Application of porous carrier material in microbial immobilization

Abstract

The invention discloses application of a porous carrier material in microbial immobilization, and belongs to the technical field of microbial immobilization. The surface of the porous support material is positively charged (pKagt; the porous carrier (2, 8) has medium hydrophilicity (the contact angle is 20-60 degrees), is used for immobilizing microorganisms, especially white-rot fungi to obtain a porous carrier-microorganism composite material, and realizes efficient organic matter degradation and stable activity of the immobilized white-rot fungi in actual wastewater under the condition of not adjusting pH when being used for wastewater treatment. The technical problem that white-rot fungi can only be used under acidic conditions in the past is solved, and a new thought is provided for application of functional microorganisms and degradation of emerging pollutants.

Description

Technical Field

[0001] The present application belongs to the technical field of microorganism immobilization, and specifically relates to the application of porous carrier materials in microorganism immobilization. Background Art

[0002] In recent years, emerging pollutants such as persistent organic pollutants (POPs), endocrine disrupting chemicals (EDCs) and pharmaceuticals and personal care products (PPCPs) have been frequently detected in water environments, posing significant risks to the ecological environment and human health. Emerging pollutants generally have complex structures such as highly branched chains, multi-ring structures and electron-withdrawing functional groups, which are difficult to degrade; and they are widely distributed and still pose risks at environmental concentrations. Wastewater containing emerging pollutants has the characteristics of large water volume, low concentration and high risk, and conventional sewage treatment facilities cannot remove these difficult-to-degrade compounds, so there is an urgent need for economical and effective methods to reduce and remove them.

[0003] To this end, a large number of functional microorganisms with special degradation capabilities have been widely studied, such as Bacillus thuringiensis B1 and Klebsiella sp. KSC, which can directly metabolize specific PPCPs as the only carbon source. In addition to degrading bacteria, white rot fungi are a type of microorganism that can degrade lignin. They have a degradation enzyme system represented by laccase and are currently the only microorganisms that can completely degrade dozens of PPCPs. Among them, Trametes versicolor (T.versicolor) has been widely studied because of its high degradation efficiency.

[0004] However, these functional microorganisms still have problems with insufficient degradation efficiency and difficulty in surviving in actual wastewater. For example, degrading bacteria can generally only remove a certain type of fixed-structured compounds and have poor removal effects on mixed pollutants. White-rot fungi face problems of slow degradation rates and bacterial competition in actual wastewater. For this reason, almost all studies adjust the wastewater to a pH of 4.5, which is beneficial for fungal growth.

[0005] Microbial (carrier) immobilization is a method of fixing microbial cells on a carrier through physical and chemical means. According to the immobilization mechanism, it can be divided into adsorption, covalent binding, embedding and encapsulation. This technology has the advantages of providing a safe and stable growth environment, high mechanical strength, and blocking toxic substances. Commonly used commercial carriers include biochar, polyethylene, polyurethane, etc., which have been widely used in sewage treatment. For example, the biological biofilm achieved on the carrier has the advantages of large biomass and long sludge age. Polyurethane is also used to fix functional microorganisms, and biofilms for degrading bacteria and fungi have been achieved. However, these carrier materials are non-porous or have a low specific surface area (<100m2 / g), which itself has almost no adsorption effect on emerging pollutants and cannot further improve the degradation efficiency of functional microorganisms. At the same time, they also have the problem of biomass washout. After the functional microorganisms are fixed, they are not tightly bound to them, and pollution will appear in the environmental medium. In general, the existing immobilized carriers have problems such as low removal efficiency and biomass washout. The fixed functional microorganisms are difficult to apply in actual wastewater. It is necessary to further study new carrier materials for the immobilization of microorganisms. Summary of the invention

[0006] 1. Problem to be solved

[0007] The present invention aims to solve the problems of low removal efficiency of existing functional microorganisms, especially white rot fungi, difficulty in surviving in actual wastewater or facing competition from environmental microorganisms. The present invention provides an application of a porous carrier material in microbial immobilization. The surface of the porous carrier material is positively charged (pK a >8) and moderately hydrophilic (contact angle of 20° to 60°), it is used to immobilize microorganisms, especially white rot fungi, to obtain a porous carrier-microorganism composite material, and when the composite material is used for wastewater treatment, it can simultaneously enhance the organic matter degradation effect and biological activity of the functional microorganisms.

[0008] 2. Technical solution

[0009] In order to solve the above problems, the technical solutions adopted in this application are as follows:

[0010] In a first aspect, the present application provides a porous carrier material, wherein the BET specific surface area of ​​the porous carrier material measured by a nitrogen isotherm method is greater than 600 m 2 g -1 , pK a >8 and the contact angle is 20°~60°.

[0011] Furthermore, the specific surface area>650m 2 g -1 .

[0012] Furthermore, the specific surface area>700m 2 g -1 .

[0013] Furthermore, pK a >9.

[0014] Furthermore, the contact angle is 30° to 50°.

[0015] Furthermore, the contact angle is 30° to 40°.

[0016] Furthermore, the above-mentioned porous carrier material includes any one of porous thienyl cyclodextrin polymer, porous thienyl chitosan polymer, and porous thienyl columnar aromatic polymer; microbial immobilization involves complex processes such as carrier adsorption, microbial degradation, and enzyme degradation, and it is necessary to avoid the use of carriers that are harmful to microorganisms. Chitosan, cyclodextrin, etc. are all bio-friendly materials, and have a large number of functional groups on the surface, which can be immobilized for specific microorganisms through surface modification.

[0017] Furthermore, the porous carrier material includes a porous thienyl cyclodextrin polymer; the cyclodextrin polymer has the advantage of fast adsorption rate and can be combined with microbial degradation to further improve the degradation performance.

[0018] Furthermore, the cyclodextrin is any one of α, β, and γ cyclodextrin.

[0019] In the second aspect, the present application provides a method for preparing the above-mentioned porous carrier material, which is a method of using cyclodextrin or chitosan or pillar aromatic hydrocarbon as a monomer, grafting a thiophene ring to form a thiophene-based monomer, and using an aromatic ring- or methoxy-containing alkylating agent as a cross-linking agent to obtain the material by Friedel-Crafts alkylation reaction under the catalysis of a Lewis acid ionic liquid catalyst. The alkylating agent forms an alkyl carbon cation under the catalysis of a Lewis acid, and the carbon cation then undergoes an electrophilic reaction with an electron-rich aromatic compound. Since the reaction can be continuously substituted, a porous material with a high degree of polymerization can be obtained.

[0020] Furthermore, the cyclodextrin is any one of α, β, and γ cyclodextrin.

[0021] Furthermore, the above-mentioned grafting of thiophene rings includes: using anhydrous NaH to replace the hydroxyl group of the monomer with sodium alcoholate, and then adding a halogenated thiophene ring for grafting.

[0022] Furthermore, the halogenated thiophene ring includes brominated thiophene or chlorinated thiophene.

[0023] Furthermore, the above-mentioned Lewis acid ionic liquid catalyst includes: an imidazole-type or quaternary ammonium-type ionic liquid, and a composite ionic liquid formed with a metal chloride.

[0024] Furthermore, the metal chloride includes ferric chloride and / or aluminum chloride.

