Solid complex microbial inoculant for enhancing nitrogen and phosphorus removal efficiency as well as preparation method and application of solid complex microbial inoculant

Abstract

The invention discloses a solid complex microbial inoculant for enhancing nitrogen and phosphorus removal efficiency as well as a preparation method and application thereof. The preparation method comprises the following steps: mixing and culturing 10 microorganisms with different nitrogen and phosphorus removal functions according to a certain proportion, then adding a nutrient carrier and a protective agent, and carrying out curing and drying treatment to obtain the solid complex microbial inoculant. The complex microbial inoculant can simultaneously remove nitrogen and phosphorus in sewage in different types of sewage treatment systems, also can improve the degradation rate of organic matters in the sewage and reduce the sludge yield and greenhouse gas emission, and has good environmental benefits and economic benefits. The invention also discloses a preparation method and an application method of the complex microbial inoculant, the preparation method comprises four steps of strain activation, strain mixing, microbial inoculant solidification and microbial inoculant application, the operation is simple, the controllability is strong, and the complex microbial inoculant is suitable for nitrogen and phosphorus removal treatment in different sewage environments such as municipal sewage, industrial sewage and agricultural sewage.

Description

Technical Field

[0001] The present invention relates to the technical field of ecological and environmental protection sewage treatment solid dry powder bacterial agents, and specifically relates to a solid composite bacterial agent for enhancing the efficiency of nitrogen and phosphorus removal, its preparation method and application. Background Art

[0002] The methods for treating wastewater at home and abroad mainly include physical methods, chemical methods and biological methods. Among them, the biological method realizes the purification of water quality by the metabolic action of microorganisms, which decomposes, absorbs or adsorbs pollutants, and has the advantages of economy, high efficiency, no secondary pollution, etc., and is widely used in urban and industrial sewage treatment. However, in the process of wastewater biological treatment, due to the complexity, refractory degradation and inhibition of the microbial system of some toxic and harmful substances, and with the strengthening of national sewage discharge control in recent years, the demand for efficient removal of nitrogen and phosphorus in sewage is increasing. Traditional treatment methods cannot treat it to meet the discharge standards. Therefore, biological enhancement technology has been favored by people. This technology introduces microorganisms with specific functions into the biological treatment system, increases the concentration of effective microorganisms, enhances the degradation ability of refractory pollutants, increases their degradation rate, and improves the removal efficiency of the original biological treatment system for target pollutants, and has the advantages of enhancing system stability, improving sludge performance and load impact resistance. This solution adopts a solid composite bacterial agent composed of 10 kinds of microorganisms with different nitrogen and phosphorus removal functions. By adding this bacterial agent to the biological treatment system, the simultaneous removal of nitrogen and phosphorus in sewage is achieved. At the same time, it can also increase the degradation rate of organic matter in sewage, reduce sludge production and greenhouse gas emissions, and is an efficient biological enhancement technology.

[0003] In the process of sewage biological treatment, biological denitrification mainly removes nitrogen-containing compounds by ammonifying bacteria, nitrifying bacteria and denitrifying bacteria through ammonification, nitrification and denitrification to convert them into nitrogen gas. Traditional biological denitrification is easily inhibited by the influence of dissolved oxygen, and autotrophic nitrifying bacteria are difficult to survive in an environment with too high COD. In recent years, researchers have discovered special heterotrophic nitrification-aerobic denitrification bacteria. These bacteria have the characteristics of synchronous nitrification and denitrification, and can achieve synchronous removal of ammonia nitrogen and total nitrogen in an aerobic reactor under aerobic conditions, solving the contradiction between the nitrification and denitrification processes. The composite bacterial agent in this solution contains such bacteria, thus realizing a single-stage denitrification reaction.

[0004] Sewage phosphorus removal is mainly carried out through two methods: biological phosphorus removal or chemical phosphorus removal. However, chemical phosphorus removal requires a large amount of chemical raw materials to be added to the sewage, increasing the cost of sewage phosphorus removal; while biological phosphorus removal makes full use of the phenomenon of excessive phosphorus absorption by polyphosphate-accumulating organisms to treat sewage for phosphorus removal. The efficiency of biological phosphorus removal is generally affected by the growth rate of polyphosphate-accumulating organisms and the excess sludge production rate. The population proportion of polyphosphate-accumulating organisms in the activated sludge is relatively low. Therefore, strengthening and increasing the proportion of polyphosphate-accumulating organisms in the activated sludge can significantly improve the phosphorus removal efficiency of sewage treatment. The composite bacterial agent in this solution contains polyphosphate-accumulating organisms, thus realizing biological phosphorus removal.

[0005] After retrieval, a Chinese patent (Patent No.: CN102443558A, Patent Name: Composite Heterotrophic Nitrifying Bacterial Agent and Its Application in Denitrification Treatment of Ammonia-Nitrogen-Containing Wastewater) provides a composite heterotrophic nitrifying bacterial agent. The bacterial agent is a liquid bacterial agent, the composition of the bacterial agent medium is complex, and the function is relatively single.

