Compound bacterial agent for nitrogen and carbon removal of leachate as well as preparation method and application of compound bacterial agent

Abstract

The invention discloses a compound bacterial agent for nitrogen and carbon removal of leachate as well as a preparation method and application of the compound bacterial agent, belongs to the technical field of microorganism application, and particularly relates to the compound bacterial agent for nitrogen and carbon removal of leachate, which comprises a lactobacillus plantarum bacterial solution, a bacillus subtilis bacterial solution, a nitrosomonas bacterial solution and a thauera bacterial solution. The invention provides application of the compound bacterial agent in nitrogen and carbon removal of leachate. The complex water quality characteristics of the leachate are fully considered, high COD is used for denitrification, efficient COD removal and stable denitrification are achieved, and specifically, the removal rate of COD is 93.4%, and the removal rate of TN is 99.3%.

Description

Technical Field

[0001] The present invention belongs to the technical field of microbial application, and in particular relates to a composite bacterial agent for denitrification and decarbonization of leachate, and a preparation method and application thereof. Background Art

[0002] Leachate is mainly high-concentration organic wastewater produced by factors such as precipitation, surface water inflow, groundwater infiltration, and the water content of the garbage itself during the processes of garbage stacking, sorting, compression, landfill and fermentation. Its water quality is complex, with extremely high COD concentrations, generally around 60,000 mg / L and sometimes even reaching 100,000 mg / L. Nitrogen exists mainly in the form of ammonia nitrogen, with ammonia nitrogen concentration reaching as high as 1,500-2,500 mg / L. There is also an imbalance in the ratio of nutrient elements, manifested in an imbalance in the carbon, nitrogen, and phosphorus required by microorganisms in the leachate, with phosphorus being particularly scarce, with a BOD5:P ratio greater than 300. The heavy metal and salt content is also high, with the leachate containing a variety of metal ions such as iron, zinc, lead, and calcium. The iron concentration can reach 2,000 mg / L, and the zinc concentration can reach 130 mg / L. At the same time, the leachate contains large amounts of sodium, potassium, calcium, and magnesium salts. The total dissolved solids (TDS) concentration is often as high as over 10,000 mg / L, and the electrical conductivity can also reach 10,000-45,000 μS / cm. These characteristics make leachate treatment difficult. If directly discharged without proper treatment, these nitrogen pollutants will cause serious and lasting damage to the soil, water and other ecological environments.

[0003] In recent years, microbial complex agents have become an important tool for leachate denitrification and carbon removal due to their high efficiency, environmental friendliness, and adaptability. However, existing technologies have limited efficiency in denitrification and carbon removal using single strains or simple complex agents, making them inadequate for treating complex leachates. Furthermore, the microorganisms in most agents lack effective synergy, resulting in incomplete and inefficient denitrification and carbon removal.

[0004] As a highly efficient biofilm reactor, the bio-rotating disc is widely used in wastewater treatment. Its denitrification and carbon removal effectiveness also depends on the performance of functional bacterial agents. However, existing technologies have limited denitrification and carbon removal efficiencies for single strains or simple compound bacterial agents, making them difficult to meet the treatment requirements of complex leachates. Summary of the Invention

[0005] To solve the above technical problems, the present invention proposes a composite bacterial agent for denitrification and carbon removal of leachate, as well as its preparation method and application. The composite bacterial agent provided by the present invention fully considers the complex water quality characteristics of the leachate, utilizes high COD for denitrification, and realizes efficient COD removal and stable denitrification, specifically, the removal rates of COD and TN are 93.4% and 99.3%, respectively.

[0006] To achieve the above object, the present invention provides a compound bacterium agent for denitrification and carbon removal of leachate, and the compound bacterium agent includes Lactobacillus plantarum ( Lactobacillus plantarum ), Bacillus subtilis ( Bacillus subtilis ), Nitrosomonas ( Nitrosomonas ), and Thauera ( Thauera ).

[0007] Preferably, the volume ratio of the Lactobacillus plantarum bacterium solution, Bacillus subtilis bacterium solution, Nitrosomonas bacterium solution, and Thauera bacterium solution in the compound bacterium agent is 4:4:1:1.

[0008] Preferably, the effective viable count of the Lactobacillus plantarum bacterium solution is 10 9 ~10 10 CFU / mL, and the Lactobacillus plantarum bacterium solution is the bacterium solution of Lactobacillus plantarum with the preservation number of CGMCC 1.16089; the effective viable count of the Bacillus subtilis bacterium solution is 10 9 ~10 10 CFU / mL, and the Bacillus subtilis bacterium solution is the bacterium solution of Bacillus subtilis with the preservation number of CGMCC 1.821.

