Preparation method and application of carrier pellet for immobilizing low-temperature denitrifying bacteria
By preparing immobilized carrier microspheres and immobilizing low-temperature denitrifying bacteria using encapsulation and cross-linking methods, the problem of low denitrification efficiency in wastewater treatment plants under low-temperature conditions was solved, achieving efficient denitrification of low-temperature wastewater and reducing operating costs.
Patent Information
- Application Number
- CN202511430971.4
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-10-06
- Publication Date
- 2026-01-09
AI Technical Summary
Under low temperature conditions, the activity of denitrifying bacteria in the denitrification zone of wastewater treatment plants decreases, leading to a decline in nitrogen removal efficiency, increased operating costs, and potential eutrophication of water bodies. Existing technologies lack effective methods for immobilizing denitrifying bacteria at low temperatures.
Immobilized carrier microspheres were prepared using materials such as polyacrylamide, polyvinyl alcohol, sodium alginate, guar gum, boric acid, and calcium chloride. Low-temperature denitrifying bacteria were immobilized by encapsulation and cross-linking methods to enhance their activity and resistance in low-temperature environments.
The concentration and resistance of low-temperature denitrifying bacteria were increased, enhancing their denitrification capacity in low-temperature wastewater. The total nitrogen removal rate was increased by 19.8% to 32.6%, reducing dependence on carbon sources and improving wastewater treatment efficiency.
Smart Images
Figure CN121294423A_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of wastewater treatment technology, specifically relating to a method for preparing carrier microspheres for immobilizing low-temperature denitrifying bacteria and its application. Background Technology
[0002] With the increasing wastewater collection rate and increasingly stringent environmental protection requirements, the negative impact of low temperatures on biological denitrification has severely affected the effluent compliance rate of wastewater treatment plants. Low temperatures significantly inhibit microbial activity. While this has little impact on the aerobic aeration zones of wastewater treatment plants because compressed air aeration introduces heat into the water, it has a greater impact on the non-aerated anoxic denitrification zones. This is because denitrifying bacteria have a narrow active temperature range (15℃~20℃), and their metabolic capacity drops sharply below this range, essentially halting the reaction below 5℃. In northern my country, wastewater temperatures are generally low in winter, typically between 5℃ and 15℃. Low temperature has become a key factor restricting the effectiveness of biological denitrification, often requiring wastewater treatment plants to reduce their load or add large amounts of external carbon sources to survive the low-temperature period. This not only increases the operating costs of wastewater treatment plants but may also lead to environmental risks such as eutrophication. To address this issue, low-temperature denitrification technology has become a research hotspot in recent years, including screening for cold-resistant bacterial strains, enhancing biological functions, developing new packing materials, and coupling physicochemical methods. These technologies aim to increase the number of denitrifying bacteria or optimize reaction conditions to withstand low-temperature environments. Exploring efficient and stable low-temperature denitrification strategies is of great significance for achieving year-round compliance with wastewater discharge standards and promoting the sustainable development of denitrification technology. It is also a key scientific issue that urgently needs to be addressed in the field of water treatment.
[0003] Low-temperature denitrifying bacteria are a type of microorganism that can maintain high biological activity at low temperatures. Introducing these bacteria into biological denitrification processes can effectively improve the denitrification efficiency of low-temperature water. However, in actual wastewater systems, the composition of pollutants is complex, free denitrifying bacteria have poor resistance to environmental toxicity, weak competition with native bacteria, and low bacterial concentrations. Therefore, special measures are needed to increase the concentration of low-temperature denitrifying bacteria in the wastewater treatment system. Since the 20th century, microbial immobilization technology has been widely used in wastewater treatment. By confining planktonic microorganisms to a limited space using physical or chemical means, the amount of microorganisms per unit space can be increased. Immobilized microorganisms have advantages such as high biomass, stability, low loss, fast reaction speed, strong resistance to toxicity, and easy separation. Methods for immobilizing microorganisms are divided into four main categories: adsorption, covalent bonding, cross-linking, and encapsulation. In practical applications, due to the different drawbacks of individual methods, composite immobilization methods are often used. Composite immobilization is a method that organically combines various immobilization methods. It can give full play to the advantages of each immobilization method, while overcoming their respective shortcomings to the greatest extent, reducing environmental interference with microorganisms, and improving the mechanical properties and service life of immobilized materials.
