CN1584051A - Method for analyzing sludge microbiological community configuration in urban sewage plant - Google Patents
Method for analyzing sludge microbiological community configuration in urban sewage plant Download PDFInfo
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Abstract
A method for analyzing sludge microbiocoenosis construction of municiple sewage plant relating PCR-DGGE technology analytical method for sludge microbiocoenosis construction of municiple sewage plant is disclose. It includes: DNA extracting, PCR amplifying, PCR product concentrating, DGGE electrophoretic separating to obtain DGGE map. It is characterized by two DNA extracting to obtain general DNA, taking 2-5g sludge sample, sludge DNA extracting by DNA extract, adding DNA extract etc. centrifugal collecting supernatant fluid in sediment, adding mixed liquor of chloroform and isopentanol, centrifugal pooling, DGGE electrophoretic separating by denaturing gradient 30%-60% polyacrylamide gel to obtain DGGE map. It achieves time saving and low cost.
Description
Technical field
The present invention relates to a kind of use polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) technology to the analytical procedure that microflora in the municipal sewage sludge constitutes, belong to the microbial ecology field.
Background technology
Zoogloea that municipal sewage sludge is formed by multiple microorganism and absorption organism and the aggregate formed of inorganics on it, contain a large amount of bacteriums, virus, protozoon, parasitic ovum and other microorganism, microbial diversity is abundant, and microflora constitutes complicated.Microflora's formation has very great learning value and actual application prospect in the analysis municipal sewage sludge: both can be by analyzing the space-time dynamic variation that obtains microbial species group structure in the mud, the changes of function trend of the judgement and the prediction mud ecosystem; Again can be according to the dynamic change of microflora's formation and specific population in the mud ecosystem, the running condition of monitoring and regulation and control sludge treatment disposing technique, thus the correct guidance effect is moved and controlled to technology.
Past is adopted classification and evaluation to the traditional cultural method of the many employings of the research of microorganism in the mud, mainly is the features such as individual morphology, cultural characteristic and Physiology and biochemistry according to microorganism, and utilization colony counting method, microscopy method etc. are carried out the research of environmental.But this traditional method exists a lot of not enough: the truth that can not reflect microorganism in the municipal sewage sludge sample of complicated component; Be difficult to obtain waiting for information about of biological community structure and spatial distribution; And pure culture method and principle are introduced from medical microbiology mostly, municipal sewage sludge sample to complicated component is not well suited for that (for example many microorganisms are in poor nutrition under natural ecological environment, and in the laboratory with the nutritious beef broth peptone sum of bacterium that comes in the working sample to live, a large amount of oligotrophic microorganismss is not suitable for growth, causes the measurement result error big); Common laboratory is usually just cultivated down at 28 ℃ when the separation and Culture bacterium, and in the mud except there being middle and high warm type bacterium, also have the low temperature modification bacterium, this moment, the low temperature modification bacterium was then out in the cold.
In recent years, the Modern Molecular Biotechnology that progressively grows up has been adopted in microbial population performance analysis in waste water and the soil, especially based on 16S r DNA nucleotide sequence variation analytical technology, screening and the microbial diversity that caused of inrichment in cultivating, separating lost because this technology can effectively be avoided tradition, the group constitutes problems such as variation, and can more directly reflect microbial diversity and population distribution situation in the sample reliably, and Modern Molecular Biotechnology is also more simple and easy, quick and accurate than traditional cultivation, separation and evaluation.These methods comprise restriction fragment length polymorphism (RFLP), random amplified polymorphism (RAPD), amplification fragment length polymorphism (AFLP), nucleic acid hybridization technique, fluorescence in situ hybridization (FISH) and denaturing gradient gel electrophoresis (DGGE) etc.Wherein denaturing gradient gel electrophoresis (DGGE) is because of stability and good reproducibility, and is highly sensitive, dominant population information can be provided in the group and can analyze advantage such as a plurality of samples simultaneously and is applied to the microbial population performance analysis in waste water and the soil.
Ecological Envionment Research Centre, Chinese Academy of Sciences utilization PCR-DGGE technology is carried out design of primers to the research of agricultural land soil microflora and has been applied for the Chinese patent design of primers scheme of microflora " a kind of PCR-DGGE research environment " (publication number CN1456684A).
