CN111661893A - Method for eliminating antibiotic resistance genes in water by using nano biochar - Google Patents
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Abstract
The invention relates to a method for eliminating antibiotic resistance genes in water by using nano biochar, belonging to the field of environmental pollution prevention and control. The raw materials for preparing the biochar have wide sources, are mainly biomass waste, and have the characteristics of simple preparation method, easy implementation, low carbon and environmental protection. The nano biochar can be used for adsorbing common resistance genes in a water body, so that the DNA structure of the biochar is damaged, and the possibility of re-propagation of the biochar is blocked; compared with common large-particle biochar, the adsorption time is obviously shortened, and the time is shortened 5/6; the adsorption rate of the resistance gene in the water body can reach 66 percent; and the operation is simple and convenient, and the popularization and the application are easy.
Description
Technical Field
The invention relates to a method for eliminating antibiotic resistance genes in water by using nano biochar, belonging to the field of environmental pollution prevention and control.
Background
The discovery of antibiotics makes great contribution to preventing and treating bacterial infection and promoting the development of agriculture and animal husbandry for human beings. However, with the long-term use and abuse of antibiotics, the problem of antibiotic resistance has become more serious and has become one of the most serious public health problems worldwide. 2011 the world health organization proposed a call to "defend against drug resistance — no action is taken today and no drug is available tomorrow". In 2016, China also made and issued "plan for inhibiting bacteria drug-resistant action (2016-2020)", aiming at strengthening the scientific management of antibiotics, inhibiting the development and spread of drug-resistant bacteria and resistance genes, ensuring the health of people and promoting the sustainable development of economy and society.
As a new type of environmental pollutant, the persistent residue, transmission and diffusion of antibiotic resistance genes in the environment are more harmful than the antibiotics themselves. Compared with traditional chemical pollutants, the resistance gene has unique biological characteristics and environmental behaviors, and can be transferred from a parent to a offspring through inheritance, namely vertical transfer, and can also be spread in and among microbial species through gene horizontal transfer (comprising three modes of conjugation, transduction and transformation). Conjugative transfer resistance is usually transferred by direct contact between resistant bacteria and indigenous bacteria, and transmission of resistance is mediated by genetic elements such as plasmids, integrants, and the like. Transduction is the introduction of resistance into microbial cells by phage. Transformation is the acquisition of free DNA by the active microorganism from the surrounding environment to acquire resistance. Different forms of resistance genes exist in the environment, including intracellular DNA carried by bacteria and viruses, and extracellular free DNA, which can transmit resistance through the three different transmission pathways. Normally, the vertical transmission and conjugation of genes must occur between living bacteria to allow transmission of resistance genes. Similarly, viruses containing resistance genes must be active to allow transduction to occur. However, transformation does not require an active or infectious donor microorganism. Competent bacteria in the environment can directly acquire extracellular free DNA to acquire resistance. Therefore, the migration and spread of the resistance gene in the environmental medium are greatly enhanced. The antibiotic resistance gene can enter soil and water environment through various ways, such as manure application, aquaculture, medical wastewater discharge and the like, the variety is as high as hundreds, and the pollution situation is severe. The resistance gene has the biological characteristics of easy replication or transmission and the physicochemical characteristics of durability, so the resistance gene can exist and transmit in the organism or in the environment for a long time, and even if donor cells carrying the resistance gene kill or die, DNA which dies or is released into the environment by metabolism still has the activity and transmits rapidly. Antibiotic resistance contamination poses a great potential threat to human health and the ecological environment, and antibiotic resistance gene contamination and the risk of spreading the antibiotic resistance gene contamination in the environment are one of the most important ecological safety and human health problems facing human beings. Especially the pathogens induced by the bacteria resistant to antibacterial drugs, are serious threats to human health and ecological safety. However, the existing conventional water treatment technologies (such as ultraviolet sterilization, chlorination, etc.) do not have obvious removal effects on a part of antibiotic resistance genes, so that the development of an efficient and environment-friendly resistance gene elimination technology becomes a key and difficult problem for water resource safe utilization and wastewater reclamation.
