Preparation and application of porous material loaded with microorganism active sludge with lipophilic and hydrophobic properties

Abstract

The application provides a preparation method of a porous material loaded with mixed microorganisms, comprising the following steps: S1, preparation of a mixed bacteria suspension; S2, preparation of an activated sludge porous material; and S3, preparation of a porous material loaded with mixed microorganisms. The application also provides a preparation method of an oil-wetting and water-repellent activated sludge porous material loaded with microorganisms, which comprises spraying a hydrophobic silica particle suspension on the porous material loaded with mixed microorganisms to obtain a modified material which is oil-wetting and water-repellent. The porous material loaded with mixed microorganisms and the oil-wetting and water-repellent activated sludge porous material loaded with microorganisms can effectively degrade medium and long chain alkanes in oil, especially n-hexadecane.

Description

Technical Field

[0001] This invention belongs to the field of environmental materials application technology, specifically relating to a method for preparing porous materials of oleophilic and hydrophobic microbial-loaded activated sludge. This invention also relates to the application of this porous material in the degradation of n-hexadecane. Background Technology

[0002] Petroleum, often referred to as the "lifeblood of industry," is not only a crucial fossil fuel in industrial production but also a raw material for many chemical products, such as solvents, fertilizers, pesticides, and plastics. During the extraction, processing, transportation, and use of petroleum, leaks and emissions of crude oil and various petroleum products cause severe petroleum pollution. Hexadecane, a significant component of long-chain alkanes in light crude oil, is characterized by low water solubility and low volatility, making it difficult to eliminate once it enters the environment, thus classifying it as a persistent pollutant. Therefore, there is an urgent need for efficient, high-performance, low-cost, and environmentally friendly technologies to address this problem. To date, research has employed various methods, including physical, chemical, and biological methods, to eliminate petroleum-derived pollution. Compared to physical and chemical methods, biological treatment methods offer significant advantages. They can treat oil-containing components through biodegradation, offering advantages such as good treatment efficiency, low cost, minimal environmental impact, and wide applicability, and have gained favor among most researchers. However, multiple studies have shown that free-floating microorganisms have long degradation times, are highly susceptible to environmental interference, and exhibit low effective concentrations per unit volume. Therefore, to address the rapid degradation of complex petroleum components caused by a single strain, microbial immobilization technology can be used to confine multiple microorganisms within a specific spatial area, achieving high microbial density and maintaining their biological activity, thereby improving microbial degradation efficiency. The carrier material plays a crucial role in the microbial immobilization process. Activated sludge, as wastewater from wastewater treatment plants, possesses bioaggregation, adsorption, and oxidation capabilities, enabling the decomposition and removal of organic pollutants from wastewater. Therefore, it can serve as a low-cost, green, environmentally friendly, and highly efficient porous material for removing oily wastewater, exhibiting good biocompatibility, low price, and providing abundant carbon sources for microorganisms, turning waste into resources. Currently, research typically focuses on immobilizing microorganisms through encapsulation and adsorption. However, the limited amount of immobilized microorganisms and weak binding forces of adsorption methods restrict their widespread application. Therefore, developing and exploring novel high-performance microbial-supported porous materials will be a major direction for future research. Summary of the Invention

[0003] To address the problems existing in the prior art, this invention provides a method for preparing an oleophilic and hydrophobic microbial-loaded porous activated sludge material and its application in the degradation of n-hexadecane. This invention employs a multi-stage concentration and fixation method to load a mixed bacterial agent onto the activated sludge porous material. On one hand, this effectively increases the number of microorganisms, improves the binding force between the microorganisms and the loaded material, and enhances the activity of the microorganisms. On the other hand, it utilizes the synergistic effect of the microorganisms to maximize their efficiency, promote microbial degradation, and achieve the goal of highly efficient degradation.

[0004] To achieve the technical objective of this invention, the technical solution adopted by this invention is as follows: This invention provides a method for preparing porous materials loaded with mixed microorganisms, comprising the following steps: S1, Preparation of mixed bacterial suspension: Bacillus thuringiensis BT, Pseudomonas aeruginosa A6, Acinetobacter rhusiopathiae A5, Nocardia alba F1, and Bacillus thuringiensis were mixed. BS The bacterial solutions were prepared separately and then mixed in equal volume ratios to form a mixed bacterial suspension. S2, Preparation of porous activated sludge material: Activated sludge was obtained through gravity sedimentation, sieving, centrifugal concentration, and sedimentation. The activated sludge was added to a sodium alginate solution, stirred, and freeze-dried. Finally, the freeze-dried activated sludge material was placed in a Ca... 2+ Cross-linking in solution yields a porous activated sludge material, named PAS. S3, Preparation of porous material loaded with mixed microorganisms: The activated sludge porous material PAS prepared in step S2 is bound with polyvinyl alcohol, then placed in the mixed bacterial suspension prepared in step S1 to adsorb the mixed bacteria, and then Ca... 2+ Cross-linking was performed to obtain a porous material loaded with mixed microorganisms.

[0005] Preferably, in step S2, the ratio of activated sludge to sodium alginate is 5.0 g: 2.0 g; and / or The Ca 2+ The ratio of the amount of water used to distilled water is 5.0 g: 100.0 mL; and / or The ratio of sodium alginate to distilled water is 5.0 g: 100.0 mL.

[0006] Preferably, in step S2, the Ca 2+ The weight percentage concentration is 0.98-1.22%; and / or Step S3 is repeated 1-6 times.

[0007] The present invention also provides a porous material loaded with mixed microorganisms, which is prepared by the above-described method.