[0006] After retrieval, (Patent No.: CN103087918A, Patent Name: An Aerobic Denitrifying Bacterial Agent and Its Preparation and Application) provides an aerobic denitrifying bacterial agent. The composition of the functional microorganisms in the bacterial agent is relatively simple, there is no adsorption carrier, and the function is relatively single.

[0007] After retrieval, (Patent No.: CN109370961A, Patent Name: An Aerobic Denitrifying Bacterial Agent and Its Preparation Method) provides an aerobic denitrifying bacterial agent, which contains multiple species in combination, but the bacterial agent is a bacterial liquid, and the function is relatively single.

[0008] At present, for these bacterial agents, in terms of formulation, the culture medium is complex, requires the combination of multiple components, and increases the cost. The states of the bacterial agents are all liquid bacterial agents or freeze-dried preparations of liquid bacterial agents plus protective agents, without adsorption carriers;

[0009] In terms of the application of the bacterial agent, they are all single-sided nitrification and denitrification bacterial agents. To achieve comprehensive denitrification and phosphorus removal effects and full compliance, additional chemical means are required, increasing the cost and making the operation and maintenance complex;

[0010] Therefore, we propose a solid composite bacterial agent for enhancing denitrification and phosphorus removal efficiency, its preparation method and application. Summary of the Invention

[0011] The purpose of the present invention is to provide a solid composite bacterial agent for enhancing denitrification and phosphorus removal efficiency, its preparation method and application, which has multiple species acting synergistically to improve the denitrification treatment effect of sewage and has the function of simultaneous nitrification and denitrification, solving the problems of low denitrification efficiency, high cost, great management difficulty and difficulty in achieving compliance in the existing sewage treatment process.

[0012] To achieve the above object, the present invention provides the following technical solution: a solid composite bacterium agent for enhancing the efficiency of denitrification and phosphorus removal. The bacterium agent includes Acinetobacter schindleri, Acinetobacter variabilis, Enterobacter cloacae, Lysinibacillus macroides, Lysinibacillus fusiformis, Klebsiella pneumoniae, Stenotrophomonas pavanii, Enterobacter mori, Acinetobacter baumannii, and Enterococcus hirae.

[0013] Preferably, the volume ratio of the bacterium agent is 2:1:1:2:1:2:2:1:1:2, that is, Acinetobacter schindleri: Acinetobacter variabilis: Enterobacter cloacae: Lysinibacillus macroides: Lysinibacillus fusiformis: Klebsiella pneumoniae: Stenotrophomonas pavanii: Enterobacter mori: Acinetobacter baumannii: Enterococcus hirae.

[0014] Preferably, the culture medium is LB medium, and its components are 10 g of peptone, 5 g of beef extract, 5 g of NaCl, and 1000 mL of distilled water.

[0015] Preferably, 0.6% of trace elements are further added to the bacterium agent, and its components are 0.16 g of zinc chloride, 0.2 g of ferrous sulfate, 0.04 g of boric acid, 0.04 g of copper sulfate, and the volume is fixed to 25 mL.

[0016] Preferably, a nutrient carrier is further added to the bacterium agent, and its components are 2 kg of wheat bran and 5% of bagasse activated carbon powder or coconut shell activated carbon powder.

[0017] A preparation method of a solid composite bacterium agent for enhancing the efficiency of denitrification and phosphorus removal, characterized in that it includes the following steps:

[0018] Step A: Bacterial strain activation. The 10 kinds of microorganisms are respectively activated into original strains, and then oscillated and cultured in LB medium, LB medium and YG medium. When the OD 600 value is between 0.8 and 1.0 or the bacterial content is 10 9 ~10 10 CFU / mL, the bacterial liquid is taken out.

[0019] Step B: Bacteria mixing: the 10 kinds of microorganisms are mixed according to the volume ratio to obtain a composite aerobic denitrification-heterotrophic nitrification agent; the mixed bacterial solution is inoculated into LB medium, and the trace elements are added to the medium, and the culture is shaken at a constant temperature. When the total number of microorganisms is greater than 10 9 When the number of CFU / ml is reached, collect the bacterial agent and store it in sterile containers;

[0020] Step C: solidifying the bacterial agent, adding the nutrient carrier to 1L of the microbial composite bacterial agent, and then fermenting and adsorbing the agent in a constant temperature incubator, adding a protective agent, stirring evenly to obtain a mixed bacterial agent, and drying the mixed bacterial agent in a freeze dryer until the water content is less than 5% to obtain a solid composite bacterial agent;

[0021] Preferably, the YG medium comprises 1 g of yeast extract, 1 g of glucose, 0.3 g of K2HPO4, 0.25 g of KH2PO4, 0.2 g of MgSO4, pH 7.2-7.4, and 1000 ml of water.

[0022] Preferably, the preparation method of the nutrient carrier is: after the wheat bran and bagasse activated carbon powder or coconut shell activated carbon powder are fully mixed and stirred, high-temperature sterilization is carried out in an autoclave for 1 hour, and then drying is carried out for standby use, and the temperature range of the high-temperature sterilization is 105-110°C.

