CN115073230B - 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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- CN115073230B CN115073230B CN202210572534.6A CN202210572534A CN115073230B CN 115073230 B CN115073230 B CN 115073230B CN 202210572534 A CN202210572534 A CN 202210572534A CN 115073230 B CN115073230 B CN 115073230B
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- C—CHEMISTRY; METALLURGY
- C05—FERTILISERS; MANUFACTURE THEREOF
- C05F—ORGANIC FERTILISERS NOT COVERED BY SUBCLASSES C05B, C05C, e.g. FERTILISERS FROM WASTE OR REFUSE
- C05F3/00—Fertilisers from human or animal excrements, e.g. manure
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- C—CHEMISTRY; METALLURGY
- C05—FERTILISERS; MANUFACTURE THEREOF
- C05F—ORGANIC FERTILISERS NOT COVERED BY SUBCLASSES C05B, C05C, e.g. FERTILISERS FROM WASTE OR REFUSE
- C05F17/00—Preparation of fertilisers characterised by biological or biochemical treatment steps, e.g. composting or fermentation
- C05F17/80—Separation, elimination or disposal of harmful substances during the treatment
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- C—CHEMISTRY; METALLURGY
- C05—FERTILISERS; MANUFACTURE THEREOF
- C05G—MIXTURES OF FERTILISERS COVERED INDIVIDUALLY BY DIFFERENT SUBCLASSES OF CLASS C05; MIXTURES OF ONE OR MORE FERTILISERS WITH MATERIALS NOT HAVING A SPECIFIC FERTILISING ACTIVITY, e.g. PESTICIDES, SOIL-CONDITIONERS, WETTING AGENTS; FERTILISERS CHARACTERISED BY THEIR FORM
- C05G3/00—Mixtures of one or more fertilisers with additives not having a specially fertilising activity
Abstract
The invention provides a method for strengthening and removing antibiotic resistance genes in aerobic compost, which comprises the following steps: mixing organic solid waste with composting auxiliary materials to obtain initial composting materials; adding micrometer zero-valent iron, hydrogen peroxide solution and ascorbic acid solution into an initial composting material, stirring and mixing to obtain an aerobic composting material, wherein the initial pH value of the aerobic composting material is 6-8, the initial water content is 60% -70%, and the initial carbon-nitrogen ratio is 25-30:1; aerobic composting is carried out on the aerobic composting materials, the composting time of the aerobic composting is 15-20 days, and a proper amount of water is supplemented every day in the high temperature period, so that the water content of the composting is ensured to be 60% -65%. The invention introduces ascorbic acid and micron zero-valent iron into Fenton reaction process, promotes the generation of ferrous iron Fe (II) in large quantity and rapidly, thereby greatly improving the strengthening and removing effect of antibiotic resistance genes in aerobic composting, accelerating the degradation speed of organic matters and shortening the composting period.
Description
Technical Field
The invention belongs to the field of organic solid waste treatment, and particularly relates to a method for strengthening and removing antibiotic resistance genes in aerobic composting.
Background
The antibiotic for animals is widely applied to large-scale livestock breeding as a feed additive, has the functions of preventing diseases and promoting the growth of livestock, but the selective pressure of the antibiotic can induce the generation of antibiotic resistance genes. Resistant bacteria in the animal gut are excreted with the feces, making the breeding waste an important reservoir for Antibiotic Resistance Genes (ARGs) in the environment. The absolute abundance of ARGs in animal feces can reach 10 11 ～10 13 cobies/g (dry weight). ARGs can enter pathogenic bacteria through a horizontal gene transfer mode by taking a movable gene element as a medium, so that the resistance of antibiotics to the pathogenic bacteria is reduced, and the human health is threatened.
Agricultural utilization of aerobic composting products is one of the important ways in which ARGs spread in the environment. Therefore, there is an urgent need to develop a technical study for effectively reducing the abundance of ARGs in the aerobic composting process. In addition, the long-time consumption problem of aerobic composting is always a main factor limiting the development of the aerobic composting, and a new technical method is also required to break the bottleneck.
