A method for preparing a novel biofilm packing material immobilizing a large number of microorganisms

Abstract

This invention discloses a method for preparing a novel biofilm packing material immobilized with a large number of microorganisms. Functional bacteria play a crucial role in water treatment for degrading specific pollutants. The invention first washes the bacterial solution with PBS buffer to prepare a bacterial suspension; weigh appropriate amounts of polyvinyl alcohol (PVA) and sodium alginate (SA) and dissolve them in an appropriate amount of distilled water, then place the solution in a constant temperature water bath; add sodium acetate and the bacterial suspension to the cooled PVA-SA mixed solution and stir thoroughly; inject the prepared mixed solution into a mold, then immerse the mold in a CaCl2 saturated boric acid solution to allow cross-linking and hardening for 4 hours to form the final material; store the resulting material at 4°C. The novel biofilm packing material provided by this invention immobilizes a large number of functional bacteria, enabling it to target specific pollutants or special environments; its sea urchin-like shape and structure allow the packing material to effectively resist external pressure.

Description

Technical Field

[0001] This invention belongs to the field of wastewater treatment technology, specifically relating to a method for preparing a novel biofilm packing material that immobilizes and embeds a large number of microorganisms. Background Technology

[0002] The essence of biofilm technology is to allow bacteria and fungi-related microorganisms to attach to and grow on biofilm packing materials, forming a film-like biological sludge—biofilm. When wastewater flows through the packing materials, the organic pollutants in the wastewater come into contact with the biofilm. The numerous microorganisms in the biofilm degrade and consume the organic pollutants in the water through their own metabolism, thereby purifying the wastewater and allowing the microorganisms themselves to multiply and proliferate.

[0003] Wastewater treatment biofilm packing materials, generally referred to as biological packing materials or packing materials, have advantages such as large specific surface area, light weight, high strength, and corrosion resistance. They are mainly used in wastewater treatment as carriers for microbial biofilm formation. Packing material types include honeycomb and mesh structures, with common types including honeycomb inclined tube packing materials, synthetic fiber balls, fiber bundles, biological ribbons, and ropes. The packing materials are mostly made of polyethylene, polypropylene and their modified materials, polyurethane foam, etc., with a specific gravity close to that of water. They are mainly cylindrical and spherical in shape, facilitating biofilm formation and preventing clumping and clogging.

[0004] Functional bacteria play a crucial role in water treatment by degrading specific pollutants. These microorganisms can catalyze the decomposition of pollutants, reducing their harmfulness or even rendering them harmless. For example, in aquaculture wastewater treatment systems, bacteria such as Rhodobulb have been found to effectively remove nutrients such as nitrogen and phosphorus, as well as chemical oxygen demand (COD), from the water.

[0005] Bioaugmentation refers to the technique of adding screened or genetically engineered bacterial strains into a treatment system to improve its treatment efficiency. Currently, in practical engineering, bioaugmentation is mainly achieved by directly adding free bacteria with degradation capabilities into the treatment unit using bacterial solutions or agents. However, the added exogenous strains compete with native microorganisms, leading to difficulties in colonization. Laboratory research has used gel particles to immobilize and embed bacterial strains, but these particles pose a risk of clogging pumps and pipelines in practical applications. Therefore, this invention proposes a novel biofilm packing material for immobilizing and embedding functional bacteria to effectively solve the above problems. Summary of the Invention

[0006] To overcome the shortcomings of existing technologies, this invention provides a method for preparing a novel biofilm packing material that immobilizes and embeds a large number of microorganisms. The prepared biofilm packing material has a sufficiently large surface area suitable for the adsorption and growth of microorganisms, which is conducive to the formation of more biofilms; it is also conducive to the floating and flowing of the packing material in water.