[0008] The present invention also provides a method for preparing a porous material of oleophilic and hydrophobic microbial-loaded activated sludge, comprising the following steps: brushing a commercially available epoxy resin dissolved in anhydrous ethanol onto a porous material loaded with mixed microorganisms, and then spraying a liquid suspension of hydrophobic silica particles of tridecafluorooctyltriethoxysilane onto the surface of the material to obtain a hydrophobic and oleophilic microbial-loaded activated sludge porous material.

[0009] Preferably, the ratio of the SiO2 nanoparticles to the anhydrous ethanol solution is 1 g: 49.5 g; and / or The ratio of the tridecafluorooctyltriethoxysilane to the SiO2 nanoparticles is 1 g: 2 g; and / or The ratio of epoxy resin to anhydrous ethanol is 1 g: 2 g.

[0010] The present invention also provides an oleophilic and hydrophobic microbial activated sludge porous material, which is prepared by the above-described method.

[0011] The present invention also provides the above-mentioned porous material loaded with mixed microorganisms, or the application of the porous material of activated sludge loaded with oleophilic and hydrophobic microorganisms in the degradation of petroleum hydrocarbons; wherein the petroleum hydrocarbons are long-chain n-alkanes, medium- and long-chain alkanes, hopanes or steranes; Preferably, the medium- to long-chain alkane is a C4-C16 alkane.

[0012] As a further preferred option, the medium-to-long chain alkane is n-hexadecane.

[0013] This application utilizes several highly efficient petroleum-degrading bacteria isolated and purified from oily sludge. These bacteria can not only degrade n-hexadecane, but also convert high-carbon n-alkanes into medium- and low-carbon alkanes, ultimately transforming them into water and carbon dioxide. Furthermore, this bacterial agent can also degrade n-alkanes, hopanes, and steranes. When this bacterial agent is loaded onto porous activated sludge materials, it can theoretically degrade long-chain n-alkanes, medium- and long-chain alkanes, hopanes, and steranes.

[0014] Preferably, the degradation temperature is 27~47℃, preferably 37℃; and / or The degradation occurs at a pH of 2-10, preferably pH 6; and / or The weight percentage concentration of the petroleum-derived alkanes is 1%-5%.

[0015] This invention provides methods for synthesizing porous materials loaded with mixed microorganisms and porous activated sludge materials loaded with oleophilic and hydrophobic microorganisms, primarily through multiple concentrations to immobilize microorganisms and selective modification using oleophilic and hydrophobic methods. Secondly, it provides applications in terms of their bioactivity, specifically for the degradation of petroleum hydrocarbons. This invention uses an oil-in-water (O / W) emulsion method to obtain honeycomb-structured porous activated sludge materials. This material transforms waste into a valuable resource, possessing strong mechanical properties, recyclability, high adsorption capacity, and hydrophobic and oleophilic properties. Simultaneously, five microbial agents for degrading petroleum hydrocarbons are mixed in equal proportions, and through multiple concentrations, the mixed microbial agents are loaded into the porous activated sludge material, strengthening the binding force between microorganisms and the porous activated sludge material, increasing the number of microorganisms per unit volume, and enhancing the bioactivity of the microorganisms per unit volume. A hydrophobic and oleophilic film is coated onto the surface of the microbial-loaded porous activated sludge material, giving it selective adsorption properties and enhancing its contact rate with n-hexadecane, achieving efficient degradation. The porous materials loaded with mixed microorganisms and the porous activated sludge materials loaded with oleophilic and hydrophobic microorganisms of this invention can be used for the degradation of petroleum hydrocarbons. This invention utilizes the adhesive polyvinyl alcohol (PVA) and the crosslinking agent Ca 2+ Multiple concentrations of the bacterial agent and selective adsorption-based hydrophobic modification play a crucial role in improving the degradation rate. The oleophilic-hydrophobic loaded mixed microbial activated sludge porous material prepared in this invention exhibits good characteristics in degrading n-alkanes, and has certain practical application value for future petroleum pollution remediation. Attached Figure Description

[0016] The accompanying drawings are provided to further illustrate the invention and form part of the specification. They are used in conjunction with embodiments of the invention to explain the invention and do not constitute a limitation thereof. In the drawings: Figure 1 This is a flowchart illustrating the preparation and degradation process of the oleophilic and hydrophobic microbial-loaded activated sludge porous material of the present invention.

[0017] Figure 2 This is a scanning electron microscope image of PAS.

[0018] Figure 3 This is a scanning electron microscope (SEM) image of MPP(@).

[0019] Figure 4 This refers to the oleophilic and hydrophobic porous material for loading microbial activated sludge prepared in Example 3.

[0020] Figure 5Figure a shows the degradation rates of free mixed bacteria, PAS(@), and OPAS-M at different degradation times under the conditions of 37℃, 120 r / min, pH=6, and a hexadecane weight percentage concentration of 3%. Figure b shows the first-order kinetics comparison of free mixed bacteria, PAS(@), and OPAS-M under the conditions of 37℃, 120 r / min, pH=6, and a hexadecane weight percentage concentration of 3%. Figure c shows the original GC images of the mixed bacterial suspension (referred to as "free bacteria" in this figure), PAS(@), and OPAS-M, respectively.

