Composite microbial carrier and preparation method thereof
By using a magnetic composite microbial carrier with a multi-layer gradient structure and electrochemically induced in-situ polymerization technology, the problems of easy inactivation of microorganisms, low mass transfer efficiency, and secondary pollution in the treatment of heavy metal polluted water in existing technologies have been solved, achieving efficient, stable, and environmentally friendly heavy metal removal.
Patent Information
- Application Number
- CN202511831247.2
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-12-07
- Publication Date
- 2026-03-20
AI Technical Summary
Existing magnetic microbial carriers for treating heavy metal-contaminated water have several drawbacks, including the desorption process being dependent on changes in the macroscopic environment, complex operation, significant damage to microorganisms, untreated heavy metals in the regenerated solution, low mass transfer efficiency, and high risk of secondary pollution.
A magnetic composite microbial carrier with a multi-layered gradient structure, comprising a magnetic-microbial core, a biocompatible hydrogel inner layer, and a stimulus-responsive electroactive outer layer, is formed through electrochemically induced in-situ polymerization. This enables in-situ electrochemical reduction/oxidative desorption of heavy metals and gentle activation of microbial activity under an external electric field.
It achieves efficient, stable, and sustainable heavy metal removal capabilities of the carrier, avoids damage to microorganisms caused by chemical regeneration, reduces operating costs, and ensures the long service life and environmental friendliness of the carrier.
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Figure BDA0005725690370000081
Abstract
Description
TECHNICAL FIELD
[0001] The present application belongs to the technical field of microbial carrier preparation for wastewater treatment, and in particular relates to a magnetic composite microbial carrier and a preparation method thereof, in particular to a magnetic composite carrier for treating heavy metal contaminated water bodies and a preparation method thereof realized by a multi-layer gradient structure and an electrochemical induction in-situ polymerization technology. BACKGROUND
[0002] Heavy metal contaminated water bodies pose a serious threat to global ecological environment and human health, and its treatment is a major challenge in the environmental field. Heavy metals have biological accumulation, high toxicity and persistence, and it is urgent to develop efficient, sustainable and environmentally friendly remediation technologies. Existing physical and chemical methods, such as chemical precipitation, adsorption, ion exchange and membrane separation, are effective under certain conditions, but generally have high cost, complex operation, low selectivity and other problems, especially the generation of heavy metal-containing sludge or regenerated waste liquid, leading to secondary pollution, and the pollution problem has not been fundamentally solved.
[0003] Bioremediation technology has attracted widespread attention due to its good environmental compatibility, low cost and sustainability. Using microorganisms to remove heavy metals through mechanisms such as biosorption, reduction and complexation is considered a green and potential strategy. In order to improve the stability of microorganisms in complex water bodies, treatment efficiency and facilitate recovery, magnetic composite microbial carrier technology has emerged. This technology combines microorganisms with magnetic materials, providing physical protection while also allowing for rapid separation with an external magnetic field, effectively addressing the problems of microorganism loss and difficulty in recovery.
[0004] Existing magnetic carriers mostly use sodium alginate, polyvinyl alcohol and other hydrogels to embed microorganisms, and are combined with magnetic nanoparticles. Some studies introduce temperature-sensitive or pH-responsive hydrogels (such as PNIPAM) to attempt intelligent control of heavy metal adsorption-desorption, triggering desorption by changing pH or temperature, reducing damage to microorganisms by strong acid or strong base, and reducing the risk of secondary pollution.
[0005] However, as application requirements continue to improve, these carriers have gradually exposed obvious limitations at the principle level. The desorption process still relies on macro-environmental changes (such as overall adjustment of pH or temperature), which is complex to operate and stressful to the internal microorganisms, affecting activity and reuse efficiency. More importantly, the desorbed heavy metals still exist in the regenerated liquid, which needs further treatment, and the real green regeneration has not been achieved, and the risk of secondary pollution and subsequent cost problems are still prominent.