[0023] Preferably, the protective agent comprises 6% starch and 2% trehalose;

[0024] The freeze-drying step is as follows: turn on the refrigerator, when the cold trap temperature reaches -40°C, evenly spread the obtained mixed bacterial solution on the material plate, the material thickness is 10mm, put the sample probe into the material, then put the material plate into the pre-freezing rack, put the pre-freezing rack into the cold trap to start pre-freezing, the time is 6-10 hours;

[0025] Continue freezing for 2-4 hours; put the pre-frozen sample into the upper part of the drying chamber, and after the cold trap temperature reaches -35℃, cover it with a glass cover, close the air release valve, start the vacuum gauge button, start the vacuum pump, and vacuum freeze-drying begins;

[0026] When the temperature of the freeze dryer gradually rises from -35℃ to 5℃, the duration is 30h-48h; when the sample is dried and the water content is below 5%, turn off the vacuum pump, turn off the vacuum gauge, slowly open the air valve, remove the glass cover, and take out the sample; grind the freeze-dried powder, pass it through a 100-mesh sieve, and you will get the solid bacterial agent; finally seal it for storage

[0027] The application of a solid composite bacterial agent for enhancing the efficiency of nitrogen removal and phosphorus removal is to introduce the composite heterotrophic nitrifying bacterial agent into a water body containing nitrogen and phosphorus at an inoculation rate of 5%-10%, and the number of live bacteria in the composite aerobic denitrification-heterotrophic nitrifying bacterial agent is 10 9 ~1010 CFU / mL, and the treatment conditions are: temperature 20 - 35 °C, pH 6 - 9, and aeration time 24 - 48 hours.

[0028] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0029] 1. By setting a solid composite bacterium agent composed of 10 microorganisms with different nitrogen and phosphorus removal functions, the present invention achieves the effects of simultaneously removing nitrogen and phosphorus in sewage, improving the degradation rate of organic matter in sewage, reducing sludge production and greenhouse gas emissions, and is an efficient biological enhancement technology;

[0030] 2. By setting the application of a solid composite bacterium agent for enhancing nitrogen and phosphorus removal efficiency in wastewater treatment, the present invention achieves the effects of not only realizing synchronous nitrification and denitrification, but also improving the phosphorus removal and degradation effects of hydrolyzing macromolecular organic substances, and is a comprehensive sewage treatment method;

[0031] 3. By setting a composite bacterium agent with characteristics such as high efficiency, shock load resistance, wide applicability, reducing treatment costs and environmental protection, the present invention achieves the effects of improving the performance and reliability of sewage treatment, and is an excellent sewage treatment material;

[0032] 4. By setting a solid composite bacterium agent with a long storage time, convenient and fast dosing, and suitable for large - scale promotion through freeze - drying technology, the present invention achieves the effects of improving the convenience and popularity of sewage treatment, and is a practical sewage treatment product. Brief Description of the Drawings

[0033] Figure 1 Schematic diagram of the composition of the solid composite bacterium agent and the composition of the culture medium of the present invention;

[0034] Figure 2 Schematic diagram of the volume ratio of the solid composite bacterium agent of the present invention;

[0035] Figure 3 Schematic diagram of the components of the LB culture medium of the present invention;

[0036] Figure 4 Schematic diagram of the components and contents of the trace elements of the present invention;

[0037] Figure 5 Schematic diagram of the components and contents of the nutrient carrier of the present invention;

[0038] Figure 6 Schematic diagram of the flow chart of the preparation method of the solid composite bacterium agent of the present invention;

[0039] Figure 7 Schematic diagram of the components of the YG culture medium of the present invention;

[0040] Figure 8 Schematic diagram of the manufacturing process flow of the nutrient carrier of the present invention;

[0041] Figure 9 Schematic diagram of the freeze-drying process flow of the present invention;

[0042] Figure 10 Schematic diagram of the application process flow of the solid composite bacterial agent of the present invention. Detailed implementation manners

[0043] Next, the technical solutions in the embodiments of the present invention will be clearly and completely described in conjunction with the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without making creative efforts shall fall within the protection scope of the present invention.

[0044] Please refer to Figures 1 to 10 , the present invention provides a technical solution: a solid composite bacterial agent for enhancing the efficiency of denitrification and phosphorus removal, including a bacterial agent and a culture medium. The bacterial agent includes Acinetobacter schindleri, Acinetobacter variabilis, Enterobacter cloacae, Lysinibacillus macroides, Lysinibacillus fusiformis, Klebsiella pneumoniae, Stenotrophomonas pavanii, Enterobacter mori, Acinetobacter baumannii, and Enterococcus hirae.

[0045] Among them, Acinetobacter schindleri, Acinetobacter variabilis, and Acinetobacter baumannii belong to aerobic denitrifying bacteria, which can use organic matter as an electron donor to reduce NO3 - to N2, while consuming the organic carbon source and reducing COD; Enterobacter cloacae, Klebsiella pneumoniae, Enterobacter mori, and Enterococcus hirae belong to heterotrophic nitrifying bacteria, which can use organic matter as an electron donor to oxidize NH4 + to NO2 - or NO3 -, while consuming organic carbon sources and reducing COD; Lysinibacillus elongatus, Lysinibacillus fusiformis, and Stenotrophomonas are phosphorus-removing bacteria that can absorb phosphates under aerobic conditions, store them as polyphosphates, and then release the phosphates under anoxic or anaerobic conditions. At the same time, they use organic matter as an electron donor to participate in processes such as denitrification or sulfate reduction, reducing COD.

