CN114291992B - Preparation combination for removing municipal sludge antibiotic resistance genes and application - Google Patents
Preparation combination for removing municipal sludge antibiotic resistance genes and application Download PDFInfo
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Abstract
The application discloses a preparation combination for removing municipal sludge antibiotic resistance genes and application thereof. The formulation combination comprises ozone and acidified zero-valent iron; the ozone is in gaseous form and is used for promoting the decomposition of the resistance genes; the acidified zero-valent iron is solid particles and is used for catalyzing the decomposition of ozone on the resistance genes. The acidified zero-valent iron is zero-valent iron particles with acid radical groups connected to the surfaces, and delocalized electrons of the acid radical groups can promote iron circulation, so that the zero-valent iron has stronger proton-electron transmission performance, and can continuously dissolve out Fe2+And further, the homogeneous oxidation of ozone is continuously catalyzed, and the ozone can be promoted to efficiently and continuously generate hydroxyl radicals in a catalytic reaction system of the sludge so as to continuously maintain the oxidizing capability, continuously maintain the decomposition effect of the antagonistic genes and greatly improve the removal effect of the antagonistic genes.
Description
Technical Field
The application relates to the technical field of municipal sludge antibiotic resistance gene purification, in particular to a preparation combination for removing municipal sludge antibiotic resistance genes and application thereof.
Background
With the rapid development of socioeconomic, a plurality of Antibiotic Resistance Genes (ARGs) with extremely high content have been detected in municipal sludge, and the ARGs are considered as emerging environmental pollutants. Municipal sewage treatment plants receive sewage from a variety of sources and bacteria, and reducing the amount of ARGs in sewage treatment plants will be the key to controlling the occurrence and spread of antibiotic resistance. Researches show that chlorination disinfection in the wastewater disinfection process enhances the antibiotic resistance of pathogens and increases the abundance of intracellular and extracellular ARGs in the effluent of a sewage treatment plant.
Researches show that ozone with strong oxidizing property and hydroxyl free radicals derived from the ozone can attack biological macromolecules such as cell membranes and intracellular proteins, lipids and DNA, cause cell structure and dysfunction such as increase of permeability of the cell membranes, peroxidation of the proteins and the lipids, DNA fragmentation and replication dysfunction and the like, and also show good effects in removing ARGs in environmental samples such as compost products and sludge. However, the efficiency of generating hydroxyl radicals by self-decomposition of ozone is poor, and the requirement of effectively removing ARGs cannot be met.
Disclosure of Invention
In view of the above, the present application aims to provide a method for effectively removing antibiotic resistance genes from municipal sludge.
In a first aspect, the embodiments of the present application disclose a combination of agents for the removal of municipal sludge antibiotic resistance genes, comprising ozone and acidified zero-valent iron; the ozone is in gaseous form for promoting the decomposition of the resistance gene; the acidified zero-valent iron is solid particles and is used for catalyzing the decomposition effect of the ozone on the resistance genes.
In embodiments herein, the acidified zero valent iron is selected from at least one of silicified zero valent iron, phosphorylated zero valent iron, phosphorous acid zero valent iron, borated zero valent iron, iodated zero valent iron, periodated zero valent iron, oxalated zero valent iron, citrated zero valent iron, malalized zero valent iron, tartated zero valent iron, succinated zero valent iron, trichloroacetated zero valent iron, dinitrobenzoate zero valent iron, ethylenediaminetetraacetate zero valent iron, and sulfamated zero valent iron; preferably phosphorylated zero-valent iron, oxalated zero-valent iron, citrated zero-valent iron or succinated zero-valent iron; more preferably zero valent iron oxalate.
In the embodiment of the application, the acidified zero-valent iron is obtained by acidifying zero-valent iron with solid acid under a solid-phase ball milling condition, wherein the solid acid accounts for 2.0-8.0% of the mole percentage of the zero-valent iron.
