CN115196616A - Magnesium salt modified biochar material and application thereof in reducing abundance of antibiotic resistance genes - Google Patents

Abstract

The invention discloses a magnesium salt modified biochar material and application thereof in reducing abundance of antibiotic resistance genes, and belongs to the technical field of environmental protection. The magnesium salt modified charcoal material is prepared by the following method: pyrolyzing rice hulls at 450-500 ℃ under the condition of complete oxygen deficiency to prepare rice hull biochar; grinding and sieving the rice hull biochar to obtain charcoal powder; mixing the charcoal powder and the magnesium salt solution, carrying out ultrasonic oscillation, carrying out suction filtration, drying a filter cake, carrying out secondary calcination fixation, cooling, and grinding to prepare the magnesium salt modified charcoal material. The magnesium salt modified biochar is used as an additive of aerobic compost, and the ARGs in the manure can be obviously reduced. The method has simple operation and low manufacturing cost, can realize large-scale industrial production, and is beneficial to reducing the ARGs in the manure.

Description

Magnesium salt modified biochar material and application thereof in reducing abundance of antibiotic resistance genes

Technical Field

The invention relates to the technical field of environmental protection, in particular to a magnesium salt modified biochar material and application thereof in reducing abundance of antibiotic resistance genes.

Background

The antibiotic as a great invention in the 20 th century is widely used for preventing and treating various diseases by livestock and poultry breeding industry, can protect the health of animals, plays an important role in promoting the growth and development of the animals, and greatly reduces the cost of the livestock and poultry breeding industry. With the widespread use of antibiotics in the poultry farming industry, there is increasing pressure on the environment from fecal excretion. Under the environmental pressure of long-term administration of antibiotics, the antibiotics can induce intestinal bacteria of animals to generate Antibiotic Resistance Genes (ARGs) and drug-resistant bacteria, so that the livestock manure becomes an important storage bank of the antibiotics and the ARGs. The ARGs refer to the fact that microorganisms have drug resistance to antibiotics, have certain resistance to inhibition and lethal effects of the antibiotics, and can perform growth and reproduction activities. The antibiotics and the ARGs are discharged out of the body along with the excrement of the livestock and the poultry, so that the antibiotics and the ARGs enter water, soil and the atmosphere, and pressure is generated on the drug resistance of microorganisms in the environment, so that ecological pollution is caused, and the human health is harmed. It is found that feeding chicken with a certain concentration of antibiotic can cause tetracycline-resistant strains in the excrement of the chicken, and the abundance of sulfanilamide resistance genes sul1 and sul2 in pig manure is closely related to the use of high-dose antibiotic. Resistant Bacteria (ARB) resistant to antibiotics in human or animal feces are excreted out of the body along with the intestinal tract, and most of the resistant bacteria are transmitted in various environments by means of gene level transfer and the like. Numerous studies have demonstrated a significant increase in the abundance of ARGs in soils chronically fertilized with manure, particularly tetracycline and sulfonamide resistance genes. Wu Dan and the like find that the detection rates of tetracycline resistance genes (tetT 1, tetA1 and tetM 2), quinolone resistance genes (qnrB 1 and qnrS 1) and sulfonamide resistance genes (sul 1 and dfrA 1) in livestock and poultry excrement of Beijing farms are all over 90 percent. Livestock and poultry manure is often applied to soil as an organic fertilizer in agricultural production, and Movable Genetic Elements (MGEs) contained in the manure are a main driving force for the abundance change of ARGs, so that certain risks are caused to the ecological environment of the soil. Research shows that the integron intI1 has obvious positive correlation with various ARGs in the cow dung composting process, and the intI1 is the main driving force for the variation of the ARGs in the compost. The ARGs, as a novel environmental pollutant, have heredity, can be copied by self, have persistence and nondegradable property in the environment and bring huge risks to the ecological environment and human health. The World Health Organization (WHO) has reported that approximately more than 200 million americans are infected with drug-resistant pathogens each year, and 23000 people die of them. The ARGs can cause a series of health problems with serious consequences, and people are urgently required to take measures to control the further spread of the ARGs.