Yet the chemistry of municipal sewage sludge and agricultural land soil, physical properties height are heterogeneous, and it is also obviously different that microbe population and population distribute, and this has just determined two kinds of environmental samples to there are differences when carrying out microflora's component analysis.Still have nothing to do both at home and abroad at present and carry out the relevant report that municipal sewage sludge microflora constitutes research in utilization PCR-DGGE technology.Because municipal sewage sludge complicated component, microbial diversity enrich.
Summary of the invention
The objective of the invention is to disclose a kind of method that municipal sewage sludge microflora constitutes of analyzing.Further aim of the present invention is the municipal sewage sludge microflora formation that the PCR-DGGE technology is applied to analyze complicated component.
For achieving the above object, the present invention in conjunction with pedotheque microbial diversity research method, finds out a kind of analytical procedure that is applicable to that microbial population constitutes in the municipal sewage sludge according to the municipal sewage sludge characteristic.Can directly extract the total genomic dna of microorganism in the municipal sewage sludge sample without pure culture, because the relative proportion of the kind of dna molecular and each molecule has reflected the kind quantity of microbial population and the relative proportion between population in the mud sample, in the DGGE collection of illustrative plates that obtains, each band can be to form from the genomic dna segment of some kinds of different microorganisms, the number of band reflects the height of this population microbial population quantity, like this, the structural information of municipal sewage sludge microflora just is recorded on the DGGE collection of illustrative plates indirectly.Therefore, analyze group's formation that the DGGE collection of illustrative plates just can be analyzed sludge microbe.Concrete steps comprise the first step DNA extraction, the second step pcr amplification: add primer, Taq enzyme, damping fluid and dNTP earlier in the dna profiling that extracts, control initial 94 ℃ of pre-sex change 5min then; 94 ℃ of sex change 45s, 60 ℃ of primer renaturation 45s, 72 ℃ of primer extension 90s, 30 circulations; 72 ℃ are extended 20min eventually.Product detects product with 1% agarose gel electrophoresis, and electrophoretic buffer is 0.5 * tbe buffer liquid.The 3rd step PCR product concentrates: merge number pipe pcr amplification after product, seal with the Parafilm film, place-70 ℃ of refrigerator freeze overnight to preserve, take out the back and drained the machine concentration 4 hours with freezing, it is in 7.6 the TE damping fluid that the dry powder that obtains is dissolved in 30 μ l pH.The 4th step DGGE electrophoretic separation gets the DGGE collection of illustrative plates, is characterized in:
The first step is directly to extract total DNA of mud at twice, at first take by weighing 2～5g mud sample, DNA extraction liquid, 40-60 μ L20mg/mL N,O-Diacetylmuramidase and 1.5mL 20% promoting agent SDS with 13.5-27ml, 65 ℃ of degree water-bath 2 hours, several down every 15-20 minute jog, centrifugal collection supernatant liquor places centrifuge tube, adds the SDS of 4.5mL DNA extraction liquid and 0.5mL 20% in the precipitation again, and centrifugal collection supernatant liquor is incorporated in supernatant liquor last time.Add isopyknic chloroform in supernatant liquor: primary isoamyl alcohol=24: 1 (volume ratio) mixed solution, centrifugal 10min gets upper aqueous layer in another centrifuge tube.Repeating to drill in upper aqueous layer, it is centrifugal do to add after chloroform-primary isoamyl alcohol mixed solution, combining water layer, and with the Virahol that accounts for 0.6 times of volume of water layer, room temperature is placed 1h, and it is in 7.6 the TE damping fluid that the centrifugal 20min of 9000r/min, collecting precipitation are dissolved in 500 μ L pH.
In the 4th step, the DGGE electrophoretic separation: at first prepare the polyacrylamide denaturing gradient gel, making its denatured gradient is 30%～60%.Using electrophoretic buffer then is 1 * TAE, voltage 150V, 60 ℃, electrophoresis 4～8h, ethidium bromide staining 20～40min.Get the DGGE collection of illustrative plates,
Advantage of the present invention is as follows:
1. because the present invention directly extracts the total DNA in the municipal sewage sludge sample, screening and inrichment in traditional culturing process have been avoided, can more directly react microbial diversity and population distribution situation in the mud sample truly, tradition is cultivated, is separated and identifies also more simple and easy, accurate.
