A method for preparing and applying melamine-based bio-manganese oxide

Abstract

This invention relates to a method for preparing and applying melamine-based biogenic manganese oxide. The method involves inoculating *Pseudomonas putida* M1 onto LB solid medium and culturing the resulting single colonies into LB liquid medium to obtain an M1 bacterial suspension. The M1 suspension is then inoculated into LEPT medium, and an aqueous MnCl2 solution is added. The mixture is incubated at 30°C in a constant-temperature shaking incubator for 16 hours. Melamine sponges are then added, and the culture continues for 3-4 days to obtain melamine-based biogenic manganese oxide. The melamine-based biogenic manganese oxide is then freeze-dried and stored at room temperature. This invention utilizes *Pseudomonas putida* M1 to oxidize divalent manganese, loading the resulting biogenic manganese oxide onto melamine to prepare melamine-based biogenic manganese oxide. This melamine-based biogenic manganese oxide has a fixed shape, is easy to recycle, reusable, simple to operate, environmentally friendly, and has high degradation efficiency, providing a new direction for addressing antibiotic pollution in water bodies.

Description

Technical Field

[0001] This invention belongs to the field of aquatic ecological environment restoration technology, specifically relating to a method for preparing and applying melamine-based bio-manganese oxide. Background Technology

[0002] Tetracycline antibiotics are a class of antibiotics based on hydroxyl tetracycline, mainly including chlortetracycline, oxytetracycline, and tetracycline. Their chromophores are a conjugated double bond system of ketone and enol groups, determining their color and characteristic UV absorption (365nm-350nm). Functional groups include dimethylamino (pKa = 7.5), phenolic hydroxyl (pKa = 6.5), tricarbonylmethane system (pKa = 3.3), and pI = 5.4 amide group. Due to their broad-spectrum antibacterial activity, low price, and excellent antibacterial performance, they are widely used in medicine, agriculture, and especially animal husbandry. However, research shows that animals cannot fully utilize tetracycline, and most of it enters the environment through body fluids. Furthermore, due to the widespread use of tetracycline, many researchers have confirmed that tetracycline antibiotics are prevalent in the environment, posing a significant potential threat to ecosystems and human safety. Water sources are a common medium for tetracycline presence, and the migration of pollutants with water flow increases the difficulty of antibiotic pollution control. Therefore, effectively removing tetracycline from water is crucial. Currently, common methods for removing tetracycline from water include physical adsorption, chemical removal, and biodegradation. However, physical methods do not fundamentally degrade the tetracycline; they only alter its storage sites. In practical applications, chemical degradation has poor recycling efficiency, requires large amounts of chemical reagents, and introduces byproducts during the reaction, causing secondary pollution. Biodegradation methods are time-consuming, produce small amounts of degradation, and are costly, making them difficult to implement in practice.

[0003] MnO2 has strong oxidizing properties and can oxidize and degrade tetracycline (TC). First, TC molecules diffuse to the MnO2 surface and are adsorbed to form surface complexes. Due to the strong oxidizing properties of Mn(IV), TC molecules lose an electron to form a free radical intermediate. Mn(IV) is reduced to Mn(III). Mn(III) then undergoes a redox reaction with TC molecules or the free radical intermediate to release Mn(II). The free radical intermediate is then converted into the final product through oxidation and other processes. The degradation process of tetracycline is complex, with up to 14 intermediate products. First, under the action of MnO2, the amino group at C2 and the hydroxyl group at C6 of tetracycline are released to generate H2O and NH3. Then, a CO and an NH3 are removed between C2 and C3. Due to the complex structure of tetracycline and the large number of derivatives, three products may be generated.

[0004] Manganese oxides are common mineral components in the natural environment, possessing a large specific surface area and high redox potential. Numerous studies have shown that manganese oxides are widely used for the adsorption of heavy metal ions and the removal of organic pollutants. Biogenic manganese oxides are generated by manganese oxide bacteria under mild conditions. They are generally nanoscale minerals with weak or short-range ordered crystals, characterized by high reactivity, high Mn valence state, large specific surface area, and numerous octahedral vacancies. Compared to chemical manganese oxides, biogenic manganese oxides can maintain high growth activity. The structure of biogenic manganese oxides contains less Mn(III), almost entirely Mn(IV), and carries a negative charge due to the presence of negative vacancy sites in its crystal structure. Its adsorption and oxidizing properties are both higher than those of chemical manganese oxides.

