CN114315441B - Method for strengthening removal of antibiotic resistance genes in aerobic compost - Google Patents
Method for strengthening removal of antibiotic resistance genes in aerobic compost Download PDFInfo
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Abstract
The invention discloses a method for strengthening removal of antibiotic resistance genes in aerobic compost, which comprises the following steps: 1. uniformly mixing the organic solid waste and auxiliary materials; 2. carrying out aerobic composting on the mixed material; 3. after the compost high-temperature period is finished, introducing ozone into the compost system when the compost enters the cooling period, and normally finishing the cooling and rotting period of the compost. The method can effectively reduce the ARGs by utilizing the higher compost body temperature in the compost high-temperature period, and further reduces the ARGs in the compost materials by combining the ozone oxidation technology aiming at the problem of the increase of the abundance of the ARGs in the compost maturity period, thereby finally reducing the abundance of the ARGs in the compost products and reducing the health risk generated after the compost soil is utilized. The technology can also reduce the abundance of potential host bacteria of the ARGs in the compost material, and can also obviously weaken the jointing and transforming capacity of microorganisms in the compost material, thereby reducing the risk of vertical gene transfer and horizontal gene transfer of the ARGs.
Description
Technical Field
The invention belongs to the field of organic solid waste treatment, and relates to a method for strengthening removal of antibiotic resistance genes in an aerobic composting system.
Background
In the large-scale and intensive livestock and poultry farming industry, antibiotics are often added to feed in sub-therapeutic doses in order to treat and prevent diseases, promote animal growth, and improve feeding efficiency. The antibiotic yield in 2013 years in China is about 16.2 ten thousand tons, about 8.4 ten thousand tons are used for livestock, and 52 percent of the antibiotic yield is consumed by the pig industry. However, antibiotics added to feed are difficult to absorb and metabolize by the animal's intestinal tract, and 58% of the antibiotics are excreted as their parent compounds or primary metabolites through feces and urine. The residual non-metabolized antibiotics in the livestock manure become important sources of antibiotics in the environment, and can cause the generation of antibiotic resistant microorganisms and Antibiotic Resistant Genes (ARGs) in the manure, so that the livestock manure becomes an important storage bank of the ARGs. The resistant strains containing the ARGs in the livestock manure are one of the most important sources of the ARGs in soil and underground water, and the discharge of a large amount of livestock manure directly causes non-point source pollution of the ARGs.
In 2004, the american scientists Rysz and Alvarez proposed Antibiotic Resistance Genes (ARGs) as a new type of environmental pollutant. Bacteria can obtain ARGs in a variety of ways, including vertical gene transfer and horizontal gene transfer. Among them, there is a clear difference between the effect of Intracellular DNA (iDNA) and extracellular DNA (eDNA) on the environment and the transfer mechanism of ARGs carried by it. Vertical gene transfer is the transfer of modified genetic information located on a bacterial chromosome into daughter cells. Horizontal gene transfer mechanisms include conjugation, transduction, and transformation, which is the process by which extracellular DNA is taken up by competent cells, resulting in recipient cells containing genetic traits for the absorbed DNA. Transduction and conjugation are processes by which intracellular DNA is mediated by phage or bacteria through flagella and transferred to recipient microorganisms.
Aerobic high-temperature composting is one of the main approaches for harmless treatment and resource utilization of livestock and poultry manure, can reduce the environmental pollution of organic wastes, and can recycle compost products in agricultural production, so that the aerobic high-temperature composting is widely concerned. Researches show that aerobic composting can also be used as a main way for controlling the risks of antibiotics and ARGs in livestock and poultry excrement, however, because composting is completed by the joint participation of various microorganisms and is greatly influenced by composting conditions and materials, the changes of different types of antibiotics and ARGs in different researches have obvious differences. However, the reduction of ARGs by conventional aerobic composting techniques is still controversial, and a lot of researchers propose that conventional aerobic composting techniques cannot effectively control the proliferation and diffusion of ARGs and Mobile Genetic Elements (MGEs), so that it is important to develop more effective techniques to promote the removal of ARGs in the composting process.
The abundance of ARGs increases during the composting period and its changes are significantly related to the structure of the microbial population in the compost material. Actinomycetes door (Actinobacteria) And (of Deformable bacteria phylum: (Proteobacteria) Are potential host bacteria of the ARGs and are the most predominant phylum level bacteria in the composting stage, so antibiotic-resistant bacteria inevitably increase in the composting stage, causing the diffusion of the ARGs at this stage. The rebound of these ARGs is a key factor contributing to the difference in the effect of ARGs reduction at the end of composting. Therefore, the development of a proper technical means for pertinently controlling the cooling period and the decomposing period ARGs is urgently neededTo enhance the removal of ARGs, but currently there is less research on this.