[0021] Figure 6 (a) Degradation rates of suspended cells, PAS(@), and OPAS-M at different n-hexadecane concentrations at 37℃, 120 r / min, pH=6, and a degradation time of 50 h. (b) Degradation rates of suspended cells, PAS(@), and OPAS-M at different temperature conditions at 120 r / min, pH=6, and a degradation time of 60 h. (c) Degradation rates of suspended cells, PAS(@), and OPAS-M at different pH conditions at 37℃, 120 r / min, and a degradation time of 60 h. Detailed Implementation

[0022] The following examples are provided to better understand the present invention, but do not limit the invention. Unless otherwise specified, the experimental methods in the following examples are conventional methods. Unless otherwise specified, the experimental materials used in the following examples were purchased from conventional biochemical reagent companies. All quantitative experiments in the following examples were performed in triplicate, and the results were averaged.

[0023] The preparation and degradation route of the oleophilic and hydrophobic microbial-loaded activated sludge porous material of the present invention are as follows: Figure 1 As shown.

[0024] Figure 1 This is a flowchart illustrating the preparation and degradation process of the oleophilic and hydrophobic microbial-loaded activated sludge porous material of the present invention.

[0025] The strain used in this invention: Bacillus thuringiensis (Bt) Bacillus thuringiensis BT), Pseudomonas aeruginosa ( Pseudomonas aeruginosa A6), Acinetobacter rufi ( Acinetobacter lwoffii (A5), Nocardia albopictus Nocardioides luteus F1), Bacillus ( Aacillus, BS ).

[0026] Among them, Bacillus thuringiensis (Bt) Bacillus thuringiensisThe study on the degradation of PAHs by Bacillus thuringiensis (BT) has been published in the literature “Study on the degradation of PAHs by Bacillus thuringiensis, Luo Xiaofang et al., Biotechnology Bulletin”.

[0027] Pseudomonas aeruginosa ( Pseudomonas aeruginosa A6), Acinetobacter rufi ( Acinetobacter lwoffii (A5), Nocardia albopictus Nocardioides luteus F1) has been published in the literature “Study on biological degradation and transform characteristics of different components in petroleum hydrocarbon used by bacterial consortium, Lihua Chen, etc., EnvironEarth Sci”.

[0028] Bacillus ( Aacillus, BS The findings have been published in the literature “Study of the Characteristics of Two Immobilized Microbial Materials in Degradation and Evolution of Petroleum Hydrocarbon, Xiaofang Luo, etc.”.

[0029] The applicant hereby undertakes to provide the above-mentioned 5 strains to the public within 20 years from the date of application.

[0030] The preparation method of the oleophilic and hydrophobic microbial-loaded activated sludge porous material of the present invention includes the following steps: I. Preparation of PAS (Porous Material for Activated Sludge) Activated sludge was obtained through gravity sedimentation, passing through a 100-mesh sieve, centrifugal concentration, and sedimentation. 5.0 g of the activated sludge was placed in a beaker, and 2 g of sodium alginate (5 wt%) solution was added. The mixture was stirred for 15 minutes, then placed into a cylindrical plastic mold with a diameter of 2.5 cm and frozen for 12 hours. Following this, it was freeze-dried for 36 hours. Finally, the freeze-dried activated sludge material was cross-linked in a 3 wt% Ca(NO3)24h2O solution for 24 hours, and the porous activated sludge material was named PAS. PAS was autoclaved at 121°C for 30 minutes and cooled to room temperature before use.

[0031] The ratio of activated sludge to sodium alginate is 5.0g:2.0g; Ca 2+The weight ratio of sodium alginate to distilled water is 5.0 g: 100.0 mL; the weight ratio of sodium alginate to distilled water is 5.0 g: 100.0 mL.

[0032] This invention prepares porous activated sludge materials using soft template technology. Sodium alginate serves as the continuous phase, and activated sludge as the dispersed phase, with a weight ratio of activated sludge to sodium alginate of 5:2. When the amount of sodium alginate is low, the resulting activated sludge pore structure is less numerous and unevenly distributed. Conversely, excessive sodium alginate content results in an overly porous activated sludge material with poor mechanical strength, making it unsuitable for loading microbial agents.

[0033] Sodium alginate requires ultra-low temperature freezing. Using a freeze dryer, the water in the sample sublimates directly in the presence of ice, creating numerous pores in the sample. However, directly freeze-drying it without first immersing it in liquid nitrogen will damage the sample and prevent it from forming a proper shape.

[0034] II. Preparation of mixed bacterial suspension: Bacillus thuringiensis (Bt) Bacillus thuringiensis BT), Pseudomonas aeruginosa ( Pseudomonas aeruginosa A6), Acinetobacter rufi ( Acinetobacter lwoffii A5), Nocardia albopictus ( Nocardioides luteus F1), Bacillus ( Aacillus, BS Five strains of bacteria were cultured in a three-stage scale-up process according to the method described in the relevant literature to prepare bacterial suspensions with a concentration of 1×10⁻⁶. 10 ~1×10 12 cfu·mL -1 The bacteria were mixed in equal volume ratios to prepare a mixed bacterial suspension. The colony activity of the mixed bacterial suspension was detected using the dilution plating method, reaching 1×10⁻⁶. 10 ~1×10 12 cfu·mL -1 Then, store at 4 ℃ until use.

[0035] 3. The sterilized hydrophilic and oleophilic activated sludge porous material is immersed in a 3% polyvinyl alcohol (PVA) solution for 20 minutes to allow the 3% PVA to fully penetrate into the activated sludge porous material. Excess PVA is removed by filtration, and the surface is rinsed with distilled water to prevent blockage of the outer wall pores. Then, it is immersed in the mixed bacterial suspension from step (2) for at least 20 minutes to allow the activated sludge porous material to fully adsorb the bacterial suspension, resulting in activated sludge porous material loaded with bacterial suspension. The activated sludge porous material loaded with bacterial suspension is then dried at 37–40°C for 1 hour. At this point, the mixed bacterial agent has been concentrated and loaded onto or encapsulated on the PVA surface. Finally, it is immersed in a solution containing 0.98–1.22% Ca by weight. 2+ Cross-linking in solution (at 4°C) for 1 h, then filtration of excess unreacted Ca-containing material.2+ Solution, dry.

