Complex microbial inoculant for metronidazole pharmaceutical wastewater as well as preparation method and application of complex microbial inoculant

Abstract

The invention relates to the technical field of wastewater treatment microorganisms, in particular to a complex microbial inoculant for metronidazole pharmaceutical wastewater as well as a preparation method and application thereof.The complex microbial inoculant comprises thalli and a protective agent, and the thalli are prepared from 16.3%-26.7% of anaerobic rope bacteria, 12.0%-20.5% of kristson bacteria, 13.0%-16.6% of Gracilinema bacteria, 2.4%-6.9% of kiwi bacteria, 2.2%-4.3% of co-edible bacillus, 1.1%-6.3% of candidate warm bacillus, 2.1%-3.7% of hot filament bacteria, 2.1%-3.1% of patella and 27.8%-38.7% of other bacteria. Aiming at the problem of difficulty in biodegradation in treatment of metronidazole pharmaceutical wastewater containing high-concentration sulfides in the prior art, the complex microbial inoculant system cooperates and cooperates with each other, and can adapt to stress of high-concentration sulfides in high-COD metronidazole pharmaceutical wastewater; the COD degradation capability is excellent, and the metabolic capability is strong.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of wastewater treatment microorganisms, in particular to a compound microbial agent for metronidazole pharmaceutical wastewater, a preparation method and application thereof. BACKGROUND

[0002] Metronidazole is a core variety of nitroimidazole antibiotics, which is widely used in clinical anti-infection treatment (such as anaerobic bacterial infection, trichomoniasis) and livestock breeding deworming. Its industrial production takes formaldehyde as the starting material, and is prepared through multi-step chemical transformations such as condensation and cyclization. Sulfuric acid is continuously used as a catalyst and formaldehyde is used as a raw material in the production process, and the wastewater generated thereby contains not only difficult-to-degrade organic pollutants such as formaldehyde and glyoxal, but also high-concentration SO4 2- When this kind of wastewater enters a biological treatment system, the organic pollutants (formaldehyde, glyoxal, etc.) will directly cause the COD (chemical oxygen demand) load of the system to increase; and the excessive SO4 2- Under anaerobic conditions, it is reduced to products including persulfate (S2O3 2- ) ions, sulfides (S 2- ) and biological sulfur (Bio-S). Sulfides are strong inhibitors of some key microorganisms in wastewater biological treatment. This inhibition is mainly due to the intracellular pH imbalance, inhibition of specific enzymes and destruction of extracellular polymeric substances caused by sulfides, which can eventually lead to cell inactivation or lysis. Excessive concentration of sulfides can have significant toxic effects on numerous microorganisms, thereby inhibiting their growth and metabolic activity, resulting in limited COD degradation capacity of the system.

[0003] Currently, for the COD effluent abnormal problem caused by the accumulation of sulfide toxicity in metronidazole pharmaceutical wastewater, enterprises generally use the method of adding chemical agents to ensure that the effluent of the biological treatment system meets the standards. With the increase of enterprise production capacity, the wastewater treatment capacity also increases, and the single dependence on chemical agent treatment method will significantly increase the operating cost of the biological treatment system. Therefore, it is particularly important to develop a new biological treatment technology that can effectively alleviate the inhibition of high-concentration sulfides in metronidazole pharmaceutical wastewater and continuously degrade COD. SUMMARY

[0004] In view of the problem of difficult biological degradation of COD in the existing metronidazole pharmaceutical wastewater containing high-concentration sulfides, the present application provides a compound microbial agent for metronidazole pharmaceutical wastewater, a preparation method and application thereof.

[0005] The first aspect of the present application provides a composite microbial agent for metronidazole pharmaceutical wastewater, which adopts the following technical scheme: a composite microbial agent for metronidazole pharmaceutical wastewater, comprising a microbial body and a protective agent, wherein the microbial body is composed of the following bacterial liquid mass percentages: Anaerolineaceae 16.3-26.7%, Christensenellaceae 12.0-20.5%, Gracilinema 13.0-16.6%, Mesotoga 2.4-6.9%, Syntrophobotulus 2.2-4.3%, Candidatus_Caldatribacterium 1.1-6.3%, Calorithrix 2.1-3.7%, Patescibacteria 2.1-3.1%, and other bacteria 27.8-38.7%.