[0025] Furthermore, the above-mentioned ionic liquid is 1-ethyl-3-methylimidazolium chloride ionic liquid.

[0026] Further, the cross-linking agent includes any one or more of biphenyl dichlorobenzyl, p-dichlorobenzyl, biphenyl dibromide, p-dibromide, dimethoxymethane, trimethyl orthoformate, and 1,4-dimethoxybenzene. Further, the cross-linking agent includes trimethyl orthoformate.

[0027] Furthermore, the reaction temperature conditions of the Friedel-Crafts alkylation reaction include: 45°C for 4 to 6 hours, then heated to 80 to 150°C for 18 to 20 hours. Furthermore, the reaction temperature conditions of the Friedel-Crafts alkylation reaction include: 45°C for 5 hours, then heated to 80 to 150°C for 19 hours. If the reaction temperature is not within the above range, the synthesized polymer will have incomplete reaction, low specific surface area, and low yield.

[0028] Furthermore, the above reaction temperature conditions include: reacting at 45°C for 5 hours, and then heating to 120°C for 19 hours.

[0029] Furthermore, the porous carrier material is a porous thienyl cyclodextrin polymer, and the preparation method thereof comprises the following steps:

[0030] Dissolve cyclodextrin in anhydrous DMF; then slowly add anhydrous NaH at 0°C and stir for 15 minutes; slowly drop 3-bromomethylthiophene (3-Bm-Th) and stir at room temperature for 24 hours; after the reaction is completed, quench the reaction with methanol; then mix the product with water and concentrate to near dryness under vacuum at 84°C; mix the obtained product with water and extract with dichloromethane; dry the organic phase with anhydrous sodium sulfate, then filter and concentrate under vacuum at 35°C, and finally obtain a light yellow compound thienyl cyclodextrin (ThCD) by silica gel chromatography;

[0031] At 0°C, ThCD was dissolved in 1-ethyl-3-methylimidazolium chloride / ferric chloride ([EMIm]Cl / FeCl 3 ) ionic liquid; under nitrogen protection, anhydrous trimethyl orthoformate (Tmof) was slowly added dropwise at room temperature, the reaction mixture was stirred and heated to 45°C for 5 hours, and then heated to 120°C for 19 hours; after cooling, the brown precipitate was washed with water and methanol until the filtrate was colorless, and then Soxhlet extraction with methanol was performed for 24 hours, and vacuum dried at 60°C for 24 hours to obtain a porous thienyl cyclodextrin polymer (Th-CDP).

[0032] Furthermore, the molar ratio of the above-mentioned 3-bromomethylthiophene to cyclodextrin is 14 to 21:1. By adjusting the ratio of the cyclodextrin grafted thiophene ring, some hydroxyl groups on the cyclodextrin can be selectively retained, thereby achieving the regulation of the hydrophilicity and hydrophobicity of the carrier surface. β-cyclodextrin has a total of 21 hydroxyl groups. When the ratio of grafted thiophene to cyclodextrin is 14:1 and 21:1, the contact angle of the prepared carrier surface increases, and the carrier surface changes from superhydrophilic to moderately hydrophilic, and has a good ability to maintain the activity of specific functional microorganisms.

[0033] Furthermore, the molar ratio of the alkylating agent to the thienyl cyclodextrin is 5 to 100:1.

[0034] In a third aspect, the present application provides an application of the above-mentioned porous carrier material in the immobilization of microorganisms, wherein the microorganisms include one or more of white rot fungi, nitrifying bacteria, denitrifying bacteria, Bacillus, Pseudomonas and activated sludge; the porous carrier material has a BET specific surface area measured by a nitrogen isotherm method of >600m 2 g -1 , pK a >8 and the contact angle is 20°~60°.

[0035] Furthermore, the above-mentioned microorganism is a white rot fungus.

[0036] Furthermore, the white rot fungus is one or more of Phanerochae techrysosporium, Trametes versicolor, Corilus versicolor, Pleurotus sajor-caju and Pleurotus ostreatus.

[0037] Furthermore, the white rot fungus is Trametes versicolor.

[0038] Furthermore, the above-mentioned porous carrier material is used in the immobilization of microorganisms by first adding the porous carrier material into the culture medium, then adding (functional) microorganisms by the mycelium liquid inoculation method, and culturing by shaking so that the microorganisms are fixed to the inside of the porous carrier material by physical adsorption.

[0039] Furthermore, the use of the porous carrier material in the immobilization of microorganisms, wherein the microorganisms are white rot fungi, specifically includes:

[0040] Preparation of mycelial liquid:

[0041] The T. versicolor agar block was inoculated into a potato dextrose (PD) medium and cultured with shaking (135 rpm) at 25°C for 4-5 days; the obtained mycelial pellets were separated from the medium, the mycelial pellets were collected and added with 0.85% sterile saline, and the mixture was stirred with a high-speed homogenizer for 15 minutes to obtain mycelial liquid, which was stored at 4°C;

[0042] Immobilized microorganisms:

[0043] The porous carrier material was added to the PD culture medium and sterilized under high pressure; 1.5 mL of mycelium liquid of the white rot fungus T. versicolor was inoculated into the culture medium, and the culture was shaken at 25° C. and 135 rpm for 5 days to prepare the immobilized white rot fungus.

[0044] In a fourth aspect, the present application provides a porous carrier-microorganism composite material, wherein: the microorganisms include one or more of white rot fungi, nitrifying bacteria, denitrifying bacteria, Bacillus, Pseudomonas and activated sludge; the BET specific surface area of ​​the porous carrier material measured by the nitrogen isotherm method is greater than 600m 2 g -1 , pK a >8 and the contact angle is 20°~60°.

[0045] Furthermore, the above-mentioned microorganism is a white rot fungus.

[0046] Furthermore, the white rot fungus is one or more of Phanerochae techrysosporium, Trametes versicolor, Corilus versicolor, Pleurotus sajor-caju and Pleurotus ostreatus.

[0047] Furthermore, the white rot fungus is Trametes versicolor.

[0048] In a fifth aspect, the present application provides a method for preparing the above-mentioned porous carrier-microorganism composite material, which comprises: adding the porous carrier material into the culture medium, then adding functional microorganisms by the bacterial liquid inoculation method, and culturing by shaking so that the microorganisms are fixed to the inside of the porous carrier material by physical adsorption.

[0049] Furthermore, the method for preparing the porous carrier-microorganism composite material, wherein the microorganism is a white rot fungus, specifically comprises:

[0050] Preparation of mycelial liquid:

[0051] The T. versicolor agar block was inoculated into the PD medium and shaken (135 rpm) was cultured at 25°C for 4-5 days; the obtained mycelial pellets were separated from the culture medium, the mycelial pellets were collected and added with 0.85% sterile saline, and the mixture was stirred with a high-speed homogenizer for 15 minutes to obtain mycelial liquid, which was stored at 4°C;

[0052] Preparation of porous carrier-microorganism composite materials:

[0053] The porous carrier material was added into the PD culture medium and sterilized under high pressure; the mycelium liquid of the white rot fungus T. versicolor was inoculated into the culture medium and cultured with shaking at 25°C and 135 rpm for 5 days to prepare a porous carrier-microorganism composite material (immobilized white rot fungus).