Biochar is a solid-phase carbonized substance obtained by high-temperature pyrolysis of biological organic matters under the anaerobic or low-oxygen condition, and biomass can form a large number of free radical compounds in the high-temperature pyrolysis process and is kept in a pore structure of the biochar for a long time, so that the biochar is called as a persistent free radical. These free radicals have extremely strong redox activity and can effectively degrade organic compounds in contact with the free radicals, including antibiotics and resistance genes thereof. However, DNA is large in molecular size (e.g., resistance genes), cannot be effectively adsorbed into biochar pores (<2nm), significantly limits its contact with biochar radicals, and thus is difficult to degrade by large-particle conventional biochar.
Disclosure of Invention
In order to solve the existing problems, the invention provides the nano biochar capable of effectively reducing the antibiotic resistance genes in the water body, the free radicals are directly exposed on the surfaces of the particles by the particles of the nano biochar, and after DNA molecules are adsorbed, the resistance genes can be oxidized to lose the activity of the resistance genes, so that the propagation and spread of the resistance genes in the environment can be effectively inhibited.
The invention provides a method for reducing the content of resistance genes or destroying the DNA structure of the resistance genes, which utilizes nano charcoal to adsorb the resistance genes.
In one embodiment of the invention, the particles of the nano biochar are no greater than 150 nm.
In one embodiment of the invention, the particles of the nano biochar are no greater than 100 nm.
In one embodiment of the invention, the resistance genes include tetA, tetB, qnrA, qnrB, ampC, ermB.
In one embodiment of the invention, the nano biochar is added into a system containing a resistance gene to adsorb the resistance gene.
In one embodiment of the present invention, the system containing the resistance gene is a water body.
In one embodiment of the invention, the concentration of the nano biochar in the system is 30-200 mg/L.
In one embodiment of the invention, the nano biochar is added into a system containing a resistance gene and then is vibrated for 20-30 hours at 100-200 rpm.
The invention provides a method for preparing the nano biochar, wherein the nano biochar takes biomass as a raw material and is prepared.
In one embodiment of the invention, the biomass includes, but is not limited to, chaff, straw, wood chips, livestock and poultry manure.
In one embodiment of the invention, the rice straws are crushed and dried to constant weight, a carbonization furnace is adopted to prepare biochar through pyrolysis under the protection of nitrogen, the pyrolysis temperature is 400-700 ℃, the pyrolysis time is 1-6 hours, and the biochar is ground and sieved by a 50-100-mesh sieve; soaking the biochar passing through a sieve of 50-100 meshes in deionized water, fully stirring until the biochar is uniformly mixed, slowly filtering biochar suspension by using a sieve of 200-500 meshes, wherein large-particle biochar is obtained when the biochar suspension does not pass through the sieve meshes, placing the sieved biochar suspension in a beaker, adding deionized water for dilution, performing ultrasonic dispersion for 0.5-1.5 hours, and standing for 1.5-2.5 hours; slowly extracting a biochar suspension liquid 5-15 cm below the liquid level by using a siphon, adding water to restore the volume of the original liquid, and then carrying out siphoning, wherein the water supplementing-siphoning step is repeated for a plurality of times until the supernatant liquid begins to become clear; and (3) carrying out ultrasonic treatment on the upper suspension liquid sucked out by the siphon for 25-35 min, then carrying out centrifugal separation to obtain a precipitate, and drying the precipitate to obtain the nano biochar.
The invention also protects the application of the method for adsorbing the resistance gene by using the biochar or the method for preparing the nano biochar in adsorbing the resistance gene in the fields of environmental protection, biology, chemical industry, medicine and agriculture.
The invention has the beneficial effects that:
1) the raw materials for preparing the biochar have wide sources, are mainly biomass waste, and have the characteristics of simple preparation method, easy implementation, low carbon and environmental protection.
2) The nano biochar can be used for adsorbing common resistance genes in a water body, so that the DNA structure of the biochar is damaged, and the possibility of re-propagation of the biochar is blocked; compared with common large-particle biochar, the adsorption time is obviously shortened, and the time is shortened 5/6; the adsorption rate of the resistance gene in the water body can reach 66 percent; and the operation is simple and convenient, and the popularization and the application are easy.
Drawings
FIG. 1 is a transmission electron micrograph of nano-biochar; wherein nano400 and nano700 represent nano biochar at 400 ℃ and 700 ℃, respectively.