[0009] Preferably, the preparation of the Nitrosomonas bacterium solution includes the following steps: inoculating activated sludge into liquid medium III at an inoculation amount of 3~7% v / v, performing the first aerobic constant temperature culture at 23~27°C and 110~150 rpm for 60~84 h, transferring and inoculating into a new liquid medium III at an inoculation amount of 3~7% v / v, performing the second aerobic constant temperature culture at 23~27°C and 110~150 rpm for 60~84 h, and inoculating into a new liquid medium III at an inoculation amount of 3~7% v / v again, performing the third aerobic constant temperature culture at 23~27°C and 110~150 rpm for 60~84 h to obtain the Nitrosomonas bacterium solution; The formula of the liquid medium III is: ammonium sulfate 0.25 g / L, ammonium chloride 0.25 g / L, sodium bicarbonate 1.6 g / L, sodium chloride 0.3 g / L, disodium hydrogen phosphate 1 g / L, magnesium sulfate 0.03 g / L, calcium chloride 7.5 g / L, trace elements 1 ml / L, adding water to make up the volume to 1 L, and the pH of the liquid medium III is 7.5.

[0010] Preferably, the preparation of the Thauera sp. bacterial liquid comprises the following steps: inoculating activated sludge into the fourth liquid medium at an inoculation amount of 3-7% v / v, performing the first anaerobic constant-temperature cultivation at 28-32 °C and 110-150 rpm for 60-84 h, transferring and inoculating into a new fourth liquid medium at an inoculation amount of 3-7% v / v, performing the second anaerobic constant-temperature cultivation at 28-32 °C and 110-150 rpm for 60-84 h, and again inoculating into a new fourth liquid medium at an inoculation amount of 3-7% v / v, performing the third anaerobic constant-temperature cultivation at 28-32 °C and 110-150 rpm for 60-84 h to obtain the Thauera sp. bacterial liquid; The formula of the fourth liquid medium is as follows: sodium humate 8 g / L, sodium fulvate 6 g / L, acetic acid 3 g / L, glucose 2 g / L, peptone 1 g / L, sodium nitrate 2 g / L, sodium chloride 0.3 g / L, disodium hydrogen phosphate 1 g / L, magnesium sulfate 0.03 g / L, trace elements 1 ml / L, adding water to make the volume up to 1 L, and the pH of the fourth liquid medium is 7.5.

[0011] The present invention also provides a preparation method of the compound microbial agent, comprising the following steps: 1) Inoculating Lactobacillus plantarum into the first liquid medium, performing anaerobic constant-temperature cultivation at 40-44 °C and 110-150 rpm for 36-60 h to obtain the Lactobacillus plantarum bacterial liquid; 2) Inoculating Bacillus subtilis into the second liquid medium, performing aerobic constant-temperature cultivation at 35-39 °C and 110-150 rpm for 12-36 h to obtain the Bacillus subtilis bacterial liquid; 3) Inoculating activated sludge into the third liquid medium at an inoculation amount of 3-7% v / v, performing the first aerobic constant-temperature cultivation at 23-27 °C and 110-150 rpm for 60-84 h, transferring and inoculating into a new third liquid medium at an inoculation amount of 3-7% v / v, performing the second aerobic constant-temperature cultivation at 23-27 °C and 110-150 rpm for 60-84 h, and again inoculating into a new third liquid medium at an inoculation amount of 3-7% v / v, performing the third aerobic constant-temperature cultivation at 23-27 °C and 110-150 rpm for 60-84 h to obtain the Nitrosomonas bacterial liquid; 4) Inoculating activated sludge into the fourth liquid medium at an inoculation amount of 3-7% v / v, performing the first anaerobic constant-temperature cultivation at 28-32 °C and 110-150 rpm for 60-84 h, transferring and inoculating into a new fourth liquid medium at an inoculation amount of 3-7% v / v, performing the second anaerobic constant-temperature cultivation at 28-32 °C and 110-150 rpm for 60-84 h, and again inoculating into a new fourth liquid medium at an inoculation amount of 3-7% v / v, performing the third anaerobic constant-temperature cultivation at 28-32 °C and 110-150 rpm for 60-84 h to obtain the Thauera sp. bacterial liquid; 5) Mix the Lactobacillus plantarum bacterial liquid obtained in step 1), the Bacillus subtilis bacterial liquid obtained in step 2), the Nitrosomonas bacterial liquid obtained in step 3), and the Thauera bacterial liquid obtained in step 4) according to a volume ratio of 4:4:1:1 to obtain a mixed bacterial liquid. Centrifuge the mixed bacterial liquid, take the precipitate, and resuspend the precipitate with a sodium chloride solution with a mass fraction of 0.9% to the same volume as the mixed bacterial liquid to obtain a compound microbial agent.

[0012] Preferably, the inoculation amount of the Lactobacillus plantarum in step 1) is 0.5 - 1.5% v / v; the formula of the first liquid medium in step 1) is: sodium humate 13.6 g / L, sodium fulvate 10.2 g / L, acetic acid 5.1 g / L, glucose 3.4 g / L, peptone 1.7 g / L, dipotassium hydrogen phosphate 2 g / L, sodium acetate trihydrate 5 g / L, ammonium citrate 2 g / L, magnesium sulfate heptahydrate 0.2 g / L, manganese sulfate tetrahydrate 0.2 g / L, sodium chloride 60 g / L, add water to make up to 1 L, and the pH of the first liquid medium is 6.2.

