CN106698652A - Method for removing antibiotics resistance gene in sewage - Google Patents

Method for removing antibiotics resistance gene in sewage Download PDF

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Publication number
CN106698652A
CN106698652A CN201611143134.4A CN201611143134A CN106698652A CN 106698652 A CN106698652 A CN 106698652A CN 201611143134 A CN201611143134 A CN 201611143134A CN 106698652 A CN106698652 A CN 106698652A
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sewage
args
membrane
resistance gene
aerobic
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CN106698652B (en
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王亚宜
朱奕静
蒋勗欣
张姚
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Tongji University
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    • CCHEMISTRY; METALLURGY
    • C02TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
    • C02FTREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
    • C02F3/00Biological treatment of water, waste water, or sewage
    • C02F3/30Aerobic and anaerobic processes
    • C02F3/301Aerobic and anaerobic treatment in the same reactor
    • CCHEMISTRY; METALLURGY
    • C02TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
    • C02FTREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
    • C02F3/00Biological treatment of water, waste water, or sewage
    • C02F3/02Aerobic processes
    • C02F3/12Activated sludge processes
    • C02F3/1236Particular type of activated sludge installations
    • C02F3/1268Membrane bioreactor systems
    • CCHEMISTRY; METALLURGY
    • C02TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
    • C02FTREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
    • C02F3/00Biological treatment of water, waste water, or sewage
    • C02F3/34Biological treatment of water, waste water, or sewage characterised by the microorganisms used
    • CCHEMISTRY; METALLURGY
    • C02TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
    • C02FTREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
    • C02F9/00Multistage treatment of water, waste water or sewage
    • CCHEMISTRY; METALLURGY
    • C02TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
    • C02FTREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
    • C02F2103/00Nature of the water, waste water, sewage or sludge to be treated
    • C02F2103/34Nature of the water, waste water, sewage or sludge to be treated from industrial activities not provided for in groups C02F2103/12 - C02F2103/32
    • C02F2103/343Nature of the water, waste water, sewage or sludge to be treated from industrial activities not provided for in groups C02F2103/12 - C02F2103/32 from the pharmaceutical industry, e.g. containing antibiotics
    • CCHEMISTRY; METALLURGY
    • C02TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
    • C02FTREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
    • C02F2301/00General aspects of water treatment
    • C02F2301/04Flow arrangements
    • CCHEMISTRY; METALLURGY
    • C02TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
    • C02FTREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
    • C02F2303/00Specific treatment goals
    • C02F2303/16Regeneration of sorbents, filters
    • YGENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02WCLIMATE CHANGE MITIGATION TECHNOLOGIES RELATED TO WASTEWATER TREATMENT OR WASTE MANAGEMENT
    • Y02W10/00Technologies for wastewater treatment
    • Y02W10/10Biological treatment of water, waste water, or sewage

Abstract

The invention relates to a method for removing antibiotics resistance gene in sewage, and belongs to the technical field of sewage purification treatment. According to the method, the relevance between membrane surface pollutant components and the antibiotics resistance gene is inspected, a biological treatment and membrane technology combined process is adopted, biological treatment, membrane technology and membrane surface pollutant adsorption intercepting effects are organically coupled, adjustment and control and operating parameters during a membrane bioreactor biological process are fixed and optimized, and the optimum concentration of membrane surface organic pollutant required by the enhanced removal of the antibiotics resistance gene in the sewage is fixed, so that removal rate of the antibiotics resistance gene in the sewage reaches 93.7 percent. The method has the advantages that chemical agents are not required to be additional cast, secondary pollution is reduced, operation management costs are reduced, the significant requirements on economical, social and environmental sustainable development are met, the technology is simple and reasonable, industrialized application requirements on sewage advanced treatment and recycling are met, and a scientific and reliable novel technology is provided for controlling the spreading of the antibiotics resistance gene in the sewage.

Description

A kind of method of antibiotics resistance gene in removal sewage
Technical field
The present invention relates to a kind of method for removing antibiotics resistance gene in sewage.Belong to the dirty water decontamination handles technology neck Domain.