[0006] By adopting the above technical scheme, the composite microbial agent comprises a microbial body and a protective agent, which can effectively deal with the inhibitory effect of sulfur compounds in metronidazole pharmaceutical wastewater on microorganisms and the problem of affecting the COD degradation rate. The protective agent can reduce the concentration of free sulfide and improve the survival rate of the microbial body, thereby creating a favorable living environment for the microbial body. The various bacteria in the microbial body synergize and do not antagonize each other, forming a highly efficient anti-sulfur stress combination. The applicant found that the interaction between Gracilinema, Mesotoga, Candidatus_Caldatribacterium, and Syntrophobotulus can target the main types of organic matter in metronidazole pharmaceutical wastewater, play a highly efficient anti-sulfur stress role, and further promote the efficient degradation of COD in metronidazole pharmaceutical wastewater; Gracilinema can preliminarily decompose macromolecular organic matter and crack complex organic matter in wastewater, making the organic matter more easily utilized by other microorganisms, thereby providing a usable substrate for the strictly anaerobic Syntrophobotulus with fermentation metabolic characteristics for further metabolism, and the mutual cooperation between the two promotes the gradual degradation of organic matter and improves the removal efficiency of COD. By adding Mesotoga, it can oxidize sulfate in an anaerobic environment, convert the oxidized sulfur SO4 2- in the environment into sulfide S 2- , and then oxidize S 2- to elemental sulfur (S 0) or low-toxicity thiosulfate, etc., reducing the concentration of sulfate in pharmaceutical wastewater, thereby alleviating the sulfur stress on other bacteria, especially Gracilinema and Comamonas bacteria. This provides a more suitable living environment for candidate thermophiles and other bacteria, enabling them to normally perform metabolic functions in metronidazole pharmaceutical wastewater. Candidate thermophiles metabolize and decompose organic matter such as sugars in the wastewater at high temperatures, using sugars in the early decomposition products of Gracilinema and other bacteria to generate energy and convert sugars to CO2 and H2O, not only directly reducing COD, but also maintaining the stability of the microbial community at high temperatures, thereby helping Gracilinema and Comamonas bacteria to continuously perform metabolic activities in the SO4 2 - conversion to S 2 - and other sulfate-reducing reactions, thereby optimizing the ecological structure of the entire microbial community through the interaction of Gracilinema, Misgurnus, candidate thermophiles, and Comamonas bacteria, enhancing the resistance of the bacterial community to sulfur stress and the synergistic effect of organic matter degradation.

[0007] In addition, Misgurnus can act as a mutualistic acetate-oxidizing bacterium, can symbiotically coexist with hydrogenotrophic methanogens, and can further symbiotically coexist with anaerobic filamentous bacteria that degrade complex organic matter and produce small-molecule organic acids, hydrogen, and other metabolic products, thereby promoting material exchange and symbiotic relationships for hydrogen to promote organic matter degradation. In summary, the interaction of the bacteria targets the main types of organic matter in metronidazole pharmaceutical wastewater, and through their unique properties and mutual cooperation, they achieve efficient sulfur stress resistance and promote efficient COD degradation. Anaeromyces and Christensenella can efficiently degrade high-COD complex organic matter, converting large-molecule organic matter into small-molecule organic matter. Anaeromyces has a unique filamentous structure that helps to enhance sludge settling performance and improve solid-liquid separation efficiency, thereby effectively increasing the removal rate of organic load and the COD degradation rate. In addition, the addition of thermophilic filamentous bacteria can synthesize extracellular polysaccharides, which play an important role in the formation of sludge aggregates, further promoting the degradation of organic pollutants. Gracilinema can enhance the system's tolerance to sulfides and water quality fluctuations. The complex bacterial agent under the bacterial agent combination of the present application has high activity, large biomass, and fast reproduction, and when added to the metronidazole pharmaceutical wastewater treatment system, it exhibits excellent degradation effect on sulfur-stressed refractory industrial wastewater, and is suitable for industrial wastewater treatment. The use of the complex bacterial agent can increase the treatment water volume and water quality, reduce sludge production, reduce operating costs, and promote wastewater discharge standards.

[0008] In one specific embodiment, the other bacteria are one or more of Syntrophobacter, Syner-01, Rectinema, Bacillus, Rhizobiaceae, Aggregatilineales, JGI-0000079-D21, SJA-15, Alkalibacter, Aminicenans, SM1A02, Ornatilinea, Oscillospiraceae, Bacteroidetes_vadinHA17, Pedomicrobium, Clostridia, Amb-16S-1323.

[0009] Preferably, the other bacteria are Syner-01, Clostridia, and Aggregatilineales in a mass ratio of 3:3:4.

[0010] By using the above technical solution, the synergistic effect with other bacteria in the complex bacterial agent can further enhance the anti-sulfide stress ability and COD degradation ability of the complex bacterial agent on metronidazole pharmaceutical wastewater, so as to cope with the treatment of metronidazole pharmaceutical wastewater containing high-concentration sulfide and refractory organic matter. Through the interaction of Syner-01, Clostridia, and Aggregatilineales in the present application, the Aggregatilineales can promote the formation of granular sludge and provide physical protection for other bacteria. The outer layer of the formed granular sludge can adsorb part of the sulfide, which can reduce the concentration of free sulfide. The inner layer of the granular sludge provides a low-toxicity and high-nutrient metabolic space for Syner-01 and Clostridia. Syner-01 can degrade polycyclic aromatic hydrocarbons and other substances, and act on the heterocyclic structure of metronidazole to convert it into small-molecule intermediates containing hydroxyl and carboxyl groups (such as acetic acid and propionic acid). As anaerobic acid-producing bacteria, Clostridia can degrade the above-mentioned small-molecule organic substances produced by Syner-01 through β-oxidation, glycolysis, and other pathways, thereby significantly improving the COD degradation rate of the wastewater and efficiently degrading metronidazole and other stubborn components containing nitroimidazole rings with poor biodegradability.

