CN106220257B - Sulfanilamide (SN) drug-fast bacteria and its livestock excrement composting technique of drug resistant gene pollution in a kind of organic fertilizer based on reduction - Google Patents
Sulfanilamide (SN) drug-fast bacteria and its livestock excrement composting technique of drug resistant gene pollution in a kind of organic fertilizer based on reduction Download PDFInfo
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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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- 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
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- 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
- C05F17/30—Preparation of fertilisers characterised by biological or biochemical treatment steps, e.g. composting or fermentation using irradiation, e.g. solar or nuclear radiation; using electric or magnetic fields
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Abstract
The invention discloses a kind of as follows based on sulfanilamide (SN) drug-fast bacteria and its livestock excrement composting technique of drug resistant gene pollution, step in organic fertilizer is reduced：(1) feces of livestock and poultry pH is adjusted to 5.0 6.5；(2) sawdust is added in, and adjusts the final moisture content of material 50% 65%；(3) by step (2) treated the abundant blending and piling of material, aerobic compost is carried out 20 30 days, then after-ripening, drying, granulation.The present invention can effectively reduce sulfanilamide (SN) drug-fast bacteria quantity and the abundance of sulfanilamide (SN) drug resistant gene in cultivation source organic fertilizer product, reduce release of the drug-resistance factor in aquaculture to farm environment, the problem of reducing environmental pollution caused by breeding waste recycling and human health potential risk, provides safeguard to cultivate the safe handling of source organic fertilizer and the sustainable development of circular agriculture.
Description
Technical field
The present invention relates to environmental protection technical field, it is more particularly to a kind of based on reduce in organic fertilizer sulfanilamide (SN) drug-fast bacteria and its
The livestock excrement composting technique of drug resistant gene pollution.
Background technology
Caused by abuse of antibiotics a large amount of antibiotics resistance genes (Antibiotic Resistance Genes,
ARGs appearance and wide-scale distribution) has become one of significant threat of 21 century human health.In concern antibiotic in the environment
Residual, return and become and while toxic effect, the distribution of ARGs, origin, environment return the influence to become and its to environment, health
Deng the focus for also becoming environmental area.Livestock and poultry cultivation night soil is the main contributions of the ARGs burdens of getting worse in environment
Person.Domestic and international researcher, which has proven to using in farmland antibiotic residue excrement, can greatly increase the quantity of ARGs and resistance bacterium in farmland
And type.However, the organic fertilizer using feces of livestock and poultry as raw material is to improve one of soil organism most effective way.Therefore,
ARGs in feces of livestock and poultry is controlled to store, it is extremely urgent to reduce the risk that it spreads to soil environment.
At present, more and more people start to substitute the direct using in farmland of cultivation source waste with commercialization organic fertilizer.
Numerous studies show to cultivate the technique for producing organic fertilizer of excrement, such as aerobic composting, anaerobic fermentation, materialization disinfection etc., remove
Fertilizer efficiency can be improved, moreover it is possible to reach the target of control and the most of pollutant of removal.Sulfonamide resistance gene is current soil and excrement
Just two kinds of most resistant genes of middle report, wherein tetracycline resistance gene can relatively easily remove in compost, but sulphur
There is very big difference in different reports for amine resistant gene.The production method of feces of livestock and poultry organic fertilizer currently on the market
Various, there is also larger differences for the drug-fast bacteria and resistant gene content of the organic fertilizer formed under different fertilizer production technology.It can
See, different organic fertilizer production technique is different to the removal ability of drug-fast bacteria in feces of livestock and poultry with resistant gene.Regrettably, mesh
The abundance of sulfonamide resistance gene declines in feces of livestock and poultry after the processing such as preceding existing part research discovery aerobic compost, but sorry
It is that there are no explicitly for resistant gene in feces of livestock and poultry and the process of drug-fast bacteria removal and transmission controe.Therefore, it finds
The method and technological parameter that sulfonamide resistance gene and drug-fast bacteria are propagated in effective control feces of livestock and poultry have highly important meaning
Justice.
Invention content
It is polluted the purpose of the present invention is to provide sulfanilamide (SN) drug-fast bacteria in a kind of organic fertilizer based on reduction and its drug resistant gene
Livestock excrement composting technique, the technique are applied to the composting process in the production of conventional cultivation source organic fertilizer, can effectively reduce cultivation
Sulfanilamide (SN) drug-fast bacteria quantity and the abundance of sulfanilamide (SN) drug resistant gene in source organic fertilizer product reduce the drug-resistance factor in aquaculture to farmland
The release of environment, environmental pollution caused by reduction breeding waste recycling and human health potential risk are asked
Topic provides safeguard to cultivate the safe handling of source organic fertilizer and the sustainable development of circular agriculture.