Background technology
At present, abuse of the antibiotic in the industries such as medical treatment, cultivation and pharmacy is constantly aggravated, and result in bacterial antibiotic Resistance is produced, is that the prevention and control and treatment of bacteriosis bring extreme difficulties.Antibiotics resistance gene (Antibiotic Resistance genes, ARGs) it is one of key factor that bacterium obtains resistance as a kind of novel environmental pollutant, Induced mutation and horizontal transfer of the generation of ARGs essentially from gene.The mutagens such as meeting such as antibiotic, heavy metal in environment Induction of bacterial mutation generation ARGs, the tolerant bacteria of this kind of acquisition resistance will be bred and be propagated between varying environment medium. ARGs entrained by tolerant bacteria is by conversion, transduceing and engaging isogenic horizontal transfer acts on, with plasmid, transposons, whole The removable Genetic elements such as zygote, mosaic gene are combined, so as in planting, inter-species Spreading and diffusion.ARGs have transportable property and Boundedness and persistence, is difficult to be degraded and remove in the environment, and ARGs enter after food chain can by microorganism as carrier, Amplification is shifted between food chain different levels, potential hazard is caused to host organism.Sewage disposal system is that ARGs enters nature ring The main path in border and potential thesaurus.Effectively ARGs is the key issue for realizing recycling sewage generated by making, sewage in removal sewage Regeneration is continued use in water resources Critical policies in global range.Propagated in control antibiotic resistance, and avoid antibiotic The potential information requirement aspect of resistance plays a significant role.
China is antibiotics production and uses big country, in waste water and municipal wastewater that antibiotics production, hospital, cultivation are produced ARGs detections abundance is higher, using traditional wastewater treatment technologies such as traditional activated sludge processes more than existing urban wastewater treatment firm, its Treatment target is primarily targeted for the removal of the Conventional pollutions such as carbon nitrogen phosphorus, and the removal ability to ARGs in sewage is extremely limited, portion The ARGs recall rates of point species, abundance and diversity have even been raised.The ARGs for failing removal in sewage treatment plant passes through Chinese effluent enters receiving water body, and higher concentration ARGs has been detected in China's difference water body and water environment.
The technology of ARGs mainly includes in existing removal sewage:Based on ultraviolet and chlorination disinfection technology, by adding aluminium The coagulating sedimentation technology that salt and molysite are realized, coagulating sedimentation and sterilization Combined Treatment, and the complicated Combined Processing of some flows Technology (including " coagulating sedimentation-biochemical treatment-peracetic acid disinfectant-high pressure CO2Sterilization-nano titanium oxide is catalyzed-precipitation " and " grid filtration-precipitate and separate-Fenton oxidation-UV/H2O2Oxidation-sterilization " etc.).These prior arts are required for setting up a large amount of Unit for treating water, equipment, and a large amount of chemical agents need to be added, substantially increase processing cost, floor space and operational management Difficulty;Meanwhile, when the water and larger variation water quality of water inlet, such as not throwing of the dosage or coagulant of adjustment disinfectant in time There is ARGs disclosure risks in dosage, the water outlet of handling process;In addition, for reach preferable ARGs removal effects and to Sewage Plant two Excessively chemical agent is added in level water outlet, it will secondary pollution is caused to storage water body.
The content of the invention
It is an object of the invention to provide antibiotic resistance base in a kind of high treating effect, the removal sewage of the easy stabilization of operation Because of technique.With this method solve current sewage purifying treatment process for ARGs remove exist treatment effect it is not good enough, treatment The problems such as high cost, operational management complexity.
In order to achieve the above object, the present invention is by the investigation to existing Sewage Plant, find membrane bioreactor (MBR) compared with The features such as solid retention time long and activated sludge concentration higher, all efficiently contribute to process Recalcitrant chemicals.In addition The film crown_interception of membrane module and the chemical adsorption of membrane contaminant effectively intercept small molecule contaminants.The present invention is used at biology ARGs in reason and membrane technology process integration removal sewage, by Optimum Regulation MBR bioprocess and operational factor, reaches preferably ARGs removal effects.
