Polyurethane adsorption-gel embedding microorganism composite immobilization method and application thereof

Abstract

The invention relates to a polyurethane adsorption-gel embedding microorganism composite immobilization method and application thereof, and belongs to the technical field of coking wastewater treatment and microorganisms. Polyurethane sponge is immersed into a mixed solution containing phenol degrading bacteria and sodium alginate, after sufficient adsorption, the polyurethane sponge is transferred into a calcium chloride-boric acid mixed solution for cross-linking curing, and finally the organic gel composite carrier with a porous structure is formed. The immobilized carrier can significantly enhance the resistance of phenol degrading bacteria to environmental stress, and improve the adaptability and stability of the phenol degrading bacteria under different water quality conditions. Meanwhile, the carrier is simple and convenient in preparation process and low in cost, has good reusability, is suitable for efficient immobilization of microorganisms in the water treatment process, and has wide popularization and application prospects.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of coking wastewater treatment and microbial technology, and more particularly, to a polyurethane adsorption-gel embedding microbial composite immobilization method and application thereof. BACKGROUND

[0002] Coking is one of the important ways for clean and efficient conversion of coal, which not only plays a key role in the production process of metallurgical coke and provides basic raw materials for the steel industry, but also can by-product coal gas, tar and crude benzene and other chemical products, with significant economic and social benefits. However, this process is also accompanied by serious environmental pollution problems, among which coking wastewater has become one of the main factors restricting the green development of the industry due to its high toxicity and complex composition.

[0003] Among the pollutants contained in coking wastewater, phenolic substances are the highest concentration of organic components, and their chemical oxygen demand (COD) contribution accounts for about 60%-80% of the total COD of the wastewater. As a typical representative, the average concentration of phenol is about 500 mg / L, and the highest can exceed 1000 mg / L. High concentration of phenol has a significant inhibitory effect on the microbial community in the wastewater biological treatment system, resulting in a decrease in treatment efficiency; at the same time, it persists in the environment, has strong toxicity to aquatic organisms and ecosystems, and can cause damage and death to plants and animals, and can harm human health through various exposure routes.

[0004] At present, the treatment technologies for coking wastewater mainly include physical methods, chemical methods, biological methods and combinations of various processes. Physical and chemical methods can effectively remove part of the pollutants, but generally have problems such as high treatment cost, high energy consumption, easy to produce secondary pollution, etc. Biological treatment method has become the core technology for coking wastewater treatment due to its mature technology, simple operation and management, low cost and environmental friendliness, etc., and especially the activated sludge method and related derivative technologies are the most widely used. However, high concentration of phenol in wastewater has a strong inhibitory and toxic effect on microbial activity, which often leads to unstable performance or even failure of the treatment system, becoming a major bottleneck in the practical application of biological treatment technology.

[0005] Microbial immobilization technology is to limit microbial cells in specific carrier materials through physical or chemical methods to improve their resistance to adverse environments, stable degradation performance and reusability. This technology can be mainly divided into adsorption method, embedding method, cross-linking method and covalent binding method according to the immobilization mechanism. Among them, cross-linking method and covalent binding method are limited in application due to high cost; adsorption method and embedding method are commonly used, but still have problems such as poor stability of the carrier, easy falling off of the microorganisms, large mass transfer resistance and gel swelling, etc. Therefore, it is of great research significance and practical application prospect to develop new microbial immobilization methods to enhance the stability and treatment efficiency of the system. SUMMARY

[0006] The present application is directed to the problems of poor carrier stability, easy microbial shedding, large mass transfer resistance and weak environmental impact resistance in the existing microbial immobilization technology. The present application provides a polyurethane adsorption-gel embedding microbial composite immobilization method and its application. The phenol-degrading bacteria are adsorbed into polyurethane sponge, and sodium alginate gel is constructed inside, thereby obtaining a phenol-degrading bacteria immobilization carrier. The carrier can significantly enhance the degradation activity of the phenol-degrading bacteria and improve the stability and reusability of the treatment system.