[0004] To enhance the activity of low-temperature denitrifying bacteria in low-temperature wastewater and strengthen their resistance to adverse environments such as low temperatures, it is necessary to provide them with immobilized attachment carriers with sufficient habitat space and encapsulate them inside the carriers. This will minimize the predation of the organisms or the extracellular antifreeze proteins secreted by the organisms by the native bacteria, thereby effectively exerting their low-temperature denitrification function and reducing the denitrification pressure on wastewater treatment plants in winter. Summary of the Invention
[0005] Currently, there is no method for immobilizing low-temperature denitrifying bacteria. To address this technical deficiency, this invention provides a method for preparing carrier microspheres for immobilizing low-temperature denitrifying bacteria and its application, which can effectively solve the problem of difficult denitrification of wastewater during low-temperature periods in northern regions.
[0006] The above-mentioned method for preparing carrier microspheres for immobilizing low-temperature denitrifying bacteria includes the following steps:
[0007] Step 1: Prepare the culture medium;
[0008] Step 2: Isolate and screen low-temperature denitrifying bacteria using the culture medium prepared in Step 1, and then purify and culture them.
[0009] Step 3: Inoculate the low-temperature denitrifying bacteria selected in Step 2 into the denitrification medium for denitrification performance testing, and select strains with high denitrification efficiency to prepare low-temperature denitrifying bacterial suspensions;
[0010] Step 4: Mix polyacrylamide (PAM), polyvinyl alcohol (PVA), sodium alginate (SA) and distilled water, heat in a water bath to prepare PAM-PVA-SA encapsulation solution, and then sterilize under high temperature and high pressure and cool.
[0011] Step 5: Add the composite bacterial suspension prepared in Step 3 and a certain amount of adsorbent to the PAM-PVA-SA encapsulation solution cooled in Step 4, and stir thoroughly to prepare a mixture of composite bacteria and encapsulation agent.
[0012] Step 6: Dissolve guar gum (GG), calcium chloride (CaCl2) and boric acid (H3BO4) in distilled water to prepare a crosslinking agent solution with a mass concentration of 2% GG-CaCl2-H3BO4;
[0013] Step 7: Use a 2ml pipette to draw up the mixture prepared in step 5 and add it dropwise to the cross-linking agent solution in step 6 to complete the immobilization of the low-temperature denitrifying bacteria;
[0014] Step 8: The immobilized carrier microspheres prepared in Step 7 can be stored in a refrigerator or directly added to the low-temperature wastewater to be denitrified.
[0015] Preferably, in step 1, the culture medium is a mixed culture medium of beef extract peptone liquid culture medium and trace element liquid culture medium, and the pH value of the mixed culture medium is adjusted to 7.2.
[0016] Preferably, in step 2, the low-temperature denitrifying bacteria can be obtained from the sludge around the roots of plants with well-developed root systems and the ability to absorb nitrogen, such as reeds, water onions, or calamus, in wetlands in cold northern regions during winter, or from the denitrifying activated sludge of wastewater treatment plants during winter.
[0017] Preferably, in step 3, the low-temperature denitrifying bacteria isolated from the sludge are inoculated into the sterilized culture medium described in step 1 for purification and culture, and one or more strains with excellent performance are selected to prepare a low-temperature denitrifying bacteria suspension.
[0018] Preferably, in step 3, the temperature of the culture medium is controlled to be 10°C to 12°C.
[0019] Preferably, in step 4, the cooling temperature after high-temperature and high-pressure sterilization is 20℃~22℃.
[0020] Preferably, the adsorbent mentioned in step 5 refers to 200-300 mesh powdered activated carbon or diatomaceous earth.
[0021] Preferably, in step 7, the dripping rate is 15 drops / min to 20 drops / min.
[0022] Preferably, in step 7, the crosslinking time after the dripping is completed is 4 hours.
[0023] Preferably, in step 8, the immobilized carrier microspheres are stored in a refrigerator at 4°C.