As ARGs and ARB contained in animal manure become the biggest threat of being applied to farmlands as organic fertilizers, in order to reduce the spread and spread of ARGs caused by animal manure, many researchers have begun to research and utilize biotechnology to cut down ARGs before manure is recycled so as to control from the source and reduce the risk of ecological environment. Composting is a biochemical process that utilizes microorganisms widely existing in nature to controllably promote the conversion of degradable organic matter in solid waste into stable humus. The compost can not only recycle and harmlessly treat solid wastes, but also obtain a large amount of organic fertilizers, and is a mode widely used for treating livestock and poultry manure due to the advantages of low cost, simple operation and the like. The aerobic composting can not only reasonably utilize livestock and poultry manure resources, but also can rapidly degrade residual antibiotics in the manure, prevent the ARGs in the composting process from being induced again, reduce the harm of the ARGs in the livestock and poultry manure to the ecological environment, and is an important technology for reducing the spatial distribution of various ARGs in the livestock and poultry manure.

In recent years, a plurality of scholars at home and abroad research and discover that the addition of Biochar (BC) in compost has certain effect on removing ARGs. He and the like add biochar in pig manure compost, so that the total number of the ARGs is reduced by 16.83 +/-4.10%. Zhou et al added corn stalk biochar in chicken manure compost, and found that it can inhibit the spread and diffusion of ARGs. However, since the BC is different in the raw material, the specific surface area, the pore structure, the type and the nature of the surface group are different from each other, and there is a problem that the adsorption amount is small and the effect of removing ARGs is poor. Therefore, it is very necessary to further modify BC to improve adsorption capacity and change chemical properties.

Disclosure of Invention

Aiming at the prior art, the invention aims to provide a magnesium salt modified biochar material and application thereof in reducing the abundance of antibiotic resistance genes.

In order to achieve the purpose, the invention adopts the following technical scheme:

the invention provides a magnesium salt modified biochar material, which is prepared by the following method:

pyrolyzing rice hulls at 450-500 ℃ under a closed anoxic condition to prepare rice hull biochar; grinding and sieving the rice hull biochar to obtain charcoal powder; mixing the charcoal powder and the magnesium salt solution, carrying out ultrasonic oscillation, carrying out suction filtration, drying a filter cake, carrying out secondary calcination fixation, cooling, and grinding to prepare the magnesium salt modified charcoal material.

Preferably, the magnesium salt solution is 0.42mol/L MgCl ₂ And (3) solution.

Preferably, the power of ultrasonic oscillation is 100Hz, and the ultrasonic oscillation time is 2h.

Preferably, the fixed temperature of the secondary calcination is 300 ℃ and the time is 1h.

In a second aspect of the present invention, there is provided a use of the above magnesium salt modified biochar material in (1) or (2):

(1) The abundance of antibiotic resistance genes in the livestock and poultry manure is reduced;

(2) And the abundance of the mobile gene elements in the livestock and poultry manure is reduced.

In the above application, the antibiotic resistance gene comprises: sul1, sul2, dfrA1, dfrA7, ermB and ermF; the mobile genetic element comprises: intI1 and intI2.

In a third aspect of the invention, there is provided a method of reducing the abundance of antibiotic resistance genes and mobile genetic elements in stool comprising the steps of:

mixing excrement to be treated with sawdust, adding the magnesium salt modified charcoal material, and uniformly mixing to obtain a compost material;

regulating the water content of the compost material to 55-65%, and carrying out aerobic composting for 40-45 days.

Preferably, the addition amount of the magnesium salt modified biochar material is 2% of the weight of the excrement to be treated.

Preferably, sawdust is used to adjust the C/N ratio in the compost material to 20.