[0007] According to a first aspect of the present application, a polyurethane adsorption-gel embedding microbial composite immobilization method is provided, comprising the following steps: (1) inoculating a phenol-degrading bacteria liquid into a sodium alginate solution, fully mixing to obtain a mixed solution, and immersing polyurethane sponge in the mixed solution to allow the polyurethane sponge to fully adsorb the mixed solution; (2) taking out the polyurethane sponge from the mixed solution, immersing the polyurethane sponge in a mixed solution containing calcium chloride and boric acid for cross-linking and curing to obtain a gel, i.e. a polyurethane adsorption-gel embedding microbial composite immobilization carrier.

[0008] Preferably, the phenol-degrading bacteria are mixed bacteria of Acinetobacter and Pseudomonas.

[0009] Preferably, the Acinetobacter has a preservation number of CCTCC NO: M20251392 Acinetobacter bereziniae G0-1, classified as Acinetobacter, Latin name: Acinetobacter bereziniae G0-1, the unit name for preserving the strain is: China Center for Type Culture Collection, address: Wuhan, Wuhan University, China, preservation date: June 16, 2025, preservation number: CCTCC NO: M20251392.

[0010] The Pseudomonas has a preservation number of CCTCC NO: M20242868 Pseudomonas monteilii PD-1, classified as Pseudomonas, Latin name: Pseudomonas monteilii PD-1, the unit name for preserving the strain is: China Center for Type Culture Collection, address: Wuhan, Wuhan University, China, preservation date: December 20, 2024, preservation number: CCTCC NO: M20242868.

[0011] According to another aspect of the present application, a microbial composite immobilization carrier obtained by the polyurethane adsorption-gel embedding microbial composite immobilization method is provided.

[0012] According to another aspect of the present application, the microorganism composite immobilized carrier is used in phenol degradation.

[0013] Preferably, the concentration of the phenol is 800 mg / L-2000 mg / L.

[0014] Preferably, the mass concentration of the inorganic salt in the solution where the phenol is located is less than or equal to 3%.

[0015] Overall, compared with the prior art, the above technical solutions conceived by the present application mainly have the following technical advantages: (1) The phenol-degrading bacteria immobilized carrier of the present application has excellent tolerance: the phenol-degrading bacteria are simultaneously fixed in the sponge skeleton and the gel network by the adsorption-embedding composite fixation method, which effectively relieves the toxic stress of high-concentration phenol on the microorganisms, and the maximum tolerance concentration of phenol can reach 2000 mg / L.

[0016] (2) The phenol-degrading bacteria immobilized carrier of the present application has balanced and efficient degradation performance: the degradation rate of the composite immobilized carrier prepared by the adsorption-embedding composite fixation method on phenol is significantly higher than that of free bacteria and the embedding fixation method alone, and is basically equivalent to that of the adsorption fixation method, which maintains high-efficiency degradation while having better cell retention and stability.

[0017] (3) The phenol-degrading bacteria immobilized carrier of the present application has strong environmental adaptability and operational stability: the composite immobilized carrier prepared by the adsorption-embedding composite fixation method and the adsorption immobilized carrier both exhibit excellent salt tolerance, with a maximum tolerance of 3% salinity, which is significantly better than that of free bacteria and the embedding immobilized carrier; in addition, the stability of the composite immobilized carrier is outstanding during repeated use, and its reusability is significantly better than that of the adsorption fixation and embedding fixation methods.

[0018] (4) The microorganism composite immobilization method of the present application includes two-stage crosslinking. The first-stage crosslinking: Ca 2+ specifically binds to G units (guluronate) on the sodium alginate molecular chain, forming the basic three-dimensional network structure of the gel; the second-stage crosslinking: boron acid molecules form boron-oxygen coordination bonds with M units (mannuronate) on the sodium alginate molecular chain in an aqueous solution, which helps to further improve the mechanical strength and stability of the carrier. BRIEF DESCRIPTION OF DRAWINGS

[0019] Figure 1 is a schematic diagram of Example 1 (a), Comparative Example 1 (b), Comparative Example 2 (c), and Comparative Example 3 (d) in the present application.

[0020] Figure 2 is a degradation data graph of Example 1 in the present application for different concentrations of phenol.