[0054] In a sixth aspect, the present application provides application of the above-mentioned porous carrier-microorganism composite material in wastewater treatment.

[0055] Furthermore, the microorganisms in the porous carrier-microorganism composite material are white rot fungi, which are used to degrade PPCPs in wastewater.

[0056] Furthermore, the above-mentioned PPCPs include one or more of diclofenac (DCF), ibuprofen (IBU), sulfamethoxazole (SMX) and carbamazepine (CBZ).

[0057] 3. Beneficial effects

[0058] Compared with the prior art, the present application has the following beneficial effects:

[0059] (1) The present application provides a porous carrier material for use in microbial immobilization, wherein the porous carrier material has a BET specific surface area of ​​more than 600 m2 as measured by a nitrogen isotherm method. 2 g -1 , pK a >8 and the contact angle is 20°~60°. By studying the effects of porous structure, surface charge and hydrophilicity on the growth metabolism and community function of immobilized microorganisms, it was found that the positively charged and moderately hydrophilic porous carrier material achieved the efficient fixation of the white rot fungus T.versicolor and its laccase, and treated the actual wastewater from the sewage plant without adjusting the pH. At the same time, it achieved the efficient degradation of PPCPs and the stable activity of T.versicolor, providing new ideas for the application of functional microorganisms and the degradation of emerging pollutants.

[0060] (2) The present application provides a porous carrier material for use in microbial immobilization to degrade emerging pollutants in wastewater under high-concentration load conditions. Compared with unfixed T.versicolor and PUF, the removal rate of PPCPs by T.versicolor fixed by porous cyclodextrin polymer is significantly improved, with an average of 81.3%. At the same time, the material control reactors B-Ι (PBCD-BD) and B-II (Th-CDP) are saturated in the later stage of the reaction, and the effluent concentration increases significantly, while the T.versicolor fixed by porous cyclodextrin always maintains a low effluent concentration, especially Tv@Th-CDP, whose effluent concentration is only 6.2% of that of the material control after 18 days. The coupling of the porous carrier and biodegradation prolongs the performance of the reactor.

[0061] (3) The porous carrier material provided in this application is used in the immobilization of microorganisms to degrade emerging pollutants in wastewater at environmental concentrations. The study found that high laccase and biological activity were detected inside the porous carrier, and the degradation of PPCPs was related to the pK aThere was a positive correlation between the two. The positively charged Tv@Th-CDP retained T.versicolor and its laccase through electrostatic action, forming an adsorption-biodegradation synergistic system. The average removal rate of PPCPs reached 90.3%, which was 17.2% higher than that of the unfixed T.versicolor. The degradation efficiency of the negatively charged Tv@PBCD-BD and Tv@GAC was affected.

[0062] (4) The present application provides a porous carrier material for the application of microbial immobilization. The effect of the hydrophilicity of the porous carrier on the biological activity was studied by metagenomic sequencing. After 10 days of continuous treatment in the reactor, the abundance of unfixed T. versicolor decreased by 97.1%, while the moderately hydrophilic Tv@Th-CDP still had 31.2% T. versicolor abundance, with almost no loss, and had a high abundance of cytochrome P450 function. Other biomaterials were negatively affected by Proteobacteria, and the super-hydrophilic surface of Tv@PBCD-BD caused bacterial overgrowth. BRIEF DESCRIPTION OF THE DRAWINGS

[0063] Figure 1 Schematic diagram of the immobilization of the white rot fungus T. versicolor on carrier materials, including Tv@PBCD-BD, Tv@Th-CDP, Tv@GAC, and Tv@PUF.

[0064] Figure 2 These are scanning electron micrographs of immobilized white rot fungus T. versicolor, including: (a) T. versicolor; (b) Tv@PBCD-BD; (c) Tv@Th-CDP; (d) Tv@GAC; (e) Tv@PUF.

[0065] Figure 3 This is a schematic diagram of the synergistic degradation of immobilized white rot fungus Tv@Th-CDP in actual wastewater. The positively charged Th-CDP achieves the simultaneous fixation of T. versicolor and its laccase through electrostatic action.

[0066] Figure 4 The results of the immobilized white rot fungi reactor removing high concentrations of PPCPs under rapid saturation conditions: Reactor A1 (T.versicolor); Reactor C1 (Tv@PBCD-BD); Reactor D1 (Tv@Th-CDP); Reactor E1 (Tv@GAC); Reactor F1 (Tv@PUF); diclofenac (DCF); ibuprofen (IBU); sulfamethoxazole (SMX); carbamazepine (CBZ).

[0067] Figure 5The results of the removal of PPCPs at environmental concentrations by the white rot fungus T. versicolor immobilized by porous cyclodextrin polymer and its material control in the reactor. Influent1 is the influent of reactor B-1, reactor C1 and reactor D1, and Influent2 is the influent of reactor B-Ⅱ. Reactor B-1 (PBCD-BD), reactor B-Ⅱ (Th-CDP); reactor C1 (Tv@PBCD-BD); reactor D1 (Tv@Th-CDP); diclofenac (DCF); ibuprofen (IBU); sulfamethoxazole (SMX); carbamazepine (CBZ).

[0068] Figure 6 The results of PPCPs removal by immobilized white rot fungus T. versicolor reactor under environmental concentration equilibrium conditions: Reactor A2 (T. versicolor), Reactor C2 (Tv@PBCD-BD); Reactor D2 (Tv@Th-CDP); Reactor E2 (Tv@GAC); Reactor F2 (Tv@PUF); diclofenac (DCF); ibuprofen (IBU); sulfamethoxazole (SMX); carbamazepine (CBZ).

[0069] Figure 7 Adenosine triphosphate (ATP) concentration, laccase activity and glucose concentration of immobilized T. versicolor in the suspension (sus) and inside the material (ex) under environmental concentration equilibrium conditions, (a) ATP concentration during immobilization; (b) ATP concentration in the reactor; (c) laccase activity; (d) glucose concentration.

[0070] Figure 8 are the parameters of the immobilized white rot fungus T. versicolor reactor suspension, environmental concentration equilibrium conditions: (a) laccase activity and adenosine triphosphate (ATP) concentration; (b) glucose concentration and pH value. Rapid saturation pre-operation conditions: (c) laccase activity and ATP concentration; (d) glucose concentration and pH value. Reactor A (T. versicolor); Reactor BI (PBCD-BD); Reactor B-II (Th-CDP); Reactor C (Tv@PBCD-BD); Reactor D (Tv@Th-CDP); Reactor E (Tv@GAC); Reactor F (Tv@PUF).

[0071] Fig. 9Species distribution in the immobilized white rot fungus T. versicolor reactor, (a) rapid saturation pre-operation conditions; (b) environmental concentration equilibrium conditions; effluent from the secondary sedimentation tank of a sewage treatment plant (GT_WWTP); reactor A (T. versicolor); reactor C (Tv@PBCD-BD); reactor D (Tv@Th-CDP); reactor E (Tv@GAC); reactor F (Tv@PUF). Suspension (s); T. versicolor particles (p).