[0013] Preferably, the inoculation amount of the Bacillus subtilis in step 2) is 0.5 - 1.5% v / v; the formula of the second liquid medium in step 2) is: sodium humate 4 g / L, sodium fulvate 3 g / L, acetic acid 1.5 g / L, glucose 1.5 g / L, peptone 5 g / L, yeast extract 5 g / L, sodium chloride 5 g / L, trace elements 1 ml / L, add water to make up to 1 L, and the pH of the second liquid medium is 7.1.

[0014] Preferably, the formula of the third liquid medium in step 3) is: ammonium sulfate 0.25 g / L, ammonium chloride 0.25 g / L, sodium bicarbonate 1.6 g / L, sodium chloride 0.3 g / L, disodium hydrogen phosphate 1 g / L, magnesium sulfate 0.03 g / L, calcium chloride 7.5 g / L, trace elements 1 ml / L, add water to make up to 1 L, and the pH of the third liquid medium is 7.5; the formula of the fourth liquid medium in step 4) is: sodium humate 8 g / L, sodium fulvate 6 g / L, acetic acid 3 g / L, glucose 2 g / L, peptone 1 g / L, sodium nitrate 2 g / L, sodium chloride 0.3 g / L, disodium hydrogen phosphate 1 g / L, magnesium sulfate 0.03 g / L, trace elements 1 ml / L, add water to make up to 1 L, and the pH of the fourth liquid medium is 7.5.

[0015] The present invention also provides the application of the compound microbial agent in denitrification and carbon removal of leachate.

[0016] Compared with the prior art, the present invention has the following advantages and technical effects: The present invention provides a compound bacterium agent for leachate denitrification and carbon removal, its preparation method and application. The compound bacterium agent fully considers the complex water quality characteristics of leachate, especially its high ammonia nitrogen, high organic matter content and variable environmental conditions. First, the Lactobacillus plantarum bacterium solution in the compound bacterium agent decomposes the organic matter in leachate through fermentation, providing sufficient carbon source and suitable acid-base environment for subsequent nitrification and denitrification processes, thus promoting the efficient operation of the entire denitrification system. The Bacillus subtilis bacterium solution, with its strong tolerance and rapid reproduction ability, maintains stable activity under harsh conditions of high ammonia nitrogen and high salinity, accelerating the initial conversion of ammonia nitrogen. The Nitrosomonas bacterium solution, as the core strain in the nitrification process, can efficiently oxidize ammonia nitrogen into nitrite and nitrate, providing necessary intermediate products for the denitrification process. And the Thauera bacterium solution reduces nitrate to nitrogen gas, ultimately achieving the complete removal of nitrogen in leachate.

[0017] The synergistic effect of the compound bacterium agent of the present invention not only optimizes the denitrification and carbon removal process, but also significantly improves the shock resistance and stability of the system. It can operate efficiently within a wide pH value range and different dissolved oxygen conditions, reducing the strict dependence on environmental conditions. In addition, the use of the compound bacterium agent of the present invention does not require complex pretreatment and post-treatment processes, greatly reducing the operation difficulty and cost. In practical applications, the compound bacterium agent of the present invention can not only significantly shorten the treatment cycle of leachate, but also effectively reduce the sludge production, reducing the burden of subsequent sludge treatment. Its high denitrification ability and environmental adaptability make it have broad application prospects in the field of leachate treatment, providing an economic, efficient and sustainable solution to the problem of treating high ammonia nitrogen wastewater. Compared with the prior art, the compound bacterium agent of the present invention shows significant innovation and practicality. The effect of treating leachate by a biological rotating disk shows that the compound bacterium agent prepared by the present invention can achieve efficient COD removal and stable denitrification. Specifically, the removal rates of COD and TN are 93.4% and 99.3% respectively, compared with the removal rates of COD and TN of 56.1% and 55.6% by ordinary activated sludge, which are increased by 37.3% and 43.7%. Brief Description of the Drawings

[0018] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required to be used in the embodiments. Obviously, the drawings in the following description are only some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can also be obtained based on these drawings.

[0019] Figure 1 It is the 16s RNA high-throughput sequencing species annotation result of the compound bacterium agent prepared in Example 1 of the present invention; Figure 2This is the effect diagram of nitrogen and carbon removal from landfill leachate by the compound bacterium agent-enhanced activated sludge prepared in Example 1 of the present invention and ordinary activated sludge. Among them, A is the effect diagram of nitrogen and carbon removal from landfill leachate by the compound bacterium agent-enhanced activated sludge prepared in Example 1, and B is the effect diagram of nitrogen and carbon removal from landfill leachate by ordinary activated sludge; Figure 3 This is the comparison diagram of the nitrogen and carbon removal effects of the compound bacterium agent-enhanced activated sludge prepared in Example 1 of the present invention and ordinary activated sludge for treating landfill leachate; Figure 4 This is the microbial composition of the activated sludge of Guangzhou Panyu Sewage Treatment Co., Ltd. (Nancun Water Quality Purification Plant); Figure 5 This is the microbial composition of the activated sludge of the industrial water treatment center of Yamen Fortune Environmental Protection Industrial Park in Jiangmen City. Detailed implementation manners

[0020] The various exemplary implementation manners of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, characteristics, and implementation schemes of the present invention.