[0036] Polyvinyl alcohol acts as a binder, enabling the mixed bacterial agents to be fully adsorbed into the porous activated sludge material.

[0037] Ca 2+ Its function is as a cross-linking agent; in solution, Ca... 2+ The cross-linking effect is better when the weight percentage is 0.98-1.22%.

[0038] This invention uses PVA to firmly load microorganisms onto the pores of activated sludge porous material, and then uses Ca... 2+ Cross-linked PVA adheres firmly to the porous activated sludge material, thereby increasing the number of microorganisms and improving the degradation rate.

[0039] In this invention, Ca(NO3) can be used. 2、 CaCl2 and other substances containing Ca 2+ The solution is used as a crosslinking agent.

[0040] 4. Repeat step 3 multiple times, for example, 1-6 times, to obtain a porous material loaded with mixed microorganisms, named MPP(@).

[0041] Repeated loading of microorganisms can strengthen the binding force between microorganisms and porous activated sludge materials, increase the number of microorganisms per unit volume, and enhance the biological activity of microorganisms per unit volume.

[0042] 5. Place 1g of SiO2 nanoparticles into 49.5g of anhydrous ethanol solution and sonicate for 30 minutes. Then add 0.5g of tridecafluorooctyltriethoxysilane to obtain a modified hydrophobic silica particle liquid suspension. Continue the reaction at room temperature with constant magnetic stirring for 2 hours. Simultaneously, mix 1g of commercially available epoxy resin and 2g of anhydrous ethanol and magnetically stir for 30 minutes. Place the epoxy resin-containing solution into the original suspension and sonicate for 30 minutes to obtain a mixed solution. While maintaining constant magnetic stirring, dissolve 1g of curing agent in 2g of anhydrous ethanol.

[0043] Before coating, the solution containing the curing agent and the mixed solution are ultrasonically mixed at room temperature for 30 minutes to form an emulsion. The emulsion is then brushed onto the PSA-M material, and the resulting hydrophobic and oleophilic modified material is called OPAS-M.

[0044] Anhydrous ethanol is used as a solvent for SiO2 nanoparticles.

[0045] Tridecafluorooctyltriethoxysilane is key to the modification of porous activated sludge materials. Its dosage ratio to SiO2 nanoparticles is 0.5 g:1 g. This is because the activated sludge porous material loaded with microorganisms only requires surface modification, with only a thin film forming on the surface. If the concentration is too high, a thicker film forms, which is not conducive to the transport of n-hexadecane. If the concentration is too low, a film structure cannot be formed, and the modification purpose is not achieved.

[0046] Epoxy resin can dissolve in anhydrous ethanol to form a hydrophobic film, which also helps to make the modification more robust.

[0047] Example 1

[0048] Preparation of PAS, a porous material for hydrophobic and oleophilic activated sludge.

[0049] Activated sludge was obtained through gravity sedimentation, passing through a 100-mesh sieve, centrifugal concentration, and sedimentation. 5.0 g of the activated sludge was placed in a beaker, and 2 g of sodium alginate (5 wt%) solution was added. The mixture was stirred for 15 minutes, then placed into a cylindrical plastic mold with a diameter of 2.5 cm and frozen for 12 hours. Following this, it was freeze-dried for 36 hours. Finally, the freeze-dried activated sludge material was cross-linked in a 3 wt% Ca(NO3)2 solution for 24 hours. This porous activated sludge material was named PAS.

[0050] The prepared hydrophobic and oleophilic activated sludge porous material has a diameter of 2 cm, a height of 0.5 cm, and a mass of 1.0 g.

[0051] Figure 2 Figure 1 shows scanning electron microscope (SEM) images of PAS. Figure 2 shows a cross-sectional SEM image of PAS, and Figure 3 shows a longitudinal SEM image of PAS.

[0052] Figure 2 The morphology of the activated sludge porous material was characterized by scanning electron microscopy (SEM). Figure a, at 1000x magnification, clearly shows roughly ordered macropores in the cross-sectional image. Figure b, at 1000x magnification, shows a cross-sectional view of the activated sludge porous material, where the pores are roughly uniform and tightly connected, exhibiting a porous honeycomb structure. This unique pore structure gives the prepared activated sludge porous material low diffusion resistance, low density, relatively high specific surface area, and abundant potential active sites, providing a favorable living environment for the growth of internal microorganisms.

[0053] Example 2

[0054] Preparation of PAS(@) loaded with mixed microbial porous material.

[0055] (1) Synthesis of hydrophobic and oleophilic activated sludge porous material PAS: Same as in Example 1.

[0056] (2) Preparation of mixed bacterial suspension: Bacillus thuringiensis (Bt) Bacillus thuringiensis BT), Pseudomonas aeruginosa ( Pseudomonas aeruginosa A6), Acinetobacter rufi ( Acinetobacter lwoffii (A5), Nocardia albopictus Nocardioides luteus F1), Bacillus ( Aacillus, BS Five strains of bacteria were cultured in a three-stage scale-up process to prepare bacterial suspensions with concentrations of 1×10⁻⁶. 10 ~1×10 12 cfu·mL -1 The bacteria were mixed in equal volume ratios to prepare a mixed bacterial suspension. The colony activity of the mixed bacterial suspension was detected using the dilution plating method, reaching 1×10⁻⁶. 10 ~1×10 12 cfu·mL -1 Then, store at 4 ℃ until use.