[0007] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0008] A method for preparing a novel biofilm packing material for immobilizing and embedding a large number of microorganisms, characterized by comprising the following steps:

[0009] Step 1: Preparation of bacterial suspension

[0010] The prepared bacterial solution was washed with PBS buffer solution to prepare a bacterial suspension with OD600 = 0.8 to 1.0;

[0011] Step 2: Preparation of PVA-SA mixed solution

[0012] Weigh out an appropriate amount of polyvinyl alcohol (PVA) and sodium alginate (SA) and dissolve them in an appropriate amount of distilled water. Then place the solution in a constant temperature water bath and stir slowly during the heating process to ensure that the two are mixed evenly.

[0013] Step 3: Preparation of Mixed Solution

[0014] Add an appropriate amount of sodium acetate and bacterial suspension to the cooled PVA-SA mixed solution, and stir thoroughly to ensure that the bacterial solution and PVA-SA solution are fully mixed. The mass fraction of the bacterial suspension is 40%.

[0015] Step 4: Preparation of immobilization materials

[0016] The mixed solution obtained in step three is injected into the mold, and then the mold is immersed in a CaCl2 saturated boric acid solution to crosslink and harden for 4 hours. The resulting material is stored in a refrigerator at 4°C.

[0017] Furthermore, in step one, the bacterial suspension is prepared by inoculating functional bacteria into LB medium and culturing them to the end of the logarithmic phase, centrifuging at 5000 rpm for 20 min, discarding the supernatant, washing the bacterial cake twice with PBS buffer, and finally resuspending it with PBS buffer.

[0018] Furthermore, the functional bacteria in step one include, but are not limited to, heterotrophic nitrifying aerobic denitrifying bacteria, anaerobic ammonia-oxidizing bacteria, and polyphosphate-accumulating bacteria.

[0019] Furthermore, the method for preparing the PBS buffer solution in step one is as follows:

[0020] Weigh 8.0g NaCl, 0.2g KCl, 1.44g Na2HPO4, and 0.24g KH2PO4 and dissolve them in 800mL of distilled water. Adjust the solution to 7.4 with HCl, and finally add distilled water to make up to 1L to obtain 0.01M PBS buffer.

[0021] Furthermore, in step two, the PVA-SA mixed solution is heated at a temperature of 80℃ to 95℃ for more than 4 hours to dissolve.

[0022] Furthermore, in step three, the mass fraction of sodium acetate is 1%.

[0023] Furthermore, in step four, the CaCl2 saturated boric acid solution contains 2% CaCl2 by mass, and the preparation method is as follows:

[0024] Weigh 40g of boric acid and dissolve it in an appropriate amount of distilled water. Heat the solution in a water bath until it is completely dissolved. Then add an appropriate amount of CaCl2 and stir continuously with a glass rod until the solution is clear. Adjust the pH with NaHCO3 and transfer the solution to a 1000ml volumetric flask and make up to volume.

[0025] Furthermore, in step four, the mixed solution is injected into the mold using a peristaltic pump or syringe.

[0026] Furthermore, the novel biofilm packing material is shaped like a sea urchin, including many sharp apexes that extend outward from the central core, presenting a radial pattern and being evenly distributed on the surface of the packing material.

[0027] Furthermore, the length of the spikes accounts for half of the entire packing material.

[0028] The beneficial effects of this invention are:

[0029] 1) The novel biofilm packing provided by the present invention is shaped like a sea urchin, with radial spikes evenly distributed on the surface of the packing. On the one hand, this shape has a sufficiently large surface area suitable for the adsorption and growth of microorganisms, which is conducive to the formation of more biofilms. On the other hand, the presence of radial spikes is also conducive to the floating and flowing of the packing in water.

[0030] 2) The novel biofilm packing provided by the present invention has radially distributed spikes evenly distributed on the surface of the packing, which makes the packing have a sufficiently large surface area, increases the contact area with sewage, and is more conducive to the mass transfer between pollutants and dissolved oxygen between microorganisms. At the same time, it also allows the microorganisms embedded inside to obtain sufficient nutrients and have good proliferation and metabolism effects.