[0006] In addition, the microorganisms and the materials in the existing carriers are only physically embedded, lacking active activation or reinforcement mechanisms. When the mass transfer efficiency is limited, it is difficult to efficiently treat high-concentration or multi-valence heavy metal complex pollution. Traditional carriers still have deficiencies in the precision of structural design and functional integration. How to build a carrier that has microbial activity, adsorption performance, magnetic response, environmental intelligent response, and electrochemical activity, and realizes precise distribution and synergistic effect of functions, has become a key bottleneck in current technology development.
[0007] Therefore, developing a new type of magnetic composite carrier can realize in-situ reduction / oxidation desorption of heavy metals under low energy consumption conditions, avoid damage to microorganisms and secondary pollution caused by chemical regeneration, and mildly activate microbial activity during the regeneration process, forming a truly in-situ, green, efficient and sustainable repair solution, which has become a technical problem that needs to be broken through in the field. SUMMARY
[0008] To achieve the above invention purposes, the present application adopts the following technical solutions:
[0009] The present application provides a magnetic composite microbial carrier, which comprises from inside to outside:
[0010] (1) a magnetic-microbial core composed of superparamagnetic Fe3O4 nanoparticles with surface functional modification and active microorganisms;
[0011] (2) a biocompatible hydrogel inner layer covering the magnetic-microbial core, formed by ion cross-linking or covalent cross-linking of sodium alginate, chitosan or polyethylene glycol-based hydrogel;
[0012] (3) a stimulus-responsive electroactive composite outer layer covering the biocompatible hydrogel inner layer, composed of a stimulus-responsive hydrogel component and an electroactive conductive polymer network, and the outer layer is embedded with heavy metal chelating groups.
[0013] Further, the average particle size of the superparamagnetic Fe3O4 nanoparticles is 5-50 nm, and the surface is functionalized by chemical grafting of citric acid.
[0014] Further, the active microorganism is Pseudomonas putida with heavy metal adsorption, enrichment or biological transformation capacity.
[0015] Further, the material of the biocompatible hydrogel inner layer is sodium alginate-calcium ion cross-linked gel.
[0016] Further, the stimulus-responsive hydrogel component is poly(N-isopropylacrylamide-co-acrylic acid) (PNIPAM-co-PAA); the electroactive conductive polymer network is one or more of polyaniline (PANI), polypyrrole (PPy), polythiophene (PTh) and a composite material of graphene, carbon nanotube; and the outer layer is compounded by interpenetrating network, blending or covalent bonding.
[0017] Further, the heavy metal chelating group is one or more of thiol (-SH), amino (-NH2), carboxyl (-COOH) or imidazole group; and the thickness of the stimulus-responsive electroactive composite outer layer is 20-100 μm.
[0018] The present application also provides a method for preparing the magnetic composite microbial carrier, comprising the following steps:
[0019] S1, preparing superparamagnetic Fe3O4 nanoparticles with citric acid modified surface;
[0020] S2, culturing and collecting active microorganisms to prepare high-concentration bacterial suspension;
[0021] S3, uniformly dispersing the magnetic nanoparticles obtained in step S1 and the bacterial suspension obtained in step S2 in sodium alginate solution, then adding dropwise into calcium chloride solution to perform ion crosslinking reaction, forming gel microspheres embedded with magnetic nanoparticles and microorganisms, i.e. magnetic-microbial-biocompatible hydrogel core;
[0022] S4, preparing multifunctional prepolymer mixture solution containing N-isopropylacrylamide, acrylic acid, crosslinking agent, initiator and aniline monomer;
[0023] S5, soaking the core microspheres obtained in step S3 in the prepolymer mixture solution obtained in step S4 to allow monomers to be fully adsorbed;
[0024] S6, placing the microspheres after adsorbing monomers as working electrode in electrolyte to form stimulus-responsive electroactive composite outer layer on the surface of the microspheres by electrochemical in-situ polymerization;
[0025] S7, electrochemically activating and freeze-drying the polymerized carrier to obtain the final product.
[0026] Further, in step S1, the specific conditions for citric acid modification are as follows: stirring Fe3O4 nanoparticles with 0.1 M citric acid solution at 80°C and 400 rpm for 2 hours.