Disclosure of Invention
The invention aims to provide a method for strengthening and removing antibiotic resistance genes in aerobic composting, which is based on the traditional aerobic composting technology and combines an ozone oxidation technology to obviously reduce ARGs in composting samples, reduce potential risks to human health after land utilization, and can not influence the smooth proceeding of the composting process and reduce the quality of composting products.
The purpose of the invention is realized by the following technical scheme:
an enhanced removal method of antibiotic resistance genes in aerobic compost comprises the following steps:
step one, uniformly mixing organic solid waste and auxiliary materials to obtain a mixed material, wherein:
the organic solid waste is animal manure;
the auxiliary materials are common agricultural solid wastes (such as one or more of wood chips, straws, rice husks and the like) or garden wastes;
aerobic composting needs proper water content, C/N ratio and oxygen supply amount to ensure composting, conditioners such as sawdust, straws, rice hulls and garden wastes are added to effectively adjust a carbon source of composting materials, the water content is reduced, the porosity of a compost body is increased, and the composting process is optimized, generally, the mixing ratio of organic solid wastes and auxiliary materials is determined according to the water content and the carbon-nitrogen ratio of the mixing materials, the water content and the carbon-nitrogen ratio of the aerobic composting reach the optimal range by calculating a reasonable ratio, namely the water content is 45% -65% and the carbon-nitrogen ratio (C/N) is 20 to 40:1;
step two, performing aerobic composting on the mixed material obtained in the step one, wherein:
generally speaking, the temperature of the aerobic composting in the heating period is between the temperature of the initial mixed material and 50 ℃, and the temperature of the aerobic composting in the heating period needs to be more than 55 ℃ for 3 days or more than 50 ℃ for 5 to 7 days, so that the harmful microorganisms in the material can be effectively killed, and the material is harmless;
in the aerobic composting process, the water content and the carbon-nitrogen ratio are not additionally controlled, and the aeration air volume is controlled to be 0.05 to 0.2m 3 Min (based on materials per cubic meter), adjusting the pile turning frequency according to the pile body temperature and the discharging condition in actual operation;
and step three, after the compost high-temperature period is finished, when the temperature is reduced to 40-47 ℃ and enters a cooling period, introducing ozone into a compost system, normally finishing the cooling and rotting periods of the compost, and meeting the requirements of measuring the abundance of total resistance genes, intracellular and extracellular resistance genes and the joint and transformation capacity of microbial communities in the sample after the rotting period, wherein:
leading ozone into the compost from the bottom of the compost under positive pressure, wherein the total adding amount of the ozone is 0.2-0.6 g/kg of compost material, the whole ozone adding time is 30-150min, and the air volume and concentration of the ozone are adjusted according to the ozone adding time;
when the temperature of the stack is reduced to room temperature, the stack reaches the rotten stage, and the rotten stage time is generally required to be 7 days or more;
the aeration quantity in the cooling and rotting period is consistent with that in the heating period and the high-temperature period;
the resistance gene comprising tetracyclines (tetM、 tetLAnd tetW）sulfonamides (a)sul1、sul2Andsul3) Macrolides (a) and (b)ermB、ermCAndermF) And quinolones (c)aac(6’)-Ib-crAndgyrA）。
compared with the prior art, the invention has the following advantages:
1. the method can effectively reduce the ARGs by utilizing the higher compost body temperature in the compost high-temperature period, and further reduces the ARGs in the compost materials by combining the ozone oxidation technology aiming at the problem of the increase of the abundance of the ARGs in the compost maturity period, thereby finally reducing the abundance of the ARGs in the compost products and reducing the health risk generated after the compost soil is utilized. The technology can also reduce the abundance of potential host bacteria of the ARGs in the compost material, and can also obviously weaken the jointing and transforming capacity of microorganisms in the compost material, thereby reducing the risk of horizontal gene transfer of the ARGs.
2. The method can effectively reduce antibiotic resistance genes in compost materials, does not influence compost maturity, does not reduce the quality of compost products, and can be used for producing organic fertilizers from the final compost products.
