CN108486238A - A kind of biological reinforced functional flora analytic method based on high-flux sequence - Google Patents
A kind of biological reinforced functional flora analytic method based on high-flux sequence Download PDFInfo
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
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Abstract
The invention discloses a kind of biological reinforced functional flora analytic method based on high-flux sequence, including following operating procedure：Using membrane bioreactor, the domestication of functional microorganism group is carried out by the way that load in water inlet is continuously improved；The sludge after each operation phase stable operation in membrane bioreactor is acquired, centrifugation carries out the mud sample of each operation phase the extraction of microorganism total DNA with enriched microorganism；Target fragment PCR amplification simultaneously purifies amplified production；To amplified production Quality Identification, and by different sample P CR amplified productions by etc. quality mix；Clip size and quantitative detection, and high-flux sequence are carried out to mixing library；Finally carry out initial data Quality Control and bioinformatic analysis.Analytic method of the present invention can from ecological point system analysis biochemical treatment process for wastewater microbiologic population structure feature, determine Core Feature flora in waste water treatment system, and screen influence biological community structure crucial water quality parameter.
Description
Technical field
The present invention relates to a kind of biological reinforced functional flora analytic method based on high-flux sequence, belongs to microorganism biological
Intensive treatment technical field.
Background technology
Environmental pollution caused by pharmacy waste water is on the rise, and typical case's pharmacy waste water such as antibiotic, vitamin, pesticide is to the mankind
Living environment cause serious pollution.Currently, bioanalysis is still the mainstream technology of pharmacy wastewater treatment.But due to
The water quality and quantity fluctuation of pharmacy waste water is larger, easily influences biological treatment effect.The research of forefathers focus mostly in treatment process and
Its parameter determine, and in pharmacy wastewater treatment system key core flora and feature flora information know little about it, to shadow
The key factor of acoustic system Bacterial community and treatment effect is unclear, therefore the regulation and control for the treatment of process are often with experience guidance
It is main.Therefore, in pharmacy waste water biochemistry strengthening treatment process key function Bacterial community deep analysis, for improve biochemical treatment
Efficiency is significant.
Although the technologies such as clone library, DGGE, TGGE and T-RFLP can also monitor and provide the information of population variation,
But since it detects the limitation of flux and analytical technology, this "black box" of pharmacy waste water bio-augmentation treatment system annoyings always
Environmental scientific research worker and a line engineers and technicians, in recent years, rapid development have flux is big, accuracy rate is high, at low cost
The advantages that high throughput sequencing technologies and bioinformatic analysis method of new generation, can realize bacterial micro-organism structure of community and
Diversity is quickly analyzed so that biological reinforced "black box" transparence is possibly realized.From ecological point system analysis pharmacy waste water
Biological community structure feature in biochemical processing process determines Core Feature flora in pharmacy wastewater treatment system, and screens shadow
The crucial water quality parameter for ringing biological community structure, illustrates the microbial mechanism of pharmacy wastewater treatment process, is given birth to pharmacy waste water
Object reinforcement process Optimum Regulation and special effective function microbial inoculum development significance are great.
Through retrieval, the research about biological community structure parsing has related open.As Chinese Patent Application No. is
201010132091.6 patent of invention disclose a kind of analysis method of biological community structure, this application, which passes through, extracts ring
RNA in the sample of border detaches small subunit (16S/18S) rRNA from RNA, and it is right to carry out reverse transcription to small subunit by random primer
High-flux sequence is directly carried out afterwards and carries out correlation analysis, although this method is microorganism active in research environment sample
And the influence that PCR amplification generates structural analysis of microbial community is avoided, but due to containing a large amount of corruption in environmental sample
The extraction of total serum IgE and follow-up molecule manipulation can seriously be inhibited by growing the substances such as acid, heavy metal ion, frequently result in experimental repeatability compared with
Difference；Since this method is needed by carrying out gel extraction, cumbersome, heavy workload limits the practical application of this method；This
Outside, this method is only used for microorganisms structure of community, and functional microorganism group can not be parsed from ecological point to environment
The stress effect of water quality parameter.Therefore, it is strong to develop the biology that a kind of convenient and flexible operation, flux is big, accuracy rate is high, at low cost
Change functional flora structural analysis, while analytic method of the functional microorganism group to ambient water quality parameter response characteristic can be disclosed
It is particularly important.
Invention content
Goal of the invention：The present invention is directed to key core flora and feature flora in pharmacy waste water bio-augmentation treatment system
The key factor of loss of learning, influence system Bacterial community and treatment effect is unclear, and existing analysis method flux is low, operates
Cumbersome, heavy workload, it is of high cost the problems such as, the present invention is turned to realizing that pharmacy waste water bio-augmentation treatment system "black box" is transparent
Target is provided a kind of strong suitable for wastewater biological using the bioinformatic analysis means based on high-flux sequence of new generation
Change the method for processing system functional flora deep analysis.This method can be from ecological point system analysis (pharmacy waste water) biochemistry
The structure feature of science and engineering Yi Zhong microbiologic populations determines Core Feature flora in (pharmacy waste water) processing system, and screens influence
The crucial water quality parameter of biological community structure.
