A high-nitrogen wastewater treatment method based on a composite microbial agent

Abstract

The present application relates to wastewater treatment technical field, disclose a kind of high-nitrogen wastewater treatment method based on composite microbial inoculum, steps include: using composite microbial inoculum to carry out biochemical treatment to high-nitrogen wastewater containing diafenthi-conazole and / or fenbuconazole;The composite microbial inoculum includes polyhydroxyalkanoate and polyvinylpyrrolidone, and the preservation number is DSM248, DSM158 and ATCC11166 respectively of mud green bacteria, sphaerophorus wax color bacillus and capsular red bacteria;Nitration and denitrification treatment is carried out to wastewater after above-mentioned treatment.The present application uses special composite microbial inoculum to pretreat wastewater, can effectively reduce the content of diafenthi-conazole and fenbuconazole in wastewater, compared with chemical method pretreatment, can reduce energy consumption and simplify operation.

Description

Technical Field

[0001] This invention relates to the field of wastewater treatment technology, and in particular to a method for treating high-nitrogen wastewater based on a compound bacterial agent. Background Technology

[0002] Difenoconazole, a triazole fungicide, is widely used in the cultivation of crops and horticultural plants to control various diseases such as brown spot, powdery mildew, leaf blight, black spot, stripe rust, and Fusarium head blight. Benzocyclamide, a benzamide protective fungicide, inhibits sclerotium division by binding to the β-subunit of tubulin and disrupting the microtubule cytoskeleton; it is mainly used in agriculture to control oomycete diseases. Both difenoconazole and benzocyclamide are highly stable and difficult to bioavailable, resulting in slow natural degradation in the environment. Once introduced into the environment, they tend to remain for extended periods, potentially impacting health when they enter the food chain and reach animals and humans. Therefore, wastewater containing difenoconazole and benzocyclamide requires the removal of these two pollutants before discharge.

[0003] Due to the inhibitory effect of difenoconazole and benzoyl amide on microorganisms, wastewater containing these two pollutants is difficult to treat directly with microorganisms. There are very few microorganisms that can tolerate and effectively degrade difenoconazole and benzoyl amide. Existing technologies all require chemical pretreatment to reduce the content of difenoconazole and benzoyl amide in the wastewater before using microorganisms for biochemical treatment. For example, in the literature "Treatment of Fungicide Production Wastewater by Micro-electrolysis, Fenton Oxidation and Biochemical Combination Process" (Liu Ruiqian, Li Tianyi, Liu Siqi, et al. Treatment of Fungicide Production Wastewater by Micro-electrolysis, Fenton Oxidation and Biochemical Combination Process [J]. Modern Pesticides, 2018, 17(03):22-26.), the wastewater was pretreated by iron-carbon micro-electrolysis and Fenton oxidation, and then treated by two-stage anaerobic-aerobic treatment and denitrification / nitrification using microorganisms to remove difenoconazole from the wastewater; in the literature "Treatment of Difenoconazole Wastewater by Electro-Fenton and Electrochemical Oxidation Combination Process" (Shao Xingyu, Zhu Wei, He Bin, et al. Treatment of Difenoconazole Wastewater by Electro-Fenton and Electrochemical Oxidation Combination Process [J]. Industrial Water Treatment, 2020, 40(06):56-59.), a pretreatment method using a coupling of an electrocatalytic Fenton oxidation reactor and an electrochemical tubular reactor was adopted. These biochemical pretreatment processes are energy-intensive and relatively complex to operate. Summary of the Invention

[0004] To address the aforementioned technical problems—namely, the existing wastewater treatment methods for wastewater containing difenoconazole and / or benzyladenine require chemical pretreatment followed by biochemical treatment, resulting in high energy consumption and complex pretreatment operations—this invention provides a method for treating high-nitrogen wastewater based on a compound microbial agent. This invention uses a special compound microbial agent to pretreat wastewater, effectively reducing the content of difenoconazole and benzyladenine in the wastewater, and lowering energy consumption and simplifying operation compared to chemical pretreatment methods.

