CN115196616B

CN115196616B - Magnesium salt modified biochar material and application thereof in reducing abundance of antibiotic resistance genes

Info

Publication number: CN115196616B
Application number: CN202210931494.XA
Authority: CN
Inventors: 王金花; 温胜芳; 姚锦乐; 刘文文; 朱鲁生; 王军; 王兰君
Original assignee: Shandong Agricultural University
Current assignee: Shandong Agricultural University
Priority date: 2022-08-04
Filing date: 2022-08-04
Publication date: 2023-08-11
Anticipated expiration: 2042-08-04
Also published as: CN115196616A

Abstract

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

Description

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

Technical Field

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

Background

The antibiotic is used widely in preventing and treating various diseases in animal and fowl raising industry, and has important effect in promoting animal growth and development and lowered cost. With the wide use of antibiotics in the poultry industry, the pressure caused by fecal excretion to the environment is increasing. Under the environmental pressure of long-term antibiotic administration, antibiotics induce the production of Antibiotic Resistance Genes (ARGs) and drug-resistant bacteria by animal intestinal bacteria, making livestock manure an important reservoir of antibiotics and ARGs. ARGs mean that microorganisms have resistance to antibiotics and have a certain resistance to the inhibitory and lethal effects of antibiotics, and can perform growth and reproductive activities. The antibiotics and ARGs can be discharged out of the body along with the excrement of livestock and poultry, so that the antibiotics and ARGs enter water, soil and atmosphere, stress is generated on microbial drug resistance in the environment, ecological pollution is caused, and human health is endangered. It has been found that raising chickens with a certain concentration of antibiotics results in the presence of tetracycline resistant strains in the manure of the chickens, and that the abundance of the sulfanilamide resistance genes sul1, sul2 in the pig manure is closely related to the use of high doses of antibiotics. As the intestinal tract is discharged, resistant bacteria (ARB) resistant to antibiotics in human or animal feces are transmitted in various environments by gene level transfer and the like. There have been extensive studies demonstrating a significant increase in ARGs abundance in soil where manure is applied for long periods of time, particularly for tetracycline and sulfonamide resistance genes. Wu Dan et al found that the detection rate of tetracycline resistance genes (tetT 1, tetA1, tetM 2), quinolone resistance genes (qnrB 1, qnrS 1) and sulfonamide resistance genes (sul 1, dfrA 1) in the livestock and poultry feces of the Beijing farm all exceeded 90%. Livestock manure is often applied to soil as an organic fertilizer in agricultural production, and a Movable Gene Element (MGEs) contained in the livestock manure is a main driving force for changing the abundance of ARGs, so that a certain risk is caused to the ecological environment of the soil. There are studies showing that the integron intI1 has a significant positive correlation with a variety of ARGs during cow dung composting, intI1 being the main driving force for ARGs changes in the compost. ARGs are novel environmental pollutants, have hereditary property, can be self-replicated, have durability and difficult degradability in the environment, and bring great risks to the ecological environment and human health. The World Health Organization (WHO) report has pointed out that about 200 or more tens of thousands of americans are infected with a drug-resistant pathogen each year, and 23000 more people die. The serious consequences of the health problems that ARGs can cause are the urgent need to take measures to control the further spread of ARGs.

ARGs and ARB contained in livestock and poultry manure become the greatest threats of applying the livestock and poultry manure as organic fertilizers to farmlands, and in order to reduce the spread and spread of the ARGs caused by the livestock and poultry manure, many scholars begin to study and utilize biotechnology to cut down the ARGs before recycling the manure so as to control 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 organics in solid waste into stable humus. The compost not only can recycle and innocently treat the solid waste, but also can obtain a large amount of organic fertilizer, 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 not only can rationalize and utilize the livestock manure resources, but also can rapidly degrade the residual antibiotics in the manure, prevent ARGs from being induced again in the composting process, reduce the harm of the ARGs in the livestock manure to the ecological environment, and is an important technology for reducing the spatial distribution of a plurality of ARGs in the livestock manure.