[0024] To address the aforementioned technical problems, this invention also provides an application of immobilized carrier microspheres for low-temperature denitrifying bacteria, wherein the immobilized carrier microspheres prepared by the above method are applied to the denitrification treatment of wastewater in cold regions during winter.
[0025] Compared with the prior art, the present invention provides a method for preparing carrier microspheres for immobilizing low-temperature denitrifying bacteria and its application, which has the following beneficial effects: (1) The present invention uses materials such as polyacrylamide, polyvinyl alcohol, sodium alginate, guar gum, boric acid, calcium chloride and adsorbent to prepare carrier microspheres for immobilizing low-temperature denitrifying bacteria. The required materials are easy to obtain and the preparation process is simple; (2) The encapsulating agent and crosslinking agent used in the present invention are carbon-containing materials. After long-term use, they can be used as carbon sources for denitrifying bacteria. They have good biocompatibility and are conducive to microbial denitrification; (3) The carrier microspheres prepared in the present invention are filled with adsorbent as a skeleton, which increases the specific surface area of the carrier, expands the habitat space of low-temperature denitrifying bacteria, increases the mass transfer channels, enhances the resistance of microorganisms to adverse environments, and improves the adsorption and degradation efficiency of nitrogen-containing substances; (4) Compared with free low-temperature denitrifying bacteria, the total nitrogen (TN) removal rate of the immobilized low-temperature denitrifying bacteria for 13℃ low-temperature wastewater is increased by 19.8% to 32.6%, which can effectively improve the denitrification capacity of low-temperature wastewater in northern regions during winter. Attached Figure Description
[0026] Figure 1 A full view of the immobilized carrier microspheres;
[0027] Figure 2 SEM images of the surface of immobilized carrier microspheres using different adsorbents;
[0028] Figure 3 SEM images of the interior of immobilized carrier microspheres using different adsorbents;
[0029] Figure 4 A comparison of the denitrification effects of free low-temperature denitrifying bacteria and immobilized low-temperature denitrifying bacteria;
[0030] Figure 5 This is a flowchart illustrating the preparation process of the carrier microspheres for immobilizing low-temperature denitrifying bacteria according to the present invention. Detailed Implementation
[0031] The technical solutions of the present invention will now be clearly and completely described with reference to the accompanying drawings of the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.
[0032] The experimental materials used in this invention, including ferrous sulfate (FeSO4), ethylenediaminetetraacetic acid (EDTA), zinc sulfate (ZnSO4), manganese chloride (MnCl2), copper sulfate (CuSO4), cobalt chloride (CoCl2), sodium molybdate (Na2MoO4), peptone, beef extract, sodium chloride (NaCl), polyvinyl alcohol (PVA, 72000), polypropylene (PAM, 8-10 million), sodium alginate (SA), guar gum (GG), boric acid (H3BO4), calcium chloride (CaCl2), sodium citrate (C6H5Na3O7), potassium nitrate (KNO3), dipotassium hydrogen phosphate (K2HPO4), and magnesium sulfate (MgSO4), were all of analytical grade. All instruments used were sterilized under high temperature and high pressure.
[0033] The specific operation methods of this invention are as follows:
[0034] (1) Preparation of culture medium:
[0035] Beef extract peptone medium: Dissolve beef extract (5.0 g / L), peptone (10.0 g / L), and sodium chloride (10.0 g / L) in distilled water, adjust the pH of the medium to 7.2, and sterilize for 20 min.
[0036] Micronutrient solution culture medium: Dissolve EDTA 15 g / L, zinc sulfate 0.2 g / L, manganese chloride 1.5 g / L, ferrous sulfate 0.02 g / L, copper sulfate 0.5 g / L, cobalt chloride 0.3 g / L, sodium molybdate 0.2 g / L, and calcium chloride 0.05 g / L in distilled water, adjust the pH to 7.2, and then sterilize for 20 min.
[0037] Denitrification medium: Dissolve sodium citrate 2 g / L, potassium nitrate 0.5 g / L, dipotassium hydrogen phosphate 0.5 g / L, magnesium sulfate 0.2 g / L, calcium chloride 0.05 g / L, and ferrous sulfate 0.02 g / L in distilled water and sterilize for 20 min.