[0057] (3) The hydrophobic and oleophilic activated sludge porous material PAS prepared in step (1) is sterilized by high pressure steam at 121°C for 30 min and then cooled to room temperature for later use.

[0058] (4) The sterilized hydrophobically modified activated sludge porous material was immersed in a 3% polyvinyl alcohol (PVA) solution for 20 min, filtered to remove excess PVA, and rinsed with distilled water to prevent blockage of the outer wall pores. It was then immersed in the mixed bacterial suspension from step (2) for 20 min to obtain activated sludge porous material loaded with bacterial suspension. The activated sludge porous material loaded with bacterial suspension was then dried at 38°C for 1 h. At this time, the mixed bacterial agent had been concentrated and loaded onto or encapsulated on the PVA surface. Finally, it was placed in a 5% Ca(NO3)2 solution (at 4°C) for crosslinking for 1 h, and the excess unreacted Ca(NO3)2 solution was filtered out and dried.

[0059] (5) Repeat step (4) 6 times to obtain a porous material loaded with mixed microorganisms, named PAS(@).

[0060] Figure 3 Figure 1 shows the scanning electron microscope (SEM) images of PAS(@). Figure 2a shows the SEM image of PAS(@) concentrated twice, Figure 3b shows the SEM image of PAS(@) concentrated four times, and Figure 4c shows the SEM image of PAS(@) concentrated six times.

[0061] like Figure 3As shown in the figure, rod-shaped and spherical particles are clearly visible, and the number of microorganisms after six concentrations is significantly greater than after a single concentration. This is because the microorganisms were successfully loaded onto the porous activated sludge material. The more times the mixed bacterial agent is loaded, the more significant the bacterial aggregation into clumps. This is mainly because the mixed bacterial agent loaded on the macroporous material does not reach its growth threshold through chemical communication within the microbial community, but instead reproduces freely until its growth space is limited. Therefore, it can be concluded that using multiple concentrations and gradual accumulation helps to increase the number of loaded microorganisms.

[0062] The average amount of bacterial suspension adsorbed in the activated sludge porous material was as follows: 1.0 g of activated sludge porous material adsorbed 4.5 g, 1.8 g, 1.7 g, 1.5 g, 1.5 g, and 1.0 g of bacterial suspension in the 1st, 2nd, 3rd, 4th, 5th, and 6th times, respectively.

[0063] Example 3

[0064] Preparation of OPAS-M, a porous material for loading microbial activated sludge, which is oleophilic and hydrophobic.

[0065] (1) Preparation of porous material loaded with mixed microorganisms: Same as in Example 2.

[0066] (2) 1 g of SiO2 nanoparticles was placed in 49.5 g of anhydrous ethanol solution and sonicated for 30 minutes. Then, 0.5 g of tridecafluorooctyltriethoxysilane was added to obtain a modified hydrophobic silica particle liquid suspension. The reaction was continued for 2 hours at room temperature under constant magnetic stirring. Simultaneously, 1 g of commercially available epoxy resin and 2 g of anhydrous ethanol were mixed and magnetically stirred for 30 minutes. The solution containing epoxy resin was placed in the original suspension and sonicated for 30 minutes to obtain a mixed solution. While maintaining constant magnetic stirring, 1 g of curing agent was dissolved in 2 g of anhydrous ethanol.

[0067] Before coating, the solution containing the curing agent and the mixed solution are ultrasonically mixed at room temperature for 30 minutes to form an emulsion. The emulsion is then brushed onto the PSA-M material, and the resulting hydrophobic and oleophilic modified material is called OPAS-M.

[0068] Figure 4 This refers to the oleophilic and hydrophobic porous material for loading microbial activated sludge prepared in Example 3.

[0069] like Figure 4 As shown, the contact angle of water droplets on the oleophilic-hydrophobic microbial-loaded porous material prepared in Example 3 is 100.1°, which is greater than 90°, indicating that the oleophilic-hydrophobic microbial-loaded porous material prepared in Example 3 is hydrophobic. This hydrophobic-oleophilic membrane has selective adsorption properties, and n-hexadecane can be rapidly adsorbed into the porous material, accelerating the microbial degradation rate.

[0070] Example 4

[0071] Activated sludge was obtained through gravity settling, passing through a 100-mesh sieve, centrifugal concentration, and sedimentation. 5.0 g of the activated sludge was placed in a beaker, and 2 g of sodium alginate (5 wt%) solution was added. The mixture was stirred for 20 minutes, then placed into a cylindrical plastic mold with a diameter of 2.5 cm and frozen for 12 hours. Following this, it was freeze-dried for 36 hours. Finally, the freeze-dried activated sludge material was cross-linked in a Ca(NO3)2 solution for 24 hours. 2+ The porous activated sludge material was named PAS with a weight ratio of 5.0 g to 100.0 mL with distilled water.

[0072] Example 5

[0073] Preparation of PAS(@) loaded with mixed microbial porous material.

[0074] (1) Synthesis of hydrophobic and oleophilic activated sludge porous material PAS: Same as in Example 1.