Current research shows that the traditional aerobic composting process has a certain removal effect on ARGs, which is mainly dependent on the own characteristics of ARGs, wherein the composting high temperature period proves to have an important role in the reduction of ARGs. The ARGs removal effect can be enhanced by extending high temperature, forced aeration or adding additives such as biochar, zeolite, special bacteria, etc. For example, chinese patent application publication No. CN114315441a discloses a method for enhanced removal of antibiotic resistance genes in aerobic compost, which introduces ozone into the heap during the cooling period of the compost, and combines ozone oxidation technology to reduce the abundance of ARGs in the compost. The Chinese patent application with publication number of CN113416097A discloses a method for efficiently removing antibiotics and resistance genes in livestock manure based on an ultra-high temperature aerobic fermentation technology, which can use fermentation products of the livestock manure as good organic fertilizers. In the Chinese patent application with publication number of CN111662108A, a method for reducing the abundance of resistance genes and type I integrants in livestock manure compost is disclosed, wherein livestock manure, a compost conditioner and lignite are mixed for composting, so that the abundance of the resistance genes and type I integrants in the compost can be reduced.
However, the existing various aerobic composting technologies have limited ARGs removal capacity, and the inhibition of the aerobic composting technologies on ARGs in a high temperature period does not truly degrade the ARGs, which directly leads to obvious rebound of the abundance of the ARGs in a composting cooling period, so that the farmland utilization of the final composting product still has a great health risk. In addition, most methods for enhancing ARGs removal cannot realize rapid decomposition of compost materials, and the time-consuming problem is still unsolved.
Disclosure of Invention
In order to solve the technical problems that the prior aerobic composting method has poor ARGs removal effect, ARGs rebound in the composting cooling period and overlong composting time period.
The invention provides a method for strengthening and removing antibiotic resistance genes in aerobic compost, which comprises the following specific technical scheme:
a method for enhancing removal of antibiotic resistance genes in aerobic composting, comprising the steps of:
mixing organic solid waste with composting auxiliary materials to obtain initial composting materials;
adding micrometer zero-valent iron, hydrogen peroxide solution and ascorbic acid solution into an initial composting material, stirring and mixing to obtain an aerobic composting material, wherein the initial pH value of the aerobic composting material is 6-8, the initial water content is 60% -70%, and the initial carbon-nitrogen ratio is 25-30:1;
and (3) carrying out aerobic composting on the aerobic composting materials, turning the composting once every 2-3 days, wherein the composting time of the aerobic composting is 15-20 days, and supplementing a proper amount of water every day in a high-temperature period, so that the water content of the composting is ensured to be 60-65%.
In some embodiments, the organic solid waste is one or two of kitchen waste and livestock manure, and the composting auxiliary material is one or more of straw, rice hull and wood dust.
In some embodiments, the micrometer zero valent iron is added in an amount of 0.5% to 2% by weight of the initial compost material.
In some embodiments, the concentration of the hydrogen peroxide solution is 10-30%, and the addition amount of the hydrogen peroxide solution is 3-9% of the weight of the initial composting material.
In some embodiments, the concentration of the ascorbic acid solution is 10-50 mmol/L, and the addition amount of the ascorbic acid solution is 0.2-0.6% of the weight of the initial composting material.
The reaction mechanism for the enhanced removal of antibiotic resistance genes in aerobic compost is as follows:
two adjacent enol type hydroxyl groups in the ascorbic acid molecular structure are easy to dissociate and release hydrogen ions H + And the ascorbic acid has strong reducibility. Ascorbic acid first coordinates with ferric Fe (III) on the surface of micrometer zero-valent iron, and forms a surface ferric Fe (III) -ascorbic acid complex on the iron-containing material. Subsequently, electron transfer from ascorbic acid to surface ferric iron Fe (III) results in the formation of surface ferrous iron Fe (II). The abundant ferrous iron Fe (II) reacts with hydrogen peroxide to generate a large amount of strong oxidative hydroxyl radicals, which are themselves oxidized to ferric iron Fe (III). Further, the ascorbic acid continuously reduces the ferric iron Fe (III) to the ferrous iron Fe (II), so that the ferric iron Fe (III) on the surface of the iron material in the composting process is drivenAnd (3) high-efficiency circulation of ferrous Fe (II). The aim of decomposing ARGs and degrading organic matters is fulfilled by a large amount of strong oxidative hydroxyl free radicals generated in the circulation process. In addition, in the Fenton-like reaction process, the ascorbic acid can be oxidized and degraded, so that the risk of secondary pollution is effectively reduced.