[0046] The volume ratio of the bacterial agent is 2:1:1:2:1:2:2:1:1:2, namely Acinetobacter schindleri: Acinetobacter variabilis: Enterobacter cloacae: Lysinibacillus elongatus: Lysinibacillus fusiformis: Klebsiella pneumoniae: Stenotrophomonas: Enterobacter mori: Acinetobacter baumannii: Enterococcus hirae. This volume ratio is optimized through multiple experiments, which can ensure the coordination and balance among various microorganisms, making the composite bacterial agent have high denitrification and phosphorus-removing efficiency and stability.

[0047] The medium is LB medium, and its components are 10 g of peptone, 5 g of beef extract, 5 g of NaCl, and 1000 mL of distilled water. LB medium is a commonly used bacterial medium that can provide nitrogen sources, carbon sources, inorganic salts and other nutrients required by various microorganisms, promoting the growth and reproduction of microorganisms.

[0048] 0.6% of trace elements are also added to the bacterial agent, and its components are 0.16 g of zinc chloride, 0.2 g of ferrous sulfate, 0.04 g of boric acid, 0.04 g of copper sulfate, and the volume is made up to 25 mL. Trace elements are essential factors for microbial metabolism and enzyme activity, which can improve the tolerance and adaptability of microorganisms, and enhance the degradation ability and anti-inhibition ability of microorganisms.

[0049] A nutrient carrier is also added to the bacterial agent, and its components are 2 kg of wheat bran and 5% of bagasse activated carbon powder or coconut shell activated carbon powder. The nutrient carrier is a solid organic matter that can provide buffer energy and protection for microorganisms, prolong the survival time and activity of microorganisms, and can also adsorb organic matter and heavy metals in water, reducing the inhibition and interference to microorganisms.

[0050] The preparation method of the present invention is as follows:

[0051] A preparation method of a solid composite bacterial agent for enhancing denitrification and phosphorus-removing efficiency according to claim 1, comprising the following steps:

[0052] Step A: Activation of bacterial strains. The 10 kinds of microorganisms are respectively activated into original strains, and then oscillating cultures are carried out in LB medium, LB medium and YG medium respectively. When the OD 600 value is between 0.8 and 1.0 or the bacterial content is 10 9 ~10 10Take out the bacterial liquid when it reaches [[X]] CFU / mL. Strain activation is to restore microorganisms from a dormant state to an active state, enhancing their biological activity and degradation ability. LB medium and YG medium are two different media that can meet the growth requirements of different microorganisms, increasing the diversity and stability of strains. OD 600 value refers to the optical density of the bacterial liquid, which is an indicator reflecting the concentration of the bacterial liquid. The bacteria content refers to the number of bacteria per milliliter in the bacterial liquid, which is an indicator reflecting the activity of the bacterial liquid. When the OD 600 value or the bacteria content reaches a certain level, it indicates that the bacterial liquid has reached a suitable state for inoculation, and the bacterial liquid can be taken out for the next treatment.

[0053] The components of the YG medium are 1 g of yeast extract, 1 g of glucose, 0.3 g of K2HPO4, 0.25 g of KH2PO4, 0.2 g of MgSO4, pH 7.2 - 7.4, and 1000 ml of water. This medium is suitable for culturing Acinetobacter schindleri, Acinetobacter variabilis, Acinetobacter baumannii, and Enterococcus hirae, and can provide sufficient carbon sources, nitrogen sources, and phosphorus sources, as well as suitable pH and temperature conditions, to promote the growth and metabolism of these microorganisms;

[0054] Step B: Strain mixing. Mix the 10 microorganisms according to the described volume ratio to obtain a composite aerobic denitrification - heterotrophic nitrification bactericide. Inoculate the mixed bacterial liquid into the LB medium, and add the described trace elements to the medium. Incubate with constant temperature and shaking. When the total number of microorganisms is greater than 10 9 CFU / ml, collect the bactericide and aliquot it into sterile containers for storage. Strain mixing is to enable different microorganisms to cooperate synergistically to achieve synchronous nitrification - denitrification and phosphorus removal, improving the efficiency and effect of sewage treatment. LB medium is a medium suitable for the growth of various microorganisms, which can provide sufficient nutrients to promote the proliferation of the bacterial liquid. Trace elements are additives that can enhance the tolerance and adaptability of microorganisms, improving the stability and activity of the bacterial liquid. Incubation with constant temperature and shaking is a cultivation method that can ensure the uniform distribution of the bacterial liquid and oxygen supply, improving the biological activity and degradation ability of the bacterial liquid. When the total number of microorganisms reaches a certain level, it indicates that the bacterial liquid has reached a suitable state for storage, and the bactericide can be collected and aliquoted into sterile containers to prevent contamination and deterioration of the bacterial liquid.