[0075] (2) Preparation of mixed bacterial suspension: Bacillus thuringiensis (Bt) Bacillus thuringiensis BT), Pseudomonas aeruginosa ( Pseudomonas aeruginosa A6) Acinetobacter rufi ( Acinetobacter lwoffii (A5), Nocardia albopictus Nocardioides luteus F1), Bacillus ( Aacillus, BS Five strains of bacteria were cultured in a three-stage scale-up process to prepare bacterial suspensions with concentrations of 1×10⁻⁶. 10 ~1×10 12 cfu·mL -1 The bacteria were mixed in equal volume ratios to prepare a mixed bacterial suspension. The colony activity of the mixed bacterial suspension was detected using the dilution plating method, reaching 1×10⁻⁶. 10 ~1×10 12 cfu·mL -1 Then, store at 4 ℃ until use.

[0076] (3) The hydrophilic and oleophilic activated sludge porous material PAS prepared in step (1) is sterilized by high pressure steam at 121°C for 30 min and then cooled to room temperature for later use.

[0077] (4) The sterilized hydrophilic modified activated sludge porous material was placed in a 3% (w / w) polyvinyl alcohol (PVA) solution and soaked for 20 min. After filtration, excess PVA was removed, and the surface was rinsed with distilled water to prevent blockage of the outer wall pores. The material was then placed in the mixed bacterial suspension from step (2) and soaked for 25 min to obtain activated sludge porous material loaded with bacterial suspension. The activated sludge porous material loaded with bacterial suspension was then dried at 40°C for 1 h. At this time, the mixed bacterial agent was concentrated and loaded onto or encapsulated on the PVA surface. Finally, the material was placed in a 4% (w / w) Ca(NO3)2 solution (at 4°C) for crosslinking for 1 h. Excess unreacted Ca(NO3)2 solution was filtered out, and the material was dried.

[0078] (5) Repeat step (4) 6 times to obtain a porous material loaded with mixed microorganisms, named PAS(@).

[0079] Example 6

[0080] Preparation of OPAS-M, a porous material for loading microbial activated sludge, which is oleophilic and hydrophobic.

[0081] (1) Preparation of porous material loaded with mixed microorganisms: Same as in Example 2.

[0082] (2) 1 g of SiO2 nanoparticles was placed in 49.5 g of anhydrous ethanol solution and sonicated for 25 minutes. Then, 0.5 g of tridecafluorooctyltriethoxysilane was added to obtain a modified hydrophobic silica particle liquid suspension. The reaction was continued for 2 hours at room temperature under constant magnetic stirring. Simultaneously, 1 g of commercially available epoxy resin and 2 g of anhydrous ethanol were mixed and magnetically stirred for 25 minutes. The solution containing epoxy resin was placed in the original suspension and sonicated for 30 minutes to obtain a mixed solution. While maintaining constant magnetic stirring, 1 g of curing agent was dissolved in 2 g of anhydrous ethanol.

[0083] Prior to coating, the solution containing the curing agent and the mixed solution are ultrasonically mixed at room temperature for 25 minutes to form an emulsion. The emulsion is then brushed onto the PSA-M material, resulting in a hydrophobic and oleophilic modified material called OPAS-M.

[0084] Example 7

[0085] Preparation of PAS(@) loaded with mixed microbial porous material.

[0086] (1) Synthesis of hydrophobic and oleophilic activated sludge porous material PAS: Same as in Example 1.

[0087] (2) Preparation of mixed bacterial suspension: Bacillus thuringiensis (Bt) Bacillus thuringiensis BT), Pseudomonas aeruginosa ( Pseudomonas aeruginosa A6) Acinetobacter rufi ( Acinetobacter lwoffii(A5), Nocardia albopictus Nocardioides luteus F1), Bacillus ( Aacillus, BS Five strains of bacteria were cultured in a three-stage scale-up process to prepare bacterial suspensions with concentrations of 1×10⁻⁶. 10 ~1×10 12 cfu·mL -1 The bacteria were mixed in equal volume ratios to prepare a mixed bacterial suspension. The colony activity of the mixed bacterial suspension was detected using the dilution plating method, reaching 1×10⁻⁶. 10 ~1×10 12 cfu·mL -1 Then, store at 4 ℃ until use.

[0088] (3) The hydrophilic and oleophilic activated sludge porous material PAS prepared in step (1) is sterilized by high pressure steam at 121°C for 30 min and then cooled to room temperature for later use.

[0089] (4) The sterilized hydrophilic modified activated sludge porous material was placed in a 3% (w / w) polyvinyl alcohol (PVA) solution and soaked for 20 min. After filtration, excess PVA was removed, and the surface was rinsed with distilled water to prevent blockage of the outer wall pores. The material was then placed in the mixed bacterial suspension from step (2) and soaked for 22 min to obtain activated sludge porous material loaded with bacterial suspension. The activated sludge porous material loaded with bacterial suspension was then dried at 39°C for 1 h. At this time, the mixed bacterial agent was concentrated and loaded onto or encapsulated on the PVA surface. Finally, the material was placed in a 4.5% (w / w) Ca(NO3)2 solution (at 4°C) for crosslinking for 1 h. Excess unreacted Ca(NO3)2 solution was filtered out, and the material was dried.

[0090] (5) Repeat step (4) 6 times to obtain a porous material loaded with mixed microorganisms, named PAS(@).

[0091] The present invention provides a porous material loaded with mixed microorganisms and a porous material loaded with oleophilic and hydrophobic microorganisms and activated sludge that can be applied to the degradation of petroleum hydrocarbons; the petroleum hydrocarbons are long-chain n-alkanes, medium- and long-chain alkanes, hopanes, or steranes.

[0092] Preferably, the medium- to long-chain alkane is a C4-C16 alkane.

[0093] As a further preferred option, the medium-to-long chain alkane is n-hexadecane.

[0094] The present invention relates to an oleophilic and hydrophobic porous material for loading microbial activated sludge for the remediation of simulated oily wastewater.