The present invention is comprised the following steps that:
A, builds anoxic/aerobic-membrane bioreactor (A/O-MBR), to anoxic/aerobic-membrane bioreactor (A/O- MBR) in inoculation municipal sewage plant aeration tank after activated sludge, prepare to be measured during synthetic municipal wastewater is intake using artificial ARGs relative abundances are 1.11 copy numbers/16S rRNA sequence bar numbers, start anoxic/aerobic-membrane bioreactor operation;
Structure anoxic in above-mentioned steps A/aerobic-membrane bioreactor (A/O-MBR), anoxic/aerobic-membrane biological reaction The ratio between dischargeable capacity of anoxic zone and aerobic zone is 2 in device:1;Increase pressure vacuum gauge before A/O-MBR systems go out water pump;It is good Membrane module in oxygen area using the polyvinylidene fluoride flat film that aperture is 0.1 μm, using the immersion method of operation, membrane module lower section Bottom perforated aeration tube is set, and membrane module medium position sets micro porous aeration head and constitutes offering oxygen way, controls the gas of aeration:Water= 50:1;Treatment sewage is drawn into inside film from the both sides cross-film of film using peristaltic pump as suction power, after membrane filtration By pressure vacuum gauge with constant membrane flux and intermittent cyclic mode water outlet, each intermittent cyclic is filtering 10min, is stopped for water outlet Have a rest 2min;
Seed sludge in above-mentioned steps A takes from Shanghai City Quyang sewage disposal plant aeration tank, seed sludge MLSS (g/L) It is that 4.8, VSS (g/L) is that 3.70, SVI (mL/g) is that 162.5, DO (mg/L) is that 6.4~6.5, pH is 3.5~4.5, temperature (DEG C) is 25 ± 1;
Water inlet in above-mentioned steps A prepares synthetic municipal wastewater using artificial, and with sodium acetate as carbon source, ammonium chloride is nitrogen Source, potassium dihydrogen phosphate is phosphorus source, control COD for 350~400mg/L, total nitrogen be 30~50mg/L, total phosphorus concentration be 10mg/L, Manual simulation's sewage concrete component:780mg/L CH3COONa, 230mg/L NH4Cl, 43mg/L KH2PO4, 8mg/L FeSO4, 102mg/L MgSO4, 42mg/L CaCl2(contain 0.2g H in every liter of liquid microelement with 0.5mL/L liquid microelements3BO3, 0.2g CoCl2·6H2O, 0.06g CuSO4·5H2O, 2g FeCl3·6H2O, 0.22g MnCl2·2H2O, 0.2g ZnSO4· 7H2O, 0.06g KI, 0.12g NiCl2);ARGs abundance average out to 1.26 × 10 in human configuration sewage8Copy number/mL.
B, the pollutants removal rate of periodic detection system adjusts operational factor, and stabilization A/O-MBR running statuses control film Flux is 15L/m2/ h, sludge retention time is set to 60d, and hydraulic detention time is set to 6h, aerobic zone MBR ponds suspended sediment concentration MLSS is 8~12g/L, and activated sludge is 250~300% from aerobic zone to anoxic zone internal reflux ratio, and reaction running temperature is controlled At 25 ± 1 DEG C, anoxic zone speed of agitator is 200~250r/min;When pressure vacuum gauge pressure difference reaches 40kPa, close and wriggle Pump stops water inlet, as a filter cycle, membrane module is changed clothes, and first carries out physical cleaning, then with 0.5%NaClOization Cleaning is learned, reduces film surface pollution thing concentration;
C, detects film surface pollution thing component, wherein bacterolysis microbial metabolic products (Soluble Microbial Products, SMP) concentration of component be 20~35mg/m2, EPS from bacteria (Extracellular Polymeric Substances, EPS) concentration of component be 150~300mg/m2When, membrane bioreactor water flow higher had both been can guarantee that, The effect that reinforcing ARGs absorption is intercepted can be also reached simultaneously, and ARGs abundance declines 2~5 orders of magnitude, ARGs clearances in water outlet Up to 93.7%;
The beneficial effects of the invention are as follows:
(1) present invention is combined by by membrane technology and biologic treating technique, makes the ARGs in sewage on the one hand can be by MBR Middle activated sludge absorption degraded, on the other hand, the appropriate pollutant of MBR faces can preferably be adsorbed and intercept ARGs, it is to avoid small The extracellular free ARGs of molecule penetrates fenestra and reveals in water outlet, can effectively remove ARGs pollutions in sewage, it is to avoid receiving water body ARGs pollutes;
(2) concentration by detecting each component in MBR film surface pollution things of the invention, it is determined that face organic pollution is to strong Optium concentration in change removal sewage needed for ARGs, preferably ensure that ARGs removal effects, compensate for traditional activated sludge process and goes out In the presence of the deficiency of ARGs disclosure risks higher in water, it is to avoid the flow of existing advanced processes technique is complicated, chemical agent throwing amount Greatly, the drawback such as operational management complexity;
(3) the whole technological process operation operation of the present invention is simple, it is easy to accomplish, and high degree of automation, it is easy to industrialization Using, compared to existing ARGs removals technique, the present invention more convenient and efficient in terms of the operational management of handling process, and need not Chemical agent additionally is added, the secondary pollution in sewage disposal process is reduced, and effectively saves operational and administrative expenses.