[0021] Figure 3 is the degradation data graph of phenol in the present application, example 1, comparative example 1-3.

[0022] Figure 4 is the salt tolerance of free bacteria (a), example 1 (b), comparative example 2 (c) and comparative example 3 (d) in the present application under 1000 mg / L phenol.

[0023] Figure 5 is the reuse performance of example 1, comparative example 2 and comparative example 3 in the present application. DETAILED DESCRIPTION

[0024] In order to make the purpose, technical scheme and advantages of the present application more clear, the present application is further described in detail below in combination with the drawings and examples. It should be understood that the specific examples described herein are only used to explain the present application and not to limit the present application. In addition, the technical features involved in each embodiment of the present application described below can be combined with each other as long as they do not conflict with each other.

[0025] In the following examples, sodium alginate, calcium chloride, boric acid and polyurethane sponge are all commercially available.

[0026] Preferably, in the examples and comparative examples, the phenol-degrading bacteria used are obtained by domesticating and culturing in a phenol-substrate culture medium of activated sludge, wherein the activated sludge is derived from a first-stage aerobic tank of a coking wastewater plant in Wuhan. The activated sludge is replaced with influent every day in a laboratory-scale sequencing batch reactor (SBR), and the composition of the influent is: MgSO40.12 g / L, KH2PO40.4 g / L, NaCl 0.5 g / L, NaHCO30.2 g / L, FeSO4·7H2O 0.02 g / L, NH4Cl 0.5 g / L, phenol 500 mg / L, trace element solution (1.5 g / L FeCl2·4H2O, 190 mg / L CoCl2·6H2O, 100 mg / L MnCl2·6H2O, 70 mg / L ZnCl2, 62 mg / L H3BO3, 36 mg / L Na2MoO4·2H2O, 24 mg / L NiCl2·6H2O, 17 mg / L CuCl2·2H2O) 1 mL / 1000 mL. The final pH is controlled at 7. The isolated and selected phenol-degrading bacteria are Acinetobacter sp. Acinetobacter bereziniae ) and Pseudomonas sp. Pseudomonas monteilii

[0027] Preferably, the preparation method of the polyurethane adsorption-gel embedded microbial composite immobilized carrier comprises the following steps: (1) Bacterial culture and mixing: Acinetobacter sp.​Acinetobacter bereziniae ) Pseudomonas Pseudomonas monteilii ) to logarithmic growth phase, adjust the OD 600 of the bacterial solution to 1.0, mix in a volume ratio of 1:1 to prepare a phenol-degrading bacteria mixed solution; (2) Mixing of the bacterial solution and the gel matrix: add the above-mentioned phenol-degrading bacteria mixed solution to a sodium alginate solution with a mass concentration of 4% at an inoculation amount of 15% (v / v), and mix thoroughly; (3) Sponge adsorption: immerse the polyurethane sponge in the mixed solution obtained in step (2), and stand for adsorption until no air bubbles are released from the inside of the sponge; (4) Cross-linking and solidification: take out the sponge, remove the excess droplets on the surface, and then immerse it in a mixed solution containing calcium chloride and boric acid for cross-linking and solidification at 4 ℃ for 6 h, and the gel solidification is completed, thereby obtaining the microbial composite immobilized carrier.

[0028] The following are specific examples Example 1 In this example, a polyurethane adsorption-gel embedding microbial composite immobilized carrier is prepared, and the specific steps are as follows: (1) Pretreatment: acid washing and alkali washing are performed on the polyurethane sponge to remove surface impurities, and then the sponge is washed with ultrapure water until pH=7, and is dried at 60 ℃ for standby; the pretreated polyurethane, sodium alginate solution, and calcium chloride-boric acid solution are sterilized, and are cooled to room temperature for standby; (2) Bacterial culture and mixing: Acinetobacter and Pseudomonas are cultured in BHI medium to logarithmic growth phase, and the OD 600 of the bacterial solution is adjusted to 1.0, and then mixed in a volume ratio of 1:1 to prepare a phenol-degrading bacteria mixed solution; (3) Mixing of the bacterial solution and the gel matrix: add the above-mentioned phenol-degrading bacteria mixed solution to a sodium alginate solution with a mass concentration of 4% at an inoculation amount of 15% (v / v), and mix thoroughly to obtain a porous embedding liquid; (4) Sponge adsorption: immerse the polyurethane sponge in the mixed embedding liquid obtained in step (3), and stand for adsorption until no air bubbles are released from the inside of the sponge; (5) Cross-linking and solidification: take out the sponge, remove the excess droplets on the surface, and then immerse it in a mixed solution containing calcium chloride-boric acid, and react at 4 ℃ for 6 h, and the gel solidification is completed, thereby obtaining the microbial composite immobilized carrier. The actual carrier prepared is shown in Figure 1 a of FIG. 1.