[0005] The specific technical solution of this invention is as follows: In a first aspect, the present invention provides a method for treating high-nitrogen wastewater based on a compound bacterial agent, comprising the following steps: S1: A compound bacterial agent is used to biochemically treat high-nitrogen wastewater containing difenoconazole and / or benzybacterium; the compound bacterial agent includes polyhydroxyalkanoates and polyvinylpyrrolidone, as well as *Chlorobium limicola*, *Cereibacter phaeroides*, and *Rhodobacter capsulatus* with accession numbers DSM248, DSM158, and ATCC11166, respectively. S2: Nitrification and denitrification are performed on the wastewater after S1 treatment.

[0006] This invention has found that three single bacteria, namely, *Sphaerophyte DSM248*, *Sphaerocephalosporium cereus* DSM158, and *Rhodops capsulatum* ATCC11166, are difficult to effectively degrade difenoconazole and benzoylmide. However, in the presence of polyhydroxyalkanoates and polyvinylpyrrolidone, these three strains can produce a synergistic effect, achieving effective degradation of difenoconazole and benzoylmide.

[0007] This invention employs a composite bacterial agent containing the above three bacterial strains, polyhydroxyalkanoates, and polyvinylpyrrolidone for biochemical treatment of wastewater. This reduces the content of difenoconazole and benzoylmethane in the wastewater, thereby decreasing their inhibitory effect on nitrifying and denitrifying bacteria. This allows for the effective removal of COD, ammonia nitrogen, and total nitrogen from the wastewater through conventional nitrification and denitrification processes. Through this method, the invention eliminates the need for chemical methods such as iron-carbon microelectrolysis or electro-Fenton oxidation, achieving effective treatment of high-nitrogen wastewater containing difenoconazole and / or benzoylmethane, reducing energy consumption and simplifying wastewater treatment operations.

[0008] As an optional implementation, in step S1, the composite microbial agent contains 5-10 wt% polyhydroxyalkanoates, 0.3-0.7 wt% polyvinylpyrrolidone, and a total content of 2.5-3.5 × 10⁻⁶ spores of *Sphaerophyte*, *Cercospora cereus*, and *Rhodophyte capsulatum*. 11The ratio of viable CFU / g of *Sphaerophyte*, *C. cereus*, and *Rhodotorula capsulatum* is 0.25–0.45:0.40–0.65:1; the polyvinylpyrrolidone includes poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV).

[0009] As an optional implementation, in step S1, the high-nitrogen wastewater is a mixture of difenoconazole wastewater, benzyl methamidophos wastewater, and blending water after pretreatment; the pretreatment process includes coagulation flotation and acidification demulsification; the blending water includes one or more of the following: air scrubbing tower wastewater, ground washing wastewater, equipment washing wastewater, circulating water drainage, initial rainwater, and domestic sewage.

[0010] As an optional implementation, in step S1, the total nitrogen content in the high-nitrogen wastewater is 200~750 mg / L, the total content of difenoconazole is 10~100 mg / L, and the content of benzybendazole is 10~100 mg / L.

[0011] As an optional implementation, in step S1, before carrying out the biochemical treatment, the pH of the high-nitrogen wastewater is adjusted to 6.8~7.2.

[0012] As an optional implementation, in step S1, during the biochemical treatment, the illuminance is 2000~3000 lux, the water temperature is 25~30℃, and the dissolved oxygen concentration is 0.1~0.2 mg / L.

[0013] As an optional implementation, step S1 includes the preparation step of the compound microbial agent: S1.1: The three bacteria were cultured in large-scale culture to obtain bacterial solutions. S1.2: After inoculating the bacterial solutions of the three bacteria into a mixed culture medium for mixed culture, the bacterial sludge is collected by centrifugation, a freeze-drying protectant solution is added to the bacterial sludge, and the mixture is freeze-dried to obtain freeze-dried bacterial powder. S1.3: Mix freeze-dried bacterial powder, polyhydroxy fatty acid ester and polyvinylpyrrolidone to obtain a compound bacterial agent.

[0014] As an optional implementation, in step S2, the nitrification and denitrification process includes a two-stage anoxic-aerobic treatment.

[0015] As an optional implementation, the method further includes the following steps: S3: Add coagulant and coagulant aid to the wastewater after S2 treatment to carry out coagulation and sedimentation.

[0016] Secondly, the present invention provides the application of a compound microbial agent in the degradation of difenoconazole and / or benzybendazole, wherein the compound microbial agent is the compound microbial agent in the method described above.