The technical solution adopted by the present invention to solve the technical problems is：
Sulfanilamide (SN) drug-fast bacteria and its livestock excrement composting technique of drug resistant gene pollution, step in a kind of organic fertilizer based on reduction
It is as follows：
(1) acidic liquid or the acid conditioner of addition are sprayed in feces of livestock and poultry, feces of livestock and poultry pH is adjusted to 5.0-
6.5；
(2) sawdust is added in, and adjust the final moisture content of material in 50%- into step (1) treated feces of livestock and poultry
65%；
(3) by step (2) treated the abundant blending and piling of material, aerobic compost is carried out 20-30 days, then after-ripening, baking
Dry, granulation；Turning is carried out during aerobic compost, and ensures that all composting materials of heap body reach 50 DEG C or more of time in heap temperature
It maintains 5 days or more.
Ensure all composting materials of heap body heap temperature reach 50 DEG C or more of time maintain the technological means of 5 days or more can
Take for：i:When 60 DEG C of heap body mean temperature, in the insulating layers such as the external plastic covering film of heap, insulating quilt for greenhouse, compost
It 6~8 days total overlay film time of period, is monitored with thermometer, during which heap body outermost layer temperature is not less than 45 DEG C.In this way by turning,
It is ensured that the time that all composting material of heap body reaches 50 DEG C or more in heap temperature maintains 5 days or more；ii:Compost is equipped with to add
Thermal carries out Heat artificially to heap body raw material, is detected with thermometer and ensures that heap body outermost layer temperature reaches 50 DEG C or more and (includes
50℃)。
The present invention reduces the specific aim measure of sulfanilamide (SN) drug-fast bacteria and its drug resistant gene pollution in organic fertilizer and is the following：
1st, feces of livestock and poultry pH is adjusted first to adjust to 5.0-6.5, can effectively reduce sulfanilamide (SN) drug-fast bacteria in organic fertilizer in this way
And its drug resistant gene pollution.
2nd, the sawdust of specific dosage is added in, the dosage of sawdust is the 10-20% of feces of livestock and poultry dry weight, and sawdust is removal resistance
The important conditioner of gene can accelerate to reduce the content of sulfanilamide (SN) drug-fast bacteria and its drug resistant gene in compost fermentation product, have
Not replaceability and it is necessary to ensure additive amount in prescribed limit, otherwise can not have effect.
3rd, ensure that all composting material of heap body reaches 50 DEG C or more of time in heap temperature and maintains 5 days or more, the present invention
Composting process parameter be different from routine, the temperature control such as 60-80 DEG C of mean temperature for referring to heap body of conventional compost, and
Without all heap body raw materials are emphasized, so in practical compost, it may appear that it is internal awfully hot, it is external due to being contacted with environment, so heat dissipation
Soon, be not achieved 50 DEG C, generally can only be that internal material high temperature can maintain a period of time, and external material be unable to maintain that it is enough
Temperature is to enough time.50 DEG C in the present invention, 5 days, it is to ensure that all composting materials will reach this minimum temperature and dimension
(including outwardly and inwardly) is held the time, is not mean temperature.If temperature not enough or hold time it is short, can by increase turning,
Sprinkling compost activator, Heat artificially or heat preservation cover up to all 50 DEG C of composting material high temperature or more and are maintained extremely (comprising 50 DEG C)
Target 5 days few.Only all composting materials of guaranteed heap body reach 50 DEG C or more of time in heap temperature and maintain 5 days or more
Can effectively reduce in organic fertilizer sulfanilamide (SN) drug-fast bacteria and its drug resistant gene pollution, temperature or time less than or not all raw material all
Reach, then cannot efficiently reduce sulfanilamide (SN) drug-fast bacteria and its drug resistant gene pollution in organic fertilizer.
Preferably, acidic liquid described in step (1) is acetic acid, sulfuric acid or phosphoric acid.
Preferably, acid conditioner described in step (1) is acid mushroom slag, vinegar grain, sulphur or ferrous sulfate.
Preferably, the dosage of sawdust is the 10-20% of feces of livestock and poultry dry weight in step (2).
Preferably, to compost photo-irradiation treatment, the intensity of illumination 1000- of photo-irradiation treatment during aerobic compost in step (3)
30000 luxs, light application time 60 hours or more.To compost photo-irradiation treatment and above-mentioned special parameter is set, can accelerate to reduce
The content of sulfanilamide (SN) drug-fast bacteria and its drug resistant gene in compost fermentation product, otherwise cannot.