[0027] Further, in step S6, the specific parameters for electrochemical in-situ polymerization are as follows: using three-electrode system, 0.5 M H2SO4 as electrolyte, applying 0.8 mA / cm 2Constant current of 0.1 mA, and the polymerization time is 25 minutes.
[0028] Further, in step S7, the specific conditions of the electrochemical activation are as follows: using 0.1M Na2SO4 solution as electrolyte, using Ag / AgCl electrode as reference electrode, performing 10 cycles of cyclic voltammetry scanning at a potential range of-0.2V to 0.8V and a scanning rate of 50mV / s.
[0029] Advantages:
[0030] (1) The present application can realize in-situ electrochemical reduction / desorption of heavy metals by constructing an electrically active conductive polymer network on the outer layer of the carrier and under the action of a weak external electric field. This mechanism avoids the dependence on chemical reagents in traditional chemical regeneration methods and eliminates the generation of secondary pollutants such as heavy metal-containing acidic waste liquid.
[0031] (2) The present application uses an external electric field or electrochemical reaction products to mildly stimulate the embedded microorganisms, effectively promoting their metabolism and enzyme activity. This enables the microorganisms to continuously and efficiently perform biological enrichment and conversion functions, solving the limitations of easy inactivation and difficult to maintain function of microorganisms in the prior art, and achieving the spatiotemporal synergistic enhancement of carrier regeneration and microbial treatment functions.
[0032] (3) The present application organically integrates multiple functions such as magnetic separation, microorganism protection, intelligent adsorption, electrochemical regeneration, and microorganism activation into one through precise magnetic-microorganism core / hydrogel inner layer / electrically active outer layer multi-layer gradient structure design. The clear structure and functional synergy of each layer ensure that the carrier has high-efficiency heavy metal removal capacity, convenient magnetic recycling characteristics, controllable regeneration performance, and long-term stable biological activity in complex water environments.
[0033] (4) The in-situ electrochemical regeneration mechanism used in the present application is mild and non-destructive, avoiding the corrosion and damage of strong acids and strong bases to the carrier material and microorganisms, significantly prolonging the service life of the carrier. At the same time, this process uses electrons as the main reagent, without the need to consume chemical reagents, reducing the long-term operation cost of heavy metal contaminated water treatment, and providing an economic, efficient, and environmentally friendly sustainable solution. DETAILED DESCRIPTION
[0034] In order to enable those skilled in the art to better understand the technical solutions in the present application, the following will further describe the present application in detail with reference to the embodiments.
[0035] Embodiment 1
[0036] 1. Preparation process
[0037] (1) Pretreatment of magnetic nanoparticles and microorganisms
[0038] Synthesis and modification of Fe3O4 nanoparticles: Fe3O4 nanoparticles with an average diameter of 5 nm were synthesized by co-precipitation. 1 g of the nanoparticles was reacted with 100 mL of 0.1 M citric acid solution at 80 °C with stirring at 400 rpm for 2 h. After the reaction, the product was separated by magnetic separation, washed with water, and dried at 60 °C under vacuum to obtain citric acid-modified Fe3O4 nanoparticles.
[0039] Microbial culture: Pseudomonas putida (CGMCC 1.3136) was cultured in LB medium at 30 °C, 180 rpm, to OD 600 = 3. The bacterial cells were collected by centrifugation, washed with sterile physiological saline for 3 times, and finally resuspended in physiological saline to adjust OD 600 to 2.5.
[0040] (2) Preparation of magnetic-microbial-hydrogel core
[0041] 100 mg of the above citric acid-modified Fe3O4 nanoparticles were mixed with 5 mL of bacterial suspension, and uniformly dispersed in 20 mL of 2% (w / v) sodium alginate solution. Ultrasonic dispersion (100 W, 3 min) and magnetic stirring (30 min) were used to ensure uniform mixing of the components. The mixture was slowly dropped into 200 mL of 0.2 M CaCl2 solution at a rate of 0.5 mL / min using a syringe, and cross-linked by stirring at 200 rpm for 2 h. After filtration and water washing, gel microspheres with a diameter of about 0.8 mm were obtained.