[0021] It should be understood that the terms used in the present invention are only for describing specific implementation manners and are not used to limit the present invention. In addition, for the numerical ranges in the present invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Each intermediate value within any stated value or stated range, as well as each smaller range between any other stated value or intermediate value within the stated range, is also included in the present invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0022] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the present invention pertains. Although the present invention only describes preferred methods and materials, any methods and materials similar or equivalent to those described herein can also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials related to the documents. In case of conflict with any incorporated document, the content of this specification shall prevail.

[0023] Without departing from the scope or spirit of the present invention, various improvements and changes can be made to the specific implementation manners of the present invention specification, which are obvious to those skilled in the art. Other implementation manners obtained from the specification of the present invention are obvious to those skilled in the art. The specification and examples of the present invention are only exemplary.

[0024] Regarding the terms "comprising", "including", "having", "containing", etc. used herein, they are all open-ended terms, that is, they are intended to include but not be limited to.

[0025] The Lactobacillus plantarum used in the present invention was purchased from the China General Microbiological Culture Collection Center, with the preservation number CGMCC 1.16089; the Bacillus subtilis used was purchased from the China General Microbiological Culture Collection Center, with the preservation number CGMCC 1.821.

[0026] The activated sludge used in the present invention was taken from Guangzhou Panyu Sewage Treatment Co., Ltd. (Nancun Water Quality Purification Plant). It was taken from an artificial engineering system - a municipal sewage treatment plant. Since the sewage quality is similar, and at present, the water treatment process in China still mainly uses the activated sludge method, the core denitrifying functional bacteria in the activated sludge of each municipal sewage treatment plant are similar. As Figure 4 shown and Figure 5 shown, in Guangzhou Panyu Sewage Treatment Co., Ltd. (Nancun Water Quality Purification Plant) ( Figure 4 ), and the industrial water treatment center of Jiangmen Yamen Fortune Environmental Protection Industrial Park ( Figure 5 ), the 16s rRNA sequencing results of the activated sludge samples taken, for Guangzhou Panyu Sewage Treatment Co., Ltd. (Nancun Water Quality Purification Plant) Nitrosomonas and Thauera bacteria, the relative abundances were 8.13% and 1.67% respectively. In the industrial water treatment center of Jiangmen Yamen Fortune Environmental Protection Industrial Park Nitrosomonas bacteria and Thauera bacteria also stably existed. Because Nitrosomonas and Thauera bacteria are the core functional bacteria in the nitrification and denitrification processes of the denitrification treatment of the sewage activated sludge method respectively, they are therefore widely present in the sewage treatment systems using the activated sludge method. Therefore, by using the method of this patent and adding a targeted culture medium, the target microorganisms can be effectively enriched. In addition, the microbial composition results of the compound microbial agent of the present invention were obtained through three parallel repeated experiments (as Figure 1 shown), which can ensure that the top four absolute dominant strains in the microbial agent are the target microorganisms of the present invention. Therefore, the present invention can ensure repeatability.

[0027] The specific composition of the trace elements in the first liquid culture medium, the second liquid culture medium, the third liquid culture medium, and the fourth liquid culture medium used in the present invention: EDTA is 7.5 g / L, CuSO4·5H2O is 0.125 g / L, CoCl2·6H2O is 0.35 g / L, Na2MoO4·2H2O is 0.32 g / L, NaSeO4·10H2O is 0.31 g / L, ZnSO4·7H2O is 0.65 g / L, MnCl2·4H2O is 0.50 g / L, NiCl2·6H2O is 0.28 g / L, H3BO4 is 0.015 g / L. The specific ratio is to add 1 mL of trace elements to each liter of distilled water and mix them evenly.

[0028] The liquid medium 1, liquid medium 2, liquid medium 3, and liquid medium 4 used in the present invention are all sterilized by high-pressure steam. The specific conditions are a high temperature of 121°C and a pressure of 103.4 kPa.

[0029] The formula of the liquid medium 1 used in the present invention is as follows: sodium humate 13.6 g / L, sodium fulvate 10.2 g / L, acetic acid 5.1 g / L, glucose 3.4 g / L, peptone 1.7 g / L, dipotassium hydrogen phosphate 2 g / L, sodium acetate trihydrate 5 g / L, ammonium citrate 2 g / L, magnesium sulfate heptahydrate 0.2 g / L, manganese sulfate tetrahydrate 0.2 g / L, sodium chloride 60 g / L. Add water to make the volume up to 1 L. The pH of the liquid medium 1 is 6.2.

[0030] The formula of the liquid medium 2 used in the present invention is as follows: sodium humate 4 g / L, sodium fulvate 3 g / L, acetic acid 1.5 g / L, glucose 1.5 g / L, peptone 5 g / L, yeast extract 5 g / L, sodium chloride 5 g / L, trace elements 1 ml / L. Add water to make the volume up to 1 L. The pH of the liquid medium 2 is 7.1.