[0031] 3) The sea urchin-like shape and structure of this invention enable the packing material to effectively resist external pressure, such as water pressure. This improves the mechanical strength of the biological packing material and can effectively resist external pressure. It plays an important role in the packing material resisting excessive hydraulic load in the sewage treatment process.

[0032] 4) The novel biofilm packing provided by this invention embeds and immobilizes a large number of functional bacteria, which can play a targeted role against specific pollutants or special environments; in addition, the shape of this invention can avoid clogging pumps and pipes in sewage treatment systems, which is beneficial to practical applications.

[0033] 5) The functional bacteria immobilized and embedded in this patent can be replaced with other functional bacteria and their corresponding carbon sources, making it applicable to a wide range of scenarios. Attached Figure Description

[0034] Figure 1 This is a structural diagram of the biofilm packing material of the present invention. Detailed Implementation

[0035] The present invention will now be described in detail with reference to specific embodiments.

[0036] (I) Example 1

[0037] This embodiment uses Acinetobacter sp. TL-3, a low-temperature HN-AD bacterium screened in the laboratory, as an example to prepare a biofilm packing material for immobilizing low-temperature HN-AD bacteria. The preparation method includes the following steps:

[0038] Step 1: Preparation of bacterial suspension

[0039] The prepared bacterial solution was washed with PBS buffer solution to prepare a bacterial suspension with OD600 = 0.8-1.0. The functional bacteria of the present invention include, but are not limited to, heterotrophic nitrifying aerobic denitrifying bacteria, anaerobic ammonia oxidizing bacteria, and polyphosphate-accumulating bacteria.

[0040] The low-temperature HB-AD bacteria Acinetobacter sp. TL-3 was inoculated into LB medium and cultured to the end-log phase. After centrifugation at 5000 rpm for 20 min, the supernatant was discarded, the bacterial cake was washed twice with PBS buffer, and finally resuspended in PBS buffer to obtain a bacterial suspension.

[0041] The method for preparing PBS buffer solution is as follows:

[0042] Weigh 8.0g NaCl, 0.2g KCl, 1.44g Na2HPO4, and 0.24g KH2PO4 and dissolve them in 800mL of distilled water. Adjust the solution to 7.4 with HCl, and finally add distilled water to make up to 1L to obtain 0.01M PBS buffer.

[0043] Step 2: Preparation of PVA-SA mixed solution

[0044] Weigh out 8% polyvinyl alcohol (PVA) and 2% sodium alginate (SA) and dissolve them in an appropriate amount of distilled water. Then place the solution in a constant temperature water bath and stir slowly during the heating process to ensure that the two are mixed evenly. The PVA-SA mixed solution should be heated at 80℃~95℃ for more than 4 hours to dissolve.

[0045] Step 3: Preparation of Mixed Solution

[0046] Add an appropriate amount of sodium acetate and bacterial suspension to the cooled PVA-SA mixed solution, and stir thoroughly to ensure that the bacterial solution and PVA-SA solution are fully mixed. The mass fraction of the bacterial suspension is 40% and the mass fraction of sodium acetate is 1%.

[0047] Using sodium acetate as an external carbon source can compensate for the lack of carbon source when treating wastewater with low C / N ratio, which is beneficial to the growth and reproduction of microorganisms. Sodium acetate is the best carbon source for Acinetobacter sp. TL-3, and the carbon source can be adjusted according to the fixed functional bacteria.

[0048] Step 4: Preparation of immobilization materials

[0049] The mixed solution obtained in step three is injected into the mold using a peristaltic pump or syringe. The mold is then immersed in a CaCl2 saturated boric acid solution to allow it to crosslink and harden for 4 hours. The resulting material is then stored in a refrigerator at 4°C.

[0050] The saturated boric acid solution containing CaCl2 has a CaCl2 mass fraction of 2%, and the preparation method is as follows:

[0051] Weigh 40g of boric acid and dissolve it in an appropriate amount of distilled water. Heat the solution in a water bath until it is completely dissolved. Then add an appropriate amount of CaCl2 and stir continuously with a glass rod until the solution is clear. Adjust the pH with NaHCO3 and transfer the solution to a 1000ml volumetric flask and make up to volume.