In addition, micrometer zero-valent iron has toxic effects on most microorganisms, can destroy cell membranes, reduce the physiological activities of cells, subcellular and proteins, finally lead to apoptosis, and can fully degrade intracellular ARGs.
Compared with the existing aerobic composting method, the method for strengthening and removing the antibiotic resistance gene in the aerobic composting has the following beneficial effects:
1. the invention introduces ascorbic acid and micron zero-valent iron into Fenton-like reaction process, promotes the generation of ferrous iron Fe (II) in large quantity and rapidly, thereby greatly improving the strengthening and removing effect of antibiotic resistance genes in aerobic composting, accelerating the degradation speed of organic matters and shortening the composting period.
2. The micrometer zero-valent iron can directly destroy cell membranes of microorganisms, reduce the physiological activities of cells, subcells and proteins, and finally cause apoptosis, thereby ensuring that ARGs in cells of the microorganisms can be fully degraded, and further improving the strengthening removal effect of antibiotic resistance genes in aerobic compost.
3. Zero-valent iron is an inherent mineral substance of soil, and hydrogen peroxide and ascorbic acid are all easily-decomposed substances, so that the invention does not cause secondary pollution.
4. The method has mild reaction conditions and simple operation, and is suitable for large-scale popularization and application.
Drawings
FIG. 1 is a graph showing the temperature change during composting of examples and comparative examples of the present invention;
FIG. 2 is a graph showing the variation of germination percentage (GI) of seeds during composting according to the examples and comparative examples of the present invention;
FIG. 3 is a graph comparing the absolute abundance of ARGs during composting according to examples and comparative examples of the present invention.
Detailed Description
In order that the above-recited objects, features and advantages of the present invention will become more readily apparent, a more particular description of the invention will be rendered by reference to the appended drawings and appended detailed description.
The invention will be described in further detail with reference to the accompanying drawings and specific examples.
Example 1
The method for strengthening and removing antibiotic resistance genes in the aerobic compost in the embodiment comprises the following steps:
taking 3 kg of pig manure, 4 kg of kitchen waste, 1 kg of corn straw and 1 kg of wood dust, crushing all materials to 1-3 cm, and mixing to obtain an initial compost material.
Adding micrometer zero-valent iron, 30% hydrogen peroxide solution and 50mmol/L solution into the mixed material to obtain aerobic composting material. Wherein: the addition amount of the micrometer zero-valent iron is 0.5% of the weight of the initial compost material, the addition amount of the hydrogen peroxide solution is 3% of the weight of the initial compost material, and the addition amount of the ascorbic acid solution is 0.5% of the weight of the initial compost material. The initial pH value of the aerobic composting material is 6.31, the initial water content is 65%, and the initial carbon-nitrogen ratio is 27.
Carrying out aerobic composting on the aerobic composting materials:
the composting process is carried out in a cylindrical reactor with a diameter of 30cm and a height of 100cm. Natural ventilation is adopted, and the pile is turned over 1 time every 3 days in the aerobic composting treatment process.
The aerobic compost enters a high temperature period on the 3 rd day, and is supplemented with about 200mL of water every day, and the water content of the compost is kept at 60% -65%. And co-composting fermentation is carried out for 15 days.
Comparative example 1
The aerobic composting method of comparative example 1 is as follows:
taking 3 kg of pig manure, 4 kg of kitchen waste, 1 kg of corn straw and 1 kg of wood dust, crushing all materials to 1-3 cm, and mixing to obtain a compost material.
Wherein: pig manure, kitchen waste, corn stalks and wood chips were the same as those in example 1. For example, 6 kg of pig manure was taken from the same pig manure storage container, 3 kg of which was used in example 1 and the other 3 kg was used in comparative example 1. In this way, it is possible to ensure that the compost material of comparative example 1 has as much as possible the composition and carbon-nitrogen ratio of the initial compost material of example 1.
Correspondingly, the pH value of the compost material is adjusted to 6.31, and the initial water content is adjusted to 65%.
Aerobic composting of the compost material is implemented:
the composting process is carried out in a cylindrical reactor with a diameter of 30cm and a height of 100cm. Natural ventilation is adopted, and the pile is turned over 1 time every 2 to 3 days in the aerobic composting treatment process.
The aerobic compost enters a high temperature period on the 3 rd day, and is supplemented with about 200mL of water every day, and the water content of the compost is kept at 60% -65%. And co-composting fermentation is carried out for 15 days.