In the examples of the present application, the solid acid is at least one selected from the group consisting of silicic acid, phosphoric acid, phosphorous acid, boric acid, iodic acid, periodic acid, oxalic acid, citric acid, malic acid, tartaric acid, succinic acid, trichloroacetic acid, periodic acid, dinitrobenzoic acid, ethylenediaminetetraacetic acid, and sulfamic acid, and is preferably phosphoric acid, phosphorous acid, oxalic acid, citric acid, or succinic acid. The "solid acid" is an acid that is solid at ordinary temperature.
In the examples of the present application, the solid phase ball milling conditions were: the ball milling time is 4h, and the ball milling rotating speed is 500 r/min.
In a second aspect, the application also discloses the application of the preparation combination of the first aspect in removing the antibiotic resistance genes in municipal sludge, and the preparation combination comprises the step of adding acidified zero-valent iron to the municipal sludge which is aerated with ozone.
In the embodiment of the application, the using amount of the acidified zero-valent iron is 2.0-16.0g/L municipal sludge, and the gas using amount of the ozone is 3L/min.
In an embodiment of the application, the application further comprises a step of performing sonication under the following conditions: the ultrasonic frequency is 20-25 KHz, and the ultrasonic power is 0.4-1.6W/mL.
Compared with the prior art, the application has at least the following beneficial effects:
the acidified zero-valent iron in the embodiment of the application is zero-valent iron particles with acid radical groups connected to the surfaces, and delocalized electrons of the acid radical groups can promote iron circulation, so that the zero-valent iron has stronger proton-electron transmission performance, and can continuously dissolve out Fe2+Further, the homogeneous oxidation of ozone is continuously catalyzed, and the ozone can be promoted to efficiently and continuously generate hydroxyl radicals in a catalytic reaction system of sludge so as to continuously maintain the oxidizing capability, continuously maintain the decomposition effect of the resistance genes and greatly improve the removal effect of the resistance genes.
Drawings
Fig. 1 is a Scanning Electron Microscope (SEM) image of a zero-valent iron oxalate sample provided by an embodiment of the present invention. In fig. 1, fig. 1a and 1c are images of 200 and 20000 times under-mirror ball-milled zero-valent iron samples, respectively, and fig. 1b and 1d are images of 200 and 20000 times under-mirror oxalic acid-milled zero-valent iron samples, respectively.
FIG. 2 shows zero-valent iron oxalate (OA-ZVI) provided by an embodiment of the present inventionbm) And ball milled Zero Valent Iron (ZVI)bm) X-ray diffraction (XRD) pattern of (a).
FIG. 3 is a Fourier transform infrared spectroscopy (FT-IR) chart of oxalic acid dihydrate, ferrous oxalate hydrate, zero-valent iron oxalate and ball-milled zero-valent iron provided by the embodiment of the invention.
FIG. 4 is a graph showing the results of copy numbers of antibiotic resistance genes tetG, sul1 and intI1 after sludge treatment in examples 1 to 3 of the present invention and comparative example 1, respectively.
FIG. 5 is a graph showing the results of copy numbers of antibiotic resistance genes tetG, sul1 and intI1 after sludge treatment in examples 4 to 6 of the present invention and comparative example 1, respectively.
FIG. 6 is a graph showing the results of copy numbers of antibiotic resistance genes tetG, sul1 and intI1 after sludge treatment in examples 7 to 9 of the present invention and comparative example 1, respectively.
FIG. 7 is a graph showing the results of copy numbers of antibiotic resistance genes tetG, sul1 and intI1 after sludge treatment in comparative examples 1 to 5 of the present invention, respectively.
Detailed Description
In order to make the objects, technical solutions and advantages of the present application more apparent, the present application is further described in detail with reference to the following embodiments. It should be understood that the specific embodiments described herein are merely illustrative of and not restrictive on the broad application.
The catalytic ozonation method is a novel method applied to removing pollutants in drinking water and wastewater. Generally, the iron simple substance is used as an effective catalyst for the reaction, and the catalyst can promote ozone to generate a large amount of hydroxyl radicals in a reaction system, so that the oxidation of the ozone is accelerated or promoted. Therefore, the method can show excellent benefits in the aspects of removing organic pollutants and inactivating pathogenic bacteria in municipal sewage. However, iron continues to be consumed in the process and is difficult to regenerate, resulting in a continuing decline in its catalytic ozone oxidation activity.