[0005] Tetracycline antibiotics are amphoteric compounds, acting as cations in acidic solutions and anions in alkaline solutions. Bio-manganese oxides, however, carry a negative charge due to the presence of negative vacancies in their crystal structure. Therefore, in acidic solutions, the positively charged tetracycline attracts bio-manganese oxides, facilitating its adsorption. In alkaline solutions, the negatively charged tetracycline and bio-manganese oxides repel each other, hindering adsorption. Furthermore, bio-manganese oxides exhibit high dispersibility, making their use complex and difficult to recycle, thus limiting their application scope and effectiveness. Summary of the Invention

[0006] The purpose of this invention is to provide a method for preparing and applying melamine-based bio-manganese oxide. This method utilizes the strong oxidizing properties of bio-manganese oxide to remove residual antibiotics from wastewater, achieving high degradation efficiency, low cost, wide applicability, simple operation, and environmental friendliness. The method employs bio-fermentation to oxidize divalent manganese by Pseudomonas bacteria M1, and then loads the resulting bio-manganese oxide onto melamine to prepare melamine-based bio-manganese oxide. This melamine-based bio-manganese oxide has a fixed shape, is easy to recycle, reusable, simple to operate, environmentally friendly, and has high degradation efficiency, providing a new direction for solving the current situation of antibiotic pollution in water bodies.

[0007] This invention is specifically achieved through the following technical solution: a method for preparing melamine-based bio-manganese oxide according to this invention includes the following steps:

[0008] (1) The putrid pseudomonad bacteria M1 preserved in glycerol was inoculated onto LB solid medium by streak plating and cultured at 30°C for 24 h.

[0009] (2) Take a single colony from the LB solid medium in step (1) and inoculate it into the LB liquid medium. Incubate it in a constant temperature shaking incubator at 30℃ and 145r / min for 12h to obtain the M1 bacterial suspension.

[0010] (3) Inoculate the M1 bacterial suspension into LEPT medium at a ratio of 1% of the inoculation amount, and add filtered sterilized MnCl2 aqueous solution to LEPT medium. Incubate in a constant temperature shaking incubator at 30℃ and 145r / min for 16h.

[0011] (4) Add melamine sponge to the LEPT medium after 16h of culture in step (3), and continue to culture in a constant temperature shaking incubator at 30℃ and 145r / min for 3-4 days to obtain melamine-based bio-manganese oxide.

[0012] (5) After removing the obtained melamine-based bio-manganese oxide, freeze-dry it and store it at room temperature.

[0013] The aforementioned method for preparing melamine-based bio-manganese oxide involves using LEPT culture medium prepared by mixing 0.5g yeast extract powder, 0.5g acid-hydrolyzed casein, 1g glucose, 2.382g HEPES, 0.0533g calcium chloride, and 1mL of trace element solution, adding deionized water, adjusting the pH of the mixture to 7.0, and then bringing the volume to 1L with deionized water. The trace element solution includes CuSO4, ZnSO4, CoCl2, and Na2MoO4.

[0014] Furthermore, the LEPT medium can be prepared by proportionally scaling up or down the volumes of yeast extract, acid-hydrolyzed casein, glucose, HEPES, calcium chloride, trace element solution, and the adjusted volume.

[0015] The aforementioned method for preparing melamine-based bio-manganese oxide involves a trace element solution prepared by adding 10 mg CuSO4·5H2O, 44 mg ZnSO4·7H2O, 20 mg CoCl2·6H2O, and 13 mg Na2MoO4·2H2O to water, stirring until homogeneous, and then diluting the solution to 1 L with water.

[0016] Furthermore, the trace element solution can be prepared by proportionally increasing or decreasing the amounts of CuSO4·5H2O, ZnSO4·7H2O, CoCl26H2O, and Na2MoO4·2H2O, as well as the volume after finalization.