[0017] Compared with the prior art, the present invention has the following advantages: (1) In the composite microbial agent used in this invention, in the presence of polyhydroxy fatty acid ester and polyvinylpyrrolidone, the synergistic effect between DSM248, DSM158, and ATCC11166 of *Rhodotorula capsulatum* can achieve effective degradation of difenoconazole and benzybacterium. This is the first time that the above three species have been found to have a synergistic effect in degrading difenoconazole and benzybacterium.

[0018] (2) The present invention uses a special bacterial agent to replace the biochemical pretreatment process in the existing difenoconazole and benzylamine wastewater treatment, which can reduce the energy consumption of wastewater treatment and make the operation simpler. Detailed Implementation

[0019] The present invention will be further described below with reference to embodiments.

[0020] First, the present invention relates to a method for treating high-nitrogen wastewater based on a compound bacterial agent, the steps of which include: S1: A compound bacterial agent is used to biochemically treat high-nitrogen wastewater containing difenoconazole and / or benzybacterium; the compound bacterial agent includes polyhydroxyalkanoates and polyvinylpyrrolidone, as well as *Chlorobium limicola*, *Cereibacter phaeroides*, and *Rhodobacter capsulatus* with accession numbers DSM248, DSM158, and ATCC11166, respectively. S2: Nitrification and denitrification are performed on the wastewater after S1 treatment.

[0021] In some specific embodiments, in step S1, the content of polyhydroxyalkanoate in the compound microbial agent is 5-10 wt%, and the content of polyvinylpyrrolidone is 0.3-0.7 wt%.

[0022] In some specific embodiments, in step S1, the total content of *Clostridium perfringens*, *Cercospora cereus*, and *Rhodotorula capsulatum* in the compound microbial agent is 2.5~3.5×10⁻⁶. 11 The CFU / g ratio of viable counts of *Sphaerophyte*, *C. cereus*, and *Rhodops capsulatum* was 0.25–0.45:0.40–0.65:1.

[0023] In some specific embodiments, in step S1, the polyvinylpyrrolidone comprises poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV).

[0024] In some specific embodiments, in step S1, the high-nitrogen wastewater is a mixture of difenoconazole wastewater, benzyl methamidophos wastewater, and blending water after pretreatment; the pretreatment process includes coagulation flotation and acidification demulsification; the blending water includes one or more of the following: air scrubbing tower wastewater, ground washing wastewater, equipment washing wastewater, circulating water drainage, initial rainwater, and domestic sewage.

[0025] In some specific embodiments, in step S1, the total nitrogen content in the high-nitrogen wastewater is 200~750 mg / L, the total content of difenoconazole is 10~100 mg / L, and the content of benzybendazole is 10~100 mg / L.

[0026] In some specific embodiments, in step S1, before carrying out the biochemical treatment, the pH of the high-nitrogen wastewater is adjusted to 6.8~7.2.

[0027] In some specific embodiments, during step S1, the illumination is 2000~3000 lux, the water temperature is 25~30℃, and the dissolved oxygen concentration is 0.1~0.2 mg / L.

[0028] In some specific embodiments, step S1, the preparation step of the compound microbial agent includes: S1.1: The three bacteria were cultured in large-scale culture to obtain bacterial solutions. S1.2: After inoculating the bacterial solutions of the three bacteria into a mixed culture medium for mixed culture, the bacterial sludge is collected by centrifugation, a freeze-drying protectant solution is added to the bacterial sludge, and the mixture is freeze-dried to obtain freeze-dried bacterial powder. S1.3: Mix freeze-dried bacterial powder, polyhydroxy fatty acid ester and polyvinylpyrrolidone to obtain a compound bacterial agent.

[0029] In some specific embodiments, step S1.1, the process of expanding culture includes: inoculating the strain onto a solid plate culture medium, inverting it for culture until a single colony grows, picking a single colony and inoculating it into a liquid culture medium, and culturing it to the logarithmic growth phase.