[0042] (3) Preparation of multifunctional prepolymer mixture
[0043] 1.5 g of NIPAM, 0.5 g of AA, 0.05 g of MBA (crosslinking agent), and 0.02 g of APS (initiator) were dissolved in 30 mL of deionized water to obtain a hydrogel monomer solution.
[0044] 0.2 g of aniline monomer was dissolved in 5 mL of 0.1 M HCl. Under stirring in an ice bath (0-5 °C), the aniline hydrochloride solution was slowly dropped into the above hydrogel monomer solution, and mixed for 30 min to obtain a prepolymer solution.
[0045] (4) Electrochemical induction in-situ polymerization to construct the outer layer
[0046] About 5 g wet weight of the core microspheres prepared in step (2) were immersed in the pre-polymerization solution and left to stand with shaking at room temperature for 2 h. After being taken out, the microspheres were used as the working electrode (wrapped with a conductive mesh to make contact), an Ag / AgCl electrode was used as the reference electrode, and a platinum wire was used as the counter electrode. The electrolyte was a 0.5 M H2SO4 solution. Electrochemical polymerization was carried out by the constant current method, a current density of 0.8 mA / cm2 was applied, and the polymerization time was 25 min. After the reaction was completed, the microspheres were washed with deionized water to obtain the carrier.
[0047] (5) Activation and post-treatment of the carrier
[0048] The carrier was rinsed in ultrapure water until neutral. Subsequently, electrochemical activation was completed by performing 10 cycles of cyclic voltammetry scanning in a 0.1 M Na2SO4 solution at a scanning rate of 50 mV / s within a potential window of -0.2 V to 0.8 V (with an Ag / AgCl electrode as the reference electrode). Finally, the final carrier was obtained by freeze-drying. The average thickness of the outer layer was measured to be about 20 μm.
[0049] 2. Performance test
[0050] Test method: 100 mg of dry carrier was put into 100 mL of Cr(VI) solution with an initial concentration of 100 mg / L (pH = 5, temperature 25°C), and shaken in a shaking bed. At a certain time point, the sample was taken, filtered through a 0.22 μm filter membrane, and the Cr(VI) concentration in the filtrate was determined by the diphenyl carbazide spectrophotometric method to calculate the adsorption capacity.
[0051] After adsorption saturation, the carrier was taken out from the solution by magnetic separation and subjected to regeneration experiment. The saturated carrier was placed in an electrolytic cell containing 0.1 M Na2SO4, and a constant potential of -0.6 V (with an Ag / AgCl electrode as the reference electrode) was applied for electrochemical desorption reduction for 30 min. After the process was completed, the adsorption capacity of the carrier for Cr(VI) was determined again to investigate the regeneration efficiency. The above adsorption-regeneration cycle was repeated for 5 times.
[0052] Microbial survival rate: After the first adsorption-regeneration cycle, the survival rate of the microorganisms inside the carrier was determined by the viable plate count method.
[0053] Example 2
[0054] This example is basically the same as Example 1, and the main difference lies in the adjustment of the following parameters:
[0055] (1) Fe3O4 nanoparticles with an average particle size of 30 nm were used for citric acid modification.
[0056] (2) The concentration of sodium alginate and the crosslinking time of CaCl2 were adjusted to prepare gel microspheres with a diameter of about 1.0 mm.
[0057] (3) The electrochemical polymerization time was adjusted to 20 minutes.
[0058] The average thickness of the final carrier outer layer was about 60 pm.
[0059] The subsequent performance tests were consistent with Example 1.
[0060] Example 3
[0061] This example was basically the same as Example 1, with the main difference being the adjustment of the following parameters:
[0062] (1) Citric acid modification was performed using Fe3O4 nanoparticles with an average particle size of 50 nm.
[0063] (2) The sodium alginate concentration and CaCl2 crosslinking time were adjusted to prepare gel microspheres with a diameter of about 1.2 mm.
[0064] (3) The electrochemical polymerization time was adjusted to 30 minutes.
[0065] The average thickness of the final carrier outer layer was about 100 pm.
[0066] The subsequent performance tests were consistent with Example 1.