(4) the method emphasis treatment novel environmental pollutant ARGs, meets the demand of advanced treatment of wastewater and reuse, meets Promote the national great demand of economic, society and environment sustainable development, using membrane Bio-reactor Technology in sewage disposal Application effectively remove ARGs in sewage, be that advanced treatment of wastewater and water quality safety are ensured and provide theories integration and technical guarantee.
Brief description of the drawings
Fig. 1 is the processing technological flow figure of ARGs in present invention removal sewage;
Fig. 2 is target resistant gene relative abundance of the present invention along journey distribution map.
Specific embodiment
A, builds anoxic/aerobic-membrane bioreactor (A/O-MBR) of the present invention, A/O-MBR reactors operating condition such as table 1;
The A/O-MBR device operating conditions of table 1
Activated sludge in Shanghai City Quyang sewage disposal plant aeration tank is taken to inoculation in A/O-MBR, seed sludge is basic Property index is shown in Table 2.
The seed sludge fundamental property index of table 2
Water inlet uses artificial preparation synthetic municipal wastewater, and (ARGs relative abundances to be measured are 1.11 copy numbers/16S in water inlet RRNA sequence bars number), by adjusting the operational factors such as aeration flow, membrane flux, solid retention time, the fortune of stabilization A/O-MBR Row state;
The present embodiment is using artificial synthesized simulated municipal wastewater as purification object (ARGs relative abundances to be measured in water inlet It is 1.11 copy numbers/16S rRNA sequence bars number), comprise the following steps:
(1) the target resistant gene using different processing units in real-time quantitative fluorescence PCR detection procedure is relatively rich Degree (ARGs copy numbers/16S rRNA sequence bars number);
(2) each component concentration in detection face organic pollution, with reference to ARGs relative abundances, determines face organic pollution To the optium concentration needed for ARGs in reinforcing removal sewage;
(3) by adjusting the operational factors such as solid retention time (SRT), aeration flow, membrane flux, and membrane module physics Cleaning and the mode such as Chemical cleaning frequency, are maintained at optimal dense needed for removal ARGs with making film surface pollution thing concentration maximum duration Degree, so that removal effect of the consolidation system for ARGs.
Bottom perforated aeration tube is set using membrane module lower section in the step A, membrane module medium position sets micropore and exposes The offering oxygen way of gas head, the gas-water ratio for controlling aeration is 50:1 or so.
Kynoar PVDF plate type membrane assemblies in the step A use the immersion method of operation, use peristaltic pump As suction power, will intake from the both sides cross-film of film and be drawn into inside film, and along suction tube in the way of constant flow rate Water outlet.Using interval water-out manners:Filtering 10min, rests 2min.
Reactor water inlet prepares synthetic municipal wastewater using artificial in the step A, and with sodium acetate as carbon source, ammonium chloride is Nitrogen source, potassium dihydrogen phosphate is phosphorus source, and it is 350~400mg/L, 30~50mg/L and 10mg/ to control COD, total nitrogen and total phosphorus concentration L, manual simulation's sewage concrete component:780mg/L CH3COONa, 230mg/L NH4Cl, 43mg/L KH2PO4, 8mg/L FeSO4, 102mg/L MgSO4, 42mg/L CaCl2(contain 0.2g in every liter of liquid microelement with 0.5mL/L liquid microelements H3BO3, 0.2g CoCl2·6H2O, 0.06g CuSO45H2O, 2g FeCl3·6H2O, 0.22g MnCl2·2H2O, 0.2g ZnSO4·7H2O, 0.06g KI, 0.12g NiCl2);ARGs abundance to be measured is about 1.26 × 10 in human configuration sewage8Copy Number/mL, ARGs relative abundances to be measured are 1.11 copy numbers/16S rRNA sequence bar numbers.