[0030] In some specific embodiments, in step S1.1, based on an added water volume of 1000 mL, the culture medium raw materials used in the expanded culture of *Sphaerophyte sphaeroides* and *Cereus cereus* include: yeast extract 0.2-0.5 g, disodium succinate 0.5-1.5 g, ammonium acetate 0.4-0.6 g, 0.05-0.2 wt% ferric citrate solution 0-10 mL, KH₂PO₄ 0.4-0.6 g, MgSO₄·7H₂O 0.2-0.5 g, NaCl 0.2-0.5 g, NH₄Cl 0.2-0.5 g, CaCl₂·2H₂O 0-0.1 g, and 0.01-0.02 wt% vitamin B. 12 The solution contains 0.2-0.5 mL of liquid, 0-1.00 mL of trace element solution, and 0.3-0.5 g of L-cysteine ​​hydrochloride. The trace element solution contains one or more of ZnSO4, MnCl2, H3BO3, CoCl2, CuCl2, NiCl2, and Na2MoO4.

[0031] In some specific embodiments, in step S1.1, based on an added water volume of 1000 mL, the culture medium raw materials used in the expanded culture of capsular red bacteria include: 8-12 g of yeast extract, 0.5-1.5 g of K2HPO4, 0.5-1.5 g of MgSO4·7H2O, and 0.3-0.5 g of L-cysteine ​​hydrochloride.

[0032] In some specific embodiments, in step S1.2, based on an added water volume of 1000 mL, the raw materials of the mixed culture medium include: 8-12 g of peptone, 4-6 g of yeast extract, 0.5-1.5 g of glucose, 0.5-1.5 g of K2HPO4, 0.5-1.5 g of MgSO4·7H2O, 0.1-0.3 g of NaCl, and 0.3-0.5 g of L-cysteine ​​hydrochloride.

[0033] In some specific embodiments, in step S1.2, based on an added water volume of 1000 mL, the raw materials of the freeze-drying protectant solution include: 9-15 g of sucrose, 15-20 g of skim milk powder, 2.5-3.5 g of tetrahydropyrimidine, and 1-2 g of betaine.

[0034] In some specific embodiments, in step S1.2, the volume ratio of the bacterial sludge to the freeze-drying protectant solution is 1:0.7~1.5.

[0035] In some specific embodiments, step S2 includes a two-stage anoxic-aerobic treatment process.

[0036] In some specific embodiments, the method further includes the following steps: S3: Add coagulant and coagulant aid to the wastewater after S2 treatment to carry out coagulation and sedimentation.

[0037] Second, the present invention relates to the application of a compound microbial agent in the degradation of difenoconazole and / or benzybendazole, wherein the compound microbial agent is the compound microbial agent in the method described above.

[0038] The present invention will now be described with reference to specific embodiments and comparative examples. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention.

[0039] Example 1: Preparation of compound microbial agents and single microbial agents The formulation of liquid culture medium I is as follows: yeast extract 0.30g, disodium succinate 1.00g, ammonium acetate 0.50g, 0.1wt% ferric citrate solution 5mL, KH2PO4 0.50g, MgSO4·7H2O 0.40g, NaCl 0.40g, NH4Cl 0.40g, CaCl2·2H2O 0.05g, 0.01wt% vitamin B 12 The solution contains 0.40 mL of liquid medium, 1.00 mL of trace element solution, 0.30 g of L-cysteine ​​hydrochloride, and 1000 mL of distilled water. The trace element solution is formulated as follows: ZnSO4·7H2O 0.10 g, MnCl2·4H2O 0.03 g, H3BO3 0.30 g, CoCl2·6H2O 0.20 g, CuCl2·2H2O 0.01 g, NiCl2·6H2O 0.02 g, Na2MoO4·2H2O 0.03 g, and 1000 mL of distilled water. The pH of liquid culture medium I is 6.8, and it is autoclaved at 121℃ for 15 min.

[0040] The formulation of solid plate culture medium I is as follows: yeast extract 0.30g, disodium succinate 1.00g, ammonium acetate 0.50g, 0.1wt% ferric citrate solution 5mL, KH2PO4 0.50g, MgSO4·7H2O 0.40g, NaCl 0.40g, NH4Cl 0.40g, CaCl2·2H2O 0.05g, 0.01wt% vitamin B 12The medium contains 0.40 mL of solution, 1.00 mL of trace element solution, 0.30 g of L-cysteine ​​hydrochloride, and 1000 mL of distilled water. The trace element solution is formulated as follows: ZnSO4·7H2O 0.10 g, MnCl2·4H2O 0.03 g, H3BO3 0.30 g, CoCl2·6H2O 0.20 g, CuCl2·2H2O 0.01 g, NiCl2·6H2O 0.02 g, Na2MoO4·2H2O 0.03 g, 20.0 g of agar, and 1000 mL of distilled water. The pH of the solid plate culture medium I is 6.8, and it is autoclaved at 121℃ for 15 min.