[0011] In one specific embodiment, the mass ratio of Gracilinema, Misgurn, Candidatus-thermotogae, Syntrophococcus is (3-5):(1.5-2):1:(0.7-1).

[0012] By adopting the above technical scheme, the applicant finds that the interaction of Gracilinema, Misgurnus, candidate thermophilic bacillus and coprophilic bacillus optimizes the ecological structure of the whole microbial community, enhances the resistance of the microbial community to sulfur stress and the synergistic effect of organic matter degradation. If the content of Gracilinema is too low, the cracking ability of complex organic matter in wastewater is reduced, thereby reducing the rate of sulfur reduction process, resulting in insufficient sulfur reduction yield, reducing Misgurnus, and reducing the ability of the whole system to resist sulfide stress. If the content of coprophilic bacillus is too high, excessive fermentation metabolism will occur under strict anaerobic conditions, generating too much H2, which may disrupt the redox balance of the system, affecting the growth and function of other bacteria, and is not conducive to the stability of the system to resist sulfide stress.

[0013] The protective agent comprises the following components by mass fraction: modified nitrogen-doped graphene quantum dots 8-12 parts, zinc oxide modified vermiculite 15-20 parts, L-cysteine 4-6 parts, and nano calcium carbonate 3-5 parts.

[0014] The bacterial cells and the protective agent are in a mass ratio of (0.25-0.5):1.

[0015] By adopting the above technical scheme, the protective agent can reduce the concentration of sulfides in the water body, protect the bacterial cells from loss and penetration inhibition by sulfides, promote mutual symbiosis between bacteria, accelerate the conversion of sulfides, and improve the COD degradation activity, thereby improving the ability of the composite microbial agent to resist sulfide stress and the COD degradation efficiency of the metronidazole pharmaceutical wastewater. Among them, the hydroxyl group of the modified nitrogen-doped graphene quantum dot forms a coordination bond with the Zn-O bond on the surface of the zinc oxide modified vermiculite, so that the quantum dots are attached to the porous structure of the vermiculite, and then the porous structure can fix the bacterial cells, so as to improve the removal rate of sulfides. The nitrogen atom of the modified nitrogen-doped graphene quantum dot forms a hydrogen bond with the -SH of L-cysteine, stabilizes the dispersion of L-cysteine, and prolongs the supply time of sulfur metabolism substrate. Nano calcium carbonate can maintain the pH of the system and adsorb heavy metals, thereby significantly improving the ability of the composite microbial agent to resist sulfide stress and the COD degradation efficiency through the protective agent, and ensuring the stability of the composite microbial agent in the wastewater treatment system. The reduction of the content of the protective agent reduces the reduction ability of the composite microbial agent to sulfides, the concentration of free sulfides is reduced to a small extent, the protection of the bacterial cells is weakened, the survival rate of the bacterial cells is reduced, the efficiency of the composite microbial agent to resist sulfide stress and degrade COD in the metronidazole pharmaceutical wastewater is poor, and the synergistic stability of the system is reduced.

[0016] In a second aspect, the application provides a preparation method of a composite microbial agent for metronidazole pharmaceutical wastewater, comprising the following steps: preparing microbial bodies: (1) selecting single strains from a strain bank for activation to obtain activated microbial liquid; (2) respectively taking the activated microbial liquid and inoculating into metronidazole pharmaceutical wastewater culture medium for expansion culture; (3) taking the microbial liquid obtained in (2) and mixing in proportion to obtain seed liquid, and the seed liquid is enriched and cultured to OD 600 >0.5, to obtain the microbial bodies; Preparation of a protective agent: modified nitrogen-doped graphene quantum dots, zinc oxide modified vermiculite, L-cysteine, and nano calcium carbonate are weighed according to the mass ratio, water is added, and stirring is performed at 30-35°C for 30-40 min to obtain the protective agent; Mixing the microbial bodies with the protective agent and standing at 30-33°C for 10-15 min to obtain the composite microbial agent for metronidazole pharmaceutical wastewater.

[0017] The metronidazole pharmaceutical wastewater culture medium is metronidazole pharmaceutical wastewater with a COD concentration of 100-500 mg / L in a target treatment system, the pH is adjusted to 7.0-7.5, and the temperature is 30-35°C.

[0018] In a third aspect, the application provides an application of the composite microbial agent in metronidazole pharmaceutical wastewater treatment, and the composite microbial agent is placed in a UASB reactor for anaerobic process treatment.