[0095] 1. Add 30 mL of deionized water and 0.175 mL of n-hexadecane (the weight percentage concentration of n-hexadecane is 3% relative to the fixed amount of bacterial agent) to a 50 mL conical flask to obtain simulated oily wastewater. Add 4.5 g of the mixed bacterial suspension prepared in step (2) of Example 2, 4.5 g of the porous material PAS(@) loaded with mixed microorganisms prepared in Example 2, and 4.5 g of the porous material OPAS-M loaded with oleophilic and hydrophobic microorganisms prepared in Example 3 to the flask. Degrade the material for 10 h, 20 h, 30 h, 40 h, 50 h, 60 h, 70 h, and 80 h at 37℃, 120 r / min, and pH=6. Quantitatively analyze the material using GC (Clarus GC-TotalChrom, PerkinElmer Instruments (Shanghai) Co., Ltd.), and calculate the degradation rate using a standard curve. The GC measurement conditions were: column temperature 180℃; maintenance for 2 min; and the temperature of the injector and FID detector were both 220℃. The fixed flow rates for nitrogen, hydrogen, and air were 20, 45, and 450 mL / min, respectively.

[0096] Figure 5 Figure a shows the degradation rates of free mixed bacteria, PAS(@), and OPAS-M at different degradation times under the conditions of 37℃, 120 r / min, pH=6, and a hexadecane weight percentage concentration of 3%. Figure b shows the first-order kinetics comparison of free mixed bacteria, PAS(@), and OPAS-M under the conditions of 37℃, 120 r / min, pH=6, and a hexadecane weight percentage concentration of 3%. Figure c shows the original GC images of the mixed bacterial suspension (referred to as "free bacteria" in this figure), PAS(@), and OPAS-M, respectively.

[0097] like Figure 5 As shown in Figure (a), under 37℃ conditions, the degradation curves of free microorganisms, PAS-M, and OPAS-M all showed a trend of first increasing and then gradually reaching equilibrium with time, with degradation rates of 58.80%, 74.37%, and 84.70% respectively at equilibrium. In contrast, the degradation rate of free mixed microorganisms was lower than that of PAS-M and hydrophobic OPAS-M, mainly because free microorganisms are greatly affected by environmental factors, resulting in significant interference and low effective concentration per unit volume. The porous material loaded with mixed microorganisms has a pore structure rich in microorganisms, which can provide a larger growth space for microbial proliferation until the free space on the carrier is completely filled, resulting in a dense number of microorganisms. At the same time, various enrichments were used, and the microbial loading method made up for the weakness of the binding force of the traditional adsorption method. Microorganisms were firmly loaded onto the pores of the material through polyvinyl alcohol, and then polyvinyl alcohol and Ca 2+Cross-linking allows microorganisms to adhere more firmly to the porous sludge material, thereby increasing the number of microorganisms and improving their degradation rate. Secondly, the surface of PAS-M is hydrophobically modified to form a hydrophobic-oleophilic film. This film utilizes its selective adsorption properties to adsorb n-hexadecane into the microbial-loaded OPAS-M composite material, increasing the contact area between microorganisms and n-hexadecane, and improving the transfer rate of hydrophobic n-hexadecane in OPAS-M. This enhances the utilization rate of microorganisms and accelerates the degradation of n-hexadecane by the microbial composite material. Figure 5 As shown in (b), according to the first-order degradation kinetics formula, the rate constants of free microorganisms PAS-M and OPAS-M are 0.0932 d. -1 0.1949 d -1 and 0.3014 d -1 The half-lives were 7.44 days, compared to 3.56 days and 2.23 days for free-living microorganisms. The results indicate that OPAS-M has the shortest half-life, 5.21 days shorter than that of free-living microorganisms. Therefore, OPAS-M can effectively shorten the degradation cycle, achieving rapid and efficient degradation. Figure 5 As shown in (c), the peak area of ​​the free mixed bacteria, PAS(@) and OPAS-M gradually decreased with the increase of degradation time, which indicates that the residual amount of n-hexadecane gradually decreased. Under the condition of 37℃, the degradation rates reached 64.1%, 89.3% and 96.75% after 80 h, respectively.

[0098] II. Under constant conditions, degradation was carried out at different n-hexadecane concentrations, temperatures, and pH values. The degradation results are shown in [see figure]. Figure 6 .

[0099] Figure 6 (a) Degradation rates of suspended cells, PAS(@), and OPAS-M at different n-hexadecane concentrations at 37℃, 120 r / min, pH=6, and a degradation time of 50 h; Figure b shows the original GC images of mixed bacterial suspension (represented as "suspended cells" in this figure), PPM(@), and H-PPM(@). like Figure 6 As shown in the figure, it can be clearly observed that H-PAS@, PAS@, and free bacteria degrade n-hexadecane synchronously. As the concentration of n-hexadecane increases, their degradation rates gradually decrease and show a continuous downward trend, but the decrease is not significant, and the degradation rates are not much different in numerical value. This indicates that H-PAS@, PAS@, and free bacteria have little effect on the degradation of n-hexadecane in simulated oil with a weight percentage concentration of 1%-5% (relative to the content of bacterial agent).