[0072] Fig.10 COG functional distribution of immobilized white rot fungus T. versicolor reactors at the level of secondary metabolite biosynthesis, transport and decomposition; (a) Rapid saturation pre-operation conditions; (b) Environmental concentration equilibrium conditions; Wastewater treatment plant secondary sedimentation tank effluent (GT_WWTP); Reactor A (T. versicolor); Reactor C (Tv@PBCD-BD); Reactor D (Tv@Th-CDP); Reactor E (Tv@GAC); Reactor F (Tv@PUF). Suspension (s); T. versicolor particles (p). DETAILED DESCRIPTION

[0073] The present application is further described below in conjunction with specific embodiments.

[0074] It should be noted that the terms such as "upper", "lower", "left", "right", "middle", etc. cited in this specification are only for the convenience of description and are not used to limit the scope of implementation. Changes or adjustments to their relative relationships should be regarded as the scope of implementation of this application without substantially changing the technical content.

[0075] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this application belongs; the term "and / or" used herein includes any and all combinations of one or more of the associated listed items.

[0076] If the specific conditions are not specified in the examples, the experiments were carried out under conventional conditions or conditions recommended by the manufacturer. If the manufacturers of the reagents or instruments are not specified, they are all conventional products that can be purchased commercially.

[0077] As used herein, the term "about" is used to provide flexibility and imprecision associated with a given term, measurement or value. The degree of flexibility for a particular variable can be easily determined by one skilled in the art.

[0078] As used herein, the term "at least one of" is intended to be synonymous with "one or more of." For example, "at least one of A, B, and C" expressly includes only A, only B, only C, and combinations of each thereof.

[0079] Concentration, amount and other numerical data can be presented in range format herein.It should be understood that such range format is only used for convenience and simplicity, and should be flexibly interpreted as not only including the numerical value clearly described as range limit, but also including all single numerical values ​​or sub-ranges contained in the range, just as each numerical value and sub-range are clearly described.For example, the numerical range of about 1 to about 4.5 should be interpreted as not only including the limit value of 1 to about 4.5 clearly described, but also including single numerals (such as 2,3,4) and sub-ranges (such as 1 to 3,2 to 4, etc.).The same principle is applicable to the scope of only narrating a numerical value, such as "less than about 4.5", which should be interpreted as including all the above-mentioned values ​​and ranges.In addition, no matter how the breadth of the described range or feature is, this explanation should be applicable.

[0080] In this application, the instruments and methods used for characterization and performance evaluation of composite materials are:

[0081] Physical and chemical adsorption instrument: ASAP 2460 (Micromeritics, USA), the specific surface area of ​​the support material was determined by nitrogen adsorption method.

[0082] Water contact angle measuring instrument: OCA20 (Dataphysics, Germany).

[0083] Zeta potential instrument: Zetasizer Nano ZS90 (Malvern, UK).

[0084] Scanning electron microscope: QUANTA 250FEG (FEI, USA). The samples were washed with PBS and fixed with glutaraldehyde, then dehydrated and freeze-dried, and sprayed with gold for microscopic detection.

[0085] Liquid chromatography (LC) and liquid chromatography-mass spectrometry (LC-MS): Agilent 1260 (Agilent, USA), API4000 (AB SCIEX, USA) were used for concentration detection of PPCPs.

[0086] UV-visible spectrophotometer: Specord 210 (Analytik Jena, Germany). Laccase activity was determined by the DMP method, and glucose concentration was detected by the DNS method.

[0087] Microplate reader: Synergy H1 (BioTek, USA), ATP concentration was detected using BacTiter-GloTM microbial cell viability assay kit.

[0088] Metagenomic sequencing: for species and functional analysis of microorganisms in reactors.

[0089] Example 1

[0090] This embodiment provides a porous carrier material and a preparation method thereof.

[0091] In this embodiment, the porous carrier material is a porous thienyl cyclodextrin polymer (Th-CDP), and its preparation method is referred to in the literature Tu, Y.; Wang, H.; Yang, P.; Xu, G.; Hu, X.; Li, A.; Xie, XA porous thienylcyclodextrin polymer synthesized in a homogeneous ionic liquid catalytic system for the rapid removal of pharmaceuticals and personal care products (PPCPs). Green Chemistry, 2022, 24 (1): 227-237. Specifically including:

[0092] 1-Ethyl-3-methylimidazolium chloride / ferric chloride ([EMIm]Cl / FeCl 3 ) Preparation of ionic liquids:

[0093] 29.92 g [EMIm]Cl and 66.20 g FeCl 3 The mixture was dried in vacuum at 60 °C for 24 h and then immediately transferred to a glove box; the reaction was carried out in a ceramic bead cooling bath (pre-cooled) to convert FeCl 3 Slowly add it to the beaker containing [EMIm]Cl and stir it continuously with a glass rod. The reaction releases heat and the two components melt into liquid. During the stirring process, the temperature of the solution rises to about 20-40°C. After stirring at room temperature for 24 hours, the Lewis acid ionic liquid [EMIm]Cl / FeCl is obtained. 3 ;

[0094] Synthesis of Thienyl Cyclodextrin (ThCD):

[0095] 2g of β-cyclodextrin (β-CD) was dissolved in anhydrous N,N-dimethylformamide (DMF); then 2.22g of 60% anhydrous NaH was slowly added at 0°C and stirred for 15min; 10.34g of 3-bromomethylthiophene (3-Bm-Th) was slowly added dropwise and then stirred at room temperature for 24h; the reaction was quenched with 5mL of methanol; then the product was mixed with 200mL of water and concentrated to near dryness under vacuum at 84°C; the obtained product was mixed with 100mL of water and extracted with 150mL of dichloromethane three times, 50mL each time; the organic phase was dried over anhydrous sodium sulfate, then filtered and concentrated under vacuum at 35°C, and finally a light yellow compound ThCD was obtained by silica gel chromatography;

[0096] Synthesis of Porous Thienyl Cyclodextrin Polymer (Th-CDP):

[0097] At 0°C, 0.8 g of ThCD was dissolved in [EMIm]Cl / FeCl 3 ; The solution N 2 After three replacements, in N 2 Under protection, 2.68 g of anhydrous trimethyl orthoformate (Tmof) was slowly added dropwise at room temperature, the reaction mixture was stirred and heated to 45°C for 5 hours, and then heated to 120°C for 19 hours; after cooling, the brown precipitate was washed with water and methanol until the filtrate was colorless, and then Soxhlet extraction with methanol was performed for 24 hours, and vacuum dried at 60°C for 24 hours to obtain a porous thienyl cyclodextrin polymer (Th-CDP).

[0098] Example 2

[0099] This example provides the effects of different grafting agents and catalyst systems on the surface charge of porous cyclodextrin polymers.

[0100] Selecting different functional groups to be grafted onto cyclodextrin will change the surface properties of the resulting polymer. Polypyrrole, polythiophene and polyfuran are commonly used conductive materials. Therefore, cyclodextrin is grafted with different aromatic heterocyclic reagents to evaluate the effect of different grafting reagents on the surface charge of porous cyclodextrin polymers.