[0019] In summary, the application has at least one of the following beneficial technical effects: 1. The composite microbial agent contains microbial bodies and a protective agent, which can effectively deal with metronidazole pharmaceutical wastewater, prevent sulfur-containing compounds from acting as microbial inhibitors to affect the COD degradation rate, reduce the concentration of free sulfide with the protective agent, improve the survival rate of the microbial bodies, and create a favorable living environment for the microbial bodies; the strains in the microbial bodies cooperate, coordinate and do not antagonize each other, forming a high-efficiency anti-sulfur stress combination. Among them, Gracilinema, Misgurn, candidate thermophilic bacillus and synedrospira interact with each other to enhance the resistance of the bacterial colony to stress, thereby improving the sulfur reduction rate and the anti-sulfide stress ability. The composite microbial agent under the combination of the microbial agent has high activity, large biomass and fast reproduction, and when added to a metronidazole pharmaceutical wastewater treatment system, it has good degradation effect on sulfur-containing stress-resistant industrial wastewater and is suitable for industrial wastewater treatment. The use of the composite microbial agent can improve the water quantity and water quality, reduce the sludge yield, reduce the operation cost, and promote the wastewater discharge to meet the standards.

[0020] 2. The synergistic bacteria, clostridium, and aggregate bacteria interact in the ratio of the application. The aggregate bacteria can promote the formation of granular sludge, and the formed granular sludge provides physical protection for other bacteria. The outer layer of the granular sludge can adsorb part of the sulfide, thereby reducing the concentration of free sulfide. The inner layer of the granular sludge provides a low-toxicity and high-nutrient metabolic space for synergistic bacteria and clostridium. This significantly improves the COD degradation rate of wastewater, and efficiently degrades stubborn components such as metronidazole containing nitro-imidazole ring with poor biodegradability.

[0021] 3. By using modified nitrogen-doped graphene quantum dots, zinc oxide modified vermiculite, L-cysteine, and nano calcium carbonate as main components of the protective agent, the anti-sulfide stress ability and COD degradation efficiency of the composite bacterial agent are significantly improved, and the stability of the composite bacterial agent in the wastewater treatment system is ensured. The reduction of the content of the protective agent reduces the reduction capacity of the composite bacterial agent to sulfide, the concentration of free sulfide decreases at a small rate, the protection of the bacterial agent is weakened, which leads to a decrease in the survival rate of the bacterial agent, and the efficiency of the composite bacterial agent in resisting sulfide stress and degrading COD in metronidazole pharmaceutical wastewater is poor, and the stability of the system is reduced. DETAILED DESCRIPTION

[0022] In the application, some of the raw materials in the examples and comparative examples are as follows: nitrogen-doped graphene quantum dots are purchased from Xianfeng Nanometer; vermiculite (200 mesh) is purchased from Lingshou County Aosen Mineral Powder Factory; nano calcium carbonate (specification 50-100 nm) is purchased from Maikelin; and other raw materials are available on the market.

[0023] Preparation Example 1 Preparation of modified nitrogen-doped graphene quantum dots: 5 g of nitrogen-doped graphene quantum dots were dispersed in 100 mL of water, 20 mL of 30% hydrogen peroxide was added, and stirring was carried out at 60°C for 3 h. After centrifugation and drying, grinding was carried out on a 400 mesh sieve to obtain modified nitrogen-doped graphene quantum dots.

[0024] Preparation Example 2 Preparation of zinc oxide modified vermiculite: (1) 10 g of vermiculite was added to 100 mL of 1 mol / L hydrochloric acid, and stirring was carried out at 60°C for 2 h. After filtration, 50 mL of 0.5 mol / L Zn(NO3)2 solution was added, and ultrasonic treatment was carried out for 30 min. After drying at 60°C, calcination was carried out at 500°C for 1 h, and grinding was carried out on a 200 mesh sieve to obtain zinc oxide modified vermiculite.

[0025] Example 1 A composite microbial agent for metronidazole pharmaceutical wastewater, comprising a microbial body and a protective agent, the microbial body is composed of the following bacterial liquid mass percentage: 2.06 mg of anaerobic string bacteria, 1.5 mg of christensen's bacteria, 1.35 mg of gracilinema bacteria, 0.675 mg of misrao bacteria, 0.405 mg of commensal bacteria, 0.45 mg of candidate thermophilic bacteria, 0.3 mg of hot silk bacteria, 0.26 mg of patella bacteria, and 3 mg of other bacteria; the protective agent is 10 mg of modified nitrogen-doped graphene quantum dots prepared in preparation example 1, 18 mg of zinc oxide modified vermiculite prepared in preparation example 2, 6 mg of L-cysteine, and 4 mg of nano calcium carbonate; wherein the other bacteria are synergistic bacteria, clostridium, and aggregated bacteria with a mass ratio of 3:3:4.