In the step A, pressure vacuum gauge is installed before A/O-MBR goes out water pump, using 40kPa as the transmembrane pressure upper limit, When pressure difference reaches 40kPa, the suction power plant peristaltic pump of MBR system is closed, as a filter cycle.The film changed clothes is first Physical cleaning is first carried out, then Chemical cleaning is carried out with 0.5%NaClO.
In the step A, A/O-MBR operating condition concrete conditions are as follows:The membrane flux of membrane module is 15L/m2/ h, solid Residence time SRT is set to 60d, and hydraulic detention time HRT is set to 6h, and aerobic zone suspended sediment concentration MLSS is 8~12g/L, living Property sludge to be back to the internal reflux ratio of anoxic zone from aerobic zone be 250~300%, reaction running temperature control lacks at 25 ± 1 DEG C Oxygen area speed of agitator is 200~250r/min.
Can be by the solid retention time (SRT), the physical cleaning of membrane module and the Chemical cleaning that adjust activated sludge frequently Rate, the degree of Optimal Control System film surface pollution.SMP (bacterolysis microbial metabolic products) concentration of component be 20~ 35mg/m2, EPS from bacteria (EPS) concentration of component is 150~300mg/m2, system both can guarantee that water flow higher, The effect that reinforcing ARGs absorption is intercepted can be also reached simultaneously.
Result to above-described embodiment is tested and analyzed, analysis result such as Fig. 2 of all kinds of resistant genes to be measured.
(1) ARGs is quantitative
All kinds of ARGs Plantago fengdouensis situations to be measured along the different units of journey detection waste water processes, sample point is set :Water inlet, anoxic zone activated sludge, aerobic zone activated sludge, the cake layer of film surface pollution thing, diaphragm, delivery port, specific inspection Survey analysis method as follows.
1) DNA is extracted
DNA is extracted according to Fast DNATMSpin Kit for soil kits producer provides method and carries out, specific steps It is as follows:
A) lyophilized mud sample or the filter membrane for shredding are put into (Lysing Matrix E tube) in cracking tube E pipes, plus Enter 978 μ L phosphate buffers (Sodium Phosphate Buffer) and 122 μ L MT buffer solutions (MT Buffer), use Mixed liquor is shaken and mixed by vortex oscillation instrument;
B) is transferred to supernatant in 2mL centrifuge tubes in 10min is centrifuged under 14000 × g centrifugal force by E pipes, adds 250 μ L Protein Precipitation Solutions (Protein Precipitation Solution), and centrifuge tube is turned upside down 10 times to mix;
C) is transferred to supernatant in 10mL centrifuge tubes in 5min is centrifuged under 14000 × g centrifugal force by 2.0mL centrifuge tubes;
D) after shaking up DNA binding solns (Binding Matrix), the 10mL centrifugations during 1.0mL mixed liquors are added c) are drawn Guan Zhong;
E) centrifuge tube is turned upside down after 2min, stands 3min, DNA is attached on Binding Matrix, and wait Silica matrix precipitates (this step can be with proper extension according to the precipitation status time);
F) 500 μ L of supernatant liquid of careful removal, it is to avoid suction out sediment;
Remaining supernatant and sediment are mixed again, 600 μ L mixed liquors is drawn to containing SPINTMIn the pipe of Filter, 1min is centrifuged with 14000 × g;
G) liquid in collecting pipe is discarded, previous action is repeated until the liquid transfer in 10mL centrifuge tubes has been centrifuged;
H) by SPINTMFilter is placed in new 2mL centrifuge tubes, adds 500 μ L SEWS-M, again suspended with pipette tips SPINTMFliter granules in pipe, 1min is centrifuged with 14000 × g;
I) again by SPINTMFilter is placed in new 2mL centrifuge tubes, any solution is added without, then with 14000 × g Centrifugation 2min, by SPIN after centrifugationTMFliter is placed in 5min at room temperature;
J) by SPINTMFliter is placed in new 1.5mL centrifuge tubes, adds 100 μ L DES solution, stands 1min, with 14000 × g is centrifuged 1min, and the solution collected in lower floor's centrifuge tube is the DNA for extracting and purifying completion.