The invention has the beneficial effects that:

according to the invention, the magnesium salt is used for modifying the biochar to prepare the magnesium salt modified biochar. The magnesium salt modified charcoal is used as an additive of aerobic compost, so that the absolute abundance of antibiotic resistance genes and movable gene elements in the livestock and poultry manure can be reduced, and the ARGs in the manure can be obviously reduced. The method has simple operation and low manufacturing cost, can realize large-scale industrial production, and is beneficial to reducing the ARGs in the manure.

Drawings

FIG. 1 is a scanning electron micrograph of BC and MBC;

wherein (a) is 1000 times BC magnification; (b, c) is BC magnification 20000 times; (d) MBC is 1000 times amplified; (e, f) is MBC 20000 times magnification.

FIG. 2 is the change in absolute abundance of ARGs and MGEs after 45 days of composting.

Detailed Description

It should be noted that the following detailed description is exemplary and is intended to provide further explanation of the disclosure. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs.

As described previously, ARGs and ARB contained in livestock manure have become one of the biggest threats to its application to farmlands as an organic fertilizer. Aerobic composting is an important technology for reducing the spatial distribution of various ARGs in livestock and poultry manure. In earlier researches, biochar added into compost has certain effect on removing the ARGs. However, the properties of the biochar are mainly influenced by raw materials and pyrolysis conditions, the proportions of lignin, cellulose and hemicellulose contained in different biomass materials are different, and the tissue structures of different crops are different, so that the prepared biochar has larger difference in pore structures; even if the biomass raw material is the same, the change of factors such as pyrolysis temperature, pyrolysis time, oxygen introduction amount and water content can cause the change of the structure and composition of the biomass charcoal. Therefore, the biomass charcoal prepared by different methods has larger performance difference, and the effect of removing the ARGs in the composting process is also obviously different. Some biomass charcoal is even not beneficial to the removal of the ARGs in the aerobic composting process.

Based on the modification, the biomass charcoal is subjected to modification treatment, so that the absolute abundance of antibiotic resistance genes and movable gene elements in the livestock and poultry manure can be stably and effectively reduced in the aerobic composting process, and the ARGs in the manure are obviously reduced.

In order to make the technical solutions of the present application more clearly understood by those skilled in the art, the technical solutions of the present application will be described in detail below with reference to specific embodiments. If the experimental conditions not specified in the examples are specified, the conditions are generally conventional or recommended by the reagent company; reagents, consumables and the like used in the following examples are commercially available unless otherwise specified.

Example 1: preparation of magnesium salt modified biochar material

1. Preparing rice hull Biochar (BC):

taking rice hulls as raw materials, placing the rice hulls in an oven, drying at 50 ℃ to constant weight, then placing in a closed container, placing in a muffle furnace for heating, setting the temperature of the muffle furnace to be 500 ℃, and continuously heating for 2 hours after reaching the final temperature; cooling to room temperature, taking out, grinding and sieving with a 100-mesh sieve to obtain the rice hull Biochar (BC).

2. Preparation of magnesium salt Modified Biochar (MBC):

weighing the prepared rice hull biochar, and putting the rice hull biochar into prepared 0.42mol/L MgCl ₂ In solution, the quality (g) of the rice hull biochar and MgCl ₂ The volume (mL) ratio of the solution is 1: 10, and the solution is subjected to ultrasonic oscillation for 2h (the temperature is 25 ℃, and the ultrasonic power is 100 Hz); and (3) after suction filtration and drying, placing the mixture in a muffle furnace for secondary calcination and fixation (300 ℃,1 h), cooling to room temperature, grinding and sieving with a 100-mesh sieve to obtain magnesium salt Modified Biochar (MBC).

Scanning electron microscope observation is carried out on the prepared rice hull biochar and the magnesium salt modified biochar, and the result is shown in figure 1.