[0041] The formulation of liquid culture medium II is as follows: yeast extract 10.0g, K2HPO4 1.0g, MgSO4·7H2O 1.0g, L-cysteine ​​hydrochloride 0.3g, and distilled water 1000mL. The pH of liquid culture medium II is 7.0, and it is autoclaved at 121℃ for 15min.

[0042] The formulation of Solid Plate Culture Medium II is as follows: yeast extract 10.0 g, K2HPO4 1.0 g, MgSO4·7H2O 1.0 g, L-cysteine ​​hydrochloride 0.3 g, agar 20.0 g, and distilled water 1000 mL. The pH of Solid Plate Culture Medium II is 7.0, and it is autoclaved at 121℃ for 15 min.

[0043] The formulation of Liquid Culture Medium III is as follows: 10.0 g peptone, 5.0 g yeast extract, 1.0 g glucose, 1.0 g K₂HPO₄, 1.0 g MgSO₄·7H₂O, 0.2 g NaCl, 0.3 g L-cysteine ​​hydrochloride, and 1000 mL distilled water. The pH of Liquid Culture Medium III is 7.0, and it is autoclaved at 121°C for 15 min.

[0044] The freeze-drying protectant solution is formulated as follows: 10.0g sucrose, 15.0g skim milk powder, 3.0g tetrahydropyrimidine, 1.5g betaine, and 1000mL distilled water. Autoclave at 115℃ for 20min.

[0045] Chlorobium limicola DSM248 (purchased from German Collection of Microorganisms and Cell Cultures GmbH) was inoculated into solid agar medium I and cultured upside down under anaerobic conditions at 25°C and tungsten lamp irradiation (3000 lux). After single colonies grew, single colonies were picked and inoculated into liquid agar medium I and cultured in a shaker at 25°C under anaerobic conditions and tungsten lamp irradiation (3000 lux) until the logarithmic growth phase was reached to obtain DSM248 bacterial suspension.

[0046] Cereibacter sphaeroides DSM158 (purchased from German Collection of Microorganisms and Cell Cultures GmbH) was inoculated into solid agar medium I and cultured upside down under anaerobic conditions at 28°C and tungsten lamp irradiation (3000 lux). After single colonies grew, single colonies were picked and inoculated into liquid agar medium I and cultured in a shaker at 28°C under anaerobic conditions and tungsten lamp irradiation (3000 lux) until the logarithmic growth phase was reached to obtain DSM158 bacterial suspension.

[0047] Rhodobacter capsulatus ATCC11166 (purchased from American Type Culture Collection) was inoculated into solid agar medium II and cultured upside down under anaerobic conditions at 30°C and tungsten lamp irradiation (3000 lux). After single colonies grew, single colonies were picked and inoculated into liquid agar medium II and cultured in a shaker at 30°C under anaerobic conditions and tungsten lamp irradiation (3000 lux) until the logarithmic growth phase was reached, thus obtaining ATCC11166 bacterial suspension.

[0048] DSM248, DSM158, and ATCC11166 bacterial suspensions were inoculated into liquid medium III and cultured together at 28°C under anaerobic conditions and tungsten lamp irradiation (3000 lux) until the total viable count reached 1.0 × 10⁻⁶. 9 Collect the bacterial sludge by centrifugation if the concentration is above CFU / mL. Add an equal volume of lyophilization protectant solution to the bacterial sludge, mix well, and then freeze-dry to obtain lyophilized bacterial powder. Mix the lyophilized bacterial powder, poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV), and polyvinylpyrrolidone (PVP) at a mass ratio of 10:1:0.06 to obtain a compound bacterial agent. Compound bacterial agents CM1 and CM2 were prepared according to the above method. The bacterial content of the two compound bacterial agents is as follows: Compound bacterial agent CM1 contains 7.6 × 10⁻⁶ DSM248, a type of *Cladosporium spp.*10 CFU / g, *Cyclocarya cereus* DSM158 7.2 × 10⁻⁶ 9 CFU / g, Rhodopseudomonas capsulatum ATCC11166 1.7×10 11 CFU / g; Compound microbial agent CM2 contains 4.5 × 10⁻⁶ DSM248 *Dendrobium nobile*. 10 CFU / g, *Cercospora cereus* DSM158 1.0×10⁻⁶ 11 CFU / g, Rhodopseudomonas capsulatum ATCC11166 1.6×10 11 CFU / g.

