Preparation of iron-copper-sodium alginate composite microspheres and application of the microspheres in inactivation of bacteria and degradation of pollutants by activated periodate

Abstract

This invention relates to the preparation of an iron-copper-sodium alginate composite microsphere and its application in activating periodate to inactivate bacteria and degrade pollutants. The microspheres utilize sodium alginate as a three-dimensional network framework, forming a stable gel structure through ionic cross-linking. A bimetallic active component composed of zero-valent iron and trace amounts of copper is uniformly dispersed within the microspheres. This process activates periodate to achieve highly efficient removal of antibiotic-resistant bacteria, resistance genes, and new pollutants from water. The iron-copper-sodium alginate microspheres are prepared via ionic cross-linking. They are highly efficient and broad-spectrum, achieving rapid sterilization through iron-copper synergy at low metal loading. They are environmentally friendly, as the solid microspheres are completely recyclable, reducing secondary pollution. They are also low-cost, being primarily iron-based with trace amounts of copper for synergistic effects, and reusable. Therefore, this invention provides a novel technical solution for bacterial inactivation and resistance gene removal in water, possessing significant theoretical importance and application prospects.

Description

Technical Field

[0001] This invention relates to the field of microbial inactivation technology, and in particular to a method for inactivating bacteria and efficiently removing resistance genes by activating periodate with chemical activators. Background Technology

[0002] With the widespread use and discharge of antibiotics, the spread of antibiotic-resistant bacteria and their resistance genes in water bodies has become a global environmental and public health crisis. Traditional water treatment processes (such as chlorination and ultraviolet light) are ineffective at inactivating antibiotic-resistant bacteria and struggle to effectively remove free and intracellular resistance genes, potentially even increasing the risk of horizontal transfer of resistance genes. Therefore, developing a novel water treatment technology that can both efficiently inactivate bacteria and effectively remove their resistance genes is of paramount importance.

[0003] Advanced oxidation processes offer significant advantages in degrading organic pollutants and inactivating microorganisms due to their ability to generate highly oxidizing active species. Periodate, as a stable and efficient oxidant, has received widespread attention in the field of advanced oxidation water treatment in recent years. It has a relatively high standard oxidation potential (E0). 0 =+1.6 V), can be activated by various means such as light, heat, ultrasound, and transition metals to produce active iodine species, free radicals or high-valence metal intermediates, thereby achieving efficient degradation of pollutants and rapid inactivation of microorganisms.

[0004] However, existing transition metal-activated periodate systems for inactivating antibiotic-resistant bacteria and resistance genes still face challenges such as limited inactivation efficiency, stringent reaction conditions (e.g., narrow pH range, large catalyst dosage), and high risk of secondary pollution, making it difficult to meet the high-efficiency purification requirements of practical water treatment scenarios. On the one hand, most research focuses on homogeneous catalytic systems. While these systems offer high efficiency, the metal catalysts exist in ionic form in the water, making them difficult to recover and reuse, easily leading to metal residues and secondary pollution, and potentially posing long-term risks to aquatic ecosystems. On the other hand, existing technologies for heterogeneous catalytic materials still require optimization. In practical applications, active site loss is common, and some materials suffer from complex preparation processes or high costs. Furthermore, there is still room for improvement in the simultaneous and efficient removal of bacteria and resistance genes, while also considering stability and environmental compatibility, of heterogeneous periodate-activated materials.

[0005] Heterogeneous catalysis based on gel microspheres, by embedding active components within them, enables catalyst separation and reuse, offering an effective solution to the aforementioned problems. However, current research on periodate-based heterogeneous catalysis systems for controlling drug-resistant bacteria remains limited, particularly lacking highly efficient, stable, and recyclable catalytic materials capable of simultaneously removing drug-resistant bacteria and resistance genes. Therefore, constructing a heterogeneous catalytic material that combines high catalytic activity, excellent stability, easy recyclability, and environmental friendliness to achieve efficient periodate activation and advanced water treatment not only has significant scientific research value but also substantial practical engineering application implications. Summary of the Invention

[0006] This invention provides iron-copper bimetallic gel microspheres embedded in sodium alginate. The microspheres use sodium alginate as a three-dimensional network framework and form a stable gel structure through ionic cross-linking; the microspheres contain a bimetallic active component composed of zero-valent iron and trace amounts of copper, wherein the mass percentage of copper in the bimetallic active component is 1-25%, preferably 1%.