2. owing to the chloroform-primary isoamyl alcohol extraction steps that repeats in DNA extraction of the present invention, to be further purified DNA, impurity such as the humic acid matter in the minimizing mud, organic molecule are to the restraining effect of nucleic acid extraction and PCR reaction process.Therefore the mud sample microbial population that first PCR-DGGE is applied to complicated component is analyzed dynamically.With traditional method just go out the result week more than one relatively, saved a large amount of time (only needing to finish in two days) and financial resources, and the disappearance of microorganism information when having avoided traditional method spawn culture and screening.
3. because the present invention has reduced the sampling amount of sample, it only is 1/5 of traditional sampling amount, make water ratio low, the loose municipal sewage sludge of sample can not form the very big mud of viscosity with the buffered soln that adds in the sample, the N,O-Diacetylmuramidase and the promoting agent SDS that make it the back adding mix, and cause the present invention simple for process.
4. the present invention need not expensive operation test kit, cost is low, can analyze a plurality of municipal sewage sludge microbial species group structures simultaneously with method of the present invention, obtain its space dynamic change, and provide good guidance and role of evaluation for the operation of municipal sewage sludge treatment process improvement.
5. applied range of the present invention, be suitable for municipal sewage sludge and after various treatment and disposal technologies mud, can carry out DGGE at the mud sample of different operation phase of sludge treatment technique and analyze, thereby instruct and estimate for the technology operation stability provides.Can analyze simultaneously at the different population bacterial classification, determine dominant bacteria and and then the research bacterial classification functional, for scientific research and technologic improvement provide new thinking and foundation.
Description of drawings
Fig. 1 is a process flow diagram of the present invention
Fig. 2-A is that municipal wastewater treatment plant is through dewatered sludge DNA extraction agarose gel electrophoresis figure
Fig. 2-B is pcr amplification agarose gel electrophoresis figure
Fig. 2-C is DGGE polyacrylamide gel electrophoresis figure
Fig. 3-A is 5 kinds of mud DNA extraction of composting process agarose gel electrophoresis figure
Fig. 3-B is 5 kinds of mud pcr amplification agarose gel electrophoresis figure
Fig. 3-C is 5 kinds of mud DGGE polyacrylamide gel electrophoresis figure
Embodiment
At first see also accompanying drawing 1, the first step DNA extraction of the present invention is carried out at twice, at first adds DNA extraction liquid, N,O-Diacetylmuramidase and granulated glass sphere and shake back adding promoting agent SDS reaction in mud sample, and is centrifugal that supernatant liquor precipitates suddenly.In precipitation, add the centrifugal collection supernatant liquors of concussion such as DNA extraction liquid then again, be incorporated in supernatant liquor last time.Then add chloroform-primary isoamyl alcohol mixed solution in supernatant liquor, the centrifuging and taking water layer obtains water layer and contains dna solution.Again water layer contain repeat in the dna solution to drill centrifugal after do adding chloroform-primary isoamyl alcohol mixed solution, contain dna solution behind the purifying, add Virahol and place 1h, centrifugal, collecting precipitation is dissolved in and obtains total DNA that mud sample extracts in the TE damping fluid.Enter then that the second step pcr amplification, the 3rd step PCR product concentrate, the 4th step DGGE electrophoretic separation gets the DGGE collection of illustrative plates.