In order to solve the above technical problems, the technical solution adopted in the present invention is：
A kind of biological reinforced functional flora analytic method based on high-flux sequence, this method is by acquiring sludge acclimatization
The sludge in each stage in journey extracts its microorganism total DNA, and carries out the high-flux sequence based on 16S rDNA, when finally using
Sequence is analyzed and the bioinformatics means such as network analysis carry out deep analysis to the biological reinforced functional flora that domestication obtains, from life
State angle system parses the structure feature of microbiologic population in biochemical processing process, determines Core Feature flora therein, and
Screening influences the crucial water quality parameter of functional microorganism structure of community, to realize the accurate tune for instructing bio-augmentation treatment system
Control.
Wherein, the above-mentioned biological reinforced functional flora analytic method based on high-flux sequence specifically includes following operation step
Suddenly：
Step 1, with membrane bioreactor, the domestication of functional microorganism group is carried out by the way that load in water inlet is continuously improved；
Step 2, it is micro- to be enriched with that the sludge after each operation phase stable operation, centrifugation in membrane bioreactor are acquired
Biology, and the extraction of microorganism total DNA is carried out to the mud sample of each operation phase；
Step 3, target fragment PCR amplification and amplified production is purified；
Step 4, to amplified production Quality Identification, and by different sample P CR amplified productions by etc. quality mix；
Step 5, clip size and quantitative detection, and high-flux sequence are carried out to mixing library；
Step 6, initial data Quality Control and bioinformatic analysis are finally carried out.
Wherein, in step 1, the service condition of membrane bioreactor is：Hydraulic detention time is 14 hours, dissolved oxygen 3
~6mg/L, pH value are 7~8, and temperature is 25 ± 2 DEG C；By be continuously improved intake in load, each operation phase monitor into
The COD and ammonia nitrogen concentration of water outlet, operation a period of time appropriate spoil disposal make in reactor sludge concentration maintain 4000mg/L~
5000mg/L, to keep the activity of sludge, the removal rate of COD and ammonia nitrogen reaches and is stably held in 90% and 99% or more, obtains
Activated sludge-functional microorganism the group stablized to growth conditions.
Wherein, in step 1, the membrane material of membrane module used in membrane bioreactor is hollow polyvinylidene fluoride film,
Aperture is 0.03 μm, outer diameter 2.2mm, internal diameter 1.0mm, membrane area 0.235m2。
Wherein, in step 2, the sample, which comes from functional microorganism group domestication process MBR reaction systems, each to be run
Sludge after stage stable operation, centrifugation is with enriched microorganism.
Wherein, it in step 2, usesSpin Kit for Soil kits progress each operation phase obtains
The mud sample arrived carries out the extraction of microorganism total DNA, and DNA purity is A260/A280=1.8~2.0 after extraction.
Wherein, in step 3, target fragment PCR amplification sequence is 16S rDNA hypervariable regions V3~V4 (341F and 806R),
The concentration of amplified production is more than 15ng/ μ L.
Wherein, in step 4, the Quality Identification of amplified production includes measuring concentration, purity and the fragment length of PCR product
Size.
Wherein, in step 5,2100 biological analysers of Agilent and quantitative PCR is used to carry out piece to mixing library respectively
Duan great little and quantitative detection.
Wherein, in step 5, high-flux sequence instrument is the MiSeq microarray datasets of Illumina companies, is 2 × 300bp two
End sequencing.
Wherein, in step 6, initial data Quality Control and bioinformatic analysis include the following steps：First, according to not same
The barcode of product splits initial data；Secondly, using tagcleaner softwares by " primer sequence " in every sequence and
Its front end is removed for adjusting " heterogeneous intervening sequence " sequence that 0~7 bp of laser signal is not waited；Furthermore by two
Terminal sequence spliced according to intersection (overlap) after using Mothur platforms carry out quality of data filtering, noise reduction, go it is embedding
Zoarium, and the non-bacterial sequence Quality Control operation of removal, and carry out OTUs calculating and classification annotation with QIIME softwares；Finally, right
The OTU data of time series sampling and the monitoring water quality data that is corresponding to it carry out the extension local similarity analysis of time series
(eLSA), the correlation of synchronization and delay based on normalization sorting data is calculated by local similarity analysis (LSA), simultaneously
Generate be similar to Spearman sequence correlation analysis related coefficient, by Cytopscape softwares carry out network visualization and
Analysis of Topological Structure.