[0031] The formula of the liquid medium 3 used in the present invention is as follows: ammonium sulfate 0.25 g / L, ammonium chloride 0.25 g / L, sodium bicarbonate 1.6 g / L, sodium chloride 0.3 g / L, disodium hydrogen phosphate 1 g / L, magnesium sulfate 0.03 g / L, calcium chloride 7.5 g / L, trace elements 1 ml / L. Add water to make the volume up to 1 L. The pH of the liquid medium 3 is 7.5.

[0032] The formula of the liquid medium 4 used in the present invention is as follows: sodium humate 8 g / L, sodium fulvate 6 g / L, acetic acid 3 g / L, glucose 2 g / L, peptone 1 g / L, sodium nitrate 2 g / L, sodium chloride 0.3 g / L, disodium hydrogen phosphate 1 g / L, magnesium sulfate 0.03 g / L, trace elements 1 ml / L. Add water to make the volume up to 1 L. The pH of the liquid medium 4 is 7.5.

[0033] Example 1 1) Lactobacillus plantarum was inoculated into the liquid medium 1 at an inoculation amount of 1% v / v, and anaerobically cultured at a constant temperature of 42°C and 130 rpm for 48 h to obtain a Lactobacillus plantarum bacterial liquid (the effective viable count was 10 9 CFU / mL).

[0034] 2) Bacillus subtilis was inoculated into the liquid medium 2 at an inoculation amount of 1% v / v, and aerobically cultured at a constant temperature of 37°C and 130 rpm for 24 h to obtain a Bacillus subtilis bacterial liquid (the effective viable count was 10 9 CFU / mL).

[0035] 3) Inoculate the activated sludge into the third liquid medium at an inoculation amount of 5% v / v, and perform the first aerobic constant-temperature culture at 25°C and 130 rpm for 72 h. Then transfer and inoculate it into a new third liquid medium at an inoculation amount of 5% v / v, and perform the second aerobic constant-temperature culture at 25°C and 130 rpm for 72 h. Again, inoculate it into a new third liquid medium at an inoculation amount of 5% v / v, and perform the third aerobic constant-temperature culture at 25°C and 130 rpm for 72 h to obtain a Nitrosomonas bacterium solution.

[0036] 4) Inoculate the activated sludge into the fourth liquid medium at an inoculation amount of 5% v / v, and perform the first anaerobic constant-temperature culture at 30°C and 130 rpm for 72 h. Then transfer and inoculate it into a new fourth liquid medium at an inoculation amount of 5% v / v, and perform the second anaerobic constant-temperature culture at 30°C and 130 rpm for 72 h. Again, inoculate it into a new fourth liquid medium at an inoculation amount of 5% v / v, and perform the third anaerobic constant-temperature culture at 30°C and 130 rpm for 72 h to obtain a Thauera bacterium solution.

[0037] 5) Mix the Lactobacillus plantarum bacterium solution, Bacillus subtilis bacterium solution, Nitrosomonas bacterium solution, and Thauera bacterium solution according to a volume ratio of 4:4:1:1 to obtain a mixed bacterium solution. Centrifuge it at 4000 r / min, take the precipitate, and resuspend the precipitate with a 0.9% mass fraction sodium chloride solution to the same volume as the mixed bacterium solution to obtain a compound microbial agent.

[0038] The results of 16S RNA high-throughput sequencing species annotation of the compound microbial agent prepared in this example are as Figure 1 shown, which shows the specific bacterial species and composition of the compound microbial agent, and shows that in addition to Lactobacillus plantarum and Bacillus subtilis, the dominant bacteria in the compound microbial agent also include Nitrosomonas bacteria and Thauera bacteria.

[0039] Example 2 1) Inoculate Lactobacillus plantarum into the first liquid medium at an inoculation amount of 0.5% v / v, and perform the anaerobic constant-temperature culture at 40°C and 110 rpm for 36 h to obtain a Lactobacillus plantarum bacterium solution (the effective viable count is 10 9 CFU / mL).

[0040] 2) Inoculate Bacillus subtilis into the second liquid medium at an inoculation amount of 0.5% v / v, and perform the aerobic constant-temperature culture at 35°C and 110 rpm for 12 h to obtain a Bacillus subtilis bacterium solution (the effective viable count is 10 10 CFU / mL).

[0041] 3) Inoculate the activated sludge into the third liquid medium at an inoculation amount of 3% v / v, and perform the first aerobic constant-temperature culture at 23 °C and 110 rpm for 60 h. Then, transfer and inoculate it into a new third liquid medium at an inoculation amount of 3% v / v, and perform the second aerobic constant-temperature culture at 23 °C and 110 rpm for 60 h. Again, inoculate it into a new third liquid medium at an inoculation amount of 3% v / v, and perform the third aerobic constant-temperature culture at 23 °C and 110 rpm for 60 h to obtain the Nitrosomonas bacteria solution.