Embodiment 1
Dewatered sludge to the Shanghai City municipal wastewater treatment plant carries out sampling analysis, and the essential property of mud is: water ratio 80%, water-soluble total nitrogen 3.0g/kgDM, total organic matter 550g/kgDM, the dried solid of total oily 30g/kg, priority pollutants does not detect, coliform value 〉=24,000MPN/100gDM.Biological community structure to dewatered sludge carries out following analysis:
The repeating of the first step DNA extracted: take by weighing the 2g mud sample in triangular flask, add 13.5mL DNA extraction liquid (100mmol/L Tris-HCl, 100mmol/L EDTA, 100mmol/L K
2HPO
4, 100mmol/LKH
2PO
4, 1.5mol/L NaCl, 1%CTAB, pH8.0), add 40 μ L N,O-Diacetylmuramidases (20mg/mL) and 2-5 sterilization granulated glass sphere again,, shake 20min on the 225r/min shaking table in 37 ℃, then adding 1.5mL concentration is 20% promoting agent SDS, 65 ℃ of water-bath 2h, every 15min shake gently several down, with the said extracted sample centrifugal 10min of 5000r/min at room temperature, collect supernatant liquor, transfer in the 50mL centrifuge tube.The SDS that adds 4.5mL DNA extraction liquid and 0.5mL 20% in the precipitation again, 65 ℃ of water-bath 10min behind the vibration 10s, the centrifugal 10min of 5000r/min, collect supernatant liquor, with supernatant liquor merging last time, with isopyknic mixed solution (24 volume chloroforms and 1 volume primary isoamyl alcohol are mixed and made into), the centrifugal 10min of 5000r/min gets upper aqueous layer in another 50mL centrifuge tube.Repeat the work of drilling in water layer, combining water layer is placed 1h with the Virahol room temperature of 0.6 times of volume, and it is in 7.6 the TE damping fluid that the centrifugal 20min of 9000r/min, collecting precipitation are dissolved in 500 μ L pH.Obtain Fig. 2-A dewatered sludge DNA extraction agarose gel electrophoresis figure.
Second step, pcr amplification and product detect: the mud DNA that extracts is carried out pcr amplification, the sequence of used primer P1 is: 5 '-CGCCCGCCGCGCGGCGGGCGGGGGGGGCCCACGGGGGGCCTACGGGAGGAGCAG-3 ', the sequence of primer P2 is: 5 '-ATTACCGCGGATGCTGG-3 ', company limited is synthetic by the rich inferior biotechnology in Shanghai.The PCR reaction system is 39 μ L ddH
2O, 5 μ L 10* reaction buffers, 1 μ L PCR primer P1,1 μ L PCR primer P2,1 μ L dNTP, 1 μ L Taq enzyme, 2 μ L templates.The pcr amplification program is: initial 94 ℃ of pre-sex change 5min; 94 ℃ of sex change 45s, 60 ℃ of primer renaturation 45s, 72 ℃ of primer extension 90s, 30 circulations; 72 ℃ are extended 20min eventually.Obtain the mud pcr amplification product, the agarose gel electrophoresis with 1% detects product, and electrophoretic buffer is 0.5 * tbe buffer liquid.See Fig. 2-B pcr amplification agarose gel electrophoresis figure.
The 3rd step, the PCR product concentrates: merge number pipe pcr amplification after product, seal with the Parafilm film and to place-70 ℃ of refrigerator freeze overnight to preserve, take out the back and drained the machine concentration 4～6 hours with freezing, it is in 7.6 the TE damping fluid that the dry powder that obtains is dissolved in 30 μ l pH, and it is standby to be stored in-30 ℃ of refrigerators.
In the 4th step, the DGGE electrophoretic separation: in order to satisfy the distinctive complicated microbe species of municipal sewage sludge, the denatured gradient of preparation polyacrylamide denaturing gradient gel rises to 30%～60%.The electrophoretic buffer that uses is 1 * TAE, voltage 150V, 60 ℃, electrophoresis 5h, ethidium bromide staining 20min.Get the DGGE collection of illustrative plates, see Fig. 2-C DGGE polyacrylamide gel electrophoresis figure.Can analyze comparison sludge microbe population diversity degree by this figure.
Embodiment 2
Can analyze a plurality of municipal sewage sludge microbial species group structures simultaneously with method of the present invention, obtain its space dynamic change, for the operation of municipal sewage sludge treatment process improvement provides good guidance and role of evaluation.Now to through certain rapidly and efficiently composting process handle each the treatment stage mud sampling analysis, the essential property of mud is as shown in table 1:
Table 1. composting process each the treatment stage mud essential property
Numbering sampling characteristics water ratio % VS%
1 last consignment of backflow material 37 37.8
2 dewatered sludges (former mud) 81 10.5
After amendment mixes
3 58 23.7
Material was handled the 1st day
The pliotherm period material is located
4 42 40.9
Managed the 8th day
Discharging handles the 16th
5 36 43.8
My god
By step of the present invention each mud sample being carried out DNA extraction, pcr amplification and DGGE below separates.