[0038] The preparation of the above three culture media corresponds to step 1.
[0039] (2) Isolation of low-temperature denitrifying bacteria:
[0040] When the river water temperature was below 10℃, mud samples were taken from the reed root system of a wetland in Harbin and from the denitrification tank of a sewage treatment plant in the city. After dilution, the mud samples were inoculated into a mixed culture medium (hereinafter referred to as mixed culture medium) composed of beef extract peptone medium and trace element medium at a volume ratio of 100:3. The samples were then incubated at 13℃ with shaking for 3 days. After that, white or blue-haloed colonies were picked and incubated at 13℃ with shaking for another 3 days for purification. After that, colonies with good growth were picked and inoculated into denitrification culture medium.
[0041] (3) Preparation of low-temperature denitrifying bacterial suspension:
[0042] The temperature of the denitrification medium was controlled at 10℃~12℃. After shaking culture of low-temperature denitrifying bacteria for 3 days, three strains with a TN removal rate greater than 60% were selected and cultured in a mixed medium at 35℃~37℃ until the logarithmic growth phase. The bacterial cells were washed with physiological saline, and bacterial suspensions of each strain were prepared with sterile water and their OD values were adjusted. 600 After reaching a concentration of 0.6 to 0.7, the three bacterial suspensions were mixed in a 1:1:1 ratio to prepare a compound low-temperature denitrifying bacterial suspension.
[0043] (4) Preparation of low-temperature denitrifying bacteria immobilization carrier microspheres:
[0044] Take 0.05g PAM, 6g PVA, and 0.5g SA respectively and add them to an Erlenmeyer flask. Slowly add sterile water to make up to 100mL. Heat and stir in a 60℃ constant temperature water bath until fully dissolved to prepare a PAM-PVA-SA embedding agent solution. Sterilize under high temperature and high pressure. After the PAM-PVA-SA embedding agent solution cools to 20℃~22℃, add 10mL of composite low temperature denitrifying bacterial suspension and 0.5g of diatomaceous earth adsorbent or activated carbon adsorbent, and stir thoroughly until homogeneous. Using a 2mL sterile dropper, slowly add the above-stirred bacterial suspension, adsorbent, and PAM-PVA-SA embedding agent mixture at a rate of 15 drops / min~20 drops / min to a sufficient amount of 2% GG-CaCl2-H3BO4 crosslinking agent solution. After crosslinking for 4h, uniform spherical particles are obtained. After washing with sterile water, carrier microspheres are obtained. In the 2% GG-CaCl2-H3BO4 crosslinking agent solution, the mass ratio of each component is GG:CaCl2:H3BO4 = 1:4:4.
[0045] The morphology of the carrier microspheres prepared according to the above steps is as follows: Figure 1 As shown, the SEM image of the small ball is as follows: Figure 2 and Figure 3 As shown. From Figure 1 As can be seen, the carrier microspheres immobilized with low-temperature denitrifying bacteria are regularly spherical, with a diameter of 4mm–6mm and a density slightly greater than water, and settle underwater. Figure 2 It can be seen that the surfaces of the immobilized carrier microspheres, whether diatomaceous earth or activated carbon, as adsorbents are rough and uneven with a large specific surface area. This is beneficial for expanding the growth surface of low-temperature denitrifying bacteria and their mass transfer contact area with the external environment. According to Figure 3 It can be observed that when diatomaceous earth is used as an adsorbent, the carrier microspheres have more microporous structures, which may also allow more low-temperature denitrifying bacteria to grow inside the diatomaceous earth immobilized carrier microspheres.
[0046] (5) Optimize immobilization conditions:
[0047] To determine the optimal ratio of immobilization materials and the optimal immobilization time, this invention investigated the effects of the dosage of encapsulating agents (PVA and SA), adsorbents (diatomaceous earth or activated carbon), and immobilization time on the removal efficiency of nitrate nitrogen (NO3-N) and TN. A 4-factor, 3-level L9(3) model was designed. 4 An orthogonal experiment was conducted, using the removal rates of NO3-N and TN as evaluation indicators to determine the optimal immobilization conditions. The orthogonal experiment factors and levels are shown in Table 1, and the orthogonal experiment analysis tables are shown in Tables 2 and 3.