[0042] 4) Inoculate the activated sludge into the fourth liquid medium at an inoculation amount of 3% v / v, and perform the first anaerobic constant-temperature culture at 28 °C and 110 rpm for 60 h. Then, transfer and inoculate it into a new fourth liquid medium at an inoculation amount of 3% v / v, and perform the second anaerobic constant-temperature culture at 28 °C and 110 rpm for 60 h. Again, inoculate it into a new fourth liquid medium at an inoculation amount of 3% v / v, and perform the third anaerobic constant-temperature culture at 28 °C and 110 rpm for 60 h to obtain the Thauera bacteria solution.

[0043] 5) Mix the Lactobacillus plantarum bacteria solution, Bacillus subtilis bacteria solution, Nitrosomonas bacteria solution, and Thauera bacteria solution according to a volume ratio of 4:4:1:1 to obtain a mixed bacteria solution. Centrifuge it at 4000 r / min, take the precipitate, and resuspend the precipitate with a 0.9% (mass fraction) sodium chloride solution to the same volume as the mixed bacteria solution to obtain the compound microbial agent.

[0044] Example 3 1) Inoculate Lactobacillus plantarum into the first liquid medium at an inoculation amount of 1.5% v / v, and perform the anaerobic constant-temperature culture at 44 °C and 150 rpm for 60 h to obtain the Lactobacillus plantarum bacteria solution (the effective viable count is 10 10 CFU / mL).

[0045] 2) Inoculate Bacillus subtilis into the second liquid medium at an inoculation amount of 1.5% v / v, and perform the aerobic constant-temperature culture at 39 °C and 150 rpm for 36 h to obtain the Bacillus subtilis bacteria solution (the effective viable count is 10 10 CFU / mL).

[0046] 3) Inoculate the activated sludge into the third liquid medium at an inoculation amount of 7% v / v, and perform the first aerobic constant-temperature culture at 27 °C and 150 rpm for 84 h. Then, transfer and inoculate it into a new third liquid medium at an inoculation amount of 7% v / v, and perform the second aerobic constant-temperature culture at 27 °C and 150 rpm for 84 h. Again, inoculate it into a new third liquid medium at an inoculation amount of 7% v / v, and perform the third aerobic constant-temperature culture at 27 °C and 150 rpm for 84 h to obtain the Nitrosomonas bacteria solution.

[0047] 4) Inoculate the activated sludge into the fourth liquid medium at an inoculation amount of 7% v / v, and perform the first anaerobic constant temperature culture at 32 °C and 150 rpm for 84 h. Then, transfer and inoculate it into a new fourth liquid medium at an inoculation amount of 7% v / v, and perform the second anaerobic constant temperature culture at 32 °C and 150 rpm for 84 h. Again, inoculate it into a new fourth liquid medium at an inoculation amount of 7% v / v, and perform the third anaerobic constant temperature culture at 32 °C and 150 rpm for 84 h to obtain the Thauera sp. bacterial liquid.

[0048] 5) Mix the Lactobacillus plantarum bacterial liquid, Bacillus subtilis bacterial liquid, Nitrosomonas bacterial liquid and Thauera sp. bacterial liquid according to a volume ratio of 4:4:1:1 to obtain a mixed bacterial liquid. Centrifuge it at 4000 r / min, take the precipitate, and resuspend the precipitate with a 0.9% (mass fraction) sodium chloride solution to the same volume as the mixed bacterial liquid to obtain the compound microbial agent.

[0049] Experimental Example 1 Use the compound microbial agent prepared in Example 1. Mix the compound microbial agent prepared in Example 1 with the activated sludge at a volume ratio of 0.5% (that is, add 5 mL of the compound microbial agent prepared in Example 1 to each liter of activated sludge) as the compound microbial agent-enhanced activated sludge treatment group, and the single activated sludge treatment group. Inoculate them into the biological rotating disk according to the effective volume of the biological rotating disk tank body respectively. The mixing ratio of the compound microbial agent-enhanced activated sludge or single activated sludge to the leachate to be treated is 2 L:8 L. Treat the leachate for 23 d, and the effective volume of the reactor is 10 L.

[0050] [[ID=ll]]Compare the nitrogen and carbon removal effects of the two groups on the landfill leachate. The water quality parameters of the influent leachate are: COD cr = 8324 mg / L; NH3-N = 2699 mg / L; NO2 - -N = 22 mg / L; NO3 - -N = 6 mg / L; TN = 2929 mg / L; the conductivity is 3890 μS / cm. Collect the influent and effluent water samples of the two experimental groups and measure multiple key indicators, including NH4 + -N, NO3 - -N, NO2 - -N, TN, COD.

[0051] As Figure 2 shown, Figure 2 in which A is the treatment of the leachate by the compound microbial agent-enhanced activated sludge treatment group. As the reaction time progresses, the concentrations of various pollutants in the compound microbial agent-enhanced group decrease rapidly and continuously. The concentrations of ammonia nitrogen, total nitrogen, etc. are almost completely degraded in about 20 d, and the COD is degraded to less than 600 mg / L; Figure 2In Group B, the leachate was treated by single activated sludge, with slow pollutant degradation and a large amount of residues. Pollutants such as ammonia nitrogen and total nitrogen still had residues above 1200 mg / L after 23 days, and the residual concentration of COD exceeded 3000 mg / L. It can be seen that the activated sludge strengthened by the compound bactericide is superior in the removal rate, thoroughness and stability of leachate pollutants.