Preferably, the light source of photo-irradiation treatment is sunlight, fluorescent lamp or energy-saving lamp.
The beneficial effects of the invention are as follows：
(1) the sulfanilamide (SN) drug-fast bacteria quantity in the Swine manure that technique using the present invention prepares and sulfanilamide (SN) drug resistance base
The abundance of cause significantly reduces, and is far above common compost work to the inhibiting rate of the growth of sulfanilamide (SN) drug-fast bacteria and the diffusion of sulfanilamide (SN) drug resistant gene
Skill, wherein sulfanilamide (SN) drug-fast bacteria quantity is only the pig that common compost prepares in the Swine manure prepared using the technique
The a ten thousandth of excrement organic fertilizer.
(2) technique of the invention has compatibility well with now widely used cultivation source organic fertilizer production technology,
The technical solution being related to by being equipped with the technique in existing aerobic composting process, which can reach, reduces sulfanilamide (SN) drug-resistance factor
Target.
Specific embodiment
Below by specific embodiment, technical scheme of the present invention is described in further detail.
In the present invention, if not refering in particular to, used raw material and equipment etc. are commercially available or commonly used in the art.
Method in following embodiments is the conventional method of this field unless otherwise instructed.
Total embodiment：Sulfanilamide (SN) drug-fast bacteria and its feces of livestock and poultry heap of drug resistant gene pollution in a kind of organic fertilizer based on reduction
Fertile technique, step are as follows：
(1) acidic liquid or the acid conditioner of addition are sprayed in feces of livestock and poultry, feces of livestock and poultry pH is adjusted to 5.0-
6.5；The acidic liquid is acetic acid, sulfuric acid or phosphoric acid；The acidity conditioner is that acid mushroom slag, vinegar grain, sulphur or sulfuric acid are sub-
Iron.
(2) sawdust is added in, and adjust the final moisture content of material in 50%- into step (1) treated feces of livestock and poultry
65%；The dosage of sawdust is the 10-20% of feces of livestock and poultry dry weight.
(3) by step (2) treated the abundant blending and piling of material, aerobic compost is carried out 20-30 days, then after-ripening, baking
Dry, granulation；Turning is carried out during aerobic compost, and ensures that all composting materials of heap body reach 50 DEG C or more of time in heap temperature
It maintains 5 days or more.
It is preferred that compost photo-irradiation treatment, the intensity of illumination 1000-30000 luxs of photo-irradiation treatment, light during aerobic compost
According to 60 hours time or more.The light source of photo-irradiation treatment is sunlight, fluorescent lamp or energy-saving lamp.
Embodiment：
First, test material explanation
Composting material：Pig manure, type are peasant household free-ranging type pork pig, and apparent is dark brown, and wetter, stink is very heavy, original pH
It is 7.4, humidity 72%；Sawdust humidity is 4%.
2nd, assay method explanation
1st, sulfanilamide (SN) drug-fast bacteria quantitative measurement
LB agar plates is selected to be used for the separation of sulfonamide resistance bacterium.Addition total concentration is 20mg l in the medium-1、50mg
l-1With 100mg l-1Sulfanilamide (SN) antibiotic solution (sulphadiazine：Sulfadimidine=1:1) for resistance screening.Using life
It manages brine (0.85%) and extracts compost sample after 2 hours, gradient dilution supernatant is taken to be applied in resistant panel and non-resistant tablet.
25 DEG C are cultivated 24 hours.Tablet of the clump count between 20-200 is counted, calculates resistant clones number (CFU/g) and resistance
Bacterium ratio.
2nd, sulfonamide resistance gene sul1 and sul2 is quantified
- 20 DEG C of preservations, freeze-dried back after sample collection.The fertilizer sample 0.25g after freeze-drying is taken, according to
Omega EZNATMSoil DNA kits (Omega companies) illustrate to extract DNA.Then using organic fertilizer genomic DNA as template,
Using the primer shown in table 1, (1 primer sequence of table is common sulfonamide resistance gene primer in the most of article reported at present
Sequence is supplied to Shanghai life work to synthesize by sequence) carry out quantitative fluorescent PCR (qPCR).Fluorescent quantitative PCR uses
The kit MightyAmp that Takara companies provideTMFor Realtime PCR Kit, ABI StepOnePlusTMIt is glimmering in real time
Fluorescent Quantitative PCR instrument.The concentration of standard plasmid DNA is measured using NanoDrop micro-spectrophotometers, plasmid is established and copies
The standard curve of shellfish number and CT value correspondences.Good (the R of all resistant gene standard curve linear dependences2>0.99), qPCR
Expanding effect (E) can be used for the quantitative analysis of target gene between 99%~110%.Utilize 16SrDNA pairs of reference gene
Resistant gene in sample is corrected：The absolute copy number of resistant gene relative abundance=resistant gene/16SrDNA genes are absolute
Copy number
Table 1 is for examination primer sequence
3rd, one pH of experiment, which is adjusted, reduces sulfanilamide (SN) drug-fast bacteria and the experimental study of drug resistant gene abundance in pig manure
1st, pig manure pH is adjusted with sulfuric acid, forms following 4 processing, be described as follows：
T1：Pig manure does not adjust pH, pH 7.4；
T2：Adjust pig manure pH to 6.38；
T3：Adjust pig manure pH to 5.35；
T4：Adjust pig manure pH to 4.82.