In recent years, many domestic and foreign scholars research and found that adding Biochar (BC) in compost has a certain effect on removing ARGs. The biological carbon is added into pig manure compost such as He, so that the total number of ARGs is reduced by 16.83% +/-4.10%. The addition of corn stalk biochar in chicken manure compost by Zhou et al has been found to inhibit the spread and diffusion of ARGs. However, since BC is different in raw material, specific surface area, pore structure, type and property of surface group are also different, and there is a problem that adsorption amount is small and removal effect on ARGs is poor. Therefore, it is necessary to further modify BC to increase the adsorption force and change the chemical properties.

Disclosure of Invention

Aiming at the prior art, the application 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 above purpose, the application adopts the following technical scheme:

in a first aspect of the application, a magnesium salt modified biochar material is provided, which is prepared by the following method:

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

Preferably, the magnesium salt solution is MgCl of 0.42mol/L ₂ A 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 application, there is provided the use of the magnesium salt modified biochar material as described in (1) or (2):

(1) Reducing the abundance of antibiotic resistance genes in the livestock and poultry manure;

(2) Reduces the abundance of movable gene elements in the livestock and poultry feces.

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

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

the water content of the composting material is regulated to 55-65%, and aerobic composting is carried out for 40-45d.

Preferably, the magnesium salt modified biochar material is added in an amount of 2% of the weight of the feces to be treated.

Preferably, the C/N ratio in the compost material is adjusted to 20:1 with sawdust.

The application has the beneficial effects that:

the application utilizes magnesium salt to modify the biochar to prepare magnesium salt modified biochar. The magnesium salt modified biochar is used as an additive of aerobic composting, so that the absolute abundance of antibiotic resistance genes and movable gene elements in the livestock manure can be reduced, and ARGs in the manure can be obviously reduced. The application has simple operation and low manufacturing cost, can realize large-scale industrialized production, and is beneficial to reducing ARGs in the manure.

Drawings

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

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

Figure 2 shows the change in absolute abundance of ARGs and MGEs 45 days after composting.

Detailed Description

It should be noted that the following detailed description is illustrative and is intended to provide further explanation of the application. 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 above, ARGs and ARBs contained in livestock manure become one of the greatest threats for their application as organic fertilizers to farmlands. Aerobic composting is an important technology for reducing the spatial distribution of a plurality of ARGs in livestock and poultry manure. In the earlier study, it was found that the addition of biochar to compost had a certain effect on the removal of ARGs. But 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 pore structures of the prepared biomass charcoal are also greatly different; even if the biomass raw materials are the same, the change of factors such as pyrolysis temperature, pyrolysis time, oxygen ventilation amount, water content and the like can cause the change of the structure and the composition of the biomass charcoal. Therefore, the biomass charcoal prepared by different methods has larger performance difference, and the effect of removing ARGs in the composting process is also obviously different. Some biomass charcoal is even unfavorable for the removal of ARGs in the aerobic composting process.

Based on the method, the biomass charcoal is modified, so that the biomass charcoal can stably and effectively reduce the absolute abundance of antibiotic resistance genes and movable gene elements in the livestock manure in the aerobic composting process, thereby obviously reducing ARGs in the manure.

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

Example 1: preparation of magnesium salt modified biochar material

1. Preparation of 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 the rice hulls in a closed container, placing the closed container 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 husk charcoal (BC).

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

weighing the rice husk charcoal prepared above, and placing into MgCl of 0.42mol/L ₂ In the solution, the mass (g) of the rice husk biochar and MgCl ₂ The volume (mL) ratio of the solution is 1:10, and the ultrasonic oscillation is carried out for 2 hours (the temperature is 25 ℃ and the ultrasonic power is 100 Hz); and (3) carrying out suction filtration, drying, placing in a muffle furnace, carrying out secondary calcination and fixation (300 ℃ for 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 rice husk biochar and the magnesium salt modified biochar prepared in the above way, and the result is shown in figure 1.