The first step DNA extraction: respectively take by weighing the 5g mud sample in triangular flask, add 27mL DNA extraction liquid (100mmol/L Tris-HCl, 100mmol/L EDTA, 100mmol/L K
2HPO
4, 100mmol/LKH
2PO
4, 1.5mol/LNaCl, 1%CTAB, pH8.0), add 60 μ L N,O-Diacetylmuramidases (20mg/mL) and 5-9 sterilization granulated glass sphere again,, shake 20min on the 225r/min shaking table in 37 ℃, then add 1.5mL20%SDS, 65 ℃ of water-bath 2h, every 20min shake gently several down, with the said extracted sample through the centrifugal 10min of room temperature 5000r/min, collect supernatant liquor, transfer in the 50mL centrifuge tube.The SDS that adds 4.5mL DNA extraction liquid and 0.5mL 20% in the precipitation again, 65 ℃ of water-bath 10min behind the vibration 10s, the centrifugal 10min of 5000r/min.Collect supernatant liquor and be incorporated in supernatant last time, mix with equal-volume chloroform-primary isoamyl alcohol, the centrifugal 10min of 5000r/min gets upper aqueous layer in another 50mL centrifuge tube.In water layer, repeat the work of drilling, combining water layer is placed 1h, the centrifugal 20min of 9000r/min with the Virahol room temperature of 0.6 times of volume, it is in 7.6 the TE damping fluid that collecting precipitation is dissolved in 500 μ L pH, records the DNA extraction agarose gel electrophoresis figure of 5 kinds of mud of accompanying drawing 3-A.
The second step pcr amplification: DNA carries out the pcr amplification the primer and the PCR reaction system is identical with embodiment 1 to extracting.The pcr amplification program is also identical with embodiment 1: initial 94 ℃ of pre-sex change 5min; 94 ℃ of sex change 45s, 60 ℃ of primer renaturation 45s, 72 ℃ of primer extension 90s, 30 circulations; 72 ℃ are extended 20min eventually.Agarose gel electrophoresis with 1% detects the PCR product, and electrophoretic buffer is 0.5 * tbe buffer liquid.Record the pcr amplification agarose gel electrophoresis figure of accompanying drawing 3-B.
The 3rd step PCR product enrichment step is identical with embodiment 1.
The 4th step DGGE electrophoretic separation: preparation polyacrylamide denaturing gradient gel, making its denatured gradient is 30%～60%.The electrophoretic buffer that uses is 1 * TAE, voltage 150V, 60 ℃, electrophoresis 5h, ethidium bromide staining 40min, the DGGE polyacrylamide gel electrophoresis figure of accompanying drawing 3-C.The DGGE collection of illustrative plates: the PCR product of 5 kinds of mud samples is separated into the electrophoretic band that band is clear, number does not wait in the DGGE kind, has tangible microbial population distributional difference between this explanation composting process different steps mud.Simultaneously can find out that by the DGGE collection of illustrative plates collection of illustrative plates of the backflow material of last consignment of and discharging is very similar, illustrate that this composting process has good circulation ratio, realizes the steady running of technology substantially.
The setting of extracting method as seen of the present invention, PCR reaction system and the operation of DGGE thereof are feasible for dynamic analysis of microbial population in the mud sample of complicated component, and the DGGE separating resulting can provide good guidance and estimate meaning the operation of this composting process.