2) DNA purity and Concentration Testing
The purity and concentration for extracting DNA sample are determined using trace protein nucleic acids instrument NanoDrop2000.Tool Body operating procedure is as follows:
A) NanoDrop2000 working interfaces are opened, detection of nucleic acids mode is selected;
B) after system completes self-inspection, 2.5 μ L DES solution are added in loading hole, covers cover plate, click blank zeroings;
C) after drying loading hole with lens wiping paper, 2.5 μ L testing samples are added in loading hole, covers cover plate, click measure Detected, recorded its concentration value, OD260/OD280 and OD260/OD230 ratios.
3) DNA sample integrality is extracted in detection
Detected using agarose gel electrophoresis and extract DNA sample integrality.
A) prepare before detecting:By sample after thawed on ice, fully mix and be centrifuged, take 2 μ L samples and detected.
B) parameter is detected:Ago-Gel concentration is 1% agarose gel, and detection voltage is 5V/cm, and detection time is 30min。
4) prepared by regular-PCR and plasmid
The ARGs fragments extracted in DNA are expanded using regular-PCR, using in agarose gel electrophoresis observation PCR primer With the presence or absence of the amplified production of target ARGs, carry out after purification, carrying out plasmid preparation for the PCR primer containing target ARGs, Prepared plasmid is used for ARGs real-time fluorescence quantitative PCRs.Common PCR primers sequence see the table below 1.
The common PCR primers sequence table of table 1
5) ARGs real-time fluorescence quantitative PCRs
A) standard curve sample is prepared
The plasmid for building through sequencing identification it is errorless after, determine plasmid OD260 values with ultraviolet specrophotometer, and as the following formula Conversion plasmid copy number:
Plasmid concentration reduction formula (copy number/μ L)=plasmid concentration (ng/ μ L) × 6.02 × 1014/ DNA molecular amount
As qPCR standard items after 10 times of gradient dilutions of the plasmid that will be built, standard curve generally comprises 4~6 standards Point, the 10 of selection standard product are distinguished by preliminary experiment-3~10-7Dilution is used to prepare standard curve.Each plasmid control parameter is shown in Table 2 below.
The plasmid standard parameter of table 2
B) quantitative fluorescent PCR reactions steps
The 25 μ L qPCR reaction systems for using include:12.5μL 2×Green qPCR Master Mix mix Dyestuff, each 0.5 μ L of upstream and downstream primer (10 μM), 2 μ L DNA masterplates, 9.5 μ L ddH2O.It is placed on 96 orifice plates of good sample are added Reacted in the type quantitative real time PCR Instruments of ABI 7500.During the result obtained after reaction can calculate sample according to standard curve Every kind of ARGs concentration.In Real-Time Fluorescent Quantitative PCR Technique, Ct values represent that the prominent optical signal in reaction tube reaches the domain of setting There is linear relationship in the period experienced during value, the Ct values of each template, starting is dense with the logarithm of the initial concentration of the template Degree is higher, and Ct values are smaller.In the standard curve made using the standard items of known initial concentration, abscissa represents PCR primer Concentration, ordinate represents Ct values.Using the Ct values of unknown sample, you can calculate the initial dense of the sample from standard curve Degree.Data analysis is completed by Step One Software (version2.0).QPCR thermal cycle conditions such as table 3 below.
The qPCR thermal cycle conditions of table 3
6) ARGs removal effects
All kinds of total ARGs abundance average out to 1.26 × 10 to be measured in system water inlet8Copy number/mL (for 1.11 copy by relative abundance Shellfish number/16S rRNA sequence bars number), ARGs abundance average out to 7.90 × 10 in water outlet6Copy number/mL, clearance is up to 93.7% (is 99.4%), to illustrate very well process the sanitary sewage containing ARGs, water outlet using the invention in terms of relative abundance ARGs abundance declines 2~5 orders of magnitude.
(2) membrane contaminant component detection
In the research process of MBR fouling membranes, face organic pollution is often divided into dissolubility microbial metabolic products (Soluble Microbial Products, SMP) and extracellular polymeric (Extracellular Polymeric Substances, EPS).The absorption of the film surface pollution thing such as SMP, EPS and interception function are conducive to removing the ARGs in sewage, by force Removal effects of the MBR to ARGs is changed.Preferably, SMP concentration of component is 20~35mg/m in controlling face organic pollution2, EPS concentration of component is 150~300mg/m2, water flow higher had both been can guarantee that, while can also reach reinforcing ARGs absorption blocking The effect cut.