[0017] In the aforementioned method for preparing melamine-based bio-manganese oxide, after adding MnCl2 aqueous solution in step (3), the final concentration of MnCl2 in LEPT medium is 1 mmol / L.

[0018] In the aforementioned method for preparing melamine-based bio-manganese oxide, step (4) involves cutting melamine sponges into the same size and then adding them to the LEPT culture medium after cultivation in step (3).

[0019] The aforementioned method for preparing melamine-based bio-manganese oxide includes manganese oxides MnO2 and Mn2O3, with MnO2 being the main component.

[0020] This invention also provides an application of the melamine-based bio-manganese oxide prepared according to the aforementioned method in the degradation of tetracycline antibiotics, particularly its use in degrading tetracycline hydrochloride in wastewater: The prepared melamine-based bio-manganese oxide is placed in antibiotic-contaminated wastewater at a final concentration of 800 mg / L. After the antibiotics are completely removed, the melamine-based bio-manganese oxide is removed and air-dried or freeze-dried, and can be reused 3-5 times. The degradation time of melamine-based bio-manganese oxide for tetracycline hydrochloride is 4-6 hours, and acidic conditions are favorable for the degradation treatment of tetracycline hydrochloride by melamine-based bio-manganese oxide. After five cycles of use, the degradation effect retention rate of melamine-based bio-manganese oxide can reach more than 50%, demonstrating its recyclability.

[0021] The experiment found that the mass of melamine-based bio-manganese oxide obtained after preparing melamine-based bio-manganese oxide from 50mg blank melamine sponge according to the aforementioned method and freeze-drying was 130mg, which is equivalent to 80mg of bio-manganese oxide being loaded on 50mg blank melamine sponge. Therefore, in the application of degrading tetracycline hydrochloride, the dosage of melamine-based bio-manganese oxide should be based on a final concentration of 800mg / L of bio-manganese oxide. For example, adding 800mg of bio-manganese oxide per liter of wastewater is equivalent to a dosage of 1300mg of melamine-based bio-manganese oxide.

[0022] Compared with existing technologies, this invention has significant advantages and beneficial effects. Through the above technical solution, this invention achieves considerable technological advancement and practicality, and has broad application value, possessing at least the following advantages:

[0023] This invention provides a method for preparing and applying melamine-based bio-manganese oxide. The method utilizes bio-fermentation and the strong oxidizing ability of Pseudomonas M1. During its growth, divalent manganese ions are added, and Pseudomonas M1 grows and oxidizes divalent manganese in a divalent manganese environment, generating high-valence bio-manganese oxide on melamine. This bio-manganese oxide is then loaded onto melamine to prepare melamine-based bio-manganese oxide. This melamine-based bio-manganese oxide is then used to degrade residual tetracycline antibiotics in the environment. Bio-manganese oxide possesses excellent properties, including adsorption and degradation functions. However, due to its dispersibility, its use is complex and difficult to recycle. Loading it onto melamine provides a fixed shape, facilitates recycling, allows for reusability, simplifies operation, is environmentally friendly, and is inexpensive. It also exhibits high efficiency in degrading tetracycline hydrochloride, making it easier to apply in practical production and providing a new direction for addressing antibiotic pollution in water bodies. Attached Figure Description

[0024] Figure 1 This is a colony morphology diagram of *Pseudomonas putida* on LB solid medium;

[0025] Figure 2 The image shows a photograph of the prepared melamine-based bio-manganese oxide.

[0026] Figure 3 This is an X-ray photoelectron spectroscopy (XPS) image of biogenic manganese oxides;

[0027] Figure 4 This is a schematic diagram of a scanning electron microscope image of blank melamine.

[0028] Figure 5 This is a schematic diagram of a scanning electron microscope image of melamine-based bio-manganese oxide;

[0029] Figure 6 It is the linear fitting curve of tetracycline hydrochloride;

[0030] Figure 7 This is a schematic diagram illustrating the degradation effects of bio-manganese oxide, melamine, and cyanamide-based bio-manganese oxide on tetracycline hydrochloride.