FIG. 1 is a scanning electron micrograph of rice hull biochar BC (a, b, c) and MBC (d, e, f) at two magnifications. As can be seen from fig. 1 (a) and (d), BC is a tubular structure as a whole, and has a regular shape, and a large number of pores exist on the surface, so that the BC has a large specific surface area. MBC FIG. 1 (d) shows a phenomenon in which voids are destroyed and somewhat collapsed before modification, and the alignment is disturbed. The original BC figure 1 (b, c) has a smooth surface without unwanted impurities, while the modified MBC has a large number of crystal grains distributed on the surface, a few cubic grains uniformly distributed on the surface, and irregular crystal aggregates at the pores, and it is judged that these crystal grains should be oxides of magnesium. MgCl for illustration ₂ The modified biochar can make MgO and other crystals attached to the surface of the biochar.

Example 2: investigation and research on reduction of antibiotic resistance gene abundance in excrement by magnesium salt modified charcoal material

1. Test materials:

compost raw materials used in the research comprise chicken manure and sawdust (the sawdust is mainly used for adjusting the carbon-nitrogen ratio), and rice hull Biochar (BC) and magnesium salt Modified Biochar (MBC) prepared in example 1 are used as compost additives.

The specific basic physicochemical properties of the composting raw material in this study are shown in table 1.

Table 1: physicochemical Properties of compost raw Material

2. And (3) test treatment:

a total of 3 treatment groups (table 2) were set, respectively:

treatment 1: taking chicken manure and sawdust as composting materials, and naming the materials as CK;

and (3) treatment 2: taking chicken manure, sawdust and rice hull Biochar (BC) as composting materials, wherein the addition amount of the rice hull biochar is 2 percent of the weight of the chicken manure, and the materials are named as BC2;

and (3) treatment: taking chicken manure, sawdust and magnesium salt Modified Biochar (MBC) as composting materials, wherein the addition amount of the magnesium salt modified biochar is 2 percent of the weight of the chicken manure and is named as MBC2;

table 2: test treatment set-up

The weight of the chicken manure used in each treatment is 10kg (fresh weight), and the properties of the chicken manure have no significant difference and are comparable. The C/N ratio of the compost material in each treatment group was adjusted to 20:1 with sawdust. Adding proper amount of distilled water to regulate the water content of the compost material in each treating group to 60%, and repeating three times for each treating set.

Aerobic composting was carried out using a rectangular foam box (length x width x height =57cm x 42cm x 30 cm) with a working volume of 70L, and two ventilation holes (2 cm x 2 cm) were made in the top and sides of the foam box, respectively, to ensure that oxygen could enter through natural ventilation during composting. Composting took 45 days. During composting, the temperature of the compost body is measured by a thermometer every day, and the compost is turned and supplemented periodically to ensure that oxygen and moisture are sufficient until the composting is finished. The stack was sampled on days 0, 1, 5, 14, 22, 37, 45. Sampling points are positioned at the periphery and the middle of the box body (five-point sampling method), the collected samples are uniformly mixed, each treatment is carried out with repeated sampling of 50g, each treated three repeated samples are uniformly mixed, namely each treatment is carried out with total sampling of 150g, and the collected samples are stored in a refrigerator with the temperature of-20 ℃ for DNA extraction.

Quantitative detection of ARGs and MGEs

(1) Preparation of reagent and culture medium

2mol/L MgCl ₂ Solution: weighing MgCl ₂ Dissolving 19g of solid in deionized water, diluting to 100mL, and sterilizing at 121 ℃ for 20min for later use.

1mol/L glucose solution: weighing 18g of glucose, dissolving in deionized water, diluting to 100mL, filtering and sterilizing with a 0.22 mu m filter membrane, and standing at 4 ℃ for later use.

250mmol/L KCl solution: weighing 1.86g of KCl solid, dissolving in deionized water, and making the volume to be 100mL.