FIG. 2 shows the signal intensity of free radicals on the surface of the nano biochar; wherein nano400 and nano700 represent nano biochar at 400 ℃ and 700 ℃, respectively.
FIG. 3 is the effect of nano biochar at 400 ℃ on the adsorption amount of resistance genes ampC and ermB and the amplification capacity thereof; wherein (a) the influence of 400 ℃ large-particle biochar on the adsorption capacity and amplification capacity of ampC and ermB; (b) the influence of the nano charcoal at 400 ℃ on the adsorption quantity and the amplification capacity of resistance genes ampC and ermB; 1. 2, 3 are PCR amplification results after ampC and ermB react with 4.5, 9.0 and 12.0mg/mL large-particle biochar respectively, CK and 4 are negative and positive controls respectively (the positive control is an unreacted original DNA template, and the negative control is that the original biochar and DNA are directly added into a PCR reaction system in sequence to eliminate the influence of the biochar on the DNA amplification process); n1, N2 and N3 show the PCR amplification results of ampC and ermB after reaction with nano biochar at concentrations of 100, 150 and 200mg/L, respectively; n4, N5 and N6 respectively represent the PCR amplification result of the supernatant as a DNA template after reaction centrifugation of 100, 150 and 200mg/L nano biochar; n7, N8 and N9 are biochar alone, negative and positive controls, respectively.
FIG. 4 is the effect of nano biochar at 700 ℃ on the adsorption amounts of resistance genes ampC and ermB and their amplification capacities; wherein (a) the influence of large-particle biochar at 700 ℃ on the adsorption capacity and amplification capacity of ampC and ermB; (b) the influence of nano charcoal at 700 ℃ on the adsorption quantity and the amplification capacity of resistance genes ampC and ermB; 1. 2, 3 are PCR amplification results after ampC and ermB react with 4.5, 9.0 and 12.0mg/mL large-particle biochar respectively, CK and 4 are negative and positive controls respectively (the positive control is an unreacted original DNA template, and the negative control is that the original biochar and DNA are directly added into a PCR reaction system in sequence to eliminate the influence of the biochar on the DNA amplification process); n1, N2 and N3 show the PCR amplification results of ampC and ermB after reaction with nano biochar at concentrations of 100, 150 and 200mg/L, respectively; n4, N5 and N6 respectively represent the PCR amplification result of the supernatant as a DNA template after reaction centrifugation of 100, 150 and 200mg/L nano biochar; n7, N8 and N9 are biochar alone, negative and positive controls, respectively.
FIG. 5 is the kinetics of elimination of calf thymus DNA in solution (a) and adsorption isotherm curve (b) by nano biochar; wherein nano400 and nano700 represent nano biochar at 400 ℃ and 700 ℃ respectively; bulk400 and bulk700 represent large particle biochar at 400 ℃ and 700 ℃ respectively.
FIG. 6 is an atomic force microscope photograph of the damage of nanocarbon to the DNA molecular structure of calf thymus in solution, wherein (a) the DNA ring structure before reaction; (b) contacting the DNA with nano charcoal at 700 ℃ for 30 min; (c) contacting the DNA with nano charcoal at 700 ℃ for 120 min; (d) and (e) nano biochar at 700 ℃ and 400 ℃ before reaction respectively; (f) contacting the DNA with nano charcoal at 700 ℃ for 30 min; (g) the DNA is contacted with nano charcoal at 700 ℃ for 120 min.
FIG. 7 is the effect of nanocarbon on calf thymus DNA structure; a, treating the mixture into a mixed solution after reaction of biochar and DNA; b, after the biochar reacts with DNA, centrifuging and cleaning the sample; c, processing the mixture into supernatant after mixing the biochar with DNA and centrifuging; 1-2 represent large-particle biochar at 700 ℃ and 400 ℃ respectively; 3-4 represent nano biochar at 700 ℃ and 400 ℃ respectively.
Detailed Description
The present invention will be further described with reference to the following specific examples, but the present invention is not limited to these examples.