[0026] Preparation of microbial body: (1) select single strain from strain library, inoculate into anaerobic test tube containing 5 ml LB liquid medium, cultivate at 35℃ constant temperature shaker for 48 h to obtain activated bacterial liquid; (2) take 300 mg / L metronidazole pharmaceutical wastewater, adjust pH to 7.2 with 1 mol / L NaOH, and then divide into 50 ml anaerobic serum bottles, sterilize at 121℃ for 20 min, and cool to 33℃ to obtain metronidazole pharmaceutical wastewater culture medium; take activated bacterial liquid, inoculate into metronidazole pharmaceutical wastewater culture medium at 33℃ with 2% inoculation amount (v / v) for 72 h; (3) take the bacterial liquid obtained in (2), mix in proportion, and transfer the mixed bacterial liquid into metronidazole pharmaceutical wastewater culture medium with 5% inoculation amount, cultivate anaerobically at 33℃ until OD 600 >0.5, to obtain the microbial body; Preparation of protective agent: take modified nitrogen-doped graphene quantum dots prepared in preparation example 1, zinc oxide modified vermiculite prepared in preparation example 2, L-cysteine, and nano calcium carbonate with a mass ratio, add 10 ml water, stir at 35℃ for 30 min, and sterilize at 121℃ for 20 min to obtain the protective agent; Mix the microbial body and the protective agent with a mass ratio of 0.3:1, stand at 33℃ for 15 min to obtain the composite microbial agent for metronidazole pharmaceutical wastewater.

[0027] Example 2 The difference between this example and example 1 is that the gracilinema bacteria is 1.125 mg, the misrao bacteria is 0.9 mg, the commensal bacteria is 0.405 mg, and the candidate thermophilic bacteria is 0.45 mg.

[0028] Example 3 The difference between this example and example 1 is that the gracilinema bacteria is 1.35 mg, the misrao bacteria is 9 mg, the commensal bacteria is 0.63 mg, and the candidate thermophilic bacteria is 0.45 mg.

[0029] Example 4 The difference between this embodiment and embodiment 1 is only that the other bacteria are synergistic bacteria, clostridium and aggregated bacteria with a mass ratio of 5:3:1.

[0030] Example 5 The difference between this embodiment and embodiment 1 is only that the other bacteria are clostridium and aggregated bacteria with a mass ratio of 6:4.

[0031] Example 6 The difference between this embodiment and embodiment 1 is only that the other bacteria are synergistic bacteria, clostridium and aggregated bacteria with a mass ratio of 6:4.

[0032] Example 7 The difference between this embodiment and embodiment 1 is only that the other bacteria are aggregated bacteria.

[0033] Example 8 The difference between this embodiment and embodiment 1 is only that the protective agent is nitrogen-doped graphene quantum dots 10 mg, zinc oxide modified vermiculite 18 mg prepared in preparation example 2, L-cysteine 6 mg, and nano calcium carbonate 4 mg.

[0034] Example 9 The difference between this embodiment and embodiment 1 is only that the protective agent is nitrogen-doped graphene quantum dots 10 mg, vermiculite 18 mg, L-cysteine 6 mg, and nano calcium carbonate 4 mg.

[0035] Example 10 The difference between this embodiment and embodiment 1 is only that the bacteria and the protective agent are mixed in a mass ratio of 0.1:1, and then placed at 33°C for 15 min to obtain a composite bacterial agent for metronidazole pharmaceutical wastewater.

[0036] Comparative example 1 A composite bacterial agent for metronidazole pharmaceutical wastewater includes bacteria, and the bacteria are composed of the following bacteria liquid mass percentages: anaerobic string bacteria 2.06 mg, christensen's bacteria 1.5 mg, gracilinema bacteria 1.35 mg, misopae bacteria 0.675 mg, cannibal bacteria 0.405 mg, candidate thermophilic bacteria 0.45 mg, hot silk bacteria 0.3 mg, patella bacteria 0.26 mg, and other bacteria 3 mg; wherein the other bacteria are synergistic bacteria, clostridium and aggregated bacteria with a mass ratio of 3:3:4.

[0037] Preparation of bacteria: (1) select single strain from the strain library, inoculate into anaerobic test tube containing 5ml LB liquid medium, incubate at 35℃ constant temperature shaker for 48h, obtain activated bacteria liquid; (2) take 300mg / L metronidazole pharmaceutical wastewater, pass through 0.22μm filter membrane, adjust pH to 7.2 with 1mol / L NaOH, divide into 50ml anaerobic serum bottles, sterilize at 121℃ for 20min, cool to 33℃ to obtain metronidazole pharmaceutical wastewater medium; take activated bacteria liquid respectively, inoculate into metronidazole pharmaceutical wastewater medium at 33℃ with 2% inoculation amount (v / v) and incubate for 72h; (3) take the bacteria liquid obtained in (2), mix in proportion, transfer the mixed bacteria liquid into metronidazole pharmaceutical wastewater medium with 5% inoculation amount, anaerobic incubate at 33℃ until OD 600 >0.5, to obtain the composite microbial agent.