[0067] Comparative Example 1: Lack of electroactive composite outer layer
[0068] The preparation process was exactly the same as Example 1, but after completing step (2) to prepare the sodium alginate-embedded magnetic-microbial core microspheres, steps (3), (4), and (5) were omitted, and the stimulus-responsive electroactive composite outer layer was not constructed. The core microspheres were directly freeze-dried to obtain the comparative carrier.
[0069] The adsorption test method was the same as Example 1. However, after adsorption saturation, a traditional chemical regeneration method was used: the saturated carrier was placed in a 0.1 M HNO3 solution and shaken for 2 h for acid desorption, then washed with deionized water to neutralize and used for the next adsorption cycle.
[0070] Comparative Example 2: Lack of microbial units
[0071] The preparation process was basically the same as Example 1, but in step (2), no microbial bacteria suspension was added, only 100 mg of citric acid-modified Fe3O4 nanoparticles were dispersed in the sodium alginate solution to form gel microspheres, and the outer layer construction was continued. Finally, a comparative carrier containing only magnetic materials and no microorganisms was obtained.
[0072] The subsequent performance tests were consistent with Example 1.
[0073] Comparative Example 3: Traditional chemical regeneration method
[0074] The preparation process is exactly the same as that of Example 1, and the carrier of the application is obtained.
[0075] The adsorption test method is the same as that of Example 1. However, after adsorption saturation, instead of electrochemical regeneration, the chemical regeneration method of Comparative Example 1 (0.1M HNO3 desorption for 2h) is used.
[0076] Effect comparison
[0077] The above performance test data of Examples 1-3 and Comparative Examples 1-3 are summarized in Table 1.
[0078] Table 1
[0079]
[0080] As can be seen from Table 1, after 5 adsorption-regeneration cycles, the adsorption capacity retention rates of the application (Examples 1-3) are all stable at more than 90% (the highest is 94.1%). This proves that the electrochemical regeneration method adopted by the application can efficiently desorb heavy metals and has little damage to the structure and function of the carrier, and has excellent recycling and economic performance. After the traditional acid regeneration of Comparative Example 1 and Comparative Example 3, the capacity retention rates drop to 58.2% and 65.5%. The strong chemical corrosion severely damages the structure of the carrier and kills the microorganisms, leading to rapid performance degradation and unstable use.
[0081] The mild electrochemical regeneration process of the application (Examples 1-3) is very friendly to the embedded microorganisms, and the survival rate is as high as 94.5%-96.3% after one cycle. This means that the microorganisms can continuously exert the biological conversion function, which is the basis for long-term and deep repair. The strong acid regeneration conditions of Comparative Example 1 and Comparative Example 3 are fatal to the microorganisms, and the survival rates are less than 15% and only 68.4% respectively. The large number of deaths of microorganisms leads to the loss of the biological remediation function of the carrier after one regeneration.
[0082] Comparing the application (Examples 1-3) with Comparative Example 2, the application can completely convert the highly toxic Cr(VI) into low-toxic Cr(III) due to the combination of adsorption and biological reduction functions, realizing the harmless of pollutants. Comparative Example 2 (without microorganisms) can only adsorb, and the total chromium concentration does not change, only the pollution location is changed, and the environmental risk still exists.
[0083] The electrochemical regeneration process of the application (Examples 1-3) uses electrons as "cleaning reagents" and does not produce any secondary pollution waste liquid, realizing the in-situ green cycle of the carrier and solving the problem of subsequent treatment. The acid waste liquid containing heavy metals is produced every time in Comparative Example 1 and Comparative Example 3, and the treatment of these dangerous liquid wastes requires high cost and brings new environmental risks.
[0084] In summary, this invention successfully achieves a balance between high efficiency, stability, and environmental friendliness in the remediation of heavy metals in water bodies through a multi-layered gradient structure design and regeneration strategy. Its core advantage lies in overcoming the technical bottlenecks of traditional methods, such as low regeneration efficiency, difficulty in maintaining microbial activity, and the risk of secondary pollution. It provides a sustainable solution that combines adsorption and biotransformation functions, enabling in-situ green recycling and regeneration, thus offering a new technological approach for the treatment of heavy metal-polluted water bodies.