[0031] Figure 8 The removal rate of tetracycline hydrochloride by melamine-based bio-manganese oxide under pH conditions of 2.5, 6, 7, and 9;

[0032] Figure 9 It is melamine-based bio-manganese oxide in 0.01 mol / L Na + K + Ca 2+ Mg 2+ Mn 2+Removal rate of tetracycline hydrochloride under the specified conditions;

[0033] Figure 10 The degradation effect of melamine-based bio-manganese oxide on tetracycline hydrochloride is maintained even after five repeated uses.

[0034] Figure 11 The graph shows the antibacterial effects of freshly prepared TC solution (A), TC solution that has not been degraded and has been kept at room temperature in the dark for 4 hours (B), TC solution that has been degraded by melamine-based bio-manganese oxide (C), and sterile water (D) on Escherichia coli. Detailed Implementation

[0035] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the present invention will be clearly and completely described below in conjunction with specific embodiments. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0036] Unless otherwise specified, the technical solutions used in the following embodiments and experiments of the present invention are all conventional techniques, and the reagents or materials used are all from commercial sources unless otherwise specified. The method for obtaining Pseudomonas putida M1 includes: isolating Pseudomonas putida M1 from the soil near manganese ore (the isolation method is existing technology and will not be described in detail), placing it in glycerol and freezing it in a refrigerator.

[0037] The method for preparing melamine-based bio-manganese oxide provided by this invention specifically includes the following steps:

[0038] (1) *Pseudomonas putida* M1 preserved in glycerol was inoculated onto LB agar using the streak plate method and incubated at 30°C for 24 h. The colonies of *Pseudomonas putida* M1 were as follows: Figure 1 As shown;

[0039] (2) Take a single colony from the LB solid medium in step (1) and inoculate it into the LB liquid medium. Incubate it in a constant temperature shaking incubator at 30℃ and 145r / min for 12h to obtain the M1 bacterial suspension.

[0040] (3) Inoculate the M1 bacterial suspension into LEPT medium at a ratio of 1% (V / V) and add filtered sterilized MnCl2 aqueous solution to LEPT medium to make the final concentration of MnCl2 1 mmol / L. Incubate for 16 h in a constant temperature shaking incubator at 30℃ and 145 r / min.

[0041] The LEPT medium is prepared by mixing 0.5g yeast extract, 0.5g acid-hydrolyzed casein, 1g glucose, 2.382g HEPES, 0.0533g calcium chloride, and 1mL of trace element solution, adding an appropriate amount of deionized water to adjust the pH of the mixture to 7.0, and then bringing the volume to 1L with deionized water. This ratio can be scaled up or down. The trace element solution, comprising CuSO4, ZnSO4, CoCl2, and Na2MoO4, is prepared by adding 10mg CuSO4·5H2O, 44mg ZnSO4·7H2O, 20mg CoCl2·6H2O, and 13mg Na2MoO4·2H2O to water, stirring until homogeneous, and then bringing the volume to 1L. This ratio can also be scaled up or down.

[0042] (4) Add melamine sponge cut into pieces of the same size to the LEPT medium after 16h of culture in step (3), and continue to culture in a constant temperature shaking incubator at 30℃ and 145r / min for 3-4 days to obtain melamine-based bio-manganese oxide.

[0043] (5) Collect the melamine-based bio-manganese oxide prepared in step (4), remove it, freeze-dry it, and store it at room temperature. The dried melamine-based bio-manganese oxide is as follows: Figure 2 As shown, melamine-based bio-manganese oxide is brownish-black.

[0044] For comparison, bio-manganese oxide was prepared using the same method but without the addition of melamine sponge. The specific steps are as follows:

[0045] (1) The putrid pseudomonad bacteria M1 preserved in glycerol was inoculated onto LB solid medium by streak plating and cultured at 30°C for 24 h.

[0046] (2) Take a single colony from the LB solid medium in step (1) and inoculate it into the LB liquid medium. Incubate it in a constant temperature shaking incubator at 30℃ and 145r / min for 12h to obtain the M1 bacterial suspension.

[0047] (3) Inoculate the M1 bacterial suspension into LEPT medium at a ratio of 1% (V / V) and add filtered sterilized MnCl2 aqueous solution to LEPT medium to make the final concentration of MnCl2 1 mmol / L. Incubate in a constant temperature shaking incubator at 30℃ and 145r / min for 3-4 days.