[0038] Comparative example 2 A composite microbial agent for metronidazole pharmaceutical wastewater, comprising bacteria and protective agent, the bacteria is composed of the following bacteria liquid mass percentage: 2.06mg of anaerobic string bacteria, 1.5mg of christensen's bacteria, 2.025mg of misrao bacteria, 0.405mg of commensal bacteria, 0.45mg of candidate thermophilic bacteria, 0.3mg of hot silk bacteria, 0.26mg of patella bacteria, 3mg of other bacteria; the protective agent is 10mg of modified nitrogen-doped graphene quantum dots prepared in preparation example 1, 18mg of zinc oxide modified vermiculite prepared in preparation example 2, 6mg of L-cysteine and 4mg of nano calcium carbonate; wherein the other bacteria are synergistic bacteria, clostridium and aggregated bacteria with a mass ratio of 3:3:4.

[0039] Preparation of bacteria: (1) select single strain from the strain library, inoculate into anaerobic test tube containing 5ml LB liquid medium, incubate at 35℃ constant temperature shaker for 48h, obtain activated bacteria liquid; (2) take 300mg / L metronidazole pharmaceutical wastewater, pass through 0.22μm filter membrane, adjust pH to 7.2 with 1mol / L NaOH, divide into 50ml anaerobic serum bottles, sterilize at 121℃ for 20min, cool to 33℃ to obtain metronidazole pharmaceutical wastewater medium; take activated bacteria liquid respectively, inoculate into metronidazole pharmaceutical wastewater medium at 33℃ with 2% inoculation amount (v / v) and incubate for 72h; (3) take the bacteria liquid obtained in (2), mix in proportion, transfer the mixed bacteria liquid into metronidazole pharmaceutical wastewater medium with 5% inoculation amount, anaerobic incubate at 33℃ until OD 600 >0.5, to obtain the bacteria; Preparation of protective agent: take modified nitrogen-doped graphene quantum dots prepared in preparation example 1, zinc oxide modified vermiculite prepared in preparation example 2, L-cysteine and nano calcium carbonate by mass ratio, add 10ml water, stir at 35℃ for 30min, sterilize at 121℃ for 20min to obtain the protective agent; Mix the bacteria and the protective agent at a mass ratio of 0.3:1, stand still at 33°C for 15 min, to obtain the composite microbial agent for metronidazole pharmaceutical wastewater.

[0040] Comparative Example 3 A composite microbial agent for metronidazole pharmaceutical wastewater, comprising bacteria and a protective agent, wherein the bacteria is composed of the following bacterial liquid mass percentage: 2.06 mg of Anaeromyces, 1.5 mg of Christensenella, 1.8 mg of Gracilinema, 0.675 mg of Misgurn, 0.405 mg of Syntrophococcus, 0.3 mg of Thermofilum, 0.26 mg of Patibacter, and 30 mg of other bacteria; the protective agent is 10 mg of modified nitrogen-doped graphene quantum dots prepared in Preparation Example 1, 18 mg of zinc oxide modified vermiculite prepared in Preparation Example 2, 6 mg of L-cysteine, and 4 mg of nano calcium carbonate; wherein the other bacteria are synergistic bacteria, clostridium, and aggregated bacteria at a mass ratio of 3:3:4.

[0041] Preparation of bacteria: (1) select single strain from a strain bank, inoculate into an anaerobic test tube containing 5 ml of LB liquid medium, and cultivate at 35°C for 48 h on a constant temperature shaker to obtain activated bacterial liquid; (2) take 300 mg / L of metronidazole pharmaceutical wastewater, adjust the pH to 7.2 with 1 mol / L NaOH, and then divide into 50 ml anaerobic serum bottles, sterilize at 121°C for 20 min, and cool to 33°C to obtain metronidazole pharmaceutical wastewater medium; take the activated bacterial liquid, inoculate into the metronidazole pharmaceutical wastewater medium at an inoculation amount of 2% (v / v), and cultivate at 33°C for 72 h; (3) take the bacterial liquid obtained in (2), mix according to the proportion, and inoculate the mixed bacterial liquid into the metronidazole pharmaceutical wastewater medium at an inoculation amount of 5%, and cultivate anaerobically at 33°C until OD 600 >0.5, to obtain the bacteria; Preparation of protective agent: take the modified nitrogen-doped graphene quantum dots prepared in Preparation Example 1, the zinc oxide modified vermiculite prepared in Preparation Example 2, L-cysteine, and nano calcium carbonate according to the mass ratio, add 10 ml of water, stir at 35°C for 30 min, sterilize at 121°C for 20 min, and obtain the protective agent; Mix the bacteria and the protective agent at a mass ratio of 0.3:1, stand still at 33°C for 15 min, to obtain the composite microbial agent for metronidazole pharmaceutical wastewater.