The resistance gene in the embodiment of the invention refers to a typical antibiotic resistance gene in a natural water body (Haihe Tianjin section), namely an aminoglycoside antibiotic resistance gene ampC and a macrolide resistance gene ermB. Specifically, bacteria are propagated from a water body and are domesticated and cultured under the selection pressure of corresponding antibiotics (gentamicin and chloramphenicol), so that bacteria with aminoglycoside resistance and macrolide resistance are obtained. Extracting DNA from the resistant bacteria, synthesizing ampC and ermB primers, taking the DNA extracted from the resistant bacteria as a template to carry out PCR amplification, carrying out gel electrophoresis test comparison on the obtained PCR product, and displaying a clear band which is the resistance gene required by the experiment.
And (3) measuring the DNA concentration by an ultraviolet spectrophotometer, wherein the Abs260nm/280nm value is the required concentration of the ARGs.
Example 1
(1) Preparation of biochar by biomass pyrolysis
Crushing and drying the rice straws to constant weight, preparing the biochar by pyrolyzing the rice straws in a carbonization furnace under the protection of nitrogen, wherein the pyrolysis temperature is 400 ℃, the pyrolysis time is 2 hours, grinding the biochar by a sieve of 100 meshes, and taking the biochar passing through the sieve of 100 meshes.
(2) Extraction of nano biochar
Soaking the obtained charcoal in deionized water, and stirring thoroughly until the charcoal is mixed uniformly; slowly filtering the biochar suspension by using a 300-mesh sieve, namely, large-particle biochar which does not pass through the sieve mesh, putting the biochar suspension which passes through the 300-mesh sieve into a beaker, slowly adding deionized water for dilution, ultrasonically dispersing for 1 hour, and then standing for 2 hours; slowly extracting the biochar suspension liquid 10cm below the liquid level by using a siphon, adding water to restore to the volume of the stock solution, and then carrying out siphoning, wherein the water supplementing-siphoning step is repeated for a plurality of times until the supernatant liquid begins to become clear; performing ultrasonic treatment on the upper suspension liquid sucked out by the siphon for 30min, and then performing centrifugal separation on the nano biochar at the rotation speed of 10000rpm for 20min to obtain a precipitate; and drying the obtained precipitate to obtain the nano biochar particles at 400 ℃.
The particle size of the nano biochar is characterized by utilizing a high-resolution transmission electron microscope: mixing a nano biochar sample with absolute ethyl alcohol, performing ultrasonic treatment for 10min, dropping a drop of suspension on a copper mesh, and observing by using an electron microscope; as can be seen from figure 1, the particle size of the nano biochar is 30-100 nm; the persistent free radical in the nano biochar is measured by electron paramagnetic resonance spectroscopy, and as shown in figure 2, the nano biochar has strong persistent free radical strength at 400 ℃.
(3) Elimination of nano charcoal on resistance gene in water
Weighing a certain mass of the nano biochar particles prepared in the step (2), and adding the nano biochar particles into 4mL of resistance gene polluted water with the pH value of 7.0 +/-0.1 (wherein the concentrations of the resistance genes ampC and ermB are respectively 3.4-3.7 × 103mg/L), placing the mixed solution in a shaking table (150rpm), mixing for 24h, fully mixing with the water body polluted by the resistance genes to achieve adsorption balance, centrifuging (12000rpm for 30min), separating, absorbing the supernatant, and measuring the concentration of the resistance genes in the solution by using an ultraviolet spectrophotometer.
And calculating the adsorption removal capacity of the nano biochar to the resistance genes according to the difference value of the initial concentration and the equilibrium concentration of the resistance genes in the solution and the quality of the biochar.
The calculation formula is as follows: the removal rate (%) - (initial concentration-equilibrium concentration)/initial concentration, wherein the concentration unit of the resistance gene is mg/L, and the mass unit of the biochar is mg.
As shown in FIG. 3, Table 1 and Table 2, the removal rates of ampC were 30.1%, 35.5% and 38.1% at the nano charcoal concentrations of 100mg/L, 150mg/L and 200mg/L, respectively; the removal rates for ermB were 28.1%, 34.6%, and 36.3%, respectively.