[0055] Step C: Bacterial agent solidification. Add the aforementioned nutrient carrier to 1 L of the microbial composite bacterial agent, then conduct fermentation and adsorption culture in a constant-temperature incubator. Add a protective agent and stir evenly to obtain a mixed bacterial agent. Dry it in a freeze dryer until the water content is below 5% to obtain a solid composite bacterial agent. Bacterial agent solidification is to enable microorganisms to form a stable biofilm on the solid carrier, improve their tolerance and adaptability, and facilitate storage and use. The nutrient carrier is a solid organic matter that can provide buffer energy and protection for microorganisms, and can also adsorb organic matter and heavy metals in water, reducing the inhibition and interference to microorganisms. Fermentation and adsorption culture is a culture method that can promote the combination and adaptation of microorganisms and the carrier, and can improve the solidification effect and activity of the bacterial agent. The protective agent is an additive that can prevent microorganisms from being damaged by water loss and ice crystal formation during freeze drying, and can improve the storage effect and stability of the bacterial agent. Freeze drying is a method that can effectively remove the water in the bacterial agent, reduce the weight and volume of the bacterial agent, facilitate storage and transportation, and can improve the use effect and efficiency of the bacterial agent. When the water content drops below 5%, it indicates that the bacterial agent has reached a state suitable for storage, and a solid composite bacterial agent can be obtained and stored in a sealed manner.

[0056] The preparation method of the aforementioned nutrient carrier is as follows: fully mix and stir wheat bran and sugarcane bagasse activated carbon powder or coconut shell activated carbon powder, then conduct high-temperature sterilization in a high-pressure sterilizer for 1 hour, and then dry it for standby. The temperature range of the high-temperature sterilization is 105 - 110 °C. This nutrient carrier can provide carbon source and energy for microorganisms, and can also serve as a fixed carrier for microorganisms, increasing the contact area and biomass of microorganisms, improving the sewage treatment efficiency, and can also adsorb organic matter and heavy metals in sewage, reducing the toxicity of sewage.

[0057] The components of the protectant are 6% starch and 2% trehalose. The freeze-drying steps are as follows: Turn on the refrigerator. When the cold trap temperature reaches -40°C, evenly spread the obtained mixed bacterial liquid on the material tray. The material thickness is about 10 mm. Place the sample probe into the material, then put the material tray on the pre-freezing rack, and put the pre-freezing rack into the cold trap to start pre-freezing for 6 - 10 hours. If the material is frozen in the refrigerator (-40°C low-temperature refrigerator, pre-frozen for 6 - 10 h), then the cold trap temperature can be lowered to -35°C, and continue to freeze for 2 - 4 h. Put the pre-frozen sample on the upper part of the drying chamber. After the cold trap temperature reaches -35°C, cover the glass cover, close the air release valve, turn on the vacuum gauge key, turn on the vacuum pump, and start vacuum freeze-drying. When the temperature of the freeze-dryer gradually rises from -35°C to 5°C, the duration is 30 h - 48 h. When the sample drying is completed and the water content is below 5%, turn off the vacuum pump, turn off the vacuum gauge, slowly open the air release valve, remove the glass cover, and take out the sample. Grind the obtained freeze-dried powder and pass it through a 100-mesh sieve to obtain the solid bacterial agent. Then store it sealed. This freeze-drying step can effectively maintain the activity and stability of microorganisms, extend the storage period of the bacterial agent, and facilitate transportation and application.

[0058] Application of a solid composite bacterial agent for enhancing denitrification and phosphorus removal efficiency: The composite heterotrophic nitrifying bacterial agent is inoculated into the water body containing nitrogen and phosphorus at an inoculation amount of 5% - 10%. The viable bacteria count in the composite aerobic denitrifying - heterotrophic nitrifying bacterial agent is 10 9 ~10 10 CFU / mL, and the treatment conditions are: temperature 20 - 35°C, pH 6 - 9, and aeration time 24 - 48 hours. This application can achieve the simultaneous removal of nitrogen and phosphorus in sewage, and at the same time improve the degradation rate of organic matter in sewage, reduce sludge production and greenhouse gas emissions, and has good environmental and economic benefits.

[0059] The following are specific examples of the present invention to further illustrate the technical solutions and technical effects of the present invention:

[0060] Example 1:

[0061] The purpose of this example is to prepare a solid composite bacterial agent for enhancing denitrification and phosphorus removal efficiency and use it to treat municipal sewage. The main pollutants in municipal sewage are ammonia nitrogen, total nitrogen, total phosphorus, and organic matter, and their removal efficiency directly affects the up-to-standard discharge of sewage and the improvement of the water environment. In this example, the technical solution of the present invention is adopted to prepare a solid composite bacterial agent composed of 10 microorganisms with different denitrification and phosphorus removal functions, and put it into the municipal sewage treatment system, achieving the simultaneous removal of nitrogen and phosphorus in sewage, and at the same time improving the degradation rate of organic matter in sewage, reducing sludge production and greenhouse gas emissions, and having good environmental and economic benefits.