[0042] Comparative Example 4 A composite microbial agent for metronidazole pharmaceutical wastewater, comprising a microbial body and a protective agent, the microbial body is composed of the following bacterial liquid mass percentage: 2.06 mg of anaerobic string bacteria, 1.5 mg of christensen bacteria, 4.35 mg of gracilinema bacteria, 0.675 mg of misrao bacteria, 0.405 mg of commensal bacteria, 0.45 mg of candidate thermophilic bacteria, 0.3 mg of hot silk bacteria, and 0.26 mg of patella bacteria; the protective agent is 10 mg of modified nitrogen-doped graphene quantum dots prepared in preparation example 1, 18 mg of zinc oxide modified vermiculite prepared in preparation example 2, 6 mg of L-cysteine, and 4 mg of nano calcium carbonate.

[0043] Preparation of microbial body: (1) select single strain from strain library, inoculate into anaerobic test tube containing 5 ml LB liquid medium, cultivate at 35℃ constant temperature shaker for 48h, obtain activated bacterial liquid; (2) take 300mg / L metronidazole pharmaceutical wastewater, adjust pH to 7.2 with 1mol / L NaOH, dispense into 50ml anaerobic serum bottle, sterilize at 121℃ for 20min, cool to 33℃ to obtain metronidazole pharmaceutical wastewater medium; take activated bacterial liquid, inoculate into metronidazole pharmaceutical wastewater medium at 33℃ with 2% inoculation amount (v / v) for 72h; (3) take the bacterial liquid obtained in (2), mix in proportion, transfer the mixed bacterial liquid into metronidazole pharmaceutical wastewater medium with 5% inoculation amount, cultivate anaerobically at 33℃ until OD 600 >0.5, i.e. the microbial body is obtained; Preparation of protective agent: take modified nitrogen-doped graphene quantum dots prepared in preparation example 1, zinc oxide modified vermiculite prepared in preparation example 2, L-cysteine, and nano calcium carbonate by mass ratio, add 10ml water, stir at 35℃ for 30min, sterilize at 121℃ for 20min to obtain the protective agent; Mix the microbial body and the protective agent with a mass ratio of 0.3:1, stand at 33℃ for 15min to obtain the composite microbial agent for metronidazole pharmaceutical wastewater.

[0044] Performance detection The composite microbial agent of the application example and the comparative example is used to treat metronidazole pharmaceutical wastewater, the treatment process is to inject the composite microbial agent into the sludge bed area of the UASB reactor, the hydraulic retention time of the influent is set to 48h, the temperature is controlled to 33℃, the upflow velocity is 0.5m / h, the dosage is 1% of the sludge concentration of the anaerobic reaction tank, and at the same time, a parallel control group is set, the sludge concentration is kept unchanged, and no microbial agent is added, the maximum value of COD before treatment is 7557.3mg / L, the average concentration of COD of the application example and the comparative example is 7529.7mg / L, the maximum concentration of sulfide before treatment is 212.4mg / L, and the average concentration of sulfide is 170.1mg / L; the treatment effect is as follows: As can be seen from the table, the above-mentioned examples of the composite microbial agent for metronidazole pharmaceutical wastewater, the composite microbial agent system cooperates together, and cooperates with each other, can well adapt to the high-concentration sulfide stress in the high-COD metronidazole pharmaceutical wastewater, and has superior reproduction ability and degradation ability. The metabolic capacity is strong, and organic pollutants such as formaldehyde and glyoxal in the waste liquid are decomposed into non-toxic substances harmless to the environment. Compared with the control group, the COD removal efficiency of the anaerobic active composite microbial agent system with a mass fraction of 1 ‰ of the microbial agent in the high-COD metronidazole pharmaceutical wastewater is significantly better than that of the anaerobic active biological system without adding any microbial agent.

[0045] As can be seen from comparative examples 1-3 and comparative examples 2-4, the interaction of Gracilinema, Misgurnus, candidate thermophilic bacillus and cannibal bacillus in the present application optimizes the ecological structure of the whole microbial community, enhances the resistance of the bacterial flora to sulfur stress and the synergistic effect of organic matter degradation. The content of Gracilinema is reduced, the cracking ability of complex organic matter in wastewater is reduced, the COD degradation rate is low, and the sulfur reduction rate is low, resulting in insufficient sulfur reduction yield, excessive cannibal bacillus content, excessive H2 generation, affecting the growth and function of other bacteria, and further affecting the oxidation-reduction balance of sulfur compounds, and reducing the resistance to sulfide stress.

[0046] As can be seen from comparative example 1 and examples 4-7, the synergistic bacteria, clostridium and aggregating bacteria in the present application interact with each other, the aggregating bacteria can reduce the concentration of free sulfide, the synergistic bacteria can degrade heterocyclic structures and convert small-molecule intermediates, and the clostridium can degrade small-molecule organic matter, which can significantly improve the COD degradation rate of wastewater and efficiently degrade components with poor biodegradability; and the content of synergistic bacteria or clostridium is reduced, and the COD degradation rate is reduced.