Table 1400 ℃ large-granular biochar antagonistic gene adsorption quantity Qe(mg/g)
| Biochar concentration (mg/mL) | 1.5 | 4.5 | 9.0 | 12.0 |
| ampC | 0.96 | 1.00 | 0.82 | 0.58 |
| ermB | 0.90 | 0.62 | 0.56 | 0.51 |
Table 2400 ℃ nano charcoal resistance gene adsorption quantity Qe(mg/g)
| Biochar concentration (mg/L) | 10 | 25 | 50 | 100 | 150 | 200 |
| ampC | 20.00 | 54.0 | 61.0 | 89.5 | 96.3 | 104.5 |
| ermB | 40.0 | 56.0 | 75.0 | 87.0 | 96.0 | 101.5 |
(4) Influence of nano biochar on resistance gene structure in water body
And respectively taking the resistance gene, the mixture of the large biochar particles at 400 ℃ and the resistance gene and the mixture of the nano biochar at 400 ℃ and the resistance gene as PCR templates, and performing a PCR amplification experiment to verify whether the DNA adsorbed on the biochar surface still has amplification capacity. Meanwhile, biochar which does not adsorb DNA is set as a blank and is added into a PCR reaction system simultaneously with the DNA for amplification and gel electrophoresis experiments, so that the biochar with the experimental dosage has no inhibition effect on the PCR amplification process.
The PCR reaction system was set to 20. mu.L and included: mu.L 10. mu. mol/L upstream/downstream primer, 10. mu.L Taq Mix (2X), 7. mu.L deionized water, 2. mu.L PCR template. The primers for PCR were:
ampC F:5’-CCTCTTGCTCCACATTTGCT-3’(SEQ ID NO.1)，
ampC R:5’-ACAACGTTTGCTGTGTGACG-3’(SEQ ID NO.2)；
ermB F:5’-AGCCATGCGTCTGACATCTA-3’(SEQ ID NO.3)；
ermB R:5’-CTGTGGTATGGCGGGTAAGT-3’(SEQ ID NO.4)。
the PCR reaction conditions are as follows: pre-denaturation at 95 ℃ for 5min, 30 cycles of denaturation at 95 ℃ for 30s, annealing at 55 ℃ for 30s, extension at 72 ℃ for 30s, and extension at 72 ℃ for 10 min. The PCR product was electrophoresed on a 1.0% agarose gel (voltage 100V) for 30min, and then photographed using a gel imaging system (Image Quant 350, GE, USA).
As shown in FIG. 3, the resistance gene adsorbed by the nanocarbon could not be amplified, indicating that structural damage occurred to the resistance gene.
Example 2
(1) Preparation of biochar by biomass pyrolysis
Crushing and drying the rice straw to constant weight, preparing the biochar by pyrolyzing the rice straw in a carbonization furnace under the protection of nitrogen, wherein the pyrolysis temperature is 700 ℃, the pyrolysis time is 2 hours, and grinding and sieving the biochar by a 100-mesh sieve to obtain the large-particle biochar.
(2) Extraction of nano biochar
Soaking the obtained charcoal in deionized water, and stirring thoroughly until the charcoal is mixed uniformly; slowly filtering the biochar suspension by using a 300-mesh sieve, namely, large-particle biochar which does not pass through the sieve mesh, putting the biochar suspension which passes through the 300-mesh sieve into a beaker, slowly adding deionized water for dilution, ultrasonically dispersing for 1 hour, and then standing for 2 hours; slowly extracting the biochar suspension liquid 10cm below the liquid level by using a siphon, adding water to restore to the volume of the stock solution, and then carrying out siphoning, wherein the water supplementing-siphoning step is repeated for a plurality of times until the supernatant liquid begins to become clear; performing ultrasonic treatment on the upper suspension liquid sucked out by the siphon for 30min, and then performing centrifugal separation on the nano biochar at the rotation speed of 10000rpm for 20min to obtain a precipitate; and drying the obtained precipitate to obtain the nano biochar particles at 700 ℃.
The particle size of the nano biochar is characterized by utilizing a high-resolution transmission electron microscope: referring to example 1, the results are shown in FIG. 2, wherein the radical strength of the nano biochar at 700 ℃ is slightly lower than that of the nano biochar at 400 ℃.
(3) Elimination of nano charcoal on resistance gene in water
Weighing a certain mass of the nano biochar prepared in the step 2, and adding the nano biochar into 4mL of resistance gene polluted water with the pH value of 7.0 +/-0.1 (wherein the concentrations of resistance genes ampC and ermB are both 3.4-3.7 × 10)3mg/L), mixing the mixed solution in a shaking table (150rpm) for 24h, centrifuging (12000rpm, 30min), separating, sucking the supernatant, and measuring the concentration of the resistance gene in the solution by using an ultraviolet spectrophotometer.