Fe can be continuously dissolved out due to zero-valent iron (ZVI)2+The homogeneous oxidation of catalytic ozone, some researches have been carried out on using zero-valent iron (ZVI) as an ozone activator under an acidic condition, and the performance of ozone for inactivating municipal sludge pathogenic bacteria is remarkably improved. But the shell layer on the surface of the zero-valent iron is extremely easy to passivate, and the Fe is limited2+The continuous dissolution of the active ingredient causes the continuous reduction of the catalytic activity of the active ingredient, and the application of the active ingredient is restricted.
To this end, the embodiments herein provide a formulation combination for use in removing a municipal sludge antibiotic resistance gene, the formulation combination comprising ozone and acidified zero-valent iron; the ozone is in gaseous form for promoting the decomposition of the resistance gene; the acidified zero-valent iron is solid particles and is used for catalyzing the decomposition effect of the ozone on the resistance genes.
The surface of the acidified zero-valent iron is connected with acid radical ions, and iron oxide crystals are formed on the surface of the acidified zero-valent iron. The ion of the acid radical can promote the iron circulation, the zero-valent iron shell layer is modified by the acid radical ion to promote the oxidation of ozone, so that the strong proton-electron transmission performance can be shown, a large amount of active free radicals are generated, and the efficiency of removing environmental pollutants is greatly improved. Simultaneously, iron oxide crystals are formed on the surface of the zero-valent iron, so that Fe can be continuously dissolved out2+So as to continuously catalyze the homogeneous oxidation of ozone and improve the oxidation effect on the ARGs.
In embodiments herein, the acidified zero-valent iron is selected from at least one of a group consisting of a silicate zero-valent iron, a phosphate zero-valent iron, a phosphite zero-valent iron, a borate zero-valent iron, an iodate zero-valent iron, a periodate zero-valent iron, an oxalate zero-valent iron, a citrate zero-valent iron, a malate zero-valent iron, a tartrate zero-valent iron, a succinate zero-valent iron, a trichloroacetated zero-valent iron, a dinitrobenzoate zero-valent iron, an ethylenediaminetetraacetate zero-valent iron, and a sulfamate zero-valent iron, preferably a phosphate zero-valent iron, an oxalate zero-valent iron, a citrate zero-valent iron, or a succinate zero-valent iron; more preferably zero-valent iron oxalate.
In the embodiment of the application, the acidified zero-valent iron is obtained by acidifying zero-valent iron with solid acid under a solid-phase ball milling condition, wherein the solid acid accounts for 2.0-8.0% of the zero-valent iron in terms of mole percentage; the solid acid is at least one selected from silicic acid, phosphoric acid, phosphorous acid, boric acid, iodic acid, periodic acid, oxalic acid, citric acid, malic acid, tartaric acid, succinic acid, trichloroacetic acid, periodic acid, dinitrobenzoic acid, ethylene diamine tetraacetic acid and sulfamic acid, and is preferably phosphoric acid, phosphorous acid, citric acid, succinic acid or oxalic acid.