FIG. 1 is a scanning electron microscope image of two kinds of rice hull biochar BC (a, b, c) and MBC (d, e, f) at a magnification. As can be seen from fig. 1 (a) and (d), BC is a tubular structure as a whole, and is regular in shape, and has a large number of pores on the surface, so that it has a large specific surface area. MBC FIG. 1 (d) destruction of voids prior to modificationSome collapse and disorder of arrangement occurs. The surface of the original BC in FIG. 1 (b, c) is smooth and free of redundant impurities, while the modified MBC has a large number of crystal particles distributed on the surface, some cubic particles are uniformly distributed on the surface, and irregular crystal aggregates are arranged at the pores, and the crystal particles are judged to be magnesium oxide. MgCl for illustration ₂ The modified biochar can enable MgO and other crystals to be attached to the surface of the biochar.

Example 2: investigation and research on reducing antibiotic resistance gene abundance in feces by magnesium salt modified biochar material

1. Test materials:

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

The specific basic physicochemical properties of the compost raw material for this study are shown in Table 1.

Table 1: physicochemical Properties of compost Material

2. Test treatment:

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

treatment 1: chicken manure and sawdust are used as compost materials and named CK;

treatment 2: taking chicken manure, sawdust and rice hull charcoal (BC) as compost materials, wherein the addition amount of the rice hull charcoal is 2% of the weight of the chicken manure, and the added amount is named BC2;

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

table 2: test treatment setup

The weight of the chicken manure used in each treatment is 10kg (fresh weight), and the properties of the chicken manure are not obviously different, so that the chicken manure is comparable. The C/N ratio of the compost material of each treatment group was adjusted to 20:1 with sawdust. The water content of the compost material of each treatment group was adjusted to 60% by adding an appropriate amount of distilled water, and each treatment was set to three replicates.

Aerobic composting is carried out by using a rectangular foam box (length multiplied by width multiplied by height=57 cm multiplied by 42cm multiplied by 30 cm) with the working volume of 70L, and two ventilation holes (2 cm multiplied by 2 cm) are respectively formed on the top and the side surface of the foam box, so that oxygen can enter through natural ventilation in the composting process. Composting takes place for 45 days. The temperature of the pile body is measured by a thermometer every day during composting, and pile turning and water supplementing are carried out regularly, so that sufficient oxygen and water are ensured until the composting is finished. The stacks were subjected to sample collection on days 0, 1, 5, 14, 22, 37, 45. The 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 repeatedly sampled by 50g, three repeated samples of each treatment are uniformly mixed, namely, each treatment is uniformly sampled by 150g, and the collected samples are stored in a refrigerator at the temperature of minus 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 The solid 19g is dissolved in deionized water, the volume is fixed to 100mL, and the sterilization is carried out for 20min at 121 ℃ for later use.

1mol/L glucose solution: 18g of glucose is weighed and dissolved in deionized water, the volume is fixed to 100mL, and the solution is filtered and sterilized by a 0.22 mu m filter membrane and is placed at 4 ℃ for standby.

250mmol/L KCl solution: 1.86g of KCl solid was weighed and dissolved in deionized water to a volume of 100mL.

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

SOC medium: substantially the same as SOB medium except that X-gal and IPTG were not added, and 20mL of 1mol/L glucose solution was added after autoclaving and cooling to 60℃or below.

(2) Target gene fragment recovery and purification

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

(3) Ligation transformation

Ligation transformation includes both pMD18-T vector ligation and DH 5. Alpha. Competent cell transformation, and is described in detail as follows:

fragments of the gene of interest were ligated into the pMD18-T vector (TaKaRa) by establishing the ligation system of Table 3. The components are added into a sterilized centrifuge tube, mixed evenly by flicking with fingers, and the system is placed in an environment of 16 ℃ for reaction for 1h after short centrifugation.