[0062] The specific steps are as follows:

[0063] Step A: Strain activation. The pure strains of Acinetobacter schindleri, Acinetobacter variabilis, Enterobacter cloacae, Lysinibacillus fusiformis, Lysinibacillus sphaericus, Klebsiella pneumoniae, Stenotrophomonas maltophilia, Enterobacter mori, Acinetobacter baumannii, and Enterococcus hirae, which were domestically screened, isolated, and identified from activated sludge respectively, were inoculated on LB agar plates and cultured for 24 hours. Then, single colonies were taken out and inoculated in LB liquid medium, and cultured with constant temperature and shaking (30 °C, 24 h, 180 r / min) to obtain the original strain liquid. Then, Acinetobacter schindleri, Acinetobacter variabilis, Acinetobacter baumannii, and Enterococcus hirae were respectively inoculated in YG liquid medium, and the other 6 microorganisms were inoculated in LB liquid medium, and cultured with constant temperature and shaking (30 °C, 24 h, 180 r / min), and the OD 600 value was tracked. When the OD 600 was between 0.8 and 1.0 or the bacterial content was 10 9 -10 10 CFU / mL, the bacterial liquid was taken out.

[0064] Step B: Strain mixing. The 10 kinds of microorganisms were mixed according to a volume ratio of 2:1:1:2:1:2:2:1:1:2 to obtain a composite aerobic denitrification - heterotrophic nitrification bacterial agent. The mixed bacterial liquid was inoculated into LB medium, and 0.6% trace elements were added to the medium, and cultured with constant temperature and shaking (30 °C, 24 h, 180 r / min). When the total number of microorganisms was greater than 10 9 CFU / ml, the bacterial agent was collected and sub-packed in a sterile container for storage.

[0065] Step C: Bacterial agent solidification. Add a nutrient carrier to 1 L of the microbial composite bacterial agent (the ratio of bacterial liquid to carrier is 2:1), then ferment and adsorb and culture in a constant temperature incubator at 30 - 35 °C for 20 - 24 hours, add a protective agent, and stir evenly to obtain a mixed bacterial agent, and dry it in a freeze dryer until the water content is less than 5% to obtain the solid composite bacterial agent. This step can effectively maintain the activity and stability of microorganisms, extend the storage period of the bacterial agent, and facilitate transportation and dosing.

[0066] Step D: Bacterial agent application. The composite heterotrophic nitrification bacterial agent was inoculated into the municipal sewage treatment system at an inoculation amount of 5% - 10%. The viable bacteria count in the composite aerobic denitrification - heterotrophic nitrification bacterial agent was 10 9 -10 10 CFU / mL, and the treatment conditions were: temperature 20 - 35 °C, pH 6 - 9, and aeration time 24 - 48 hours. This step can achieve the simultaneous removal of nitrogen and phosphorus in sewage, improve the degradation rate of organic matter in sewage, reduce sludge production and greenhouse gas emissions, and has good environmental and economic benefits.

[0067] The following are the application effects and advantages of this embodiment:

[0068] In order to verify the application effects and advantages of this embodiment, a comparative experiment was conducted in this embodiment. The composite bacterial agent of the present invention was compared with the commonly used single bacterial agent on the market, and they were respectively put into the municipal sewage treatment system. Indicators such as ammonia nitrogen, total nitrogen, total phosphorus, COD, sludge volume, and greenhouse gas emissions in the sewage were observed and measured. The results are shown in the following table:

[0069]

[0070]

[0071] As can be seen from the above table, the application of the composite bacterial agent of the present invention in sewage treatment not only realizes the simultaneous removal of nitrogen and phosphorus in sewage, but also can improve the degradation rate of organic matter in sewage, reduce sludge production and greenhouse gas emissions. Compared with the control group, it has significant advantages and achieves the expected purpose of this embodiment.

[0072] Example 2:

[0073] The purpose of this embodiment is to prepare a solid composite bacterial agent for enhancing the efficiency of nitrogen and phosphorus removal and use it to treat industrial sewage. The main pollutants in industrial sewage are ammonia nitrogen, total nitrogen, total phosphorus, and organic matter, and their removal efficiency directly affects the up-to-standard discharge of sewage and the improvement of the water environment. In this embodiment, the technical solution of the present invention is adopted to prepare a solid composite bacterial agent composed of 10 kinds of microorganisms with different nitrogen and phosphorus removal functions, and it is put into the industrial sewage treatment system, realizing the simultaneous removal of nitrogen and phosphorus in sewage, and at the same time can improve the degradation rate of organic matter in sewage, reduce sludge production and greenhouse gas emissions, and has good environmental and economic benefits.

[0074] The specific steps are as follows:

[0075] Step A: Activation of bacterial strains, which is the same as in Example 1 and will not be elaborated here.

[0076] Step B: Mixing of bacterial strains, which is the same as in Example 1 and will not be elaborated here.

[0077] Step C: Solidification of the bacterial agent, which is the same as in Example 1 and will not be elaborated here.

[0078] Step D: Application of the bacterial agent. The composite heterotrophic nitrifying bacterial agent is inoculated into the industrial sewage treatment system at an inoculation amount of 6%-10%. The viable bacteria count in the composite aerobic denitrifying-heterotrophic nitrifying bacterial agent is 10 9 ~10 10CFU / mL, and the treatment conditions are: temperature 20 - 35 °C, pH 6 - 9, and aeration time 24 - 48 hours. This step can achieve the simultaneous removal of nitrogen and phosphorus in sewage, improve the degradation rate of organic matter in sewage, reduce sludge production and greenhouse gas emissions, and has good environmental and economic benefits.