[0007] This invention discloses a method for preparing iron-copper-sodium alginate composite microspheres, comprising the following steps:

[0008] (1) Zero-valent iron was prepared by potassium borohydride reduction method: ferrous sulfate solution was reacted with freshly prepared potassium borohydride solution to reduce the precipitate, and the resulting precipitate was washed with ethanol and deoxygenated water to obtain zero-valent iron particles.

[0009] (2) The zero-valent iron particles and soluble copper salt (copper sulfate) are dispersed together in deoxygenated water at a set iron / copper mass ratio (75:25~100:0, preferably 99:1) and stirred to form a uniform metal suspension.

[0010] (3) Dissolve sodium alginate in water to prepare a colloidal solution with a mass percentage of 4-6%, preferably 5.67%; mix the metal suspension with the sodium alginate solution and stir continuously until a uniform composite sol is obtained;

[0011] (4) The composite sol was added dropwise to a mixed solution of calcium chloride and barium chloride with a mass percentage of 2%; microspheres were formed after the sol droplets came into contact with the cations; the microspheres were solidified and repeatedly washed with ultrapure water and then sealed in anhydrous ethanol for storage; the water content of the iron-copper-sodium alginate composite microspheres was determined, and the concentrations added were all calculated as dry weight.

[0012] Furthermore, in step (2), the total mass concentration of iron / copper in the metal suspension is 13~15 g / L, preferably 13.25 g / L; in step (3), the metal suspension and sodium alginate solution are mixed at a volume ratio of 60:40~70:30, preferably 64.7:35.3.

[0013] The iron-copper-sodium alginate composite microspheres obtained in this invention can be reused with activated periodate to activate periodate for inactivating bacteria and degrading pollutants. While inactivating bacteria, they can also remove resistance genes.

[0014] The above-mentioned method involves dissolving periodate in the water containing bacteria and / or pollutants to be treated to form a preliminary mixed system; adding iron-copper-sodium alginate composite microspheres to the preliminary mixed system to form a final mixed system for sterilization, removal of resistance genes and / or degradation of pollutants.

[0015] Preferably, the periodate is one or both of sodium periodate or potassium periodate.

[0016] Preferably, the concentration of iron-copper-sodium alginate composite microspheres in the mixed system is 25~125 mg / L.

[0017] Preferably, the periodate concentration in the mixed system is 0.2~0.6 mmol / L.

[0018] Preferably, the temperature is maintained at room temperature (25±2 °C) during sterilization and / or pollutant degradation.

[0019] Preferably, the pH is maintained near neutral during sterilization and / or pollutant degradation processes, maintaining the pH of the system at 7.21.

[0020] Preferably, the bacteria refer to Escherichia coli, enterotoxin-producing Escherichia coli, and Bacillus subtilis, etc. The bacterial concentration is 0~9×10⁻⁶. 8 Total bacterial count / mL.

[0021] The contaminants are selected from one or more of antibiotics, quaternary ammonium salts, preservatives, etc. These include one or more of ampicillin (AMP), sulfadiazine (SDZ), phenacetin (PNCT), ibuprofen (IBP), p-chloro-m-cresol (PCMX), benzalkonium chloride (BZC), potassium sorbate (PS), sodium benzoate (SB), and potassium cinnamate (PC).

[0022] Compared with the prior art, the beneficial effects of the present invention include at least the following:

[0023] This invention achieves highly efficient activation of periodate by preparing iron-copper-sodium alginate composite microspheres. Existing periodate activation technologies mostly rely on soluble metal salts as homogeneous catalysts, which suffer from problems such as metal ion residue, difficulty in recovery, and easy secondary pollution. This invention, for the first time, uses sodium alginate to encapsulate iron-copper bimetals to form heterogeneous gel microspheres, allowing for rapid separation and recycling of the catalyst after the reaction through simple filtration or sedimentation. Traditional advanced oxidation processes often require harsh conditions such as extreme pH, ultraviolet radiation, or high temperatures, while this invention can operate efficiently and rapidly at near-neutral pH, room temperature, and ambient pressure, eliminating the need for such harsh conditions. The catalyst is added directly in solid form, requiring no complex equipment modifications, and possesses significant advantages for engineering applications. Compared with existing single-function disinfection or oxidation technologies, the synergistic catalytic system constructed in this invention can achieve: (1) efficient inactivation of drug-resistant bacteria (7.12 log removal within 6 minutes) and efficient removal of resistance genes (removal rate of more than 66.18% within 6 minutes); (2) efficient degradation of a variety of typical new pollutants (such as antibiotics, quaternary ammonium salts, and preservatives). Attached Figure Description