Drawings
FIG. 1 is a graph showing the absolute abundance change of the antibiotic resistance gene in the ozone-treated and control groups at the end of composting;
FIG. 2 is a graph of the absolute abundance of mobile genetic elements in ozone-treated and control groups at the end of composting;
figure 3 is a graph of the frequency of engagement and transformation of microflora in the ozone treatment and control groups at the end of composting.
Detailed Description
The technical solutions of the present invention are further described below with reference to the following examples, but the present invention is not limited thereto, and any modifications or equivalent substitutions may be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention.
Example 1
The embodiment provides a method for strengthening removal of antibiotic resistance genes in aerobic compost, which comprises the following specific steps:
step one, completely mixing the livestock and poultry manure and the corn straws according to the proportion of 5.
And step two, performing aerobic composting on the mixed material obtained in the step one, wherein the heating period and the high temperature period of the aerobic composting are required to be completed smoothly (the temperature of a compost body is more than 55 ℃ and lasts for 3 days or more than 50 ℃ and lasts for 5 to 7 days). The water content and carbon-nitrogen ratio are not adjusted in the composting process, and the aeration amount is controlled to be 0.08L min -1 kg -1 And (5) stacking, wherein the stack is manually turned once every 5 days in the heating period and the high-temperature period.
Step three, when the temperature of the compost at the end of the high-temperature period is reduced to 40 ℃ (the temperature reduction period), introducing ozone into the compost system by adopting an ozone generator, wherein the total adding amount of the ozone is 0.4g kg -1 Composting material with ozone flow rate of 3L min -1 The ozone content is 40mg L -1 kg -1 The reaction time was 30min. Then the temperature of the pile is reduced and the pile is decomposed normally (the decomposing period needs 7 days), the aeration rate is consistent with the temperature rise period and the high temperature period, and the temperature is reducedAnd manually turning the pile once every 7 days in a warm period and a decomposition period. And (3) measuring the abundance of total resistance genes, intracellular and extracellular resistance genes and the connection and transformation capability of microbial communities in samples of the experimental group and the control group at the end of composting. The resistance gene comprising tetracyclines (tetM、 tetLAnd tetW）sulfonamides (a)sul1、sul2Andsul3) Macrolides (a) and (b)ermB、ermCAndermF) And quinolones (c) ((c))aac(6’)-Ib-crAndgyrA）。
example 2
In this example, the compost raw material used in the compost test was pig manure, which was obtained from a large pig farm in halbant, and had a water content of about 72%, and the conditioner was corn stalks and had a water content of about 8.5%. This example was carried out in a laboratory and monitored for tetracyclines (IV)tetM、 tetLAnd tetW）sulfonamides (a)sul1、sul2Andsul3) Macrolides (a) and (b)ermB、ermCAndermF) And quinolones (c)aac(6’)-Ib-crAndgyrA) While monitoring mobile genetic elementsintI1AndIS613. The method comprises the following specific steps:
step one, completely mixing pig manure (with the water content of 72%) and corn straw (with the water content of 8.5%) according to the proportion of 5.
Step two, placing the mixed material into a composting reactor for aerobic composting, wherein the water content of the material is not regulated in the composting process, and the aeration amount is controlled to be 0.08L min -1 kg -1 And (4) stacking, wherein the stack is manually turned once every 5 days in the temperature rise period and the high temperature period, and the stack is manually turned once every 7 days in the temperature reduction period and the decomposition period.
And step three, when the composting process shows obvious temperature rise period, high temperature period, temperature reduction period and rotten period, and the temperature of the compost is more than 55 ℃ and lasts for 3 days or more than 50 ℃ and lasts for 5 to 7 days, the high temperature period can be considered to be smoothly completed. When the temperature of the compost is reduced to 40 ℃, an ozone generator is adopted to introduce ozone into the compost system, and the total adding amount of the ozone is 0.4g kg -1 Composting material with ozone flow rate of 3L min -1 The ozone content is40mg L -1 kg -1 The reaction time was 30min.
And step four, after the ozone oxidation is finished, cooling and decomposing the pile (the decomposing period needs 7 days), wherein the aeration quantity is consistent with the heating period and the high-temperature period.
And step five, determining the abundance of total resistance genes, intracellular and extracellular resistance genes and the connection and transformation capability of microbial communities in the samples of the experimental group and the samples of the control group after composting is finished.