2nd, the excrement composting that pH will be mixed up, quiescent culture 5 days.Each processing is repeated 3 times.
Pig manure sample after culture carries out sulfanilamide (SN) drug-fast bacteria quantity and sul1, sul2 sulfanilamide (SN) drug resistant gene abundance are surveyed
Fixed, as a result record is as shown in table 2.Analytical table 2 is it is found that compared with the original swine manure treatment handled without toning acid, initial pH is
Drug-fast bacteria quantity substantially reduces in acid swine manure treatment, is only the 0.01%-0.02% of control group；Adjust acid processing can be one
Determine to reduce sulfonamide resistance gene abundance in pig manure in degree, sul1 and sul2 relative abundances are substantially less than it in wherein T2 processing
His processing group.To sum up, carrying out culture again after adjusting pig manure raw material pH to acidity can help to reduce sulfanilamide (SN) drug resistant gene in pig manure
And drug-fast bacteria, the drug-fast bacteria and drug resistant gene reduced in feces of livestock and poultry enter soil.
Sulfanilamide (SN) drug-fast bacteria and sulfanilamide (SN) drug resistant gene sul1 and sul2 variation in the lower pig manure of 2 difference pH of table processing
4th, the addition of two sawdusts of experiment reduces the experimental study of sulfanilamide (SN) drug-fast bacteria quantity and drug resistant gene abundance in pig manure
1st, wood sawdust with fresh pig manure is mixed according to constant weight percentage, be described as follows：
T1：Pig manure is not handled；
T2：Sawmilling and fresh pig manure are mixed according to weight percent 10%；
2nd, the excrement composting that will mix up pH stands light culture 5 days.Each processing is repeated 3 times.
Pig manure sample after culture carries out sulfanilamide (SN) drug-fast bacteria quantity and sul1, sul2 sulfanilamide (SN) drug resistant gene abundance are surveyed
Fixed, as a result record is as shown in table 2.Analytical table 2 is it is found that addition 10% sawdust processing can significantly reduce sulfanilamide (SN) drug-fast bacteria in pig manure
Quantity；Acid processing is adjusted to mainly promote the reduction of sulfonamide resistance gene sul2 abundance in pig manure, but the increase of sul1 abundance can be caused.
All in all, adding a certain amount of sawdust in pig manure raw material contributes to sulfanilamide (SN) drug-fast bacteria and sul2 drug resistant genes in control pig manure to pass
It broadcasts.
2 sawdust of table adds the influence to pig manure sulfanilamide (SN) drug-fast bacteria and sulfanilamide (SN) drug resistant gene sul1 and sul2
5th, the removal experimental study of sulfanilamide (SN) drug-fast bacteria and drug resistant gene in pig manure is tested under three different temperatures
1st, different temperatures gradient is set using artificial incubator, temperature gradient is described as follows：
P-6：6 DEG C of temperature；
P-30：30 DEG C of temperature；
P-40：40 DEG C of temperature；
P-50：Temperature 50 C；
P-60：Temperature 60 C.
2nd, by pig manure raw material, composting, standing are protected from light culture 5 days under different cultivation temperatures.Each processing is repeated 3 times.
Pig manure sample after culture carries out sulfanilamide (SN) drug-fast bacteria quantity and sul1, sul2 sulfanilamide (SN) drug resistant gene abundance are surveyed
Fixed, as a result record is as shown in table 3.Analytical table 3 is it is found that high temperature can also effectively reduce drug-fast bacteria quantity in pig manure, but temperature needs
Reach 50 DEG C or more and as the temperature increases, sulfanilamide (SN) drug-fast bacteria quantity drastically declines；It is raiseeed after 50 DEG C or more high-temperature process
Sul1 and sul2 sulfonamide resistances gene is greatly reduced in poultry manure.To sum up, maintain temperature of charge 50 DEG C or more 5 days, ability
Effectively reduce drug-fast bacteria and drug resistant gene.