As shown in FIG. 4, Table 3 and Table 4, the removal rates of ampC were 29.6%, 46.8% and 66.4% at the nano charcoal concentrations of 100mg/L, 150mg/L and 200mg/L, respectively; the removal rates for ermB were 35.1%, 49.7%, and 66.7%, respectively.
Table 3700 ℃ large-particle biochar antipodal gene adsorption quantity Qe(mg/g)
| Biochar concentration (mg/mL) | 1.5 | 4.5 | 9.0 | 12.0 |
| ampC | 1.39 | 1.64 | 1.00 | 0.63 |
| ermB | 0.85 | 1.56 | 1.01 | 0.07 |
Table 4700 ℃ nano charcoal antagonistic gene adsorption quantity Qe(mg/g)
| Biochar concentration (mg/L) | 10 | 25 | 50 | 100 | 150 | 200 |
| ampC | 25.0 | 40.0 | 69.0 | 83.0 | 101.7 | 104.5 |
| ermB | 30.0 | 38.0 | 59.0 | 80.5 | 100.0 | 99.5 |
(4) Influence of nano biochar on resistance gene structure in water body
Referring to example 1, the difference is that the mixture of 400 ℃ biochar macroparticle and resistance gene is replaced with the mixture of 700 ℃ biochar macroparticle and resistance gene, and the mixture of 400 ℃ nanocarbon and resistance gene is replaced with the mixture of 700 ℃ nanocarbon and resistance gene.
The result is shown in fig. 3, the resistance gene on the large-particle biochar has an obvious bright band in the result of gel electrophoresis, which indicates that the large-particle biochar does not affect the amplification, and the resistance gene on the nano biochar has no bright band in the result of gel electrophoresis, which indicates that the nano biochar causes structural damage to the resistance gene and is difficult to amplify.
Example 3
Elimination of extracellular mode DNA (simulation of extracellular DNA with calf thymus DNA with DNA secondary structure, easy concentration setting and determination before and after experiment) using 400 ℃ and 700 ℃ nano biochar in examples 1 and 2: respectively weighing 5mg of the nano biochar in the example 1 or 2, adding the nano biochar into 4mL of calf thymus DNA aqueous solution with the pH value of 7.0 +/-0.1 (the DNA concentration gradient is 2-48mg/L), placing the mixed solution in a shaking table (150rpm), mixing for 24h, centrifuging (12000rpm for 30min), separating, sucking the supernatant, and measuring the concentration of the resistance gene in the solution by using an ultraviolet spectrophotometer.
(1) Elimination of kinetic experiments
The experimental steps are as follows: respectively weighing 10mg of the large-particle biochar in examples 1 and 2 and 5mg of the nano biochar in examples 1 and 2, adding the large-particle biochar and the nano biochar into 4mL of aqueous solution of the resistance gene with the pH value of 7.0 +/-0.1 (the concentration of the resistance gene is 10mg/L), placing the mixed solution in a shaking table (150rpm), mixing, sampling at different time points (1, 5, 10, 30, 120, 320 and 600min), performing centrifugal separation, sucking supernatant, and determining the concentration of the resistance gene in the solution by using an ultraviolet spectrophotometer.
As can be seen from FIG. 5, the nano biochar solutions at 400 ℃ and 700 ℃ can reach equilibrium within 120 min; while the balance can be achieved only after 720min of large-particle biochar.
(2) Isothermal adsorption experiment
Isothermal adsorption experiment steps: 10mg of the large-particle biochar of examples 1 and 2 and 5mg of the nano biochar of examples 1 and 2 were weighed, respectively, added to 4mL of an aqueous solution of the resistance gene having a pH of 7.0. + -. 0.1 (the concentration of the resistance gene was 2 to 48mg/L), the mixture was mixed in a shaker (150rpm) for 24 hours, centrifuged (12000rpm, 30min) for separation, and the supernatant was extracted to measure the concentration of the resistance gene in the solution using an ultraviolet spectrophotometer.