[0048] (4) Centrifuge the product obtained in step (3), take the lower precipitate, freeze-dry it, and obtain bio-manganese oxide.

[0049] The LEPT medium described in step (3) is prepared by mixing 0.5g yeast extract, 0.5g acid-hydrolyzed casein, 1g glucose, 2.382g HEPES, 0.0533g calcium chloride, and 1mL of trace element solution, adjusting the pH to 7.0, and then bringing the volume to 1L with deionized water. The trace element solution includes CuSO4, ZnSO4, CoCl2, and Na2MoO4, and is prepared by adding 10mg CuSO4·5H2O, 44mg ZnSO4·7H2O, 20mg CoCl2·6H2O, and 13mg Na2MoO4·2H2O to water, stirring until homogeneous, and then bringing the volume to 1L. The preparation can be scaled up or down according to this ratio.

[0050] The prepared melamine-based bio-manganese oxide and bio-manganese oxide were characterized and analyzed, and a tetracycline hydrochloride degradation experiment was conducted:

[0051] (1) Characterization analysis:

[0052] X-ray photoelectron spectroscopy (XPS) can be used in chemical research to provide information on molecular structure and atomic valence states, and can also provide information on the elemental composition and content, chemical state, molecular structure, and chemical bonds of various compounds for materials research. Figure 3 The XPS spectra of the prepared bio-manganese oxide at the Mn2p3 / 2 and Mn3s core levels are shown. Peak separation analysis was performed on the high-resolution spectra of Mn2p3 / 2 and Mn3s. The Mn2p3 / 2 peak was split into two sub-peaks: Mn(Ⅳ) at 642.2 eV and Mn(Ⅲ) at 643.4 eV. The Mn(Ⅳ) satellite peak at 642.2 eV is relatively strong, indicating that the material contains a large amount of Mn(Ⅳ). In addition, the Mn3s XPS spectrum showed that the Mn3s multiple splitting value (ΔE) before and after calcination was 4.9. According to the relationship AOS = 8.956-1.126 (ΔE), the average oxidation state (AOS) of Mn in the sample was 3.4. The results indicate that the material mainly contains Mn(Ⅳ) and Mn(Ⅲ) with high oxidation states, indicating that the manganese oxides in the prepared melamine-based bio-manganese oxide are MnO2 and Mn2O3, and MnO2 is the main component.

[0053] Observation of blank melamine (i.e., melamine sponge) using scanning electron microscopy Figure 4 ) and melamine-based bio-manganese oxide ( Figure 5 ),from Figure 4 and Figure 5 It can be seen that blank melamine has a three-dimensional porous network structure and uniform composition, while melamine-based bio-manganese oxide maintains the three-dimensional porous network structure of melamine, and the bio-manganese oxide is uniformly loaded onto melamine along with the bacterial cells.

[0054] Establishment of a standard curve for tetracycline hydrochloride concentration:

[0055] A 25 μg / mL tetracycline hydrochloride (TC) solution was prepared and its spectrum was scanned in the range of 250–400 nm using a UV spectrophotometer. TC exhibited maximum absorption wavelengths at 277 nm and 358 nm. This invention selected a wavelength of 358 nm for quantitative determination. TC solutions with concentrations of 15 μg / mL, 20 μg / mL, 25 μg / mL, 30 μg / mL, 35 μg / mL, and 40 μg / mL were prepared. The absorbance A of different concentrations of TC was measured using a UV spectrophotometer at λ = 358 nm. A standard curve was prepared, and the linear regression equation A = 0.0251cTC + 0.1361 was obtained, with a correlation coefficient R. 2 =0.9976. The concentration of TC at different absorbances can be calculated using a linear regression equation. The standard curve for TC concentration is shown below. Figure 6 As shown.

[0056] Degradation efficiency of TC by melamine-based bio-manganese oxide, bio-manganese oxide, and blank melamine:

[0057] Experimental group: Take 130 mg of the prepared melamine-based bio-manganese oxide small pieces and add them to 100 mL of 40 ug / mL TC solution. React at room temperature in the dark. After the reaction starts, take samples every 30 min, centrifuge and then perform ultraviolet spectrophotometric determination.