[0052] As Figure 3 shown, after 23 days of treatment, the activated sludge strengthened by the compound bactericide can effectively degrade COD and ammonia nitrogen in the landfill leachate. The total nitrogen and COD removal rates are 99.3% and 93.4% respectively, while those of the ordinary activated sludge method are only 55.6% and 56.1%. It can be seen that the COD removal rate and total nitrogen removal rate of the activated sludge strengthened by the compound bactericide are significantly higher than those of the activated sludge.

[0053] In summary, the compound bactericide provided by the present invention for denitrification and carbon removal of leachate has strong shock resistance and stability in the face of its high ammonia nitrogen, high organic matter content and changing environmental conditions through the synergistic effect of the compound bactericide. In addition, the use of the compound bactericide does not require complex pretreatment and post-treatment processes, greatly reducing the operation difficulty and cost. In practical applications, this compound bactericide can not only significantly shorten the treatment cycle of leachate, but also effectively reduce the sludge production and the burden of subsequent sludge treatment. Its high-efficiency denitrification and carbon removal ability and environmental adaptability make it have broad application prospects in the field of leachate treatment, providing an economic, efficient and sustainable solution for solving the problem of high ammonia nitrogen wastewater treatment.

[0054] The above-described embodiments are only descriptions of the preferred embodiments of the present invention, and do not limit the scope of the present invention. Without departing from the design spirit of the present invention, various deformations and improvements made by those of ordinary skill in the art to the technical solutions of the present invention shall fall within the protection scope determined by the claims of the present invention.

Claims

1. A compound bactericide for denitrification and carbon removal of leachate, characterized in that, The compound bacterium agent is composed of the bacterial liquid of Lactobacillus plantarum ( Lactobacillus plantarum ), the bacterial liquid of Bacillus subtilis ( Bacillus subtilis ), the bacterial liquid of Nitrosomonas ( Nitrosomonas ), and the bacterial liquid of Thauera ( Thauera ).

2. The compound microbial agent according to claim 1, wherein In the compound microbial agent, the volume ratio of the Lactobacillus plantarum bacterial liquid, Bacillus subtilis bacterial liquid, Nitrosomonas bacterial liquid and Thauera bacterial liquid is 4:4:1:

1.

3. The compound microbial agent according to claim 1, wherein The effective viable count of the Lactobacillus plantarum bacterial liquid is 10 9 ~10 10 CFU / mL. The Lactobacillus plantarum bacterial liquid is the bacterial liquid of Lactobacillus plantarum with the preservation number CGMCC 1.16089; the effective viable count of the Bacillus subtilis bacterial liquid is 10 9 ~10 10 CFU / mL. The Bacillus subtilis bacterial liquid is the bacterial liquid of Bacillus subtilis with the preservation number CGMCC1.

821.

4. The compound microbial agent according to claim 1, wherein The preparation of the Nitrosomonas bacterial liquid comprises the following steps: inoculating activated sludge into the third liquid medium at an inoculation amount of 3-7% v / v, performing first aerobic constant-temperature culture at 23-27°C and 110-150 rpm for 60-84 h, transferring and inoculating into a new third liquid medium at an inoculation amount of 3-7% v / v, performing second aerobic constant-temperature culture at 23-27°C and 110-150 rpm for 60-84 h, and inoculating into a new third liquid medium again at an inoculation amount of 3-7% v / v, performing third aerobic constant-temperature culture at 23-27°C and 110-150 rpm for 60-84 h to obtain the Nitrosomonas bacterial liquid; The formula of the third liquid medium is: ammonium sulfate 0.25 g / L, ammonium chloride 0.25 g / L, sodium bicarbonate 1.6 g / L, sodium chloride 0.3 g / L, disodium hydrogen phosphate 1 g / L, magnesium sulfate 0.03 g / L, calcium chloride 7.5 g / L, trace elements 1 ml / L, adding water to make up the volume to 1 L, and the pH of the third liquid medium is 7.

5.

5. The compound microbial agent according to claim 1, wherein The preparation of the Thauera bacterial liquid comprises the following steps: inoculating activated sludge into the fourth liquid medium at an inoculation amount of 3-7% v / v, performing first anaerobic constant-temperature culture at 28-32°C and 110-150 rpm for 60-84 h, transferring and inoculating into a new fourth liquid medium at an inoculation amount of 3-7% v / v, performing second anaerobic constant-temperature culture at 28-32°C and 110-150 rpm for 60-84 h, and inoculating into a new fourth liquid medium again at an inoculation amount of 3-7% v / v, performing third anaerobic constant-temperature culture at 28-32°C and 110-150 rpm for 60-84 h to obtain the Thauera bacterial liquid; The formula of the fourth liquid medium is: sodium humate 8 g / L, sodium fulvate 6 g / L, acetic acid 3 g / L, glucose 2 g / L, peptone 1 g / L, sodium nitrate 2 g / L, sodium chloride 0.3 g / L, disodium hydrogen phosphate 1 g / L, magnesium sulfate 0.03 g / L, trace elements 1 ml / L, adding water to make up the volume to 1 L, and the pH of the fourth liquid medium is 7.