[0052] (II) Example 2

[0053] Preparation of biofilm packing material for immobilized polyphosphate bacteria

[0054] Step 1: Preparation of bacterial suspension:

[0055] Polyphosphate-accumulating bacteria were inoculated into LB medium and cultured at room temperature until the end of the logarithmic phase. The culture was centrifuged at 5000 rpm for 20 min, the supernatant was discarded, the bacterial cake was washed twice with PBS buffer, and finally resuspended in PBS buffer to obtain a bacterial suspension.

[0056] Polyphosphate-accumulating bacteria include, but are not limited to, Acinetobacter spp., Aeromonas spp., and Pseudomonas spp.

[0057] Step 2: Preparation of PVA-SA mixed solution:

[0058] Weigh out 8% polyvinyl alcohol (PVA) and 2% sodium alginate (SA) and dissolve them in an appropriate amount of distilled water. Then place the solution in a constant temperature water bath and stir slowly during the heating process to ensure that the two are mixed evenly. The heating temperature of the PVA-SA mixed solution is 80℃~95℃ and the mixing and dissolution time is more than 4 hours.

[0059] (3) Preparation of mixed solution:

[0060] Add 1% carbon source and 40% bacterial suspension to the cooled PVA-SA mixed solution and stir thoroughly to ensure that the bacterial solution and PVA-SA solution are fully mixed.

[0061] Carbon sources include, but are not limited to, acetic acid, propionic acid, and formic acid;

[0062] (4) Preparation of immobilized materials:

[0063] The above mixed solution was poured into a mold, and then the mold was immersed in a CaCl2 saturated boric acid solution to allow it to crosslink and harden for 4 hours. The resulting material was stored in a refrigerator at 4°C for later use.

[0064] The novel biofilm packing material is shaped like a sea urchin, comprising numerous sharp apexes extending radially outward from the central core and evenly distributed across the packing surface. The length of the spikes accounts for half of the total packing length. The structural diagram of the biofilm packing material of this invention is shown below. Figure 1 As shown.

[0065] These spikes not only help the packing material float in water but also facilitate the formation of a larger biofilm, thereby improving the biological treatment effect. Furthermore, the sea urchin-like shape increases the contact area between the packing material and the wastewater, thus enhancing its mass transfer capacity. The bacteria embedded within the packing material receive ample oxygen and nutrients, ensuring the proliferation of functional bacteria and the effective degradation of pollutants. Finally, the sea urchin-like shape and structure allow the packing material to effectively resist external pressures, such as water pressure, which improves the mechanical strength of the biological packing material and plays a crucial role in resisting excessively high hydraulic loads during wastewater treatment.

[0066] The novel biofilm packing material obtained by this invention can be applied to the treatment of low-temperature, low-C / N wastewater in high-altitude and cold regions. It can prevent the functional pure bacteria screened in the laboratory from being preyed upon and consumed by other microorganisms in the wastewater in actual applications, thereby ensuring the quantity of functional bacteria and thus ensuring the denitrification efficiency of functional bacteria in the actual environment.

[0067] In practical use, the biofilm packing is added to the aeration tank. From the perspective of fluidization, the filling rate is generally required to be <67%. From the perspective of operation energy consumption and operation and maintenance management, the filling rate is generally required to be >15%, preferably >30%. From the perspective of further upgrading and increasing the quantity in the future, the filling rate should be controlled at 30-45% to leave some room for upgrading and increasing the quantity.

[0068] In the description of this invention, unless otherwise explicitly specified and limited, the terms "set," "install," "connect," "link," and "fix" should be interpreted broadly. For example, they can refer to a fixed connection, a detachable connection, or an integral part; they can refer to a mechanical connection or an electrical connection; they can refer to a direct connection or an indirect connection through an intermediate medium; they can refer to the internal communication of two components or the interaction between two components. Those skilled in the art can understand the specific meaning of the above terms in this invention based on the specific circumstances.