Understood through analysis:
(1) ARGs throughout manages the removal in unit
(Fig. 2) is understood according to analysis result, from aerobic zone activated sludge to system exiting water process, all kinds of target ARGs phases Substantially on a declining curve to abundance, total removal rates of all kinds of target ARGs in whole waste water processes is up to 93.7%.
ARGs in sewage has obtained certain going being intake from reactor into the biological treatment flow of MBR aerobic zones Remove, but removal effect is not obvious enough;And from face cake layer to the retention of the film of reactor water outlet and face organic pollution Adsorb in the handling process of retention, ARGs abundance is remarkably decreased, it is seen then that biological treatment, film retention and the organic dirt of face The combination of the absorption retention of dye thing can effectively remove the ARGs pollutions in sewage.
Wherein, relative abundances of the Partial Species ARGs (such as sul II etc.) in face mud layer is dirty higher than aerobic zone activity Relative abundance in mud, it may be possible to because the boring aeration equipment of MBR Aerobic Ponds forms higher concentration dissolved oxygen at face, and Face easily adsorbs organic nutrient substance in itself so that the resistance bacterium that part carries ARGs largely breeds on face, while removable Dynamic Genetic elements promote the horizontal transfer and amplification of ARGs, then cause ARGs relative abundances and are presented not at face mud layer Raised with degree.
(2) ARGs and intI1 correlations in MBR Aerobic Ponds sludge
It is to obtain exogenous ARGs by Horizontal transfer that bacterium produces the one of the main reasons of antibiotic resistance, mostly Number ARGs is located on the removable Genetic elements such as integron, transposons and plasmid.Although integron is immovable in itself, can be whole Close on the plasmid or chromosome of bacterium, promote ARGs in bacterium kind and interspecies level is shifted.Research at present has found at least 5 Class integron, wherein plasmid intI1 is most commonly seen.By studying ARGs and intI1 correlations in MBR Aerobic Pond sludge, can The animal migration of ARGs in solution MBR Aerobic Pond sludge.
Bian studies the correlation of intI1 relative abundances and target ARGs Plantago fengdouensis with Pearson one-tailed tests, as a result Refer to table 4 below.Correlation between single antibiotics resistance gene relative abundance and intI1 abundance is studied to find, sul I, It is in notable positive correlation (P between tetC, tetX, ereA gene and intI1<0.01), the Pearson correlations system of wherein tetX Number R values maximum (R=0.755).It is also equal between sulfamido sul, Tetracyclines tet and total ARGs relative abundances and intI1 abundance Notable positive correlation (P is presented<0.01), Pearson relative coefficients R values are respectively 0.518,0.543 and 0.749, show MBR The being widely present property of ARGs and high transportable property in Aerobic Pond activated sludge.
ARGs and intI1 correlations in the MBR Aerobic Pond sludge of table 4
(3) ARGs relative abundances clearance and film surface pollution thing correlation
Because SMP and EPS contains more negative electricity functional group (such as SO4 -、PO4 3-,-COOH etc.), and nearly all activated sludge table Surface charge is all negative electricity, therefore SMP and EPS are nearly all negatively charged.And both phosphate group with negative electricity on DNA molecular, gland is fast Amino on purine, guanine and cytimidine can be protonated and positively charged again, in the mistake of opsonigenous substance adsorption of DNA molecule Cation (such as Ca in Cheng Zhong, MBR system2+、Mg2+) played between the phosphate group and SMP and EPS negative electrical charges of DNA molecular Bridge joint is acted on.In membrane contaminant SMP and EPS may by with antimicrobial surface, the Electrostatic Absorption of extracellular Low-molecular-weight DNA Fragments and Absorption of the bridge joint effect reinforcing to ARGs is intercepted.R language Mantel check analyses film surface pollution thing concentration of component and ARGs are relative The correlation results of abundance (copy number/16S rRNA sequence bars number) clearance refer to table 5 below, it can be seen that film surface pollution thing The concentration of middle SMP and EPS is in notable positive correlation (P with ARGs relative abundances clearance<0.05, R>0).Analysis result shows this Invented technology not only make use of Inherent advantage of the MBR techniques in terms of small molecule contaminants and treatment Recalcitrant chemicals are intercepted, And fully grasp and effectively using membrane contaminant to the absorption interception function of ARGs, ARGs pollutions in removal sewage are provided Efficient processing method.