[0101] The preparation method is as in Example 1, except that the grafting agents include: 3-bromomethylthiophene (3-Bm-Th, 10.34 g), 3-bromomethylpyrrole (3-Bm-Py, 11.43 g), 3-bromomethylfuran (3-Bm-FF, 8.93 g); benzyl bromide (BnBr, 9.48 g); and the catalyst system includes: [EMIm]Cl / FeCl 3 、FeCl 3 ; The alkylating agent is: BCMBP+DCX (biphenyl dichlorobenzyl, 10.44 g; p-dichlorobenzyl, 2.43 g).

[0102] The surface charge of the prepared porous cyclodextrin polymer was measured, and the results are shown in Table 1, which shows that compared with the benzene ring structure, a positively charged porous cyclodextrin polymer can be prepared by grafting aromatic heterocyclic compounds under the Lewis acid ionic liquid catalysis system. And in the aromatic heterocyclic compound, when the thiophene ring is used as the grafting monomer, the pK a Higher (>8), this is because after grafting, the S atom on the thienyl cyclodextrin and the α-H atom position of the thiophene ring carry a certain amount of positive charge, and the catalytic system of the ionic liquid may make the surface properties of the polymer charged. Therefore, 3-Bm-Th is preferred as the grafting agent, [EMIm]Cl / FeCl 3 Ionic liquids are catalytic systems.

[0103] Table 1

[0104] serial number Cyclodextrin Grafting reagent Alkylating agents catalyst Surface charge 1 β 3-Bm-Th BCMBP+DCX <![CDATA[[EMIm]Cl / FeCl 3 ]]> Positive 2 β 3-Bm-Py BCMBP+DCX <![CDATA[[EMIm]Cl / FeCl 3 ]]> neutral 3 β 3-Bm-FF BCMBP+DCX <![CDATA[[EMIm]Cl / FeCl 3 ]]> neutral 4 β BnB BCMBP+DCX <![CDATA[[EMIm]Cl / FeCl 3 ]]> neutral 5 β BnB BCMBP+DCX <![CDATA[FeCl 3 ]]> Negative

[0105] Note: Surface charge positive: pK a >8; Surface charge neutrality: 6≤pK a ≤8; surface charge negative: pK a <6.

[0106] Example 3

[0107] This example provides the effect of different alkylating agents on the specific surface area of ​​porous cyclodextrin polymers.

[0108] The preparation method refers to Example 1, except that the different alkylating agents include: trimethyl orthoformate (Tmof, 2.68 g), dimethoxymethane (FDA, 1.92 g), biphenyl dichlorobenzyl + p-dichlorobenzyl (BCMBP, 4.75 g; DCX, 1.10 g), biphenyl dichlorobenzyl (BCMBP, 6.33 g), p-dichlorobenzyl (DCX, 4.41 g), 1,4-dimethoxybenzene (1,4-Dimethoxybenzene, 3.48 g).

[0109] The specific surface area of ​​the prepared porous cyclodextrin polymer was measured. The results are shown in Table 2. Tmof and BCMBP+DCX are used as alkylating agents, and the porous cyclodextrin polymer obtained has a high specific surface area; while the specific surface areas obtained by other alkylating agents are relatively low.

[0110] Table 2

[0111] serial number Cyclodextrin Grafting reagent Alkylating agents catalyst Specific surface area 6 β 3-Bm-Th Tmof <![CDATA[[EMIm]Cl / FeCl 3 ]]> high 7 β 3-Bm-Th FDA <![CDATA[[EMIm]Cl / FeCl 3 ]]> medium 8 β 3-Bm-Th BCMBP+DCX <![CDATA[[EMIm]Cl / FeCl 3 ]]> high 9 β 3-Bm-Th BCMBP <![CDATA[[EMIm]Cl / FeCl 3 ]]> medium 10 β 3-Bm-Th DCX <![CDATA[[EMIm]Cl / FeCl 3 ]]> medium 11 β 3-Bm-Th 1,4-Dimethoxybenzene <![CDATA[[EMIm]Cl / FeCl 3 ]]> Low

[0112] Note: Specific surface area: >500m 2 g -1 ; Medium specific surface area: 200m 2 g-1 -500m 2 g -1 ; Low specific surface area: <200m 2 g -1 .

[0113] Example 4

[0114] This example provides the effect of different reaction temperatures on the specific surface area of ​​porous cyclodextrin polymers.

[0115] The preparation method refers to Example 1 (No. 12 is Example 1), the difference being that different final reaction temperatures are 60°C, 80°C, 100°C, 120°C and 150°C, and a constant temperature of 80°C is used for the reaction for 24 hours.

[0116] The specific surface area of ​​the prepared porous cyclodextrin polymer was measured. The results are shown in Table 3. The reaction temperature has a great influence on the polymerization reaction of thienyl cyclodextrin, and programmed temperature increase can be conducive to obtaining a higher specific surface area.

[0117] Table 3

[0118] serial number Cyclodextrin Grafting reagent Alkylating agents catalyst Temperature / ℃ <![CDATA[Specific surface area / m 2 g -1 > 12 β 3-Bm-Th Tmof <![CDATA[[EMIm]Cl / FeCl 3 ]]> 45→120 730 13 β 3-Bm-Th Tmof <![CDATA[[EMIm]Cl / FeCl 3 ]]> 45→60 56 14 β 3-Bm-Th Tmof <![CDATA[[EMIm]Cl / FeCl 3 ]]> 45→80 650 15 β 3-Bm-Th Tmof <![CDATA[[EMIm]Cl / FeCl 3 ]]> 45→100 695 16 β 3-Bm-Th Tmof <![CDATA[[EMIm]Cl / FeCl 3 ]]> 45→150 736 17 β 3-Bm-Th Tmof <![CDATA[[EMIm]Cl / FeCl 3 ]]> 80 527

[0119] Note:

[0120] 45℃→120℃: programmed temperature rise, first react at 45℃ for 5h, then heat to 120℃ for 19h;

[0121] 80℃: Keep the reaction at 80℃ for 24h.

[0122] Example 5

[0123] This example provides the effect of different substitution degrees of the grafting reaction on the hydrophilicity of the porous cyclodextrin polymer.

[0124] The preparation method refers to Example 1 (No. 12 is Example 1), the difference being that the grafting ratios are 21:1, 14:1, 7:1, and 3:1.

[0125] The contact angle of the prepared porous cyclodextrin polymer was measured. The results are shown in Table 4. Controlling the number of hydroxyl groups on cyclodextrin can achieve the regulation of the hydrophilicity and hydrophobicity of the polymer material. When the grafting ratio is greater than 14, the surface of the porous cyclodextrin polymer is moderately hydrophilic.

[0126] Table 4

[0127] serial number Cyclodextrin Grafting reagent Grafting ratio Alkylating agents catalyst Temperature / ℃ Contact angle / ° 12 β 3-Bm-Th 21 Tmof <![CDATA[[EMIm]Cl / FeCl 3 ]]> 45→120 38 18 β 3-Bm-Th 14 Tmof <![CDATA[[EMIm]Cl / FeCl 3 ]]> 45→120 26 19 β 3-Bm-Th 7 Tmof <![CDATA[[EMIm]Cl / FeCl 3 ]]> 45→120 9 20 β 3-Bm-Th 3 Tmof <![CDATA[[EMIm]Cl / FeCl 3 ]]> 45→120 5

[0128] Note:

[0129] Grafting ratio: molar ratio of thiophene reagent and cyclodextrin;

[0130] Hydrophilic surface: contact angle <20°; moderately hydrophilic surface: contact angle 20°~60°; hydrophobic surface: contact angle >60°.