[0058] Control group 1: 80 mg of the prepared powdered bio-manganese oxide was added to 100 mL of 40 ug / mL TC solution and reacted at room temperature in the dark. After the reaction started, samples were taken every 30 min, centrifuged and then measured by ultraviolet spectrophotometry.

[0059] Control group 2: Take 50mg of blank melamine sponge, cut it into small pieces with the same volume as the melamine-based bio-manganese oxide in the experimental group, add it to 100mL of 40ug / mL TC solution, and react at room temperature in the dark; after the reaction starts, take a sample every 30min, centrifuge it and perform ultraviolet spectrophotometric determination.

[0060] It should be noted that in the experiment, it was found that the mass of melamine-based bio-manganese oxide obtained after preparing melamine-based bio-manganese oxide from 50mg blank melamine sponge according to the aforementioned method and freeze-drying was 130mg, which is equivalent to 80mg of bio-manganese oxide being loaded on 50mg blank melamine sponge. Therefore, in the comparative experiment, the experimental group selected 130mg of the prepared melamine-based bio-manganese oxide, the control group 1 selected 80mg of the prepared powdered bio-manganese oxide, and the control group 2 selected 50mg of blank melamine sponge.

[0061] After measuring the absorbance at 358 nm in the experimental group, control group 1, and control group 2, the residual concentration of TC after degradation of different materials at different times was calculated using a linear regression equation. A graph was plotted with degradation time on the x-axis and residual TC concentration on the y-axis. The results are shown below. Figure 7 As shown, the blank melamine sponge has no effect on TC and will not adsorb or degrade TC. The removal rates of TC by melamine-based bio-manganese oxide and bio-manganese oxide at different times were calculated, and the results are shown in Table 1 below:

[0062] Table 1. Removal rate of TC by different materials at different times

[0063] Removal rate in the first 30 minutes 4h removal rate Bio-manganese oxides 56.7％ 95.5％ melamine-based bio-manganese oxide 68.7％ 95.7％

[0064] Depend on Figure 7 As shown in Table 1, the removal rate of TC by simple bio-manganese oxide reached 56.7% within the first 30 minutes and 95.5% within 4 hours, demonstrating high TC removal efficiency. The melamine-based bio-manganese oxide prepared in this invention can improve the degradation rate of bio-manganese oxide, achieving a tetracycline removal rate of 68.7% within the first 30 minutes and 95.7% within 4 hours. Compared to simple bio-manganese oxide, the removal rate within the first 30 minutes is increased by 21.2%, and the final removal rate is similar to that of simple bio-manganese oxide, indicating that the melamine-based bio-manganese oxide prepared in this invention effectively maintains the TC degradation activity of bio-manganese oxide. Furthermore, the TC solution after 4 hours of degradation showed no characteristic peak at 358 nm, indicating that TC was absent from the solution at this time.

[0065] Analysis of the effect of different pH values ​​on the degradation of total cyanide (TC) by melamine-based bio-manganese oxides:

[0066] Prepare a 40 μg / mL TC solution and take four aliquots for the experiment. Each aliquot reaction vessel contains 100 mL of the 40 μg / mL TC solution. Adjust the pH to 2.5, 6, 7, and 9 using HCl and NaOH solutions, respectively. Add 130 mg of prepared melamine-based bio-manganese oxide granules to each TC solution and react at room temperature in the dark. Measure and record the absorbance at regular intervals. Calculate the residual TC concentration at different times and pH using the linear regression equation of TC, and calculate the removal rate of TC by melamine-based bio-manganese oxide at different times and pH. Plot a graph with reaction time on the x-axis and removal rate on the y-axis, as shown below. Figure 8As shown, pH affects the removal rate of TC by melamine-based bio-manganese oxides; the lower the pH, the faster the degradation rate of TC and the higher the removal rate. At initial pH values ​​of 2.5 and 6.0, the degradation rate and removal rate of TC are very high, reaching 95.57% and 87.13% respectively within 1 hour. At pH values ​​of 7.0 and 9.0, the removal rate of TC is significantly lower than under acidic conditions, reaching only 65.64% and 40.60% respectively after 5 hours. However, at initial pH values ​​of 2.5 and 6.0, the removal rates reach 96.09% and 96.62% respectively after 5 hours.