1. Composting temperature determination of example 1 and comparative example 1:
the compost heap temperature of example 1 and comparative example 1 was measured by: the average value was calculated by inserting a probe thermometer into the core and measuring each time every day in the morning and evening, and the daily room temperature was recorded.
As shown in FIG. 1, comparative example 1 reached a maximum temperature of 65℃on day 4 and example 1 reached a maximum temperature of 72℃on day 5. Both groups of compost last for 5-7 days at the temperature of more than 50 ℃ and smoothly complete the high-temperature period.
During the cool down period, example 1 had a faster temperature drop, and had reached room temperature at 15, indicating that the microbial activity was low and the entire process of composting (medium, high and cool down periods) was completed. The slow cooling speed of comparative example 1 indicates that the nutrients in the compost are still not degraded completely, and the composting process is not completed yet.
2. Seed germination percentage (GI) measurement of example 1 and comparative example 1:
compost samples of example 1 and comparative example 1 were collected on days 0, 1, 3, 5, 7, 10, and 15, respectively. Fresh samples were mixed with deionized water at a ratio of 1:3 (mass/volume), placed on a constant temperature shaker at 20℃for 2h of shaking, and allowed to stand for 0.5h. 1 piece of qualitative filter paper is placed at the bottom of a 9cm diameter culture dish, 20 Shanghai green seeds are evenly placed on the qualitative filter paper, and 5mL of leaching solution is added. Cover the culture dish cover and place the culture dish cover in a constant temperature incubator at 25 ℃ for 48 hours. Counting the number of germinated seeds and the length of main roots.
The germination rate of the seeds is the most reliable index for evaluating the decomposition degree of the organic fertilizer. According to the standard of organic fertilizer NY/T525-2021, the fertilizer maturity can be judged when the germination rate of seeds is more than or equal to 70 percent.
As shown in fig. 2, the GIs of example 1 and comparative example 1 both tended to rise during composting. Example 1 had a GI value greater than 70% on day 7 and consistently higher than 70% after that, reaching 111.76% on day 15, thus it was determined that example 1 reached a state of maturity within 15 days. The GI value of comparative example 1 fluctuated up to only 68.25% on day 15 during composting, and did not meet the standard regulatory limits.
3. ARGs assay of example 1 and comparative example 1:
compost samples of example 1 and comparative example 1 were collected on days 0, 1, 3, 5, 10, 15, respectively.
Samples were freeze-dried, ground and sieved, and DNA was extracted using Rapid DNA Spin Kit for Soil kit (MP Co., U.S.) and the DNA extraction effect was examined using Nanodrop-2000 c. PCR amplification was achieved on Smart Chip Real-time PCR Systems high throughput fluorescent quantitative reaction platforms. The co-designed primers included 360 ARGs. The quantitative system needs 100 mu L of solution, and the concentration of each reagent is as follows: fluorescent quantitative reagent 1×light Cycler 480Green I Master Mix (Roche Co., USA), 5 ng/. Mu.L of DNA, 1. Mu.g/. Mu.L of BSA, 1. Mu. Mol/L of primer and nucleic-free PCR-Grade water. 360 ARGs were classified into 10 categories, tetracycline, fosfomycin, MLSB, quinolones, beta lactate, vancomycin, multidrug, sulfonamide, aminoglycoside, and others.
As can be seen from fig. 3, example 1 has a stronger removal effect on ARGs during the high temperature period than comparative example 1. The absolute abundance of initial ARGs for example 1 and comparative example 1 were 1.16x10 10 COPies/g (dry weight), example 1 minimizes ARGs absolute abundance at day 5 to 2.46×10 8 cobies/g (dry weight) removal rate was 98Percent of the total weight of the composition. The lowest value of ARGs abundance for comparative example 1 occurs at day 10 at 1.02X10 9 The removal rate was only 91% by copies/g (dry weight). The maximum removal of ARGs for example 1 was 7% higher than for comparative example 1.
The phenomenon that the abundance of ARGs bounces during the cooling period was observed for both groups of compost, but example 1 had a significant inhibitory effect on ARGs bounces. Comparative example 1 rebounded to 4.07 x 10 on day 15 compared to the lowest abundance value of ARGs for each group 9 The amplification was 300% for copies/g (dry weight), whereas example 1 was raised back to 7.54×10 8 The gain was only 207% for copies/g (dry weight).