[0131] Example 6

[0132] This example provides the surface charge and hydrophilicity of porous cyclodextrin polymers prepared from different supramolecular compounds.

[0133] The preparation method refers to Example 1 (No. 12 is Example 1), the difference being that the supramolecular compound includes: β-cyclodextrin (2g), α-cyclodextrin (1g), γ-cyclodextrin (3g), chitosan (3g), and pillar aromatics (4g).

[0134] The surface charge and contact angle of the prepared porous cyclodextrin polymers were measured. The results are shown in Table 5. The surface charges of the porous cyclodextrin polymers prepared from different supramolecular compounds are all positive, and the surfaces are all moderately hydrophilic.

[0135] Table 5

[0136] serial number Supramolecular compounds Grafting reagent Alkylating agents catalyst Temperature / ℃ Surface charge Contact angle / ° 12 β-Cyclodextrin 3-Bm-Th Tmof <![CDATA[[EMIm]Cl / FeCl 3 ]]> 45→120 Positive 38 21 α-Cyclodextrin 3-Bm-Th Tmof <![CDATA[[EMIm]Cl / FeCl 3 ]]> 45→120 Positive 36 22 γ-Cyclodextrin 3-Bm-Th Tmof <![CDATA[[EMIm]Cl / FeCl 3 ]]> 45→120 Positive 45 23 Chitosan 3-Bm-Th Tmof <![CDATA[[EMIm]Cl / FeCl 3 ]]> 45→120 Positive 33 24 Pillar aromatics 3-Bm-Th Tmof <![CDATA[[EMIm]Cl / FeCl 3 ]]> 45→120 Positive 59 25 β-Cyclodextrin 3-Bm-Th Tmof <![CDATA[FeCl 3 ]]> 45→120 Positive 55 26 β-Cyclodextrin 3-Bm-Th BCMBP+DCX <![CDATA[[EMIm]Cl / FeCl 3 ]]> 45→120 Positive 52

[0137] Note:

[0138] Surface charge positive: pK a >8; Surface charge neutrality: 6≤pK a ≤8; surface charge negative: pK a <6;

[0139] Hydrophilic surface: contact angle <20°; moderately hydrophilic surface: 20°≤contact angle≤60°; hydrophobic surface: contact angle >60°.

[0140] Comparative Example 1

[0141] This comparative example provides a porous cyclodextrin polymer (PBCD-BD) with a super-hydrophilic surface.

[0142] The preparation method of porous cyclodextrin polymer with super hydrophilic surface (PBCD-BD) refers to Tu, Y.; Xu, G.; Jiang, L.; Hu, X.; Xu, J.; Xie, X.; Li, A. Amphiphilic hyper-crosslinked porous cyclodextrin polymer with high specific surface area for rapid removal of organic micropollutants. Chemical Engineering Journal 2020, 382, ​​123015. β-cyclodextrin was grafted with a certain number of benzyl groups, and cross-linked with biphenyl dichlorobenzyl and p-dichlorobenzyl to obtain a polymer with a specific surface area of ​​1445 m 2 g -1 The PBCD-BD retains most of the hydroxyl groups of cyclodextrin, and the polymer surface is superhydrophilic.

[0143] Comparative Example 2

[0144] This comparative example provides a commercial immobilized carrier material, granular activated carbon.

[0145] In this comparative example, granular activated carbon (GAC, DARCO) was purchased from Sigma and had a microporous structure with a specific surface area of ​​612 m 2 g -1 .

[0146] Comparative Example 3

[0147] This comparative example provides a commercial immobilization carrier material, polyurethane foam (PUF).

[0148] In this comparative example, polyurethane foam (PUF) was purchased from Henan Tianyi Environmental Protection Technology Co., Ltd. It has a non-porous structure and is usually used for loading biofilms.

[0149] Example 7

[0150] This embodiment provides porous thienyl cyclodextrin polymer (Th-CDP) (No. 12), porous cyclodextrin polymer (PBCD-BD), granular activated carbon (GAC) and polyurethane foam (PUF) as carrier materials for the application of microbial immobilization.

[0151] In this example, the surface properties of the carrier material are shown in Table 6.

[0152] Table 6

[0153] Carrier material <![CDATA[Specific surface area m 2 g -1 > Contact angle° <![CDATA[pK a ]]> Th-CDP No. 12 730 38 9.5 PBCD-BD 1445 9 5.5 GAC 612 49 4.4 PUF 0.6 123 5.9

[0154] In this example, the microorganism is the white rot fungus Trametes versicolor.

[0155] The carrier material fixes the microorganisms by first adding the carrier material to the culture medium, then adding the functional microorganisms by the mycelium liquid inoculation method, and then culturing with vibration to fix the microorganisms to the inside of the carrier by physical adsorption. The schematic diagram is shown in Figure 1 As shown, specifically including:

[0156] Preparation of mycelial liquid:

[0157] Put 4 1cm 2 The T. versicolor agar blocks of different sizes were inoculated into 100 mL of PD medium and cultured with shaking (135 rpm) at 25°C for 4-5 days; the obtained mycelial balls were separated from the culture medium, the mycelial balls were collected and added with 8 mL of 0.85% sterile saline, and the mixture was stirred with a high-speed homogenizer for 15 min to obtain mycelial liquid, which was stored at 4°C;

[0158] Immobilization of microorganisms (preparation of porous carrier-microorganism composite materials):

[0159] 1 g of the above carrier material was added to 500 mL of PD culture medium and sterilized by high pressure; 1.5 mL of mycelium liquid of white rot fungus T. versicolor was inoculated into the culture medium and cultured with shaking at 25° C. and 135 rpm for 5 days to prepare immobilized white rot fungus.

[0160] Scanning electron microscopy images of immobilized white rot fungi Figure 2 As shown, (a) is T.versicolor; (b) is Tv@PBCD-BD; (c) is Tv@Th-CDP; (d) is Tv@GAC; (e) is Tv@PUF. Figure 2 It can be seen that the mycelium of T.versicolor is strip-shaped with a width of about 1μm (the diameter of the mycelium ball is 6-7mm). After fixing T.versicolor with PBCD-BD with super-hydrophilic surface, the biomaterial particles formed are smaller (3mm in diameter) because of its better dispersibility and tighter binding. In contrast, the particles Tv@Th-CDP (6-7mm in diameter) and Tv@GAC (8-9mm in diameter) produced by T.versicolor fixed with moderately hydrophilic Th-CDP and GAC are larger, and the materials are agglomerated inside them. However, due to its macroscopic pore structure, the PUF-immobilized T.versicolor adheres and grows inside it. It can be seen that the porous granular carrier and the block PUF have different morphologies after fixing T.versicolor. As for the biomaterial particles, due to dispersibility, the more hydrophobic the carrier, the larger the particles formed.

[0161] Example 8

[0162] This example provides the application of the immobilized white rot fungi obtained in Example 7 in actual wastewater to degrade PPCPs in the wastewater.