The target ARGs relative abundances clearance of table 5 and film surface pollution thing correlation analysis result
Preferably, SMP concentration of component is 20~35mg/m in controlling face organic pollution2, EPS concentration of component is 150 ~300mg/m2, system both can guarantee that water flow higher, while can also reach the effect that reinforcing ARGs absorption is intercepted.

Claims (1)

1. it is a kind of remove sewage in antibiotics resistance gene method, it is characterised in that:
A, builds anoxic/aerobic-membrane bioreactor, is exposed to anoxic/aerobic-membrane bioreactor inoculation municipal sewage plant Activated sludge in gas pond, is intake using artificial synthetic municipal wastewater of preparing, the antibiotic resistance to be measured in making municipal sewage intake Gene relative abundance is 1.11copies/16S RNA, starts anoxic/aerobic-membrane bioreactor operation;
Structure anoxic/aerobic-membrane bioreactor in above-mentioned steps A, wherein, anoxic in anoxic/aerobic-membrane bioreactor Area's dischargeable capacity:Aerobic zone dischargeable capacity=2:1;Membrane module in aerobic zone is flat using the Kynoar that aperture is 0.1 μm Plate film, using the immersion method of operation, membrane module lower section sets bottom perforated aeration tube, and membrane module medium position sets micropore and exposes Gas head constitutes offering oxygen way, controls the gas of aeration:Water=50:1;Using peristaltic pump as suction power, by treatment sewage from The both sides cross-film of film is drawn into inside film, after membrane filtration water outlet by pressure vacuum gauge with constant flow rate and intermittent cyclic mode Water outlet, each intermittent cyclic is filtering 10min, rests 2min;Increase pressure vacuum gauge before water outlet;
MLSS is 4.8g/L in activated sludge in inoculation municipal sewage plant aeration tank in above-mentioned steps A, and VSS is 3.70g/ L, SVI are 162.5mL/g, and DO is 6.4~6.5mg/L, and pH is 3.5~4.5, and temperature is 25 ± 1 DEG C;
Water inlet in above-mentioned steps A prepares synthetic municipal wastewater using artificial, is that, with sodium acetate as carbon source, ammonium chloride is nitrogen source, Potassium dihydrogen phosphate is phosphorus source, control COD for 350~400mg/L, total nitrogen be 30~50mg/L, total phosphorus concentration be 10mg/L, manually Simulation sewage concrete component:780mg/L CH3COONa, 230mg/L NH4Cl, 43mg/L KH2PO4, 8mg/L FeSO4, 102mg/L MgSO4, 42mg/L CaCl2With 0.5mL/L liquid microelements;Wherein, 0.2g is contained in every liter of liquid microelement H3BO3, 0.2g CoCl2·6H2O, 0.06g CuSO45H2O, 2g FeCl3·6H2O, 0.22g MnCl2·2H2O, 0.2g ZnSO4·7H2O, 0.06g KI, 0.12g NiCl2;ARGs abundance average out to 1.26 × 10 in human configuration sewage8copies/ mL;
B, the pollutants removal rate of periodic detection system adjusts operational factor, and steady operational status control membrane flux for 15L/m2/ H, sludge retention time is set to 60d, and hydraulic detention time is set to 6h, and aerobic zone membrane bioreactor suspended sediment concentration MLSS is 8~12g/L, activated sludge is 250~300% from aerobic zone to anoxic zone internal reflux ratio, and reaction running temperature control is 25 ± 1 DEG C, anoxic zone speed of agitator is 200~250r/min;When pressure vacuum gauge pressure difference reach 40kPa when, close peristaltic pump stop into Water, as a filter cycle, membrane module is changed clothes, and first carries out physical cleaning, then carries out Chemical cleaning with 0.5%NaClO, is obtained To film surface pollution thing;
C, detects film surface pollution thing component, and wherein bacterolysis microbial metabolic products concentration of component is 20~35mg/m2, carefully The outer polymers compositions concentration of mycetocyte is 150~300mg/m2When, membrane bioreactor water flow higher had both been can guarantee that, while Also the effect that reinforcing antibiotics resistance gene absorption is intercepted can be reached, antibiotics resistance gene abundance declines 2~5 numbers in water outlet Magnitude, antibiotics resistance gene clearance is up to 93.7%.
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CN112349345A (en) * 2020-11-06 2021-02-09 邹小明 Novel method for quantitatively evaluating response characteristic of antibiotic resistance gene to antibiotic pollution in environment

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