Furthermore, on day 15, example 1 has reached a state of maturity, at which point the ARGs abundance still has a gradual decrease. While the rebound build-up of comparative example 1 remained significant in the later stages and 15 days did not reach the end of composting, there was a risk of further rebound in ARGs abundance.
The invention has been described above in sufficient detail with a certain degree of particularity. It will be appreciated by those of ordinary skill in the art that the descriptions of the embodiments are merely exemplary and that all changes that come within the true spirit and scope of the invention are desired to be protected. The scope of the invention is indicated by the appended claims rather than by the foregoing description of the embodiments.
Claims (4)
1. A method for enhancing removal of antibiotic resistance genes in aerobic composting, which is characterized in that the method for enhancing removal of antibiotic resistance genes in aerobic composting comprises the following steps:
mixing organic solid waste with composting auxiliary materials to obtain initial composting materials;
adding micrometer zero-valent iron, hydrogen peroxide solution and ascorbic acid solution into an initial composting material, stirring and mixing to obtain an aerobic composting material, wherein the initial pH value of the aerobic composting material is 6-8, the initial water content is 60% -70%, and the initial carbon-nitrogen ratio is 25-30:1;
carrying out aerobic composting on the aerobic composting materials, turning the composting once every 2-3 days, wherein the composting time of the aerobic composting is 15-20 days, and supplementing a proper amount of water every day in a high-temperature period, so that the water content of the composting is ensured to be 60% -65%;
the addition amount of the micrometer zero-valent iron is 0.5-2% of the weight of the initial composting material; the addition amount of the hydrogen peroxide solution is 3% -9% of the weight of the initial composting material; the addition amount of the ascorbic acid solution is 0.2-0.6% of the weight of the initial composting material.
2. The method for enhancing removal of antibiotic resistance genes in aerobic composting according to claim 1, wherein the organic solid waste is one or two of kitchen waste and livestock manure, and the composting auxiliary material is one or more of straw, rice hull and wood dust.
3. The method for enhancing the removal of antibiotic resistance genes from aerobic composting according to claim 1, wherein the concentration of the hydrogen peroxide solution is 10-30%, and the addition amount of the hydrogen peroxide solution is 3% of the weight of the initial composting material.
4. The method for enhancing removal of antibiotic resistance gene in aerobic composting according to claim 1, wherein the concentration of the ascorbic acid solution is 10-50 mmol/L, and the addition amount of the ascorbic acid solution is 0.5% of the weight of the initial composting material.
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Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101792205A (en) * | 2010-02-10 | 2010-08-04 | 哈尔滨工业大学 | Fenton and Fenton-like system fortifier and using method thereof |
| CN108558493A (en) * | 2018-03-02 | 2018-09-21 | 南京农业大学 | A method of estrogen and antibiotic in oxidation composting technique removal feces of livestock and poultry |
| CN110683873A (en) * | 2019-10-22 | 2020-01-14 | 上海原本生物科技有限公司 | Method for efficiently degrading antibiotics in pig manure by using sludge charcoal |
| CN111662108A (en) * | 2020-06-02 | 2020-09-15 | 江苏省农业科学院 | Method for reducing abundance of resistance genes and I-type integron in livestock and poultry manure compost |
| CN114315441A (en) * | 2022-01-18 | 2022-04-12 | 哈尔滨工业大学 | Method for strengthening removal of antibiotic resistance genes in aerobic compost |
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- 2022-05-25 CN CN202210572534.6A patent/CN115073230B/en active Active
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101792205A (en) * | 2010-02-10 | 2010-08-04 | 哈尔滨工业大学 | Fenton and Fenton-like system fortifier and using method thereof |
| CN108558493A (en) * | 2018-03-02 | 2018-09-21 | 南京农业大学 | A method of estrogen and antibiotic in oxidation composting technique removal feces of livestock and poultry |
| CN110683873A (en) * | 2019-10-22 | 2020-01-14 | 上海原本生物科技有限公司 | Method for efficiently degrading antibiotics in pig manure by using sludge charcoal |
| CN111662108A (en) * | 2020-06-02 | 2020-09-15 | 江苏省农业科学院 | Method for reducing abundance of resistance genes and I-type integron in livestock and poultry manure compost |
| CN114315441A (en) * | 2022-01-18 | 2022-04-12 | 哈尔滨工业大学 | Method for strengthening removal of antibiotic resistance genes in aerobic compost |
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