[0024] Figure 1 These are images of the prepared iron-copper-sodium alginate composite microspheres;

[0025] Figure 2 The inactivation effects of different reaction systems on Escherichia coli in water;

[0026] Figure 3 The effect of iron-copper-sodium alginate composite microspheres with different copper loading rates on inactivation efficiency;

[0027] Figure 4 The effect of different concentrations of iron-copper-sodium alginate composite microspheres on inactivation efficiency;

[0028] Figure 5 The effect of different concentrations of periodate on inactivation efficiency;

[0029] Figure 6 The effect of the iron-copper-sodium alginate composite microsphere / periodate system on the elimination of intracellular DNA and resistance genes;

[0030] Figure 7 The effect of the iron-copper-sodium alginate composite microsphere / periodate system on the elimination of extracellular DNA and resistance genes;

[0031] Figure 8 The inactivation effect of the iron-copper-sodium alginate composite microsphere / periodate system on Escherichia coli in water at different pH levels;

[0032] Figure 9The degradation effect of the iron-copper-sodium alginate composite microsphere / periodate system on different new pollutants;

[0033] Figure 10 It is the detection of free radicals during the reaction process of the iron-copper-sodium alginate composite microsphere / periodate system;

[0034] Figure 11 It is used to detect high-valence metal species during the reaction process of the iron-copper-sodium alginate composite microsphere / periodate system. Detailed Implementation

[0035] The present application will be further described in detail and completely below with reference to embodiments. The embodiments described in this application are only some embodiments of the present invention, and not all embodiments. Moreover, the embodiments described herein are only used to explain the present application and are not intended to limit the present application.

[0036] Preparation of iron-copper-sodium alginate composite microspheres:

[0037] (1) Zero-valent iron was prepared by potassium borohydride reduction method: ferrous sulfate solution was reacted with freshly prepared potassium borohydride solution to reduce the precipitate, and the resulting precipitate was washed with ethanol and deoxygenated water to obtain zero-valent iron particles.

[0038] (2) The zero-valent iron particles and soluble copper salt (copper sulfate) are dispersed together in deoxygenated water at a set iron / copper mass ratio (75:25~100:0, preferably 99:1), and a uniform metal suspension is formed by stirring. The total iron / copper mass concentration is 13~15 g / L, preferably 13.25 g / L.

[0039] (3) Dissolve sodium alginate in water to prepare a colloidal solution with a mass concentration of 4-6%, preferably 5.67%; mix the metal suspension and sodium alginate solution at a volume ratio of 70:30-60:40, preferably 64.7:35.3, and continue stirring until a uniform composite sol is obtained;

[0040] (4) The composite sol was added dropwise to a 2% calcium chloride and barium chloride mixed solution; after the sol droplets came into contact with the cations, microspheres were formed; after the microspheres were solidified, they were repeatedly washed with ultrapure water and then sealed in anhydrous ethanol for storage; the water content of the iron-copper-sodium alginate composite microspheres was determined, and the concentrations added were all calculated as dry weight.

[0041] Example 1

[0042] Inactivation effects of different reaction systems on Escherichia coli in water