[0047] As can be seen from comparative example 1 and examples 8-10 and comparative example 1, the modified nitrogen-doped graphene quantum and the porous structure of zinc oxide modified vermiculite can fix bacterial bodies, improve the adsorption of sulfur compounds, improve the removal rate of sulfides, prolong the supply time of sulfur metabolism substrates, and further degrade for a long time; significantly improve the anti-sulfide stress ability and COD degradation efficiency of the composite microbial agent, and ensure the stability of the composite microbial agent in the wastewater treatment system. The content of the protective agent is reduced, the reduction ability of the composite microbial agent to sulfides is reduced, the concentration of free sulfides is reduced, the protection effect on the bacterial bodies is weakened, the survival rate of the bacterial bodies is reduced, the efficiency of the composite microbial agent to degrade COD and resist sulfide stress in the metronidazole pharmaceutical wastewater is poor, and the stability of the system is reduced.

[0048] The above are preferred embodiments of the present application, and are not intended to limit the protection scope of the present application, therefore: any equivalent changes made on the basis of the structure, shape, principle of the present application should be covered within the protection scope of the present application.

Claims

1. A composite microbial agent for metronidazole pharmaceutical wastewater, characterized in that, The bacteria body and the protective agent, the bacteria body is composed of the following bacteria liquid mass percentage: 16.3-26.7% of anaerobic string bacteria, 12.0-20.5% of Kristensen bacteria, 13.0-16.6% of Gracilinema bacteria, 2.4-6.9% of Misrao bacteria, 2.2-4.3% of Syncomycetes bacteria, 1.1-6.3% of candidate thermophilic bacteria, 2.1-3.7% of hot silk bacteria, 2.1-3.1% of patella bacteria, 27.8-38.7% of other bacteria.

2. The compound microbial agent for metronidazole pharmaceutical wastewater according to claim 1, characterized in that: The other bacteria are one or more of mutual bacteria, synergistic bacteria, straight silk bacteria, bacillus, rhizobium, aggregate bacteria, JGI-0000079-D21 bacteria, SJA-15 bacteria, alkaliphilic bacteria, Aminicenans bacteria, SM1A02 bacteria, decorative line bacteria, Oscillospira bacteria, Bacteroidetes_vadinHA17 bacteria, soil microbacteria, Clostridium, Amb-16S-1323 bacteria.

3. The compound microbial agent for metronidazole pharmaceutical wastewater according to claim 1, characterized in that: The mass ratio of the Gracilinema bacteria, Misrao bacteria, candidate thermophilic bacteria and Syncomycetes bacteria is (3-5):(1.5-2):1:(0.7-1).

4. The compound microbial agent for metronidazole pharmaceutical wastewater according to claim 1, characterized in that: The protective agent includes the following components: 8-12 parts of modified nitrogen-doped graphene quantum dots, 15-20 parts of zinc oxide modified vermiculite, 4-6 parts of L-cysteine, and 3-5 parts of nano calcium carbonate.

5. The compound microbial agent for metronidazole pharmaceutical wastewater according to claim 1, characterized in that: The bacteria body and the protective agent are in a mass ratio of (0.25-0.5):

1.

6. The preparation method of the complex microbial agent for metronidazole pharmaceutical wastewater according to any one of claims 1-5, characterized in that: The method comprises the following steps: preparing the bacteria: (1) selecting single strain from a strain library to obtain activated bacteria solution; (2) inoculating the activated bacteria solution into a metronidazole pharmaceutical wastewater culture medium to perform expansion culture; (3) mixing the bacteria solution obtained in the step (2) according to a proportion to obtain a seed solution, and performing enrichment culture on the seed solution until OD 600 >0.5, so as to obtain the bacteria. Preparation of protective agent: take modified nitrogen-doped graphene quantum dots, zinc oxide modified vermiculite, L-cysteine and nano calcium carbonate by mass ratio, add water, stir at 30-35℃ for 30-40min, get protective agent; Mix the bacteria body and the protective agent, stand at 30-33℃ for 10-15min, get the composite microbial agent for metronidazole pharmaceutical wastewater.

7. The preparation method of the complex microbial agent for metronidazole pharmaceutical wastewater according to claim 6, characterized in that: The metronidazole pharmaceutical wastewater culture medium is the pharmaceutical wastewater with COD concentration of 100-500mg / L in the target treatment system, the pH is adjusted to 7.0-7.5, and the temperature is 30-35℃.

8. The application of the composite microbial agent of any one of claims 1-5 in the treatment of metronidazole pharmaceutical wastewater, characterized in that: Put the composite microbial agent into the UASB reactor for anaerobic process treatment.

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