5.

6. The preparation method of the compound microbial agent according to any one of claims 1 to 5, characterized in that, Comprising the following steps: 1) Inoculating Lactobacillus plantarum into the first liquid medium, performing anaerobic constant-temperature culture at 40-44°C and 110-150 rpm for 36-60 h to obtain the Lactobacillus plantarum bacterial liquid; 2) Inoculating Bacillus subtilis into the second liquid medium, performing aerobic constant-temperature culture at 35-39°C and 110-150 rpm for 12-36 h to obtain the Bacillus subtilis bacterial liquid; 3) Inoculate the activated sludge at an inoculation amount of 3 - 7% v / v into Liquid Medium III, and conduct the first aerobic constant-temperature cultivation at 23 - 27°C and 110 - 150 rpm for 60 - 84 h. Then, transfer and inoculate it at an inoculation amount of 3 - 7% v / v into a new Liquid Medium III, and conduct the second aerobic constant-temperature cultivation at 23 - 27°C and 110 - 150 rpm for 60 - 84 h. Again, inoculate it at an inoculation amount of 3 - 7% v / v into a new Liquid Medium III, and conduct the third aerobic constant-temperature cultivation at 23 - 27°C and 110 - 150 rpm for 60 - 84 h to obtain Nitrosomonas bacterium liquid; 4) Inoculate the activated sludge at an inoculation amount of 3 - 7% v / v into Liquid Medium IV, and conduct the first anaerobic constant-temperature cultivation at 28 - 32°C and 110 - 150 rpm for 60 - 84 h. Then, transfer and inoculate it at an inoculation amount of 3 - 7% v / v into a new Liquid Medium IV, and conduct the second anaerobic constant-temperature cultivation at 28 - 32°C and 110 - 150 rpm for 60 - 84 h. Again, inoculate it at an inoculation amount of 3 - 7% v / v into a new Liquid Medium IV, and conduct the third anaerobic constant-temperature cultivation at 28 - 32°C and 110 - 150 rpm for 60 - 84 h to obtain Thauera bacterium liquid; 5) Mix the Lactobacillus plantarum bacterium liquid obtained in step 1), the Bacillus subtilis bacterium liquid obtained in step 2), the Nitrosomonas bacterium liquid obtained in step 3), and the Thauera bacterium liquid obtained in step 4) according to a volume ratio of 4:4:1:1 to obtain a mixed bacterium liquid. Centrifuge it, take the precipitate, and resuspend the precipitate with a sodium chloride solution with a mass fraction of 0.9% to the same volume as the mixed bacterium liquid to obtain a compound microbial agent.

7. According to the preparation method described in claim 6, wherein In step 1), the inoculation amount of the Lactobacillus plantarum is 0.5 - 1.5% v / v; the formula of Liquid Medium I in step 1) is as follows: sodium humate 13.6 g / L, sodium fulvate 10.2 g / L, acetic acid 5.1 g / L, glucose 3.4 g / L, peptone 1.7 g / L, dipotassium hydrogen phosphate 2 g / L, sodium acetate trihydrate 5 g / L, ammonium citrate 2 g / L, magnesium sulfate heptahydrate 0.2 g / L, manganese sulfate tetrahydrate 0.2 g / L, sodium chloride 60 g / L. Add water to make the volume up to 1 L, and the pH of Liquid Medium I is 6.

2.

8. The preparation method according to claim 6, wherein In step 2), the inoculation amount of the Bacillus subtilis is 0.5 - 1.5% v / v; the formula of Liquid Medium II in step 2) is as follows: sodium humate 4 g / L, sodium fulvate 3 g / L, acetic acid 1.5 g / L, glucose 1.5 g / L, peptone 5 g / L, yeast extract 5 g / L, sodium chloride 5 g / L, trace elements 1 ml / L. Add water to make the volume up to 1 L, and the pH of Liquid Medium II is 7.

1.

9. The preparation method according to claim 6, characterized in that, The formula of the liquid medium III described in step 3) is as follows: ammonium sulfate 0.25 g / L, ammonium chloride 0.25 g / L, sodium bicarbonate 1.6 g / L, sodium chloride 0.3 g / L, disodium hydrogen phosphate 1 g / L, magnesium sulfate 0.03 g / L, calcium chloride 7.5 g / L, trace elements 1 ml / L, and add water to make up the volume to 1 L. The pH of the liquid medium III is 7.5; the formula of the liquid medium IV described in step 4) is as follows: sodium humate 8 g / L, sodium fulvate 6 g / L, acetic acid 3 g / L, glucose 2 g / L, peptone 1 g / L, sodium nitrate 2 g / L, sodium chloride 0.3 g / L, disodium hydrogen phosphate 1 g / L, magnesium sulfate 0.03 g / L, trace elements 1 ml / L, and add water to make up the volume to 1 L. The pH of the liquid medium IV is 7.

5.

10. Application of the compound microbial agent according to any one of claims 1 to 5 in denitrification and carbon removal of leachate.

Images