[0048] Since the amount of PAM used in the preparation process is very small, accounting for only 0.45% of the mixture of encapsulating agent and bacterial suspension, while the crosslinking agent is in excess throughout the preparation process, the influence of PAM and crosslinking agent is not considered in this step.
[0049] Table 1. Factor Level Table for Orthogonal Experiment
[0050]
[0051] Table 2. Orthogonal experimental analysis of diatomaceous earth as an adsorbent for immobilization.
[0052]
[0053]
[0054] Table 2 shows the effects of four factors on NO3 by comparing the magnitudes of the ranges R. - The effect of α-N removal rate and TN removal rate is greater with a larger R value. It was found that when diatomaceous earth is used as an adsorbent to prepare carrier microspheres, the effect on NO3- removal rate is significantly reduced. - The order of influence on NO3 removal rate is C>A>B>D, while the order of influence on TN removal rate is A>C>B>D. This is because a small amount of nitrite nitrogen accumulates during the denitrification process, which makes NO3... - The range R of the nitrogen removal rate differs from the range R of the TN removal rate. Since TN is the indicator for evaluating wastewater nitrogen removal efficiency, the order of influence of the four factors is determined to be A>C>B>D based on the relationship between the R values of the TN removal rate. The K value reflects the magnitude of the influence of different levels of the factor on the removal rate, and the corresponding levels for each factor are selected as A1C1B1D1 based on the K value.
[0055] Similarly, based on the experimental data in Table 3, when using activated carbon as an adsorbent to prepare carrier spheres, the order of influence of these four factors on the TN removal rate is B>C>D>A. Then, based on the K value, the corresponding levels of the factors are selected as B1C1D1A3.
[0056] Table 3. Orthogonal experimental analysis of activated carbon as an adsorbent for immobilization.
[0057]
[0058]
[0059] Analysis of Tables 2 and 3 reveals that the adsorbent material significantly impacts the denitrification of immobilized microspheres, leading not only to variations in denitrification efficiency but also to differences in the magnitude of the influence of operating conditions on the denitrification rate. For low-temperature denitrifying bacterial carrier microspheres prepared using diatomaceous earth as the adsorbent, the optimal denitrification effect is achieved when the mass concentrations of PVA, SA, and diatomaceous earth in the encapsulating agent are 6 g / 100 mL, 0.5 g / 100 mL, and 0.5 g / 100 mL, respectively, and the cross-linking fixation time is 4 h. For low-temperature denitrifying bacterial carrier microspheres prepared using activated carbon as the adsorbent, the optimal denitrification effect is achieved when the mass concentrations of PVA, SA, and activated carbon in the encapsulating agent are 8 g / 100 mL, 0.5 g / 100 mL, and 0.5 g / 100 mL, respectively, and the cross-linking fixation time is 4 h.
[0060] Example:
[0061] The effectiveness of the present invention is verified through the following embodiments:
[0062] Example 1: This example is a comparative example of a method for preparing carrier microspheres for immobilizing low-temperature denitrifying bacteria and its application. The method is carried out according to the following steps:
[0063] (1) Prepare beef extract peptone culture medium and trace element culture medium according to the steps described in the specific implementation method, isolate three kinds of low-temperature denitrifying bacteria, and prepare 10 mL of compound low-temperature denitrifying bacteria suspension.
[0064] (2) 100 mL of nitrified liquid collected from the aeration tank of a wastewater treatment plant in Harbin was placed in a sterilized conical flask, and the TN concentration and NO3 concentration of parallel water samples were measured simultaneously. - -N concentration and NO2 - -N concentration;
[0065] (3) Add 10 mL of bacterial suspension prepared in (1) to the conical flask containing nitrification solution described in (2), seal the mouth of the flask with cotton, and then place the conical flask in a constant temperature shaking incubator at 13℃ for 3 days.
[0066] (4) After the culture is completed, the concentrations of TN and NO3 in the water sample from the conical flask are measured. - -N concentration and NO2 - -N concentration.