[0043] Under the experimental conditions, 50 mL of water sample containing *E. coli* (DH5α, i.e., antibiotic-resistant bacteria, ARB) was added to a 150 mL Erlenmeyer flask, with an initial bacterial concentration of 1.31 × 10⁻⁶. 7 Total bacterial count / mL. Add 0.4 mL of a prepared 52 mmol / L sodium periodate solution (to a concentration of 0.4 mmol / L) to the water sample and mix thoroughly with a magnetic stirrer. Then add 100 mg / L of iron-copper-sodium alginate composite microspheres with a 1% copper loading rate. No adjustment of reaction temperature or pH is required. Under the set reaction conditions, samples were taken at time points of 0, 1, 2, 3, 4, 5, and 6 minutes. The samples were inoculated onto solid culture media using the dilution plating method and incubated at 37°C for 18–24 h. Bacterial viability was then measured. Results are as follows: Figure 2 As shown, after 6 minutes of treatment, the inactivation rate of ARB reached 99.9999% (7.12 log). Under the same conditions, four reaction systems were selected as control systems, including: persulfate / iron-copper-sodium alginate composite microsphere system, percarbonate / iron-copper-sodium alginate composite microsphere system, H2O2 / iron-copper-sodium alginate composite microsphere system, and ferrate / iron-copper-sodium alginate composite microsphere system. Bacterial viability was measured at the same reaction time intervals, and the results are shown below. Figure 2 As shown, the inactivation effects of the above systems on ARB were 0.78 log (83.57%), 0.86 log (86.13%), 0.64 log (77.31%), and 0.74 log (81.96%), respectively. In summary, except for the periodate / iron-copper-sodium alginate composite microsphere system, all other systems showed poor inactivation effects.

[0044] Example 2

[0045] Effect of different copper loading rates on inactivation efficiency of iron-copper-sodium alginate composite microspheres

[0046] Iron-copper-sodium alginate composite microspheres with a copper loading (i.e., the mass percentage of copper in the bimetallic compound) of 1%, 2%, 3%, 4%, 5%, 15%, and 25% at a concentration of 100 mg / L were added to water samples, and experiments were conducted according to the method in Example 1. The results are as follows: Figure 3 When no copper was loaded (0% copper), after 6 minutes of system treatment, the ARB inactivation count reached 4.92 log, corresponding to an inactivation rate of 99.9988%. As the copper loading rate increased, the inactivation effect showed a trend of first increasing and then decreasing, reaching the highest at 1% copper loading (7.12 log). Therefore, 1% copper loading rate was selected for subsequent experiments.

[0047] Example 3

[0048] Effect of different concentrations of iron-copper-sodium alginate composite microspheres on inactivation efficiency

[0049] Add 0.4 mL of a prepared 52 mmol / L sodium periodate solution to the water sample (to make the concentration 0.4 mmol / L), mix well, and then add 25 mg / L, 50 mg / L, 75 mg / L, 100 mg / L, and 125 mg / L of iron-copper-sodium alginate composite microspheres respectively. The experiment was carried out according to the method in Example 1. Figure 4 The results showed that the ARB inactivation rate gradually increased with the increase of the iron-copper-sodium alginate composite microsphere concentration, and when the concentration reached 100 mg / L, ARB could be completely inactivated (7.12 log). Based on the principle of low concentration and high efficiency, the optimal concentration of the iron-copper-sodium alginate composite microspheres was selected as 100 mg / L.

[0050] Example 4

[0051] Effect of different concentrations of periodate on inactivation efficiency

[0052] Add 0.2 mL, 0.3 mL, 0.4 mL, 0.5 mL, and 0.6 mL of a prepared 52 mmol / L periodate solution (to achieve concentrations of 0.2 mmol / L, 0.3 mmol / L, 0.4 mmol / L, 0.5 mmol / L, and 0.6 mmol / L, respectively) to water samples, and conduct the experiment according to the method in Example 1. The results show that the bacterial inactivation efficiency significantly increases with increasing periodate concentration. See [details omitted]. Figure 5 When the periodate concentration increased from 0.2 mmol / L to 0.3 mmol / L, the inactivation efficiency of E. coli increased from 1.63 log (97.68%) to 3.20 log (99.94%). Further increasing the periodate concentration to 0.4, 0.5, and 0.6 mmol / L resulted in complete inactivation of ARB within 6 minutes, 6 minutes, and 5 minutes, respectively. Similarly, the optimal periodate concentration was determined to be 0.4 mmol / L.

[0053] Example 5

[0054] The effect of the iron-copper-sodium alginate composite microsphere / periodate system on the elimination of intracellular DNA and resistance genes.