[0049] DSM248, DSM158, and ATCC11166 bacterial suspensions were inoculated into liquid medium III and cultured together at 28°C under anaerobic conditions and tungsten lamp irradiation (3000 lux) until the total viable count reached 1.0 × 10⁻⁶. 9 Collect the bacterial sludge by centrifugation if the concentration is above CFU / mL. Add an equal volume of freeze-drying protectant solution to the sludge, mix well, and then freeze-dry to obtain the compound bacterial agent CM3, in which the content of DSM248 (a type of *Cladophora stylosa*) is 1.0 × 10⁻⁶. 11 The CFU / g content of *C. cereus* DSM158 was 1.4 × 10⁻⁶. 11 The CFU / g content of capsular rhodopsin ATCC11166 was 1.2 × 10⁻⁶. 11 CFU / g.

[0050] DSM248 bacterial culture was inoculated into liquid medium III and cultured under anaerobic conditions at 28°C with tungsten lamp irradiation (3000 lux) until the viable count reached 1.0 × 10⁻⁶. 9 Collect the mycelial sludge by centrifugation if the concentration is above CFU / mL. Add an equal volume of lyophilization protectant solution to the sludge, mix well, and then freeze-dry to obtain lyophilized mycelial powder. Mix the lyophilized mycelial powder, PHBV, and PVP at a mass ratio of 10:1:0.06 to obtain a DSM248 content of 3.1 × 10⁻⁶. 11 Single-strain inoculum DSM248 with CFU / g.

[0051] DSM158 bacterial culture was inoculated into liquid medium III and cultured under anaerobic conditions at 28°C with tungsten lamp irradiation (3000 lux) until the viable count reached 1.0 × 10⁻⁶. 9 Collect the bacterial sludge by centrifugation if the concentration is above CFU / mL. Add an equal volume of lyophilization protectant solution to the sludge, mix well, and then freeze-dry to obtain lyophilized bacterial powder. Mix the lyophilized bacterial powder, PHBV, and PVP at a mass ratio of 10:1:0.06 to obtain *C. cereus* DSM158 with a content of 3.4 × 10⁻⁶. 11Single-strain inoculum DSM158 with CFU / g.

[0052] ATCC11166 bacterial culture was inoculated into liquid medium III and cultured under anaerobic conditions at 28°C with tungsten lamp irradiation (3000 lux) until the viable count reached 1.0 × 10⁻⁶. 9 CFU / mL or higher, collect the bacterial sludge by centrifugation. Add an equal volume of lyophilization protectant solution to the sludge, mix well, and then freeze-dry to obtain lyophilized bacterial powder. Mix the lyophilized bacterial powder, PHBV, and PVP at a mass ratio of 10:1:0.06 to obtain capsular red bacterium ATCC11166 with a content of 3.0 × 10⁻⁶. 11 ATCC11166, a single-strain inoculum of CFU / g.

[0053] Example 2: Degradation ability of single-strain and compound-strain agents on difenoconazole and benzylamanidin The degradation abilities of the three compound bacterial agents and three single bacterial agents prepared in Example 1 against difenoconazole and benzyladenine were tested respectively. The test methods are as follows: The formulation of liquid culture medium IV is as follows: 8.0 g peptone, 4.0 g yeast extract, 0.8 g glucose, 0.8 g K₂HPO₄, 0.8 g MgSO₄·7H₂O, 0.2 g NaCl, 0.3 g L-cysteine ​​hydrochloride, 100 mg difenoconazole, 100 mg benzyl benzoate, and 1000 mL distilled water. The pH of liquid culture medium IV is 7.0, and it is autoclaved at 121°C for 15 min.