[0079] The following are the application effects and advantages of this example:

[0080] To verify the application effects and advantages of this example, a comparative experiment was conducted in this example. The composite bacterial agent of the present invention was compared with the commonly used single bacterial agents on the market and was respectively put into the industrial sewage treatment system. The indexes such as ammonia nitrogen, total nitrogen, total phosphorus, COD, sludge volume and greenhouse gas emissions in the sewage were observed and measured. The results are shown in the following table:

[0081]

[0082] As can be seen from the above table, the application of the composite bacterial agent of the present invention in industrial sewage treatment not only achieves the simultaneous removal of nitrogen and phosphorus in sewage, but also can improve the degradation rate of organic matter in sewage, reduce sludge production and greenhouse gas emissions. Compared with the control group, it has significant advantages and achieves the expected purpose of this example.

[0083] Example 3: The purpose of this example is to prepare a solid composite bacterial agent for enhancing the efficiency of nitrogen and phosphorus removal and use it to treat agricultural sewage. The main pollutants in agricultural sewage are ammonia nitrogen, total nitrogen, total phosphorus and organic matter, and their removal efficiency directly affects the up-to-standard discharge of sewage and the improvement of water environment. This example adopts the technical solution of the present invention to prepare a solid composite bacterial agent composed of 10 kinds of microorganisms with different nitrogen and phosphorus removal functions and puts it into the agricultural sewage treatment system, achieving the simultaneous removal of nitrogen and phosphorus in sewage, improving the degradation rate of organic matter in sewage, reducing sludge production and greenhouse gas emissions, and having good environmental and economic benefits.

[0084] The specific steps are as follows:

[0085] Step A: Activation of bacterial strains, which is the same as in Example 1 and will not be elaborated here.

[0086] Step B: Mixing of bacterial strains, which is the same as in Example 1 and will not be elaborated here.

[0087] Step C: Solidification of bacterial agent, which is the same as in Example 1 and will not be elaborated here.

[0088] Step D: Application of bacterial agent. The composite heterotrophic nitrification bacterial agent is inoculated into the agricultural sewage treatment system at an inoculation amount of 7% - 10%. The viable bacteria count in the composite aerobic denitrification - heterotrophic nitrification bacterial agent is 10 9 ~10 10CFU / mL, and the treatment conditions are: temperature 20 - 35°C, pH 6 - 9, and aeration time 24 - 48 hours. This step can achieve the simultaneous removal of nitrogen and phosphorus in sewage, improve the degradation rate of organic matter in sewage, reduce sludge production and greenhouse gas emissions, and has good environmental and economic benefits.

[0089] The following are the application effects and advantages of this embodiment:

[0090] To verify the application effects and advantages of this embodiment, a comparative experiment was conducted in this embodiment. The composite bacterial agent of the present invention was compared with the commonly used single bacterial agents on the market, and they were respectively put into the agricultural sewage treatment system. Indexes such as ammonia nitrogen, total nitrogen, total phosphorus, COD, sludge volume, and greenhouse gas emissions in the sewage were observed and measured. The results are shown in the following table:

[0091]

[0092] As can be seen from the above table, the application of the composite bacterial agent of the present invention in agricultural sewage treatment not only realizes the simultaneous removal of nitrogen and phosphorus in sewage, but also improves the degradation rate of organic matter in sewage, reduces sludge production and greenhouse gas emissions. Compared with the control group, it has significant advantages and achieves the expected purpose of this embodiment.

[0093] Note: The water sample detection methods used in the above embodiments are as follows: NH4 + -N (ammonia nitrogen) is determined by Nessler's reagent spectrophotometry (HJ 535 - 2009); NO3 - -N (nitrate nitrogen) is determined by ultraviolet spectrophotometry (trial) (HJ / T 346 - 2007); TN (total nitrogen) is determined by alkaline potassium persulfate digestion ultraviolet spectrophotometry (HJ 636 - 2012); TP (total phosphorus) is determined by ammonium molybdate spectrophotometry (GB / T 11893 - 1989). When detecting NH4 + -N, NO3 - -N, TN, and TP, the supernatant of the bacterial culture solution after centrifugation (8000 r / min, 2 min) is used.

[0094] Although the embodiments of the present invention have been shown and described, for those of ordinary skill in the art, it can be understood that various changes, modifications, substitutions, and variations can be made to these embodiments without departing from the principles and spirit of the present invention. The scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A solid composite bacterial agent for enhancing the efficiency of denitrification and phosphorus removal, comprising a bacterial agent and a culture medium, characterized in that: The microbial agent includes Acinetobacter schindleri, Acinetobacter variabilis, Enterobacter cloacae, Lysinibacillus macroides, Lysinibacillus fusiformis, Klebsiella pneumoniae, Stenotrophomonas pavanii, Enterobacter mori, Acinetobacter baumannii, and Enterococcus hirae.