[0055] Repeat Example 1, extracting intracellular DNA from samples taken at each time point according to the instructions in the DNA extraction kit. The concentration of bacterial intracellular DNA was measured using a micro spectrophotometer, and the extracted intracellular DNA samples were analyzed by qPCR. The results are as follows: Figure 6Within the 6-minute reaction time, the intracellular DNA concentration generally showed a decreasing trend, from 51.06 ng / µL in the untreated state to 20.35 ng / µL at the end of the reaction; correspondingly, the intracellular tetA resistance gene abundance decreased from 1.44 × 10⁻⁶. 9 Reduced to 4.88×10 8 The copy number per milliliter indicates that the sterilization process is accompanied by a significant decrease in gene abundance.

[0056] Example 6

[0057] The effect of the iron-copper-sodium alginate composite microsphere / periodate system on the elimination of extracellular DNA and resistance genes.

[0058] Repeat Example 1, extract extracellular DNA from samples taken at each time point according to the steps in the DNA extraction kit instructions, determine the bacterial extracellular DNA concentration using an ultra-micro spectrophotometer, and perform qPCR analysis on the extracted extracellular DNA samples. Figure 7 The results showed that the extracellular DNA concentration initially increased and then decreased with prolonged treatment time, reaching a peak of 4.11 ng / µL at 1 minute and eventually dropping to 0.29 ng / µL, indicating that the system effectively disrupted the bacterial cell membrane, leading to DNA leakage. Similar to the changes in extracellular DNA concentration, the abundance of corresponding resistance genes also exhibited a similar trend, generally decreasing from 1.40 × 10⁻⁶. 4 The copy number / mL decreased to 3.66 × 10⁻⁶. 3 Copy number per milliliter.

[0059] Example 7

[0060] Inactivation effect of iron-copper-sodium alginate composite microspheres / periodate system on Escherichia coli in water at different pH levels

[0061] Example 1 was repeated, and the pH of the dispersion was adjusted to 3, 5, 7.2, and 9 using 0.1 M HCl and NaOH. The results are as follows. Figure 8 Acidic conditions (pH=3 and 5) promote the inactivation of ARB in the iron-copper-sodium alginate composite microsphere / periodate system, while alkaline conditions inhibit the inactivation efficiency, but still achieve an inactivation rate of 99.90%.

[0062] Example 8

[0063] Degradation effect of iron-copper-sodium alginate composite microspheres / periodate system on different new pollutants

[0064] Under experimental conditions, 50 mL of the following novel pollutants at a concentration of 10 mg / L were added to a 150 mL Erlenmeyer flask: ampicillin (AMP), sulfadiazine (SDZ), phenacetin (PNCT), ibuprofen (IBP), p-chloro-m-cresol (PCMX), benzalkonium chloride (BZC), potassium sorbate (PS), sodium benzoate (SB), and potassium cinnamate (PC). Then, periodate and iron-copper-sodium alginate composite microspheres were added sequentially to initiate the reaction, as in Example 1. Samples were taken sequentially at time points of 0, 1, 2, 3, 4, 5, and 6 minutes. The degradation effect of the novel pollutants was analyzed by high-performance liquid chromatography (HPLC). The results are as follows: Figure 9 At the reaction endpoint, the removal rates of the above nine pollutants reached 67.35%, 46.81%, 49.72%, 72.94%, 79.44%, 100%, 79.61%, 51.97%, and 100%, respectively. This indicates that the iron-copper-sodium alginate composite microsphere / periodate system also has a good removal effect on different types of new pollutants.

[0065] Example 9

[0066] Detection of free radicals during the reaction of the iron-copper-sodium alginate composite microsphere / periodate system

[0067] A mixed solution of 990 μL each of iodate and iron-copper-sodium alginate composite microspheres was prepared, and 10 μL each of 5,5-dimethyl-1-pyrrolidine-N-oxide (DMPO) and 2,2,6,6-tetramethylpiperidine-1-oxide (TEMP) was added to form a mixed solution. The superoxide anion and singlet oxygen radicals were measured using electron spin resonance spectroscopy (ESR; Bruker EMX / Plus, Germany). The generation of superoxide anions and singlet oxygen radicals was detected. The results are shown in the figure below. Figure 10 .

[0068] Example 10

[0069] Detection of high-valence metal species during the reaction of iron-copper-sodium alginate composite microspheres / periodate system

[0070] Example 1 was repeated, with 100 μmol / L methyl phenyl sulfoxide (PMSO) added as a probe before the reaction began to detect the formation of high-valent manganese species during the reaction. The results are as follows: Figure 11 As shown, PMSO was efficiently converted to methylphenyl sulfone (PMSO2) within a reaction time of 6 minutes, with a conversion rate exceeding 90%. The results indicate that high-valence metal species were generated during the reaction.