Table 3: connection system composition

And taking out competent cells, thawing in ice bath, adding all the connected carrier products into DH5 alpha cells with the concentration of 50-100 mu L, flicking and uniformly mixing, 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 medium preheated at 37℃was added to the centrifuge tube, and the culture was performed under shaking at 37℃and 180rpm for 1 hour to allow the relevant resistance marker gene on the plasmid to be expressed sufficiently. Afterwards, the transformed DH5 alpha is coated on SOB culture medium containing corresponding antibiotics, and is cultivated for 12-16 hours in an inverted dark state at 37 ℃. Then, the mixture was placed in a refrigerator at 4℃to observe the color development. Selecting white bacterial colony in a centrifuge tube, adding SOC for culturing for 3-5 h, then taking 1mL of culture bacterial liquid, delivering to Shanghai biological engineering Co., ltd, detecting whether the inserted target gene fragment exists, and extracting plasmids from the rest bacterial liquid.

(4) Extraction and identification of plasmids

The plasmid was extracted using a TIAN Pure Midi Plasmid Kit (well known as century) kit and was prepared according to the procedures described in the specification.

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

(5) Preparation of a Standard Curve

The extracted plasmid was diluted with a gradient of 10-fold to prepare a series of concentrations of plasmid standard. 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 between-3 and-3.5, and the correlation coefficient R ² The amplification efficiency is ensured to be between 90 and 120 percent when the amplification efficiency is more than 0.99.

The gene copy number was calculated as:

in c ₀ The initial concentration of plasmid DNA (. Mu.g/. Mu.L) is shown, X being the size of the gene fragment of interest.

(6) Content detection of resistance genes in compost samples

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

Table 4: qPCR reaction system

And (3) according to the Ct value of each compost sample obtained by detection, carrying the Ct value into a standard curve to obtain the initial copy number of the sample, namely absolute abundance.

3. Test results:

FIG. 2 shows the absolute abundance changes of ARGs and MGEs before and after composting, and it can be seen from the figure that MBC2 has a significant reduction effect on sulfonamide resistance genes (sul 2, dfrA1, dfrA 7), macrolide resistance genes (ermF, ermB) and MGEs integrant genes (intI 1) as a whole, and the absolute abundance is significantly lower than that of the control group. Besides the ermB gene, absolute abundance of sulfonamides, macrolide resistance genes and integrins of the rest MBC2 treated groups are also significantly lower than that of BC2 treated groups. Whereas the absolute abundance of the sul1, sul2, dfrA7, ermF genes was significantly higher in the BC2 treated group than in the control group. In conclusion, the MBC with the addition amount of 2 percent can obviously promote the reduction of ARGs abundance as a composting additive, and the reduction effect of the MBC on ARGs is obviously superior to that of common biochar.

The above description is only of the preferred embodiments of the present application and is not intended to limit the present application, but various modifications and variations can be made to the present application by those skilled in the art. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application should be included in the protection scope of the present application.

Claims

1. The application of the magnesium salt modified biochar material in the following (1) or (2):

(2) The abundance of movable gene elements in the livestock and poultry feces is reduced;

the antibiotic resistance genes include: sul1, sul2, dfrA1, dfrA7, ermB and ermF; the mobile genetic element comprises: intI1 and intI2;

the magnesium salt modified biochar material is prepared by the following method:

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

the magnesium salt solution is MgCl of 0.42mol/L ₂ A solution;

the power of ultrasonic oscillation is 100HZ, and the ultrasonic oscillation time is 2h;

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

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

mixing the feces to be treated with sawdust, adding the magnesium salt modified biochar material according to claim 1, and uniformly mixing to obtain a compost material;

the water content of the composting material is regulated to 55-65%, aerobic composting is carried out, and the composting time is 40-45d;

the adding amount of the magnesium salt modified biochar material is 2% of the weight of the feces to be treated;

the C/N ratio in the compost material was adjusted to 20:1 with sawdust.