[0067] Example 2: A method for preparing carrier microspheres for immobilizing low-temperature denitrifying bacteria and its application is carried out according to the following steps:
[0068] (1) Same as (1) in Example 1;
[0069] (2) Take 0.05g PAM, 6g PVA and 0.5g SA respectively and add them to an Erlenmeyer flask. Slowly add sterile water to make up to 100mL. Heat and stir in a 60℃ constant temperature water bath until fully dissolved to prepare the solution.
[0070] The PAM-PVA-SA embedding agent solution was sterilized by high temperature and high pressure. After the PAM-PVA-SA embedding agent solution cooled to 20℃, 10mL of composite low-temperature denitrifying bacterial suspension and 0.5g of diatomaceous earth adsorbent were added, and the mixture was stirred thoroughly until homogeneous. Using a 2mL sterile dropper, the above-stirred bacterial suspension, adsorbent and PAM-PVA-SA embedding agent mixture was slowly added dropwise to a sufficient amount of the 2% GG-CaCl2-H3BO4 crosslinking agent solution prepared in step 6. Crosslinking was carried out for 4 hours to obtain uniform spherical particles. After washing with sterile water, the carrier microspheres were obtained. In the 2% GG-CaCl2-H3BO4 crosslinking agent solution, the mass ratio of each component was GG:CaCl2:H3BO4 = 1:4:4.
[0071] (3) Same as (2) in Example 1;
[0072] (4) All the carrier microspheres prepared in this embodiment (2) are inserted into the conical flask described in this embodiment (3) using sterile forceps, and then the conical flask is placed in a constant temperature shaking incubator at 13℃ for 3 days.
[0073] (5) Same as (4) in Example 1.
[0074] Example 3: This example differs from Example 2 in that, in step (2) of this example, 0.5g of powdered activated carbon with a particle size of 200 mesh is used instead of diatomaceous earth as the adsorbent. Everything else is the same as in Example 2. A comparison of the denitrification effects of the three examples is shown in the figure below. Figure 4 As shown. By Figure 4It is evident that, compared to free low-temperature denitrifying bacteria, the denitrification effect of the carrier microspheres prepared in this invention is significantly higher, regardless of whether diatomaceous earth or activated carbon is used as the adsorbent. In terms of NO3-N removal rate, the latter two are 29.9% and 28.0% higher than the former, respectively, and in terms of TN removal rate, they are 32.6% and 19.8% higher, respectively. Furthermore, the NO2-N accumulation of the denitrifying bacteria during the denitrification process is significantly reduced after solidification. After the culture is completed, the NO2-N accumulation in the free low-temperature denitrifying bacteria system is 1.18 mg / L, while the NO2-N accumulation in the diatomaceous earth carrier microsphere system and the activated carbon carrier microsphere system is reduced to 0.49 mg / L and 0.70 mg / L, respectively. The reasons are analyzed as follows: In Example 1, the low-temperature denitrifying bacteria are in a free state in the conical flask, making them susceptible to interference from heterotrophic bacteria carried in by the water sample, especially in the first half of the cultivation process. During this period, some dissolved oxygen remains in the water sample, and the heterotrophic bacteria still possess a certain metabolic capacity. Besides preying on the free denitrifying bacteria and their secreted extracellular antifreeze proteins, they also compete with the denitrifying bacteria for carbon sources. In treating low-temperature wastewater, it is often necessary to add excessive carbon sources to compensate for the reduced denitrification efficiency caused by the decreased enzyme activity of denitrifying bacteria due to low temperatures. Since no external carbon source was added during the implementation of this example, the proliferation rate and metabolic capacity of the free low-temperature denitrifying bacteria were reduced, thus lowering the denitrification efficiency. In Examples 2 and 3, most of the low-temperature denitrifying bacteria were immobilized inside the carrier spheres. This not only prevented them from being preyed upon by external heterotrophic bacteria but also reduced the mass transfer rate due to the immobilization material, slowing down the loss of carbon sources to the heterotrophic bacteria to some extent, which helped improve the proliferation rate and denitrification rate of the denitrifying bacteria. Therefore, it can be seen that applying carrier microspheres immobilizing low-temperature denitrifying bacteria to low-temperature wastewater treatment can effectively improve the denitrification efficiency of wastewater treatment plants in northern winters and reduce the denitrification pressure of low-temperature wastewater treatment.