[0054] 5g of bacterial agent was reconstituted with 15mL of sterile water and inoculated into 100mL of liquid culture medium IV. The medium was cultured for 5 days under anaerobic conditions at 28℃ and tungsten lamp irradiation (illuminance of 3000 lux). The contents of difenoconazole and benzybendazole were detected, and the removal rate was calculated accordingly. The results are shown in Table 1.

[0055] Table 1. Degradation effects of single-strain and compound-strain agents on difenoconazole and benzyladenine. The test results in Table 1 show that when using three single bacteria—DSM248 (a type of *Sphaerocera*), DSM158 (a type of *Cercospora*), and ATCC11166 (a type of *Rhodophyta*)—the removal rates of difenoconazole and benomyl were all below 5%. The compound bacterial agents CM1 and CM2, which combined these three bacteria, achieved removal rates of over 80% for difenoconazole and benomyl, significantly higher than those using single bacteria. Furthermore, although compound bacterial agent CM3 also used the same three bacteria, the removal rates of difenoconazole and benomyl remained low because it did not contain PHBV and PVP. The above results indicate that in the presence of PHBV and PVP, *Sphaerophyte DSM248*, *Sphaerocephalus cereus* DSM158, and *Rhodops capsulatum* ATCC11166 can exhibit a synergistic effect, effectively degrading difenoconazole and benzoylmide. Furthermore, when PHBV and PVP are absent from the system, difenoconazole and benzoylmide have a significant inhibitory effect on the complex bacterial community composed of the above three bacteria.

[0056] Example 3: Application Effect of Compound Microbial Agent in Actual Wastewater Treatment The wastewater treatment process in this embodiment is as follows (daily treatment capacity is 600m³). 3 / d): S1: Pretreatment is carried out on the wastewater from the production of difenoconazole, the wastewater from the production of benzybamid, and domestic sewage. The pretreatment process is as follows: the wastewater is fed into a coagulation and flotation tank, and polyaluminum chloride and polyacrylamide are added at dosages of 80 mg / L and 5 mg / L, respectively. Coagulation and flotation are carried out to remove suspended solids and emulsified oil from the wastewater. The scum enters the physicochemical sludge tank, and the clear liquid enters the respective acidification and demulsification tanks for acidification and demulsification.

[0057] S2: The pretreated wastewater from the production of difenoconazole, wastewater from the production of benzoylmide, and domestic sewage are introduced into the mixing and equalization tank at a volume ratio of 1:1:2 to mix and adjust the pH to 6.8~7.2.

[0058] S3: The effluent from the mixing and equalization tank enters the microbial pretreatment tank, where it undergoes biochemical treatment using a compound bacterial agent. The microbial pretreatment tank employs the device described in patent CN202022679748.2. Before being used for wastewater treatment, the wastewater is inoculated with the compound bacterial agent CM2 from Example 1 of this invention and a biofilm is formed. During wastewater treatment, the illuminance in the microbial pretreatment tank is 3000 lux, the temperature is controlled within the range of 25~30℃, and the dissolved oxygen concentration is controlled within the range of 0.1~0.2 mg / L. 200g of the compound bacterial agent CM2 is added every other day.

[0059] S4: The effluent from the microbial pretreatment unit enters the primary A / O tank. First, nitrification is performed in the anoxic zone using facultative anaerobic bacteria (activated sludge), followed by denitrification in the aerobic zone using aerobic bacteria (activated sludge). The mixed liquor from the aerobic zone is then returned to the anoxic zone. The effluent from the aerobic zone enters the primary sedimentation tank, where gravity settling separates the sludge from the water. The supernatant enters the secondary A / O tank, while some of the sludge is returned to the primary A / O tank.

[0060] S5: In the secondary A / O tank, nitrification is first performed in the anoxic zone using facultative anaerobic bacteria (activated sludge), followed by denitrification in the aerobic zone using aerobic bacteria (activated sludge). The mixed liquor from the aerobic zone is then returned to the anoxic zone. The effluent from the aerobic zone enters the secondary sedimentation tank, where gravity settling separates the sludge from the water. The supernatant enters the final settling tank, while part of the sludge is returned to the secondary A / O tank.

[0061] S6: Add polyaluminum chloride and polyacrylamide to the final settling tank at dosages of 70 mg / L and 3 mg / L, respectively. Pump the sludge from the final settling tank to the sludge tank, and send the effluent to the monitoring tank. Once the water quality meets the standards, it can be discharged.