[0067] Analysis of the effects of different cations on the removal capacity of melamine-based bio-manganese oxides:

[0068] Prepare a 40 μg / mL TC solution and take six aliquots for the experiment. Each aliquot reaction vessel contains 100 mL of the 40 μg / mL TC solution. Add NaCl, KCl, CaCl2, MgCl2, and MnCl2 solutions to five of the TC solutions, respectively, to bring the final concentrations of NaCl, KCl, CaCl2, MgCl2, and MnCl2 to 0.01 mol / L. The last TC solution, without any metal salt solution, serves as a blank control. Add 70 mg of melamine-based bio-manganese oxide granules to each of the six TC solutions and react at room temperature in the dark. Measure the absorbance at regular intervals. Calculate the remaining TC concentration using the TC linear regression equation, and calculate the TC removal rate of melamine-based bio-manganese oxide in the blank control and under different cations. Plot a graph with reaction time on the x-axis and the TC removal rate of melamine-based bio-manganese oxide on the y-axis. Figure 9 As shown. Figure 9 This indicates that K + Na + It promoted the degradation of TC by melamine-based bio-manganese oxides, with removal rates of 82.39% and 69.53% at 6 hours, respectively, which were higher than the removal rates of the blank control group. Mn 2+ Mg 2+ Ca 2+ The melamine-based bio-manganese oxide exhibited an inhibitory effect on the degradation of TC. At 6 hours, the removal rates were 56.92%, 41.98%, and 31.52%, respectively, all lower than the removal rate of the blank control group. This indicates that monovalent cations promote the degradation of TC by melamine-based bio-manganese oxide, while divalent cations inhibit it. Furthermore, the strength of the promoting effect is: K... + >Na + The strength of the inhibitory effect is as follows: Ca 2+ >Mg 2+ >Mn 2+ .

[0069] Analysis of the recyclability of melamine-based bio-manganese oxide:

[0070] 130 mg of the prepared melamine-based bio-manganese oxide was added to 100 mL of 40 μg / mL TC solution and reacted at room temperature in the dark for 4 hours as the first degradation. The melamine-based bio-manganese oxide after the first TC degradation was freeze-dried and then placed in a fresh 100 mL of 40 μg / mL TC solution for another 4 hours at room temperature in the dark as the second degradation. This process was repeated, with each freeze-drying of the melamine-based bio-manganese oxide after TC degradation followed by a 100 mL 40 μg / mL TC solution, for a total of five cycles. The absorbance of the melamine-based bio-manganese oxide after each TC degradation was measured. The remaining TC concentration was calculated using the linear regression equation of TC, and the removal rate for each degradation was calculated (reaction time was 4 hours). Using the removal rate of the first degradation as a baseline, the removal rate of each degradation was divided by the removal rate of the first degradation to obtain the degradation retention rate (the first degradation retention rate was 1). The results are as follows: Figure 10 As shown. By Figure 10 It can be seen that the degradation efficiency of TC by melamine-based bio-manganese oxide was 98.58% during the second degradation, and then gradually decreased. During the fifth degradation, the degradation efficiency was 53.47%, which means that more than half of the degradation efficiency was still maintained.

[0071] Antibacterial activity test of degradation products:

[0072] (a) Take 130 mg of the prepared melamine-based bio-manganese oxide and add it to 50 mL of 40 ug / mL TC solution. React at room temperature in the dark for 4 h.

[0073] (b) Prepare another 100 mL of 40 ug / mL TC solution and place it at room temperature in the dark for 4 h;

[0074] (c) Take out the E. coli stored in glycerol at -20℃, take 100ul and put it into LB liquid medium, and activate it in a shaking incubator at 28℃ and 160r / min for 12h.