[0069] The content of this invention is not limited to the embodiments listed. Any equivalent modifications made by those skilled in the art to the technical solutions of this invention by reading this specification are covered by the claims of this invention.

Claims

1. A method for preparing a novel biofilm packing material for immobilizing and embedding a large number of microorganisms, characterized in that: Includes the following steps: Step 1: Preparation of bacterial suspension The prepared bacterial culture was washed with PBS buffer solution to prepare a bacterial suspension with OD600 = 0.8 to 1.0; The bacterial suspension in step one is prepared by inoculating functional bacteria into LB medium and culturing them to the end of the logarithmic phase, centrifuging at 5000 rpm for 20 min, discarding the supernatant, washing the bacterial cake twice with PBS buffer, and finally resuspending it with PBS buffer. Step 2: Preparation of PVA-SA mixed solution Weigh out an appropriate amount of polyvinyl alcohol (PVA) and sodium alginate (SA) and dissolve them in an appropriate amount of distilled water. Then place the solution in a constant temperature water bath and stir slowly during the heating process to ensure that the two are mixed evenly. Step 3: Preparation of Mixed Solution Add an appropriate amount of sodium acetate and bacterial suspension to the cooled PVA-SA mixed solution, and stir thoroughly to ensure that the bacterial solution and PVA-SA solution are fully mixed. The mass fraction of the bacterial suspension is 40%. Step 4: Preparation of immobilization materials The mixed solution obtained in step three is injected into the mold, and then the mold is immersed in a CaCl2 saturated boric acid solution to crosslink and harden for 4 hours. The resulting material is then stored in a refrigerator. The novel biofilm packing material is shaped like a sea urchin, including many sharp apexes that extend outward from the central core, radiating outward and evenly distributed on the surface of the packing material. The length of the spikes accounts for half of the entire packing material.

2. The method for preparing a novel biofilm packing material for immobilizing and embedding a large number of microorganisms according to claim 1, characterized in that: The functional bacteria in step one include one or more of heterotrophic nitrifying aerobic denitrifying bacteria, anaerobic ammonia oxidizing bacteria, and polyphosphate-accumulating bacteria.

3. The method for preparing a novel biofilm packing material for immobilizing and embedding a large number of microorganisms according to claim 1, characterized in that: The method for preparing the PBS buffer solution in step one is as follows: Weigh 8.0g NaCl, 0.2g KCl, 1.44g Na2HPO4, and 0.24g KH2PO4 and dissolve them in 800mL of distilled water. Adjust the pH of the solution to 7.4 with HCl, and finally add distilled water to make up to 1L to obtain 0.01M PBS buffer.

4. The method for preparing a novel biofilm packing material for immobilizing and embedding a large number of microorganisms according to claim 1, characterized in that: In step two, the PVA-SA mixed solution is heated to 80℃~95℃ and mixed and dissolved for more than 4 hours.

5. The method for preparing a novel biofilm packing material for immobilizing and embedding a large number of microorganisms according to claim 1, characterized in that: In step three, the mass fraction of sodium acetate is 1%.

6. The method for preparing a novel biofilm packing material for immobilizing and embedding a large number of microorganisms according to claim 1, characterized in that: In step four, the CaCl2 in the saturated boric acid solution has a CaCl2 mass fraction of 2%, and the preparation method is as follows: Weigh 40g of boric acid and dissolve it in an appropriate amount of distilled water. Heat the solution in a water bath until it is completely dissolved. Then add an appropriate amount of CaCl2 and stir continuously with a glass rod until the solution is clear. Adjust the pH with NaHCO3 and transfer the solution to a 1000ml volumetric flask and make up to volume.

7. The method for preparing a novel biofilm packing material for immobilizing and embedding a large number of microorganisms according to claim 6, characterized in that: In step four, the mixed solution is injected into the mold using a peristaltic pump or syringe.

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