[0029] Comparative Example 1 Compared with Example 1, the polyurethane adsorption-gel embedding microbial composite immobilized carrier is changed to a sterile composite immobilized carrier, and the specific operation steps are as follows: The sterile sponge was immersed in a 4% sodium alginate solution and allowed to adsorb into the sponge without bubble separation. After removing the excess droplets on the surface, the sponge was immersed in a calcium chloride-boric acid mixed solution and reacted at 4°C for 6 h to complete the gel solidification, thereby obtaining a sterile composite immobilized carrier. The actual carrier obtained is shown in Fig. b. Figure 1

[0030] Comparative Example 2 Compared with Example 1, the polyurethane adsorption-gel embedding microbial composite immobilized carrier was changed to an adsorption immobilized carrier, and the specific operation steps were as follows: The phenol-degrading bacteria mixture was added to sterile normal saline at an inoculation amount of 15% (v / v) and uniformly mixed. The polyurethane sponge was immersed in the mixture and allowed to adsorb into the sponge without bubble separation, thereby obtaining an adsorption immobilized carrier. The actual carrier obtained is shown in Fig. c. Figure 1

[0031] Comparative Example 3 Compared with Example 1, the polyurethane adsorption-gel embedding microbial composite immobilized carrier was changed to a gel embedding immobilized carrier, and the specific operation steps were as follows: The phenol-degrading bacteria mixture was added to a 4% sodium alginate solution at an inoculation amount of 15% (v / v) and uniformly dispersed to obtain a porous embedding liquid. Then, the embedding liquid was dropped into a calcium chloride-boric acid mixed solution using a sterile syringe, and crosslinked at 4°C for 6 h to obtain a gel embedding immobilized carrier. The actual carrier obtained is shown in Fig. d. Figure 1

[0032] Example 2 This example investigated the effect of different initial phenol concentrations on the degradation performance of the polyurethane adsorption-gel embedding microbial composite immobilized carrier.

[0033] The composite immobilized carrier prepared in Example 1 was inoculated in 100 mL of inorganic salt medium at pH 7.0, and the phenol concentration was set to 800, 1000, 1200, 1400, 1600, 1800 and 2000 mg / L, respectively. The culture was oscillated at 30°C and 150 rpm, and samples were taken at different time points. The 4-aminoantipyrine spectrophotometry was used to determine the residual phenol concentration to evaluate the degradation performance. The results are shown in Fig. Figure 2 The removal of phenol by the composite immobilized carrier includes two stages of adsorption and biodegradation. As the initial concentration of phenol increases, the adsorption rate of the carrier to phenol increases, but the biodegradation rate gradually slows down. The carrier shows good tolerance in the experimental concentration range.

[0034] Example 3 This example compared the degradation performance of different immobilized carriers and free bacteria to phenol.​​​

[0035] Example 1, Comparative Example 2, Comparative Example 3 and the free bacteria were inoculated into 100 mL of inorganic salt medium containing 1400 mg / L phenol (pH 7.0), and degradation experiments were carried out at 30°C and 150 rpm. Samples were taken periodically and the phenol concentration was determined by the 4-aminoantipyrine method. The inoculation amount of the free bacteria was equivalent to the amount of bacteria loaded in each immobilized carrier. The results are shown in Table 1. Figure 3 As shown in Table 1, the composite immobilized carrier and the adsorption immobilized carrier had the best removal effect on phenol, with a degradation rate of more than 98%, which was significantly higher than that of the free bacteria group (44.74%), indicating that the immobilization treatment effectively enhanced the degradation stability and efficiency of the bacteria.