Claims
1. A magnetic composite microbial carrier, characterized in that, The carrier comprises, from the inside out, the following: (1) Magnetic-microbial core: composed of surface-functionalized superparamagnetic Fe3O4 nanoparticles and active microorganisms; (2) Biocompatible hydrogel inner layer: covering the magnetic-microbial core, formed by sodium alginate, chitosan or polyethylene glycol-based hydrogel through ionic crosslinking or covalent crosslinking; (3) Stimulus-responsive electroactive composite outer layer: covering the biocompatible hydrogel inner layer, composed of a stimulation-responsive hydrogel component and an electroactive conductive polymer network, and the outer layer is embedded with heavy metal chelating groups.
2. The magnetic composite microbial carrier according to claim 1, characterized in that, The superparamagnetic Fe3O4 nanoparticles have an average particle size of 5 nm to 50 nm, and their surface is functionalized by chemical grafting citric acid.
3. The magnetic composite microbial carrier according to claim 1, characterized in that, The active microorganism is *Pseudomonas putida*, which has the ability to adsorb, enrich, or biotransform heavy metals.
4. The magnetic composite microbial carrier according to claim 1, characterized in that, The inner layer of the biocompatible hydrogel is made of sodium alginate-calcium ion crosslinked gel.
5. The magnetic composite microbial carrier according to claim 1, characterized in that, The stimulus-responsive hydrogel component is poly(N-isopropylacrylamide-co-acrylic acid) (PNIPAM-co-PAA); the electroactive conductive polymer network is a composite material of one or more of polyaniline (PANI), polypyrrole (PPy), and polythiophene (PTh) with graphene and carbon nanotubes; and the outer layer is composited through interpenetrating networks, blending, or covalent bonding.
6. The magnetic composite microbial carrier according to claim 1, characterized in that, The heavy metal chelating group is one or more of thiol (-SH), amino (-NH2), carboxyl (-COOH) or imidazole; the thickness of the stimulus-responsive electroactive composite outer layer is 20 μm to 100 μm.
7. A method for preparing a magnetic composite microbial carrier as described in any one of claims 1-6, characterized in that, Includes the following steps: S1. Preparation of superparamagnetic Fe3O4 nanoparticles with citric acid-modified surface; S2. Cultivate and collect active microorganisms to prepare a high-concentration bacterial suspension; S3. The magnetic nanoparticles obtained in step S1 and the bacterial suspension obtained in step S2 are uniformly dispersed in sodium alginate solution, and then added dropwise to calcium chloride solution to carry out ion cross-linking reaction, forming gel microspheres embedded with magnetic nanoparticles and microorganisms, namely magnetic-microorganism-biocompatible hydrogel core. S4. Prepare a multifunctional prepolymer mixture containing N-isopropylacrylamide, acrylic acid, crosslinking agent, initiator and aniline monomer; S5. Immerse the core microspheres obtained in step S3 in the prepolymer mixture obtained in step S4 to allow the monomers to be fully adsorbed. S6. The microspheres after adsorption of monomers are used as working electrodes and placed in the electrolyte. Stimulation-responsive electroactive composite outer layer is formed on the surface of the microspheres by electrochemical induction in situ polymerization. S7. The polymerized support is electrochemically activated and freeze-dried to obtain the final product.
8. The method according to claim 7, characterized in that, In step S1, the specific conditions for citric acid modification are as follows: Fe3O4 nanoparticles are reacted with 0.1M citric acid solution at 80°C and stirred at 400 rpm for 2 hours.
9. The method according to claim 7, characterized in that, In step S6, the specific parameters for the electrochemically induced in-situ polymerization are as follows: a three-electrode system is used, with 0.5 M H2SO4 as the electrolyte, and an application rate of 0.8 mA / cm². 2 The constant current was applied, and the polymerization time was 25 minutes.
10. The method according to claim 7, characterized in that, In step S7, the specific conditions for electrochemical activation are as follows: using 0.1M Na2SO4 solution as the electrolyte, using an Ag / AgCl electrode as the reference electrode, and performing 10 cycles of cyclic voltammetry scans at a scan rate of 50mV / s within a potential range of -0.2V to 0.8V.