At the end of composting, there was a clear difference between intracellular and extracellular ARGs in the experimental and control groups. As can be seen from FIG. 1, the absolute abundance of total ARGs in the control group was 4.84X 10 8 Copies g -1 DW 1.33X 10 higher than that in the experimental group 8 Copies g -1 DW. At the end of composting the extracellular ARGs in the test group were 0.041 logs lower than the control group and the intracellular ARGs were 2.02 logs lower than the control group. Thus, the ozone treatment can effectively reduce the total ARGs and the intracellular ARGs at the end of composting. After the ozone treatment, only 4 target ARGs are detected at the end of composting, such as aac (6') -ib-cr, sul3 and the like, which shows that the ozone post-treatment can effectively reduce the types of the iARGs, further control the rebound of the ARGs at the later stage of composting and reduce the risk of transferring the level of the iARGs to other microorganisms and pathogenic bacteria.
At the end of composting, there was a difference in the intracellular and extracellular distribution of the two MGEs detected. As can be seen from FIGS. 2 and 3, the ozonated group removed all intracellular MGEs and also reduced extracellular int 1. In addition, the ozone-treated group had a significantly lower frequency of junction at the end of composting than the control group by 18.3 ± 4.3%, P, relative to the control group<0.001, and the transformation frequency is also significantly lower than that of the control group by 6.7 +/-1.4 percent, P<0.05. The cooling period of the compost is 0.4g kg -1 The ozone treatment of (a) can significantly reduce the ability of microbial communities to engage and convert ARGs at the end of composting, thereby mitigating ecological and environmental risks caused by microbial transfer through horizontal gene transfer at the end of composting.
In conclusion, the invention can not only effectively reduce the ARGs by utilizing the higher temperature of the compost in the high-temperature period of the compost, but also further reduce the ARGs in the compost material by combining the ozone oxidation technology aiming at the problem of the increase of the abundance of the ARGs in the composting period, inhibit the rebound phenomenon of the ARGs in the composting period, finally reduce the abundance of the ARGs in the compost product, and reduce the jointing and transformation frequency of microbial communities at the end of the composting, thereby reducing the health risk generated after the land utilization of the compost.
Claims (10)
1. The application of an aerobic composting method in the reinforced removal of antibiotic resistance genes is characterized in that the aerobic composting method comprises the following steps:
step one, uniformly mixing organic solid waste and auxiliary materials to obtain a mixed material;
step two, performing aerobic composting on the mixed material obtained in the step one;
and step three, after the composting high-temperature period is finished, when the temperature is reduced to 40-47 ℃, introducing ozone into a composting system, wherein the total adding amount of the ozone is 0.2-0.6 g/kg of composting material, and after ozone oxidation is finished, the compost normally finishes the cooling and rotting period.
2. The use of the aerobic composting process of claim 1 for the enhanced removal of antibiotic resistance genes wherein in step one the organic solid waste is livestock manure.
3. The use of the aerobic composting process as claimed in claim 1, wherein in step one the auxiliary material is agricultural solid waste or garden waste.
4. The use of the aerobic composting method of claim 3 for the enhanced removal of antibiotic resistance genes wherein the agricultural solid waste is one or more of wood chips, straw, rice hulls.
5. The application of the aerobic composting method in the reinforced removal of antibiotic resistance genes as claimed in claim 1, wherein in the step one, the water content of the mixed material is 45% -65%, the carbon-nitrogen ratio is 20-40: 1.
6. the application of the aerobic composting method in the reinforcement removal of antibiotic resistance genes as claimed in claim 1, wherein in the second step, the temperature of the aerobic composting is between the temperature of the initial mixed material and 50 ℃, and the high temperature period is more than 55 ℃ for 3 days or more than 50 ℃ for 5 to 7 days.
7. The application of the aerobic composting method in the antibiotic resistance gene reinforcement removal as claimed in claim 1, wherein in the second step, the aeration air volume is controlled to be 0.05 to 0.2m 3 /min。
8. The application of the aerobic composting method in the antibiotic resistance gene reinforcement removal of claim 1, wherein in the third step, ozone is introduced into the compost from the bottom of the compost under positive pressure, and the whole ozone adding time is 30-150min.
9. The use of the aerobic composting process according to claim 1 for the enhanced removal of antibiotic resistance genes wherein in step three the period of maturity is 7 days and more.
10. The use of the aerobic composting method as claimed in claim 1 for the enhanced removal of antibiotic resistance genes, wherein in the third step, the aeration rate in the cooling and rotting period is controlled to be 0.05 to 0.2m 3 /min。
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