2. The solid composite bacterial agent for enhancing the efficiency of denitrification and phosphorus removal according to claim 1, wherein: The volume ratio of the microbial agent is 2:1:1:2:1:2:2:1:1:2, that is, Acinetobacter schindleri: Acinetobacter variabilis: Enterobacter cloacae: Lysinibacillus macroides: Lysinibacillus fusiformis: Klebsiella pneumoniae: Stenotrophomonas pavanii: Enterobacter mori: Acinetobacter baumannii: Enterococcus hirae.

3. The solid composite bacterial agent for enhancing the denitrification and phosphorus removal efficiency according to claim 1, characterized in that: The medium is LB medium, and its components are 10 g of peptone, 5 g of beef extract, 5 g of NaCl, and 1000 mL of distilled water.

4. A solid composite bacterial agent for enhancing the efficiency of denitrification and phosphorus removal according to claim 1, characterized in that: 0.6% of trace elements are also added to the microbial agent, and its components are 0.16 g of zinc chloride, 0.2 g of ferrous sulfate, 0.04 g of boric acid, 0.04 g of copper sulfate, and the volume is made up to 25 mL.

5. A solid composite bacterial agent for enhancing the efficiency of denitrification and phosphorus removal according to claim 1, characterized in that: A nutrient carrier is also added to the microbial agent, and its components are 2 kg of wheat bran and 5% of bagasse activated carbon powder or coconut shell activated carbon powder.

6. A method for preparing a solid composite bacterial agent for enhancing the efficiency of denitrification and phosphorus removal according to claim 1, characterized in that: It includes the following steps: Step A: Strain activation. Activate the 10 microorganisms into original strains respectively, and then culture them by shaking in LB medium, LB medium and YG medium. When the OD 600 value is between 0.8 and 1.0 or the bacterial content is 10 9 to 10 10 CFU / mL, take out the bacterial liquid; Step B: Bacterial strain mixing. Mix the 10 kinds of microorganisms according to the volume ratio to obtain a composite aerobic denitrification-heterotrophic nitrification bactericide; inoculate the mixed bacterial solution into LB medium, add the trace elements to the medium, and culture it under constant temperature and shaking. When the total number of bacteria is greater than 10 9 CFU / ml, collect the bactericide and dispense it into sterile containers for storage; Step C: Solidification of the microbial agent. Add the above-mentioned nutrient carrier to 1 L of the microbial complex agent, then ferment and adsorb and culture in a constant temperature incubator, add a protective agent, stir evenly to obtain a mixed microbial agent, and dry it in a freeze dryer until the water content is below 5% to obtain a solid composite microbial agent.

7. The preparation method of a solid composite bacterial agent for enhancing the efficiency of denitrification and phosphorus removal according to claim 1, characterized in that: The components of the YG medium are 1 g of yeast extract, 1 g of glucose, 0.3 g of K2HPO4, 0.25 g of KH2PO4, 0.2 g of MgSO4, pH 7.2 - 7.4, and 1000 ml of water.

8. The preparation method of a solid composite bacterial agent for enhancing the efficiency of denitrification and phosphorus removal according to claim 1, characterized in that: The preparation method of the nutrient carrier is: fully mix and stir wheat bran and bagasse activated carbon powder or coconut shell activated carbon powder, then perform high-temperature sterilization in a high-pressure sterilizer for 1 hour, and then dry it for standby. The temperature range of the high-temperature sterilization is 105 - 110°C.

9. The preparation method of a solid composite bacterial agent for enhancing the efficiency of denitrification and phosphorus removal according to claim 1, characterized in that: The components of the protective agent are 6% of starch and 2% of trehalose; The freeze-drying step is: Turn on the refrigerating machine. When the temperature of the cold trap reaches -40°C, evenly spread the obtained mixed bacterial liquid on the material tray, with the material thickness being 10 mm. Place the sample probe into the material, then place the material tray on the pre-freezing rack, and put the pre-freezing rack into the cold trap to start pre-freezing for 6 - 10 hours; Continue to freeze for 2 - 4 h; Put the pre-frozen sample in the upper part of the drying chamber. After the cold trap temperature reaches -35°C, cover the glass cover, close the air release valve, press the vacuum gauge key, start the vacuum pump, and start vacuum freeze-drying; When the temperature of the freeze dryer gradually rises from -35°C to 5°C, the duration is 30 h - 48 h; When the sample drying is completed and the water content is below 5%, turn off the vacuum pump, turn off the vacuum gauge, slowly open the air release valve, remove the glass cover, and take out the sample; Grind the obtained freeze-dried powder, pass through a 100-mesh sieve to obtain the solid microbial agent; Finally, store it sealed.

10. Use of a solid composite bacterial agent for enhancing denitrification and phosphorus removal efficiency according to claim 1, characterized in that: The composite heterotrophic nitrifying bacteria agent is inoculated into the nitrogen- and phosphorus-containing water body at an inoculation amount of 5% - 10%, and the viable bacteria count in the composite aerobic denitrifying-heterotrophic nitrifying bacteria agent is 10 9 ~10 10 CFU / mL, and the treatment conditions are: temperature 20 - 35°C, pH 6 - 9, and aeration time 24 - 48 hours.

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