Sulfanilamide (SN) drug-fast bacteria and sulfanilamide (SN) drug resistant gene sul1 and sul2 Plantago fengdouensis in pig manure after the fermentation of 3 different temperatures of table
5th, four illumination of experiment promote the removal experimental study of sulfanilamide (SN) drug-fast bacteria and drug resistant gene in pig manure
1st, it using following 2 processing of illumination box design, is described as follows：
It is protected from light：24 hours dark,
Illumination：Illumination in 24 hours, 0 hour dark, light source are LED energy-conserving light sources
2nd, by pig manure raw material be protected from light or illumination condition under composting, quiescent culture 5 days.Each processing is repeated 3 times.
Pig manure sample after culture carries out sulfanilamide (SN) drug-fast bacteria quantity and sul1, sul2 sulfanilamide (SN) drug resistant gene abundance are surveyed
Fixed, as a result record is as shown in table 4.Analytical table 4 is it is found that photo-irradiation treatment reduces the quantity of sulfanilamide (SN) drug-fast bacteria in pig manure；Illumination master
Promote the reduction of sulfonamide resistance gene sul2 abundance in pig manure, but the increase of sul1 abundance can be caused.In general, in pig manure
Preparation illumination system assists in removing the sulfanilamide (SN) drug-fast bacteria and sul2 drug resistant genes in pig manure in composting fermentation process.
4 light of table assists the influence to pig manure sulfanilamide (SN) drug-fast bacteria and sulfanilamide (SN) drug resistant gene sul1 and sul2
Embodiment described above is a kind of preferable scheme of the present invention, and not the present invention is made in any form
Limitation also has other variants and remodeling under the premise of without departing from the technical solution recorded in claim.
Claims (5)
1. sulfanilamide (SN) drug-fast bacteria and its livestock excrement composting technique of drug resistant gene pollution, feature in a kind of organic fertilizer based on reduction
It is, step is as follows：
(1) acidic liquid or the acid conditioner of addition are sprayed in feces of livestock and poultry, feces of livestock and poultry pH is adjusted to 5.0-6.5；
(2) sawdust of the dosage for the 10-20% of feces of livestock and poultry dry weight is added in into step (1) treated feces of livestock and poultry, and is adjusted
The whole final moisture content of material is in 50%-65%；
(3) by step (2) treated the abundant blending and piling of material, carry out aerobic compost 20-30 days, then after-ripening, dry, make
Grain；Turning is carried out during aerobic compost, and ensures that all composting materials of heap body reach 50 DEG C or more of time maintenance in heap temperature
At 5 days or more.
2. livestock excrement composting technique according to claim 1, it is characterised in that：Acidic liquid is described in step (1)
Acetic acid, sulfuric acid or phosphoric acid.
3. livestock excrement composting technique according to claim 1, it is characterised in that：Acid conditioner described in step (1)
For acid mushroom slag, vinegar grain, sulphur or ferrous sulfate.
4. the livestock excrement composting technique according to claims 1 or 2 or 3, it is characterised in that：Aerobic compost in step (3)
Period to compost photo-irradiation treatment, the intensity of illumination 1000-30000 luxs of photo-irradiation treatment, light application time 60 hours or more.
5. livestock excrement composting technique according to claim 4, it is characterised in that：The light source of photo-irradiation treatment for sunlight,
Fluorescent lamp or energy-saving lamp.
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| CN109592863A (en) * | 2018-12-28 | 2019-04-09 | 华东师范大学 | A method of reducing Multiple Classes of Antibiotics resistant gene abundance in sludge organism conversion process |
| CN111662108B (en) * | 2020-06-02 | 2023-05-09 | 江苏省农业科学院 | Method for reducing abundance of resistance genes and type I integrants in livestock manure compost |
| CN112159262A (en) * | 2020-09-28 | 2021-01-01 | 福建农林大学 | Composting method for reducing resistance genes |
| CN113582753A (en) * | 2021-07-14 | 2021-11-02 | 齐全农牧集团股份有限公司 | Manure composting fermentation process |
| CN114262715B (en) * | 2021-12-28 | 2023-07-28 | 中国环境科学研究院 | Method for evaluating environmental health risk of resistance genes in compost products |
| CN114907153A (en) * | 2022-05-19 | 2022-08-16 | 西北农林科技大学 | Method for reducing abundance of sulfanilamide and beta-lactam resistance genes and intI1 in pig manure |
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