A zero-valent iron oxalate (OA-ZVI)bm) The preparation examples are:
the planetary ball mill for preparing the oxalic acid zero-valent iron sample consists of a stainless steel ball milling tank and steel balls. 5.60g of commercial zero valent iron powder (100 mesh, alatin) was mixed with 0.252, 0.504, 0.756 and 1.008g oxalic acid in 2%, 4%, 6% and 8% mole percent (OA/ZVI) of zero valent iron in a ball mill jar containing steel balls. And (3) assembling the ball milling tank into a ball mill, setting the rotating speed of the ball mill to be 500rpm, and performing ball milling for 4 hours. Ball-milled zero-valent iron oxalate sample (OA-ZVI)bm) Sealing with a centrifuge tube, vacuumizing for storage, and ball-milling commercial zero-valent iron powder (without oxalic acid) by the same method and conditions to obtain a control sample, namely ball-milled zero-valent iron (ZVI)bm)。
SEM analysis: a small amount of sample (OA-ZVI) was placed in a 10mL centrifuge tubebmAnd ZVIbm) Ultrasonically dispersing in absolute ethyl alcohol, dropwise adding the solution onto clean aluminum foil paper, cutting off small pieces of aluminum foil paper with better sample dispersion after the ethyl alcohol is completely volatilized, pasting the aluminum foil paper on a carbon conductive adhesive of a sample table, and observing the surface morphology of the sample by using a cold field emission scanning electron microscope (FE-SEM, Hitachi 5-4800, Hitachi, Japan, 5kV acceleration voltage). The results are shown in FIG. 1, where ball milled Zero Valent Iron (ZVI) is observed in FIGS. a and cbm) Chloronicated zero-valent iron (OA-ZVI)bm) All are irregular spherical. Comparing fig. b and d, the surface of the oxalated zero-valent iron became extremely rough after the oxalic acid co-ball milling modification.
XRD analysis: oven-dried powder sample (OA-ZVI)bmAnd ZVIbm) The sample groove is filled with a sample groove, the sample groove is compacted by a glass slide, and then an X-ray diffraction pattern of the sample is measured by a DIMAX-RB type target-rotating X-ray diffractometer in a scanning range of 10-80 degrees by CuKa rays, the wavelength lambda of the CuKa rays is 0.1452nm, and the steps of the 10 DEG/min are counted. The result is shown in figure 2, the XRD characterization result is compared with the peak position of the standard substance, and ZVIbmDiffraction peaks at 44.7 and 65 ℃ ascribed to the standard substance alpha-Fe0(JCPDS,No.87-722)。OA-ZVIbmAnd ZVIbmThe samples did not differ significantly in crystal structure. Mixing OA-ZVIbmThe spectrum of (a) does not show similar peaks when compared with standard cards of ferric oxalate and ferrous oxalate. Thus, the zero-valent iron sample is processed by oxalic acidAfter ball milling modification, the components of the sample still mainly comprise iron simple substance.
Fourier transform infrared spectroscopy: and (3) tabletting by using spectral-grade KBr to test a background peak, mixing the sample and KBr according to a ratio of 1:100 to prepare a tablet, and measuring the FT-IR spectrum of the tablet. The scanning range of the spectrum is 4000-500 cm-1Resolution of 4cm-1. As shown in FIG. 3, 1630cm in the Fourier transform infrared spectrum of zero-valent iron oxalate-1、1360cm-1、1315cm-1And 820cm-1The main peak belongs to the characteristic peak of ferrous oxalate hydrate. Moreover, since 1655 is in-1Va (C ═ O) where oxalate anion is superimposed at cm, and OA-ZVIbmAt 1630cm-1The peak becomes wider and stronger. The results show that the surface shell layer of zero-valent iron is successfully modified by oxalic acid.
Referring to the above examples, the present application also investigated the effect of different oxalic acid to zero valent iron mole percentages on the prepared zero valent iron oxalate and ozone in the removal of municipal sludge resistance genes, and these prepared zero valent iron oxalate and ozone were treated simultaneously with municipal sludge to compare the removal effects.
One specific processing example is as follows: in 50mL of sludge, continuously generating ozone by using an ozone generator, adjusting the current of the ozone generator to be 0.40-0.45A, adjusting the gas flow to be 3L/min, adjusting the using amount of a catalyst to be 2.0-16.0g/L, placing a reaction system on a magnetic stirrer, adjusting the rotating speed to be 200-2000rpm, treating For 6h, extracting and purifying metagenomic DNA (deoxyribonucleic acid) according to the operation steps of a Fast DNA Spin Kit For Soil Kit after freeze drying a sludge sample, and performing absolute quantitative detection on the abundance of antibiotic resistance genes by using a fluorescent quantitative PCR (polymerase chain reaction) technology, wherein two antibiotic resistance genes including tetG (tetG), sul1 and transposon intI1 are detected in the embodiment.