[0062] The process described above was continued for 15 days, during which the effluent quality of each pool was continuously monitored. The results are shown in Table 2 (Table 2 records the highest values ​​of each pollutant detected during the continuous operation).

[0063] Table 2 Wastewater Treatment Effect As can be seen from the test results in Table 2, the compound microbial agent of the present invention can be used for the pretreatment of high-nitrogen wastewater containing difenoconazole and benzylamine, effectively reducing the content of difenoconazole and benzylamine in the wastewater, thereby reducing their inhibitory effect on nitrifying and denitrifying bacteria in the subsequent A / O tank, so that COD and nitrogen in the wastewater can be further removed through nitrification and denitrification.

Claims

1. A method for treating high-nitrogen wastewater based on a compound bacterial agent, characterized in that the steps include... include: S1: A compound bacterial agent is used to biochemically treat high-nitrogen wastewater containing difenoconazole and / or benzybacterium; the compound bacterial agent includes polyhydroxyalkanoates and polyvinylpyrrolidone, as well as *Chlorobium limicola*, *Cereibacter phaeroides*, and *Rhodobacter capsulatus* with accession numbers DSM248, DSM158, and ATCC11166, respectively. S2: Nitrification and denitrification are performed on the wastewater after S1 treatment.

2. The method according to claim 1, characterized in that, In step S1, the composite microbial agent contains 5-10 wt% polyhydroxyalkanoates, 0.3-0.7 wt% polyvinylpyrrolidone, and a total content of 2.5-3.5 × 10⁻⁶ spores of *Agrophytes spp.*, *Cercospora cereus*, and *Rhodophyton capsulatum*. 11 The ratio of viable CFU / g of *Sphaerophyte*, *C. cereus*, and *Rhodotorula capsulatum* is 0.25–0.45:0.40–0.65:1; the polyvinylpyrrolidone includes poly(3-hydroxybutyrate-co-3-hydroxyvalerate).

3. The method according to claim 1, characterized in that, In step S1, the high-nitrogen wastewater is a mixture of difenoconazole wastewater, benzyl methamidophos wastewater, and blending water after pretreatment; the pretreatment process includes coagulation flotation and acidification demulsification; the blending water includes one or more of the following: gas scrubbing tower wastewater, ground washing wastewater, equipment washing wastewater, circulating water drainage, initial rainwater, and domestic sewage.

4. The method according to claim 1 or 3, characterized in that, In step S1, the total nitrogen content in the high-nitrogen wastewater is 200~750 mg / L, the total content of difenoconazole is 10~100 mg / L, and the content of benzybendazole is 10~100 mg / L.

5. The method according to claim 1, characterized in that, In step S1, before carrying out the biochemical treatment, the pH of the high-nitrogen wastewater is adjusted to 6.8~7.

2.

6. The method according to claim 1, characterized in that, In step S1, during the biochemical treatment, the illuminance is 2000~3000 lux, the water temperature is 25~30℃, and the dissolved oxygen concentration is 0.1~0.2 mg / L.

7. The method according to claim 1 or 2, characterized in that, In step S1, the preparation steps of the compound microbial agent include: S1.1: The three bacteria were cultured in large-scale culture to obtain bacterial solutions. S1.2: After inoculating the bacterial solutions of the three bacteria into a mixed culture medium for mixed culture, the bacterial sludge is collected by centrifugation, a freeze-drying protectant solution is added to the bacterial sludge, and the mixture is freeze-dried to obtain freeze-dried bacterial powder. S1.3: Mix freeze-dried bacterial powder, polyhydroxy fatty acid ester and polyvinylpyrrolidone to obtain a compound bacterial agent.

8. The method according to claim 1, characterized in that, In step S2, the nitrification and denitrification process includes a two-stage anoxic-aerobic treatment.

9. The method according to claim 1, characterized in that, It also includes the following steps: S3: Add coagulant and coagulant aid to the wastewater after S2 treatment to carry out coagulation and sedimentation.

10. The application of a compound microbial agent in the degradation of difenoconazole and / or benzybendazole, characterized in that, The compound microbial agent is the compound microbial agent in the method described in any one of claims 1 to 9.

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