[0163] In this example, four refractory PPCPs, diclofenac (DCF), ibuprofen (IBU), sulfamethoxazole (SMX), and carbamazepine (CBZ), were selected as target pollutants, and a 0.5 L continuous flow upflow reactor was built with an aeration rate of 200 L min -1 , the difficult-to-degrade PPCPs in the effluent of the secondary sedimentation tank of a continuous treatment sewage plant were studied, and the degradation effect and biological activity of immobilized white rot fungi were studied.

[0164] Schematic diagram of the synergistic degradation of immobilized white rot fungi Tv@Th-CDP in actual wastewater Figure 3 As shown, the positively charged, moderately hydrophilic porous thienyl cyclodextrin polymer (Th-CDP) retains T. versicolor and its laccase (negatively charged) through electrostatic interaction, forming an adsorption-biodegradation synergistic system.

[0165] (1) Removal of high concentrations of PPCPs under rapid saturation conditions

[0166] Experimental method: 10 mg L -1 The hydraulic retention time (HRT) of the above four PPCPs is 12h.

[0167] The degradation efficiency of four difficult-to-degrade PPCPs is as follows Figure 4 As shown, reactor A1 is T.versicolor, reactor C1 is Tv@PBCD-BD, reactor D1 is Tv@Th-CDP, reactor E1 is Tv@GAC, and reactor F1 is Tv@PUF. Figure 4 It can be seen that compared with the unfixed T.versicolor and PUF, the porous carrier significantly improved the degradation performance of T.versicolor, especially the T.versicolor fixed by porous cyclodextrin polymers (Tv@PBCD-BD and Tv@Th-CDP), which removed an average of 81.3% of PPCPs.

[0168] (2) Removal of PPCPs at environmental concentrations

[0169] Experimental method: After 3 days of high concentration loading, the addition amount of the four PPCPs was reduced to 10 μg L -1 , HRT is 1d.

[0170] The results of the removal of PPCPs at environmental concentrations by the white rot fungus T. versicolor immobilized by porous cyclodextrin polymer and the porous cyclodextrin polymer in the reactor are shown in Figure 5 As shown, Influent1 is the influent of reactor B-1, reactor C1 and reactor D1; Influent2 is the influent of reactor B-II; B-1 is PBCD-BD, B-II is Th-CDP, C1 is Tv@PBCD-BD, and D1 is Tv@Th-CDP. Figure 5 It can be seen that in the later stage of the reaction, the effluent concentration of PPCPs in the T.versicolor reactors (reactor C1 and reactor D1) fixed with porous cyclodextrin polymers was much lower than that of their material control groups (reactor B-Ι and reactor B-Ⅱ). This coupling of adsorption and biodegradation prolonged the performance of the T.versicolor reactor, especially Tv@Th-CDP. The average effluent concentration of PPCPs on the 18th day was only 6.2% of that of its material control, achieving higher PPCPs biodegradation.

[0171] (3) Removal of PPCPs under environmental concentration equilibrium conditions

[0172] Experimental method: The concentration of the four PPCPs added was 10 μg L -1 , HRT is always 1d.

[0173] The degradation efficiency of four difficult-to-degrade PPCPs is as follows Figure 6 As shown, reactor A2 is T.versicolor, reactor C2 is Tv@PBCD-BD, reactor D2 is Tv@Th-CDP, reactor E2 is Tv@GAC, and reactor F2 is Tv@PUF. After correlation analysis, the degradation efficiency of PPCPs is related to the pK a There was a positive correlation between the removal of PPCPs by the positively charged Tv@Th-CDP and the removal of PPCPs by the positively charged Tv@Th-CDP was 90.3% on average, which was 17.2% higher than that of the unfixed T.versicolor. The removal effects of the negatively charged Tv@PBCD-BD and Tv@GAC were both affected.

[0174] Example 9

[0175] This example provides a study on the parameter changes during the degradation of PPCPs by the immobilized white rot fungi in Example 8.

[0176] (1) ATP concentration, laccase activity, and glucose concentration in the suspension (sus) and inside the material (ex)

[0177] Experimental method: During the immobilization process and reactor study, water samples were collected from the culture medium or reactor regularly, and biomaterial particles or material samples were taken out, weighed, and ultrasonically extracted twice, adding 7.5 mL PBS (0.01 mol / L) each time. -1 , pH 7.2-7.4), ultrasonic extraction for 30 min, and internal extract was obtained.

[0178] Figure 7 The adenosine triphosphate (ATP) concentration, laccase activity and glucose concentration of immobilized T. versicolor in the suspension (sus) and inside the material (ex) under environmental concentration equilibrium conditions. (a) ATP concentration during the immobilization process; (b) ATP concentration in the reactor; (c) laccase activity; (d) glucose concentration. Figure 7 It can be seen that high laccase and biological activities were measured inside the porous carrier material. The positively charged Tv@Th-CDP retained T.versicolor laccase (negatively charged) through electrostatic action, forming an adsorption-biodegradation synergistic system.

[0179] (2) Laccase, ATP, glucose concentration and pH value in the suspension of the immobilized white rot fungus T. versicolor reactor Experimental method: During the reactor study, water samples were collected from the reactor regularly to determine the biological activity indicators and water quality parameters.

[0180] Figure 8 The laccase, ATP, glucose concentration and pH value in the suspension of the reactor for immobilizing the white rot fungus T. versicolor were investigated. The factors affecting the carrier-biocomposite synergistic degradation were studied by correlation analysis. The pH value of the reactor inlet water was relatively stable, with an average of 7.6, and efficient degradation and stable activity were achieved without adjusting the pH. pH and ATP were key factors, and electrostatic interaction and the activity of T. versicolor had an important influence on the synergistic degradation of the carrier-biocomposite.

[0181] (3) Species distribution in the immobilized white rot fungus T. versicolor reactor

[0182] Experimental method: The suspension and biomaterial particles (mycelium balls) in the reactor were taken out for metagenomic sequencing. The solution samples were filtered through MCE (0.2μm) and then extracted from the filter membrane. The distribution of the top 20 communities in abundance in the reactor was analyzed at the species level.

[0183] Species distribution in the immobilized white rot fungi reactor Fig. 9 As shown, under the fast saturation pre-operation condition ( Fig. 9a), compared with the bulk composite biomaterial Tv@PUF, the abundance of T.versicolor fixed by the porous particle carrier is significantly higher. On the 17th day, the abundance of T.versicolor in the particles of reactors C1 and D1 (Tv@PBCD-BD and Tv@Th-CDP) decreased, but still dominated, while the particles of reactor A1 (unfixed T.versicolor) had lysed, indicating that immobilization promoted the maintenance of T.versicolor activity in the reactor. Under environmental concentration equilibrium conditions ( Fig. 9 b) The hydrophilicity of the carrier affected the abundance of T. versicolor. After 10 days of treatment, the abundance of reactor A2 (unfixed T. versicolor) decreased by 97.1%. The super-hydrophilic surface of Tv@PBCD-BD caused an increase in the abundance of Proteobacteria, while the moderately hydrophilic Tv@Th-CDP still had 31.2% T. versicolor abundance, with almost no loss. The dominant species in the reactor did not change significantly, providing a more stable environment for the growth of T. versicolor.