Solid SOB medium: to 900mL of deionized water were added 20g of tryptone, 5g of yeast extract and 0.5g of NaCl, and the mixture was stirred to completely dissolve the tryptone and yeast extract. Then, add 10mL 250mmol/L KCl solution, mix well, na (OH) ₂ The pH value is adjusted to 7, the volume is adjusted to 1L, and then 15g of agar powder is added. Sterilizing at 121 deg.C for 20min. After being taken out, the mixture is cooled to about 50 ℃, and then 5mL of 2mol/L MgCl is added ₂ 1mL of 100mg/L ampicillin solution, 2mL of 2 mg/mL X-gal solution and 800. Mu.L of 50mg/mL IPTG were mixed well and poured into a petri dish. After the condensation, the mixture is placed in a 37 ℃ environment in a dark place for 1 to 3 hours.

SOC culture medium: the same as the SOB medium except that no X-gal and IPTG were added, and that 20mL of 1mol/L glucose solution was added when the medium was cooled to 60 ℃ or lower after autoclaving.

(2) Recovering and purifying target gene fragment

And (3) purifying and recovering a qualitative PCR product of a target gene in the compost sample by using a TIANGEN Universal DNAPurification Kit according to an operation method of the instruction. After the completion, 5 mu L of product is sampled, electrophoresis is carried out for 25min under the conditions of 110V voltage and 80mA current, EB staining is carried out for 10min, and observation is carried out under a gel imaging system, so that each target gene fragment is successfully recovered and purified.

(3) Ligation transformation

The connection transformation comprises two parts of pMD18-T vector connection and DH5 alpha competent cell transformation, which are as follows:

the fragments of the gene of interest were ligated into the pMD18-T vector (TaKaRa) by establishing the ligation system of Table 3. Adding the components into a sterilized centrifuge tube, gently and uniformly mixing by fingers, centrifuging for a short time, and placing the system in a 16 ℃ environment for reacting for 1h.

Table 3: connecting system assembly

Taking out the competent cells, placing the competent cells in an ice bath for unfreezing, taking all the ligation carrier products, adding the ligation carrier products into DH5 alpha cells filled with 50-100 mu L, flicking and uniformly mixing, then carrying out ice bath for 30min, then carrying out water bath at 42 ℃ for 60-90 s, immediately carrying out ice bath for 2-3 min, and not shaking a centrifuge tube during the ice bath. Then, 600. Mu.L of SOC culture medium preheated to 37 ℃ is added into the centrifuge tube, and the mixture is subjected to shaking culture for 1h under the conditions of 37 ℃ and 180rpm of rotation speed, so that the related resistance marker genes on the plasmids are fully expressed. Then, the transformed DH 5. Alpha. Was applied to SOB medium containing the corresponding antibiotic and cultured in the dark at 37 ℃ for 12 to 16 hours under inversion. Then, the mixture was placed in a refrigerator at 4 ℃ to observe color development. Selecting white bacterial colonies in a centrifuge tube, adding SOC for culturing for 3-5 h, then taking 1mL of culture bacterial liquid, sending the culture bacterial liquid to Shanghai biological engineering Limited company, detecting whether an inserted target gene fragment exists, and using the rest bacterial liquid to extract plasmids.

(4) Extraction and identification of plasmids

The Plasmid was extracted using the TIAN Pure Midi Plasmid Kit (Kangji century) according to the procedures of the instructions.

The plasmid is detected by a trace nucleic acid protein analyzer, the quality purity is judged by the indicating values of A260/A280 and A260/A230, the ratio of A260/A280 is ensured to be 1.6-2.0, the ratio of A260/A230 is more than 2.0, and further purification or re-extraction is needed if the plasmid does not reach the standard.

(5) Preparation of the Standard Curve

And diluting the extracted plasmid by taking 10 times as gradient to prepare the series concentration of plasmid standard products. And (3) amplifying by taking each dilution gradient of the plasmid standard as a template, and drawing a standard curve by taking the logarithm of the initial copy number as an abscissa and the Ct value as an ordinate. The slope of the standard curve is required to be between-3 and-3.5, and the correlation coefficient R ² Is more than 0.99, and ensures that the amplification efficiency is between 90 and 120 percent.