[0100] like Figure 6As shown in (a), the degradation rate of n-hexadecane gradually decreased with increasing concentration, exhibiting a continuous downward trend, but the decrease was not significant (1%-3%), and the numerical differences in degradation rates were not substantial. The downward trend was more pronounced at higher concentrations; at 5%, the degradation rate was 51.4%. These results indicate that low concentrations of OPAS-M resulted in a higher degradation rate of n-hexadecane, but at high concentrations, some microorganisms in OPAS-M lost their biological activity under the influence of high concentrations of n-hexadecane, leading to a lower degradation rate. Figure 6 As shown in (b), the degradation performance of OPAS-M on n-hexadecane under different temperature conditions was investigated. The results showed that at 120 r / min and pH=6, the degradation rate of OPAS-M first increased and then decreased with increasing temperature. The degradation rate of n-hexadecane reached its maximum at 37℃, and remained above 48% between 27 and 47℃. This is mainly because microbial degradation is generally catalyzed by dioxygenases, and excessively high temperatures inhibit the activity of these enzymes, leading to a decrease in the degradation rate. The high degradation rate of n-hexadecane by OPAS-M, exceeding that at lower temperatures, may be due to the porous asphalt material providing a sanctuary for microorganisms. As the temperature increases, the solubility of n-hexadecane increases, which promotes the contact area between microorganisms and n-hexadecane, increasing its bioavailability and enabling the production of microorganisms under harsh conditions, thus maintaining its strong biodegradability in the environment. Figure 6 As shown in (c), the degradation rate of OPAS-M first increases and then decreases with increasing pH. The degradation rate reaches its maximum at pH=6. Under alkaline conditions, the degradation rate of n-hexadecane by OPAS-M is higher than that under acidic conditions, and the degradation rate remains above 54% at pH 2–10. As mentioned above, extremely acidic or alkaline conditions will have a destructive effect on the utilization capacity of microorganisms. Hydrogen ions in some acidic media readily bind to the amino or carboxyl groups of enzymes, thereby altering the original structural characteristics of the enzyme and weakening its ability to bind to n-hexadecane. In addition, acidic or alkaline conditions also affect the solubility and transport of nutrients, leading to strong biodegradation of n-hexadecane by microorganisms under neutral pH conditions.

[0101] Finally, it should be noted that the above descriptions are merely preferred embodiments of the present invention and are not intended to limit the present invention. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments or make equivalent substitutions for some of the technical features. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for preparing porous materials loaded with mixed microorganisms, characterized in that: Includes the following steps: S1, Preparation of mixed bacterial suspension: Bacillus thuringiensis BT, Pseudomonas aeruginosa A6, Acinetobacter rhusiopathiae A5, Nocardia alba F1, and Bacillus thuringiensis were mixed. BS Bacterial solutions were prepared separately, with concentrations of 1×10⁻⁶. 10 ~1×10 12 cfu·mL -1 The bacteria were mixed in equal volume ratios to prepare a mixed bacterial suspension, and the colony activity of the mixed bacterial suspension was 1×10⁻⁶. 10 ~1×10 12 cfu·mL -1 ; S2, Preparation of porous activated sludge material: Activated sludge was obtained through gravity sedimentation, sieving, centrifugal concentration, and sedimentation; the above activated sludge was added to sodium alginate solution, stirred, and freeze-dried; finally, the freeze-dried activated sludge material was placed in Ca... 2+ Cross-linking in solution yields a porous activated sludge material, named PAS. S3, Preparation of porous material loaded with mixed microorganisms: The activated sludge porous material PAS prepared in step S2 is bound with polyvinyl alcohol, then placed in the mixed bacterial suspension prepared in step S1 to adsorb the mixed bacteria, and then Ca is added. 2+ Cross-linking is carried out in solution to obtain porous materials loaded with mixed microorganisms; In step S2, the ratio of activated sludge to sodium alginate is 5.0g:2.0g; The Ca 2+ Ca in solution 2+ The ratio of distilled water to water used is 5.0 g: 100.0 mL; The ratio of sodium alginate to distilled water in the sodium alginate solution is 5.0 g: 100.0 mL; In step S3, the Ca 2+ The weight percentage concentration is 0.98-1.22%; Step S3 is repeated 1-6 times.

2. A porous material loaded with mixed microorganisms, characterized in that: It is prepared by applying the method described in claim 1.

3. A method for preparing porous materials of oleophilic and hydrophobic microbial-loaded activated sludge, characterized in that: The porous material loaded with mixed microorganisms as described in claim 2 is brushed with epoxy resin dissolved in anhydrous ethanol, and then sprayed on the surface of the material with a liquid suspension of hydrophobic silica particles modified with tridecafluorooctyltriethoxysilane to obtain a hydrophobic and oleophilic microbial activated sludge porous material. Nanoparticles of SiO2 were placed in anhydrous ethanol solution and sonicated for 30 minutes. Then, tridecafluorooctyltriethoxysilane was added to obtain a modified hydrophobic silica particle liquid suspension. Wherein: the ratio of the amount of SiO2 nanoparticles to the anhydrous ethanol solution is 1 g : 49.5 g; The ratio of tridecafluorooctyltriethoxysilane to SiO2 nanoparticles is 1 g: 2 g; The ratio of epoxy resin to anhydrous ethanol is 1 g: 2 g.

4. A porous material for supporting microbial activated sludge, characterized in that: It is prepared by applying the method described in claim 3.

5. The application of the porous material supported on mixed microorganisms as described in claim 2, or the porous material supported on oleophilic and hydrophobic microbial activated sludge as described in claim 4, in the degradation of petroleum hydrocarbons; characterized in that: The petroleum hydrocarbons are long-chain n-alkanes, medium- to long-chain alkanes, hopanes, or steranes; The degradation temperature is 27 ~ 47℃; The pH during the degradation is 2 to 10; The weight percentage concentration of the petroleum-derived alkanes is 1%-5%.

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