Claims
1. A method for preparing carrier microspheres for immobilizing low-temperature denitrifying bacteria, characterized in that, Includes the following steps: Step 1: Prepare the culture medium; Step 2: Isolate and screen low-temperature denitrifying bacteria using the culture medium prepared in Step 1, and then purify and culture them. Step 3: Inoculate the low-temperature denitrifying bacteria selected in Step 2 into the denitrification medium for denitrification performance testing, and select strains with high denitrification efficiency to prepare low-temperature denitrifying bacterial suspensions; Step 4: Mix polyacrylamide (PAM), polyvinyl alcohol (PVA), sodium alginate (SA) and distilled water, heat in a water bath to prepare PAM-PVA-SA encapsulation solution, and then sterilize under high temperature and high pressure and cool. Step 5: Add the composite bacterial suspension prepared in Step 3 and a certain amount of adsorbent to the PAM-PVA-SA encapsulation solution cooled in Step 4, and stir thoroughly to prepare a mixture of composite bacteria and encapsulation agent. Step 6: Dissolve guar gum (GG), calcium chloride (CaCl2) and boric acid (H3BO4) in distilled water to prepare a crosslinking agent solution with a mass concentration of 2% GG-CaCl2-H3BO4; Step 7: Use a 2ml pipette to draw up the mixture prepared in step 5 and add it dropwise to the cross-linking agent solution in step 6 to complete the immobilization of the low-temperature denitrifying bacteria; Step 8: The immobilized carrier microspheres prepared in Step 7 can be stored in a refrigerator or directly added to the low-temperature wastewater to be denitrified.
2. The method for preparing carrier microspheres for immobilizing low-temperature denitrifying bacteria according to claim 1, characterized in that: In step 1, the culture medium is a mixture of beef extract peptone liquid medium and trace element liquid medium, and the pH of the mixture is adjusted to 7.
2.
3. The method for preparing carrier microspheres for immobilizing low-temperature denitrifying bacteria according to claim 1, characterized in that: In step 2, low-temperature denitrifying bacteria can be obtained from the sludge around the roots of plants with well-developed root systems and the ability to absorb nitrogen, such as reeds, water onions, or calamus, in wetlands in cold northern regions during winter, or from the denitrifying activated sludge of wastewater treatment plants during winter.
4. The method for preparing carrier microspheres for immobilizing low-temperature denitrifying bacteria according to claim 1, characterized in that: In step 3, the low-temperature denitrifying bacteria isolated from the sludge are inoculated into the sterilized culture medium described in step 1 for purification and culture, and one or more strains with excellent performance are selected to prepare a low-temperature denitrifying bacterial suspension.
5. The method for preparing carrier microspheres for immobilizing low-temperature denitrifying bacteria according to claim 1, characterized in that: In step 3, the temperature of the culture medium is controlled at 10℃~12℃.
6. The method for preparing carrier microspheres for immobilizing low-temperature denitrifying bacteria according to claim 1, characterized in that: In step 4, the cooling temperature after high-temperature and high-pressure sterilization is 20℃~22℃.
7. The method for preparing carrier microspheres for immobilizing low-temperature denitrifying bacteria according to claim 1, characterized in that: The adsorbent mentioned in step 5 refers to 200-300 mesh powdered activated carbon or diatomaceous earth.
8. The method for preparing carrier microspheres for immobilizing low-temperature denitrifying bacteria according to claim 1, characterized in that: In step 7, the dripping rate is 15 drops / min to 20 drops / min.
9. The method for preparing carrier microspheres for immobilizing low-temperature denitrifying bacteria according to claim 1, characterized in that: In step 7, the crosslinking time after the dripping is completed is 4 hours.
10. The method for preparing carrier microspheres for immobilizing low-temperature denitrifying bacteria according to claim 1, characterized in that: In step 8, the immobilized carrier microspheres are stored in a refrigerator at 4°C.