The gene copy number was calculated as:

in the formula c ₀ Indicates the initial concentration of plasmid DNA (. Mu.g/. Mu.L), and X is the size of the desired gene fragment.

(6) Content detection of resistance genes in compost samples

And (3) taking the extracted sample DNA as a template to carry out a real-time fluorescent quantitative PCR experiment. The reaction system is shown in Table 4, and the reaction procedure is as follows: pre-denaturation at 95 ℃ for 15min; denaturation 95 ℃ below zero, 30s, annealing temperature, 1min, extension 72 ℃,30s,45 cycles.

Table 4: qPCR reaction system

And (4) according to the Ct value of each compost sample obtained by detection, substituting the Ct value into a standard curve to obtain the initial copy number of the sample, namely the absolute abundance.

3. And (3) test results:

fig. 2 shows the changes in absolute abundance of ARGs and MGEs before and after composting, and it can be seen from the figure that MBC2 has a significant effect of reducing sulfonamide resistance genes (sul 2, dfrA1, dfrA 7), macrolide resistance genes (ermF, ermB), and MGEs integrant gene (intI 1) as a whole, and the absolute abundance thereof is significantly lower than that of the control group. Besides the ermB gene, the absolute abundance of the sulfanilamide, macrolide resistance genes and the integrant genes of the other MBC2 treatment groups is also obviously lower than that of the BC2 treatment group. The absolute abundance of sul1, sul2, dfrA7 and ermF genes in the BC2 treated group is obviously higher than that of the control group. In conclusion, the use of 2% of MBC as a compost additive can obviously promote the reduction of the abundance of the ARGs, and the reduction effect of the MBC on the ARGs is obviously better than that of the common biochar.

The above description is only a preferred embodiment of the present application and is not intended to limit the present application, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, improvement and the like made within the spirit and principle of the present application shall be included in the protection scope of the present application.

Claims (9)

1. The magnesium salt modified biochar material is characterized by being prepared by the following method:

pyrolyzing rice hulls at 450-500 ℃ under a closed anoxic condition to prepare rice hull biochar; grinding and sieving the rice hull biochar to obtain charcoal powder; mixing the charcoal powder and the magnesium salt solution, carrying out ultrasonic oscillation, carrying out suction filtration, drying a filter cake, carrying out secondary calcination fixation, cooling, and grinding to prepare the magnesium salt modified charcoal material.

2. The magnesium salt modified biochar material of claim 1, wherein the magnesium salt solution is 0.42mol/L MgCl ₂ And (3) solution.

3. The magnesium salt modified biochar material of claim 1, wherein the power of ultrasonic oscillation is 100HZ, and the time of ultrasonic oscillation is 2h.

4. The magnesium salt modified biochar material of claim 1, wherein the temperature for the secondary calcination fixing is 300 ℃ and the time is 1h.

5. The use of the magnesium salt-modified biochar material of any one of claims 1-4 in (1) or (2) below:

(1) The abundance of antibiotic resistance genes in the livestock and poultry manure is reduced;

(2) And the abundance of the mobile gene elements in the livestock and poultry manure is reduced.

6. The use according to claim 5, wherein the antibiotic resistance genes comprise: sul1, sul2, dfrA1, dfrA7, ermB and ermF; the mobile genetic element comprises: intI1 and intI2.

7. A method of reducing the abundance of antibiotic resistance genes and mobile genetic elements in stool comprising the steps of:

mixing the excrement to be treated with sawdust, adding the magnesium salt modified biochar material of any one of claims 1-4, and uniformly mixing to obtain a compost material;

regulating the water content of the compost material to 55-65%, and carrying out aerobic composting for 40-45 days.

8. The method of claim 7, wherein the magnesium salt modified biochar material is added in an amount of 2% by weight of the manure to be treated.

9. The method according to claim 7, characterized in that sawdust is used to adjust the C/N ratio in the compost material to 20.

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