacterial solution is 1:3 to 1:3.

5.

5. The method for preparing a modified graphene oxide immobilized microbial composite material according to claim 1, characterized in that: In step S30, the mass ratio between the modified graphene oxide GO2 and the bacterial solution is 1:8 to 1:

10.

6. The method for preparing a modified graphene oxide immobilized microbial composite material according to claim 4, characterized in that: In step S30, the viable count of *Microcystis aeruginosa* in the bacterial solution is controlled to be 10. 9 CFU / mL, viable count of Pseudomonas kunmingense was 10. 10 The bacterial culture was mixed with modified graphene oxide (GO2) at CFU / mL and cultured with shaking at 30°C for 24–36 h. After centrifugation at 4°C and 5000 rpm for 10 min, the mixture was dried to obtain the composite material.

7. A method for preparing a modified graphene oxide immobilized microbial composite material according to any one of claims 1-6, characterized in that: In step S30, the drying is freeze-drying. The freeze-drying method is as follows: pre-freeze at -45 to -50℃ for 2.7 to 3.0 hours, then evacuate the vacuum until the vacuum degree reaches below 0.15 mbar, and then begin the next drying cycle. The vacuum degree is set to 0.28 mbar, the temperature is set to 20℃, and the heating time is 3.0 to 3.5 hours. After reaching this point, continue heating to 30℃ for 7.0 to 7.5 hours. After reaching this point, slowly heat up to 32℃ for 13 to 15 hours. The final drying temperature is 32℃, and the drying time is 20 to 22 hours, resulting in the dried modified graphene oxide immobilized microbial composite material.

8. A modified graphene oxide immobilized microbial composite material, characterized in that: The modified graphene oxide immobilized microbial composite material, prepared by any one of claims 1-7, comprises *Gnaphalium affine* and *Pseudomonas kunmingense*.

9. The application of a modified graphene oxide immobilized microbial composite material in the simultaneous denitrification and phosphorus removal of landfill leachate, characterized in that: The modified graphene oxide immobilized microbial composite material of claim 8 was inoculated into the SBR reactor at a mass ratio of 20%, and the landfill leachate was filled for aeration and activation. After activation, the SBR reactor was controlled to cycle through aerobic and anoxic stages, and aerobic and anoxic treatments were repeatedly performed. The pH in the SBR reactor was controlled during the reaction process, and the changes in ammonia nitrogen, total nitrogen, and total phosphorus content in the landfill leachate before and after treatment were measured.

10. The application of the modified graphene oxide immobilized microbial composite material according to claim 9 in the simultaneous denitrification and phosphorus removal of landfill leachate, characterized in that: The aerobic phase was maintained for 12 hours, with dissolved oxygen controlled at 3 mg / L; the hypoxic phase was maintained for 6 hours, with dissolved oxygen controlled at 0.2 mg / L; and this aerobic and hypoxic treatment was repeated for 5 days; or During the reaction, the pH in the SBR reactor was controlled at 7.5–8.2.

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