[0075] (d) Using the Oxford cup method, the bacterial suspension activated for 12 hours in step (c) was diluted 100-fold, and 100 μL of the bacterial suspension was spread on each petri dish. Four Oxford cups were placed in each petri dish, and 100 μL of freshly prepared 40 μg / mL TC solution, 100 μL of TC solution from step (b) after being placed at room temperature in the dark for 4 hours, 100 μL of TC solution from step (a) after degradation by melamine-based bio-manganese oxide, and 100 μL of sterile water were added to each Oxford cup, respectively, for antibacterial experiments. The culture medium was placed in a 28℃ incubator for 15 hours, and the diameter of the inhibition zone was observed. The results are as follows. Figure 11 As shown. Figure 11 This indicates that the TC solution after degradation by melamine-based bio-manganese oxide in step (a) Figure 11 (C) and sterile water ( Figure 11 Neither TC nor D showed any inhibitory effect on Escherichia coli, and the freshly prepared TC solution ( Figure 11 (A) and TC solution after being placed at room temperature in the dark for 4 hours ( Figure 11 Both B and C showed significant antibacterial effects, indicating that after 4 hours of degradation by melamine-based bio-manganese oxides, there were no active TC molecules in the TC solution.

[0076] The above description is merely an embodiment of the present invention and is not intended to limit the present invention in any way. The present invention can also have other embodiments based on the above structure and function, which will not be listed hereafter. Therefore, any simple modifications, equivalent changes, and alterations made by those skilled in the art to the above embodiments based on the technical essence of the present invention without departing from the scope of the present invention shall still fall within the scope of the present invention.

Claims

1. A method for preparing melamine-based bio-manganese oxide for degrading tetracycline hydrochloride, characterized in that... Includes the following steps: (1) The putrid Pseudomonas malodorans M1 preserved in glycerol was inoculated onto LB solid medium by streak plating and cultured at 30°C for 24 h. (2) Take a single colony from the LB solid medium in step (1), inoculate it into the LB liquid medium, and incubate it in a constant temperature shaking incubator at 30℃ and 145r / min for 12h to obtain the M1 bacterial suspension. (3) Inoculate the M1 bacterial suspension into LEPT medium at a ratio of 1% of the inoculation amount, and add filtered sterilized MnCl2 aqueous solution to the LEPT medium to make the final concentration of MnCl2 1 mmol / L. Incubate in a constant temperature shaking incubator at 30℃ and 145 r / min for 16 h. The LEPT medium is obtained by mixing 0.5 g yeast extract powder, 0.5 g acid hydrolyzed casein, 1 g glucose, 2.382 g HEPES, 0.0533 g calcium chloride and 1 mL trace element solution, adding deionized water, adjusting the pH of the mixture to 7.0, and then making up to 1 L with deionized water. The trace element solution is obtained by adding 10 mg CuSO4·5H2O, 44 mg ZnSO4·7H2O, 20 mg CoCl26H2O and 13 mg Na2MoO4·2H2O to water, stirring evenly, and making up to 1 L. (4) Add melamine sponge to the LEPT medium after 16h of culture in step (3), and continue to culture in a constant temperature shaking incubator at 30℃ and 145r / min for 3-4 days to obtain melamine-based bio-manganese oxide. The manganese oxides in the prepared melamine-based bio-manganese oxide include MnO2 and Mn2O3. (5) After removing the obtained melamine-based bio-manganese oxide, freeze-dry it and store it at room temperature.

2. The method for preparing melamine-based bio-manganese oxide as described in claim 1, characterized in that... Prepare the LEPT medium by proportionally scaling up or down the volumes of yeast extract, acid-hydrolyzed casein, glucose, HEPES, calcium chloride, trace element solution, and adjusted volume.

3. The method for preparing melamine-based bio-manganese oxide as described in claim 1, characterized in that... Prepare the trace element solution by proportionally increasing or decreasing the amounts of CuSO4·5H2O, ZnSO4·7H2O, CoCl26H2O, and Na2MoO4·2H2O and the volume after adjustment.

4. The method for preparing melamine-based bio-manganese oxide as described in claim 1, characterized in that... Step (4) After cutting the melamine sponge into the same size, add it to the LEPT medium after 16 hours of culture in step (3).

5. The method for preparing melamine-based bio-manganese oxide as described in claim 1, characterized in that... The main component of the manganese oxide in the prepared melamine-based bio-manganese oxide is MnO2.

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