Claims (1)
1. analyze the method that municipal sewage sludge microflora constitutes for one kind, comprise the first step DNA extraction;
The second step pcr amplification: add primer and reaction system in the DNA that extracts, the pcr amplification program is: initial 94 ℃ of pre-sex change 5min; 94 ℃ of sex change 45s, 60 ℃ of primer renaturation 45s, 72 ℃ of primer extension 90s, 30 circulations; 72 ℃ are extended 20min eventually, and product detects product with 1% agarose gel electrophoresis, and electrophoretic buffer is 0.5 * tbe buffer liquid;
The 3rd step PCR product concentrates: merge number pipe pcr amplification after product, seal with the Parafilm film, place-70 ℃ of refrigerator freeze overnight to preserve, take out the back and drained the machine concentration 4 hours with freezing, it is in 7.6 the TE damping fluid that the dry powder that obtains is dissolved in 30 μ l pH; The 4th step DGGE electrophoretic separation gets the DGGE collection of illustrative plates, it is characterized in that:
The first step DNA extraction is directly to extract total DNA of mud at twice, at first take by weighing 2～5g mud sample, DNA extraction liquid with 13.5-27ml, 40-60 μ L20mg/mL N,O-Diacetylmuramidase and 1.5mL20%SDS, 65 ℃ of degree water-bath 2 hours, several down every 15-20 minute jog, centrifugal collection supernatant liquor places centrifuge tube, the SDS that adds 4.5mL DNA extraction liquid and 0.5mL 20% in the precipitation again, centrifugal collection supernatant liquor is incorporated in supernatant last time, in supernatant liquor, add isopyknic mixed solution of forming by 24: 1 volume ratios by chloroform and primary isoamyl alcohol, centrifugal 10min gets upper aqueous layer in another centrifuge tube; Repeat the work of drilling in precipitation: add after the chloroform isoamyl alcohol mixed solution centrifugally, combining water layer adds the Virahol of 0.6 times of volume, and room temperature is placed 1h, and it is in 7.6 the TE damping fluid that the centrifugal 20min of 9000r/min, collecting precipitation are dissolved in 500 μ L pH;
In the 4th step, the DGGE electrophoretic separation: at first prepare denatured gradient and be 30%～60% polyacrylamide gel, using electrophoretic buffer then is 1 * TAE, voltage 150V, 60 ℃, electrophoresis 4～8h, ethidium bromide staining 20～40min, the DGGE collection of illustrative plates.
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Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN100427598C (en) * | 2006-06-12 | 2008-10-22 | 南京大学 | Lacustrine deposit microorganism general DNA extraction and biomass identification method |
| CN101392248B (en) * | 2008-06-20 | 2011-06-29 | 哈尔滨工业大学 | Extraction method of anaerobic activated sludge DNA |
| CN101619355B (en) * | 2009-07-02 | 2012-02-08 | 黑龙江大学 | Detection method of germs in traditional zymotic soybean paste |
| CN102507885A (en) * | 2011-09-28 | 2012-06-20 | 首都师范大学 | Method for forecasting comprehensive quality of complex water environment by applying number of microorganisms in bottom sediment |
| CN102559857A (en) * | 2010-12-28 | 2012-07-11 | 郭飞宏 | Analytical method for structures of microbial communities in composite filler in tower-type earthworm ecological filter |
| CN113462572A (en) * | 2021-08-11 | 2021-10-01 | 上海清涟环境科技有限公司 | Indigenous microbial agent and preparation method thereof |
| CN114686610A (en) * | 2022-04-13 | 2022-07-01 | 华北理工大学 | Method for detecting microbial community structure change in SNAD process operation process |
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Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN100427598C (en) * | 2006-06-12 | 2008-10-22 | 南京大学 | Lacustrine deposit microorganism general DNA extraction and biomass identification method |
| CN101392248B (en) * | 2008-06-20 | 2011-06-29 | 哈尔滨工业大学 | Extraction method of anaerobic activated sludge DNA |
| CN101619355B (en) * | 2009-07-02 | 2012-02-08 | 黑龙江大学 | Detection method of germs in traditional zymotic soybean paste |
| CN102559857A (en) * | 2010-12-28 | 2012-07-11 | 郭飞宏 | Analytical method for structures of microbial communities in composite filler in tower-type earthworm ecological filter |
| CN102507885A (en) * | 2011-09-28 | 2012-06-20 | 首都师范大学 | Method for forecasting comprehensive quality of complex water environment by applying number of microorganisms in bottom sediment |
| CN102507885B (en) * | 2011-09-28 | 2014-05-07 | 首都师范大学 | Method for forecasting comprehensive quality of complex water environment by applying number of microorganisms in bottom sediment |
| CN113462572A (en) * | 2021-08-11 | 2021-10-01 | 上海清涟环境科技有限公司 | Indigenous microbial agent and preparation method thereof |
| CN114686610A (en) * | 2022-04-13 | 2022-07-01 | 华北理工大学 | Method for detecting microbial community structure change in SNAD process operation process |
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