Isothermal adsorption curves of the nano biochar and the large-particle biochar (control) on calf thymus DNA in the solution are shown in fig. 5, the adsorption capacity of the two types of nano biochar on the calf thymus DNA is 100 times of the control adsorption capacity of the large-particle biochar, and the adsorption removal capacity of the nano biochar on the DNA is obviously higher than that of the large-particle biochar.
(3) Influence of nano biochar on resistance gene structure in water body
Detecting and verifying the oxidation damage capability of the nano biochar to extracellular DNA by utilizing an atomic force microscope. And (3) observing the DNA morphological structure of the nano biochar with different adsorption time by using an atomic force microscope. As shown in FIG. 6, after the contact reaction of DNA and the nano-organisms for 30min, the circular structure of DNA is destroyed and obvious damage and breakage occur, while after the contact reaction of DNA and the nano-organisms for 120min, the structure of DNA is further destroyed and DNA fragments are adsorbed and aggregated on the surface of the charcoal particles to form larger aggregates, so that the DNA is inactivated and amplification cannot occur.
Secondly, sequentially sucking 10 mu L of samples which reach the balance in the elimination kinetics experiment to be used as gel electrophoresis templates. Combining the DNA template with gel dye, adding the combined DNA template into 1% agarose gel, operating at 100eV voltage for 40min, and photographing the gel; the samples were: reacting biochar with DNA to obtain a mixed solution; after the biochar reacts with DNA, centrifugally cleaning a biochar sample; mixing biochar with DNA, and centrifuging to obtain supernatant.
The results are shown in FIG. 7: the DNA on the large-particle biochar has an obvious bright band in a gel electrophoresis result, which indicates that the large-particle biochar does not damage the DNA structure, and the DNA on the nano biochar does not have a bright band in a gel electrophoresis result, which indicates that the nano biochar damages the DNA structure.
Although the present invention has been described with reference to the preferred embodiments, it should be understood that various changes and modifications can be made therein by those skilled in the art without departing from the spirit and scope of the invention as defined in the appended claims.
SEQUENCE LISTING
<110> university of south of the Yangtze river
<120> method for eliminating antibiotic resistance genes in water by using nano biochar
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Claims (10)
1. A method for reducing the content of resistance genes or destroying the DNA structure of the resistance genes is characterized in that the resistance genes are adsorbed by nano biochar.
2. The method of claim 1, wherein the particles of nano biochar are no greater than 150 nm.
3. The method of claim 1, wherein the resistance genes comprise tetA, tetB, qnrA, qnrB, ampC, ermB.
4. The method of claim 1, wherein the nano biochar is added to a system containing a resistance gene.
5. The method according to claim 4, wherein the concentration of the nano biochar in the system is 30-200 mg/L.
6. The method according to claim 4 or 5, wherein the nano biochar is added into a system containing a resistance gene and then is shaken at 100-200 rpm for 20-30 h.
7. A method for preparing nano biochar is characterized in that the nano biochar is prepared by taking biomass as a raw material.
8. The method of claim 7, wherein the biomass includes but is not limited to chaff, straw, wood chips, livestock and poultry manure.
9. The method according to claim 7 or 8, characterized in that the biomass of claim 8 is pulverized, dried, pyrolyzed at 300-700 ℃ for 1-6 hours, and the pyrolyzed biomass is ground and sieved with a 50-100 mesh sieve; soaking the biochar passing through a sieve of 50-100 meshes in deionized water, uniformly mixing, sieving through a sieve of 200-500 meshes to obtain a biochar suspension, ultrasonically dispersing the biochar suspension for 0.5-1.5 hours, and standing for 1.5-2.5 hours; extracting a biochar suspension liquid 5-15 cm below the liquid level; and (4) carrying out ultrasonic treatment on the suspension for 25-35 min, then carrying out centrifugal separation to obtain a precipitate, and drying the precipitate to obtain the nano biochar.
10. The method of any one of claims 1 to 9, which is applied to the adsorption of resistance genes in the fields of environmental protection, biology, chemical industry, medicine and agriculture.
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| CN113651388A (en) * | 2021-08-18 | 2021-11-16 | 南开大学 | Method for weakening antibiotic resistance gene transfer between Cu (II) and bacteria by using soluble biochar |
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Application publication date: 20200915 |