[0184] (4) COG functional distribution at the level of secondary metabolite biosynthesis, transport and decomposition in the immobilized white rot fungus T. versicolor reactor

[0185] Experimental methods: The top 20 abundant COG functions (biosynthesis, transport and decomposition of secondary metabolites) in the reactor were analyzed by metagenomic sequencing.

[0186] The results of COG functional distribution at the level of secondary metabolite biosynthesis, transport and decomposition in the immobilized white rot fungus T. versicolor reactor are shown in Figure 2. Fig.10 As shown in Figure 2, cytochrome P450 (COG2124) is the key degradation function of T. versicolor and is generally considered to be involved in the degradation of pollutants together with laccase. Fig.10 a) The abundance of COG2124 increased significantly in the porous carrier-immobilized T. versicolor compared to the unimmobilized T. versicolor and PUF. Fig.10 b) After 10 days of treatment, the activity maintenance ability of Tv@Th-CDP enables it to still have a high abundance of cytochrome P450 function.

[0187] Example 10

[0188] This example provides the use of porous thienyl cyclodextrin polymer (Th-CDP) (No. 12) as a carrier material in the immobilization of microorganisms.

[0189] In this embodiment, the microorganisms include nitrifying bacteria, denitrifying bacteria, Bacillus, Pseudomonas and activated sludge.

[0190] The fixation of microorganisms on carrier materials is accomplished by first adding the carrier material into the culture medium, then adding functional microorganisms by bacterial liquid inoculation, and then culturing with shaking so that the microorganisms are fixed to the inside of the carrier by physical adsorption.

[0191] The morphology, degradation efficiency and bioactivity of the prepared microbial composite materials are shown in Table 7.

[0192] Table 7

[0193] name Carrier microorganism Biomaterial morphology Degradation efficiency Activity maintenance ability NB@Th-CDP Th-CDP Nitrifying bacteria Attachment growth Higher better DB@Th-CDP Th-CDP Denitrifying bacteria Attachment growth Higher better Bt@Th-CDP Th-CDP Bacillus Attachment growth Very high very good P@Th-CDP Th-CDP Pseudomonas Attachment growth Very high very good AS@Th-CDP Th-CDP Activated sludge Attachment growth Very high very good

[0194] Note:

[0195] Degradation efficiency is very high: >80%; relatively high: 60% to 80%; relatively poor: 40% to 60%; very poor: <40%;

[0196] The activity maintenance ability is very good: after the immobilized microorganisms are added to the reactor for 10 days, the abundance loss is less than 10%; good: the abundance loss is 10% to 30%; poor: the abundance loss is 30% to 60%; very poor: the abundance loss is greater than 60%.

Claims

1. Application of a porous carrier material in microbial immobilization, characterized in that: The microorganisms include one or more of white rot fungi, nitrifying bacteria, denitrifying bacteria, Bacillus, Pseudomonas and activated sludge; the BET specific surface area of ​​the porous carrier material is greater than 600m 2 g -1 , pK a >8 and the contact angle is 20°~60°.

2. The use of a porous carrier material in microbial immobilization according to claim 1, characterized in that: The microorganism is a white rot fungus.

3. A porous carrier-microorganism composite material, characterized in that: The microorganisms include one or more of white rot fungi, nitrifying bacteria, denitrifying bacteria, Bacillus, Pseudomonas and activated sludge; the BET specific surface area of ​​the porous carrier material is greater than 600m 2 g -1 , pK a >8 and the contact angle is 20°~60°.

4. The porous carrier-microorganism composite material according to claim 3, characterized in that: The microorganism is a white rot fungus.

5. The porous carrier material according to any one of claims 1 to 4, characterized in that The porous carrier material includes any one of a porous thienyl cyclodextrin polymer, a porous thienyl chitosan polymer, and a porous thienyl pillar aromatic polymer.

6. The method for preparing the porous carrier material according to claim 5, characterized in that: The preparation method comprises: using cyclodextrin or chitosan or pillar aromatic hydrocarbon as monomer, grafting thiophene ring to form thiophene monomer, and using aromatic ring or methoxyl group-containing alkylating agent as crosslinking agent to obtain the monomer by Friedel-Crafts alkylation reaction under the catalysis of Lewis acid ion liquid catalyst.

7. The method for preparing a porous carrier material according to claim 6, characterized in that: The grafting of thiophene rings comprises: using anhydrous sodium hydride to replace the hydroxyl group of the monomer with sodium alkoxide, and then adding a halogenated thiophene ring for grafting; the halogenated thiophene ring comprises brominated thiophene or chlorinated thiophene; and / or The Lewis acid ionic liquid catalyst includes: a composite ionic liquid formed by an imidazole type or quaternary ammonium type ionic liquid and a metal chloride; the metal chloride is ferric chloride or aluminum chloride; the ionic liquid is 1-ethyl-3-methylimidazolium chloride ionic liquid; and / or The cross-linking agent includes any one or more of biphenyl dichlorobenzyl, p-dichlorobenzyl, biphenyl dibromide, p-dibromide, dimethoxymethane, trimethyl orthoformate, and 1,4-dimethoxybenzene; The reaction temperature conditions of the Friedel-Crafts alkylation reaction include: reacting at 45° C. for 4 to 6 hours, and then heating to 80 to 150° C. for 18 to 20 hours.

8. The method for preparing a porous carrier material according to claim 7, characterized in that: The porous carrier material is a porous thienyl cyclodextrin polymer, and the preparation method comprises the following steps: Dissolve cyclodextrin in anhydrous DMF; then slowly add anhydrous NaH at 0°C and stir for 15 minutes; slowly drop 3-bromomethylthiophene and stir at room temperature for 24 hours; after the reaction is completed, quench the reaction with methanol; then mix the product with water and concentrate to near dryness under vacuum at 84°C; mix the obtained product with water and extract with dichloromethane; dry the organic phase with anhydrous sodium sulfate, then filter and concentrate under vacuum at 35°C, and finally obtain a light yellow compound thienyl cyclodextrin by silica gel chromatography; At 0°C, ThCD was dissolved in 1-ethyl-3-methylimidazolium chloride / ferric chloride ionic liquid; under nitrogen protection, anhydrous trimethyl orthoformate (Tmof) was slowly added dropwise at room temperature, the reaction mixture was stirred and heated to 45°C for 5 hours, and then heated to 120°C for 19 hours; after cooling, the brown precipitate was washed with water and methanol until the filtrate was colorless, and then Soxhlet extraction with methanol was performed for 24 hours, and vacuum drying was performed at 60°C for 24 hours to obtain a porous thienyl cyclodextrin polymer.

9. The method for preparing a porous carrier material according to claim 8, characterized in that: The molar ratio of the 3-bromomethylthiophene to cyclodextrin is 14-21:1; the molar ratio of the alkylating agent to thienyl cyclodextrin is 5-100:

1.

10. Use of the porous carrier-microorganism composite material according to claim 3 or 4 in wastewater treatment.

11. The use according to claim 11, characterized in that: The microorganisms in the porous carrier-microorganism composite material are white rot fungi, which are used to degrade PPCPs in wastewater.

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