[0036] Example 4 This example evaluates the effect of salinity on the degradation of phenol by different immobilized carriers and free bacteria.

[0037] Under different salinity conditions (mass concentration of 1%-5%), each immobilized carrier and free bacteria were inoculated into 100 mL of inorganic salt medium with a phenol concentration of 1000 mg / L, and were cultured at 30°C and 150 rpm. The residual phenol concentration was detected periodically to compare the salt tolerance of different immobilized carriers and free bacteria. According to the equal amount principle, the amount of free bacteria was the same as the volume of free bacteria contained in different immobilized carriers. The results are shown in Table 2. Figure 4 As shown in Table 2, b) in Example 1( Figure 4 and c) in Comparative Example 2( Figure 4 had the best salt tolerance and still maintained degradation activity at a higher salinity (mass concentration of 3%); a) in the free bacteria( Figure 4 had the second best salt tolerance (mass concentration of 2%); and d) in Comparative Example 3( Figure 4 was the most sensitive to salinity (mass concentration of 1%).

[0038] Example 5 This example evaluates the reuse ability of different immobilized carriers.

[0039] Different immobilized carriers were placed in 100 mL of inorganic salt medium containing 1000 mg / L of phenol (pH 7.0) and were subjected to cyclic degradation at 30°C and 150 rpm. Each cycle was considered to be completed when the removal rate of phenol reached more than 95%. After each cycle, the immobilized carrier was added to fresh medium to start the next cycle. The results are shown in Table 3. Figure 5As shown, after experiencing multiple cycles, the composite immobilized carrier (Example 1) showed the best reuse stability, the degradation rate first increased, reached a peak at the end of the 3rd cycle, which was 40.67 mg / (L·h), then decreased and remained stable; the adsorption immobilized carrier (Comparative Example 2) continuously decreased; the embedding immobilized carrier (Comparative Example 3) had a low initial activity, the degradation rate was only 4.65 mg / (L·h), and the degradation rate increased in the subsequent cycles, and reached a peak at the end of the 4th cycle, which was 29.11 mg / (L·h), and then the degradation rate decreased.

[0040] Those skilled in the art will readily understand that the above description is only the preferred embodiment of the present application, and is not intended to limit the present application, and any modifications, equivalent replacements and improvements made within the spirit and principle of the present application shall be included in the protection scope of the present application.

Claims

1. A polyurethane adsorption-gel-embedded microbial complex immobilization method, characterized by, The method comprises the following steps: (1) inoculating phenol-degrading bacteria into a sodium alginate solution, mixing thoroughly to obtain a mixed solution, and immersing a polyurethane sponge in the mixed solution so that the polyurethane sponge absorbs the mixed solution; (2) taking out the polyurethane sponge from the mixed solution, immersing the polyurethane sponge in a mixed solution containing calcium chloride and boric acid to cross-link and solidify to obtain a gel, i.e. a polyurethane adsorption-gel-embedded microbial composite immobilized carrier.

2. The polyurethane adsorptive-gel embedded microbial composite immobilization method according to claim 1, wherein, The phenol-degrading bacteria are mixed bacteria of Acinetobacter and Pseudomonas.

3. The polyurethane adsorptive-gel embedded microbial composite immobilization method according to claim 2, characterized in that, The preservation number of the Acinetobacter is CCTCC NO: M20251392, and the preservation number of the Pseudomonas is CCTCC NO: M20242868.

4. The microbial composite immobilized carrier prepared by the method of any one of claims 1-3.

5. The microbial composite immobilized carrier of claim 4 in the application of phenol degradation.

6. Use according to claim 5, characterized in that, The concentration of the phenol is 800 mg / L-2000 mg / L.

7. The use according to claim 5, wherein the compound is ###0002### The mass concentration of inorganic salts in the solution in which the phenol is located is less than or equal to 3%.

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