[0071] The examples described above are only for illustrating the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described in detail with reference to the above embodiments, those skilled in the art should understand that modifications or equivalent substitutions can still be made to the specific embodiments of the present invention. Any or equivalent substitutions that do not depart from the spirit and scope of the present invention should be covered within the scope of protection of the claims of the present invention.

Claims

1. Sodium alginate-encapsulated iron-copper bimetallic gel microspheres, characterized in that, The microspheres use sodium alginate as a three-dimensional network framework and form a stable gel structure through ionic cross-linking; a bimetallic active component consisting of zero-valent iron and trace amounts of copper is uniformly dispersed inside, wherein the mass percentage of copper in the bimetallic active component is 1~25%, preferably 1%.

2. The process for the preparation of sodium alginate-encapsulated iron-copper bimetallic gel microspheres as claimed in claim 1, characterized in that, Includes the following steps: (1) Zero-valent iron was prepared by potassium borohydride reduction method: ferrous sulfate solution was reacted with freshly prepared potassium borohydride solution to reduce the precipitate, and the resulting precipitate was washed with ethanol and deoxygenated water to obtain zero-valent iron particles. (2) The zero-valent iron particles and soluble copper salt (copper sulfate) are dispersed together in deoxygenated water at a set iron / copper mass ratio (75:25~100:0, preferably 99:1) and stirred to form a uniform metal suspension. (3) Dissolve sodium alginate in water to prepare a colloidal solution with a mass percentage of 4-6%, preferably 5.67%; mix the metal suspension with the sodium alginate solution and stir continuously until a uniform composite sol is obtained; (4) The composite sol was added dropwise to a mixed solution of calcium chloride and barium chloride with a mass percentage of 2%; microspheres were formed after the sol droplets came into contact with the cations; the microspheres were solidified and repeatedly washed with ultrapure water and then sealed in anhydrous ethanol for storage; the water content of the iron-copper-sodium alginate composite microspheres was determined, and the concentrations added were all calculated as dry weight.

3. The method of claim 2, wherein, In step (2), the total mass concentration of iron / copper in the metal suspension is 13.25 g / L; in step (3), the metal suspension and sodium alginate solution are mixed at a volume ratio of 60:40~70:30, preferably 64.7:35.

3.

4. The application of the sodium alginate-embedded iron-copper bimetallic gel microspheres as described in claim 1, in combination with activated periodate, is used to activate periodate to inactivate bacteria and degrade pollutants, and can remove resistance genes while inactivating bacteria.

5. Use according to claim 4, characterized in that, The application method involves dissolving periodate in the water containing bacteria and / or pollutants to be treated, forming a preliminary mixing system; then adding iron-copper-sodium alginate composite microspheres to the preliminary mixing system to form a final mixing system for sterilization, removal of resistance genes, and / or degradation of pollutants.

6. Use according to claim 5, characterized in that, The periodate is one or both of sodium periodate or potassium periodate.

7. Use according to claim 5, characterized in that, The concentration of iron-copper-sodium alginate composite microspheres in the mixed system is 25~125 mg / L; the concentration of periodate in the mixed system is 0.2~0.6 mmol / L.

8. Use according to claim 5, characterized in that, The temperature is maintained at room temperature (25±2 ℃) during sterilization and / or pollutant degradation; the pH is maintained at a near-neutral pH during sterilization and / or pollutant degradation.

9. The use according to claim 5, characterized in that, The bacteria referred to are Escherichia coli, enterotoxin-producing Escherichia coli, and Bacillus subtilis, etc.

10. The use according to claim 5, characterized in that, The contaminants are selected from one or more of antibiotics, quaternary ammonium salts, preservatives, etc.; more preferably, they include one or more of ampicillin (AMP), sulfadiazine (SDZ), phenacetin (PNCT), ibuprofen (IBP), p-chloro-m-cresol (PCMX), benzalkonium chloride (BZC), potassium sorbate (PS), sodium benzoate (SB), and potassium cinnamate (PC).

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