In a further embodiment, ultrasonic treatment is performed in the catalytic reaction process, a GM-1200D ultrasonic cell disruption instrument (manufactured by Nanjing Shunhima instruments and Equipment Co., Ltd.) is used, the frequency range of an ultrasonic probe is 20-25 KHz, the ultrasonic treatment process conditions are set to be 20-80W, the ultrasonic working time is 20s, and the ultrasonic interval time is 10 s. After the treatment, the mud sample macrogenomic DNA was extracted and the resistance gene was quantitatively detected in the same manner.
In the examples of the present invention, the changes in gene abundance before and after treatment were examined for three antibiotic resistance genes: tetracycline antibiotic resistance gene tetG, sulfonamide antibiotic resistance gene sul1, and transposon intro 1, in order of magnitude of gene abundance reduction (log)10) The results of the removal of the target resistance gene are shown in tables 1 to 3 and FIGS. 4 to 7. In tables 1 and 2, the mole percentage of oxalic acid to zero-valent iron in the preparation of oxalated zero-valent iron is specified in the catalyst types, and "-" indicates that the item is absent.
TABLE 1
The relevant treatment conditions for the specific examples using zero-valent iron oxalate prepared by the above method as a catalyst to catalyze ozone oxidation and further to assist sonication are shown in table 1. Table 1 lists the zero-valent iron oxalate (OA-ZVI) in different mole percentages of oxalic acid and zero-valent ironbm) The catalyst dosage, ultrasonic power and other conditions are also listed. In table 1, comparative example 1 is a municipal sludge sample without any addition of any substance as a control; comparative example 2 ball milled Zero Valent Iron (ZVI)bm) Catalyzing ozone treatment to perform sludge treatment; comparative example 3 sludge was treated with only zero-valent iron oxalate; comparative example 4 sludge was treated with ozone only; comparative example 5 sludge was treated with sonication alone.
TABLE 2
(ns: no significant difference; < 0.05; < 0.01; < 0.001.)
TABLE 3
| Examples | tetG removal efficiency | sul1 removal efficiency | intI1 removal efficiency |
| Example 1 | 20.69％ | 16.22％ | 13.90％ |
| Example 2 | 28.41％ | 22.37％ | 19.23％ |
| Example 3 | 37.59％ | 22.71％ | 21.79％ |
| Example 4 | 18.12％ | 10.61％ | 13.02％ |
| Example 5 | 21.51％ | 18.01％ | 19.31％ |
| Example 6 | 22.52％ | 8.92％ | 10.33％ |
| Example 7 | 28.61％ | 15.03％ | 16.18％ |
| Example 8 | 38.92％ | 27.76％ | 30.00％ |
| Example 9 | 42.68％ | 29.26％ | 31.33％ |
| Comparative example 1 | 0％ | 0％ | 0％ |
| Comparative example 2 | 15.77％ | 12.59％ | 11.77％ |
| Comparative example3 | -5.76％ | 0.78％ | 4.63％ |
| Comparative example 4 | 15.49％ | 10.81％ | 14.35％ |
| Comparative example 5 | 0.59％ | 8.28％ | 10.50％ |
The gene copy numbers (i.e., gene abundances) of the target antibiotic resistance genes tetG, sul1 and int I1 in municipal sludge after treatment of examples and comparative examples are listed in Table 2, and the gene copy numbers of each column were analyzed by a variance homogeneity test and labeled with significant differences. After the sludge is treated by the zero-valent iron oxalate provided by the embodiments 1-9, the abundance of the antibiotic resistance gene of the sludge is obviously reduced.
The removal efficiency of the paradoxin resistance genes tetG, sul1, and intI1 of each example and comparative example was further calculated, and the results are shown in table 3, where the removal efficiency is (number of copies of gene before treatment-number of copies of gene after treatment)/number of copies of gene before treatment × 100%.
The results show that the concentrations of the three resistance genes in example 1 are obviously reduced compared with those in comparative example 1, and the effect of removing the resistance genes can be obviously improved by using the zero-valent iron oxalate in the examples of the application. Comparative example 2 since the ball-milled zero-valent iron was used to treat the sludge, it is shown that the effect of treating the sludge with the acidified zero-valent iron is better than that of the ball-milled zero-valent iron. In contrast, comparative example 3 only uses the acidified zero-valent iron provided in the examples of the present application to treat sludge, and the effect of removing the resistance gene is very limited, thereby further illustrating that the treatment of acidified zero-valent iron needs to be coupled with ozone to promote the oxidation of ozone. While comparative example 4 only treated the sludge with ozone and comparative example 5 only treated the sludge with ultrasound, both of them had inferior removal efficiency of the resistance gene to example 1.
In summary, the embodiment of the application utilizes zero-valent iron oxalate to catalyze the ozone oxidation of the antibiotic resistance genes in the sludge, and utilizes the adsorption and oxidation of the surface oxides of the zero-valent iron to the resistance genes to limit and reduce the abundance of the resistance genes in the sludge.
The above description is only for the preferred embodiment of the present application, but the scope of the present application is not limited thereto, and any changes or substitutions that can be easily conceived by those skilled in the art within the technical scope of the present application should be covered within the scope of the present application.
Claims (10)
1. A combination of agents for use in the removal of municipal sludge antibiotic resistance genes, wherein the combination of agents comprises ozone and acidified zero valent iron; the ozone is in gaseous form for promoting the decomposition of the resistance gene; the acidified zero-valent iron is solid particles and is used for catalyzing the decomposition effect of the ozone on the resistance genes; the acidified zero-valent iron is obtained by acidifying zero-valent iron under a solid-phase ball milling condition by using solid acid, wherein the solid acid accounts for 2.0-8.0% of the zero-valent iron in mole percentage.
2. The formulation combination of claim 1, wherein the acidified zero valent iron is selected from at least one of zero valent iron silicate, phosphorylated zero valent iron, phosphorous acid zero valent iron, borated zero valent iron, iodated zero valent iron, periodated zero valent iron, oxalated zero valent iron, citrated zero valent iron, malated zero valent iron, tartrated zero valent iron, succinated zero valent iron, trichloroacetated zero valent iron, dinitro benzoic acid zero valent iron, ethylenediaminetetraacetic acid zero valent iron, and sulfamized zero valent iron.
3. The formulation combination according to claim 2, wherein the acidified zero valent iron is selected from phosphorylated zero valent iron, oxalated zero valent iron, citrated zero valent iron or succinated zero valent iron.
4. The formulation combination according to claim 3, wherein the acidified zero valent iron is zero valent iron oxalate.
5. A formulation combination according to claim 4, wherein the solid acid is selected from at least one of silicic acid, phosphoric acid, phosphorous acid, boric acid, iodic acid, periodic acid, oxalic acid, citric acid, malic acid, tartaric acid, succinic acid, trichloroacetic acid, dinitrobenzoic acid, ethylenediaminetetraacetic acid and sulfamic acid.
6. A formulation combination according to claim 5, wherein the solid acid is selected from phosphoric acid, phosphorous acid, citric acid, succinic acid or oxalic acid.
7. The formulation combination of claim 4, wherein the solid phase ball milling conditions are: the ball milling time is 4h, and the ball milling rotating speed is 500 r/min.
8. Use of the combination of agents of any one of claims 1 to 7 for the removal of antibiotic resistance genes from municipal sludge, comprising the step of adding acidified zero valent iron to the ozone-infused municipal sludge.
9. The use of claim 8, wherein the acidified zero valent iron is used in an amount of 2.0 to 16.0g/L municipal sludge and the ozone is used in an amount of 0.5 to 3L/min.
10. The use according to claim 9, characterized in that it further comprises a step of ultrasound treatment, the conditions of which are: the ultrasonic frequency is 20-25 KHz, and the ultrasonic power is 0.4-1.6W/mL.
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