The above-mentioned biological reinforced functional flora analytic method based on high-flux sequence in analyzing microbial community structures and
Analyzing microbial community is to the application in terms of ambient water quality parameter response characteristic.
Compared with the prior art, it is had the beneficial effect that possessed by technical solution of the present invention：
First, analytic method of the present invention using the DNA extracted in environmental sample rather than is easy to degradation of rna as research pair
As, it is ensured that the repeatability and comparability of experimental result；
Secondly, analytic method combination new-generation sequencing technology of the present invention avoids and needs culture point existing for existing method
From the cumbersome problem of, gel extraction, analytic method of the present invention have convenient and flexible operation, flux is big, accuracy rate is high, at
This low advantage；
Finally, Time-Series analysis (eLSA) and network analysis method are used in analytic method of the present invention, can be not only used for solving
Biological community structure is analysed, and can be from ecological point deep analysis functional microorganism group to the sound of ambient water quality parameter
Answer feature, it can be achieved that pharmacy waste water bio-augmentation treatment system accurate, high efficiency regulatory, have broad application prospects.
Description of the drawings
Fig. 1 is the network analysis for the response characteristic that microorganism changes environmental parameter (A) and penicillin concn (B)；
Fig. 2 is that (solid line) exclusion (dotted line) analysis together occurs altogether in microorganism in penicillin wastewater MBR system for handling；
Fig. 3 is belonging to horizontal distribution situation for each mud sample microorganism under the conditions of different salinity；
Fig. 4 is the network analysis for the response characteristic that microorganism changes environmental parameter (salinity).
Specific implementation mode
Technical scheme of the present invention is described further below in conjunction with attached drawing, but the scope of protection of present invention is simultaneously
It is not limited to this.
Embodiment 1
(1) take returned sludge will first by strainer filtering sundries therein from city domestic sewage treatment plant secondary settling tank
It is inoculated into MBR reactors, a concentration of 3500~4500mg/L of seed sludge；The service condition of MBR is：Hydraulic detention time
(HRT) it is 14 hours, dissolved oxygen is 3~6mg/L, and pH value is 7~8, and temperature is room temperature (25 ± 2 DEG C)；It is continuously improved in water inlet
Load, COD (from 200mg/L to 1000mg/L) and ammonia nitrogen (from 20mg/L to 250mg/L) are negative in stepping up first into water
Lotus, then the influent concentration (from 100mg/L to 1000mg/L) of penicillin is continuously improved in each stage, while reducing Portugal in water inlet
The concentration (from 1000mg/L to 0mg/L) of grape sugar, until carbon source is completely replaced as penicillin, each operation phase monitoring disengaging
The COD and ammonia nitrogen concentration of water, operation a period of time appropriate spoil disposal make in reactor sludge concentration maintain 4000mg/L~
5000mg/L, to keep the activity of sludge, the removal rate of COD and ammonia nitrogen reaches and is stably held in 90% and 99% or more, raw
The activated sludge that long status is stablized, as functional microorganism group；
(2) functional microorganism group is tamed into the sludge in process MBR reaction systems after each operation phase stable operation, from
Heart precipitation is used with enriched microorganismSpin Kit for Soil kits carry out each stage sludge micro-
The extraction of biological total DNA；
(3) by above-mentioned DNA sample progress 16S rDNA hypervariable regions V3~V4 (341F and 806R) PCR amplification, (three flat
Row), the concentration of amplified production should be greater than 15ng/ μ L；
(4) Nanodrop2000 and agarose gel electrophoresis is used to carry out pcr amplification product purity and fragment length size
Measurement, using Qubit carry out pcr amplification product concentration measurement, sample P CR amplified productions are pressed according to measured concentration difference
Etc. quality mixed；
(5) mixed library uses Agilent2100 biological analysers and quantitative PCR to carry out library fragments size respectively
With the accurate quantification of concentration, 0.1mol/LNaOH to surely measured mixing library carry out denaturation formed it is single-stranded, finally by
The MiSeq microarray datasets of Illumina companies carry out high-flux sequence using the sequencing strategy of 2 × 300bp；
(6) initial data Quality Control and bioinformatic analysis are finally carried out：First, according to barcode pairs of different samples
Initial data is split；Secondly, " primer sequence " in every sequence and its front end are used for adjusting using tagcleaner softwares
Equal " heterogeneous intervening sequence " sequence is not removed 0~7 bp of section laser signal；Furthermore by two terminal sequences according to weight
It closes and carries out quality of data filtering using Mothur platforms after part (overlap) is spliced, noise reduction, remove chimera, and go
Unless the Quality Controls such as bacterial sequences operate, and OTUs calculating and classification annotation are carried out with QIIME softwares；Finally, to time series
The OTU data of sampling and the monitoring water quality data that is corresponding to it have carried out the extension local similarity analysis (eLSA) of time series,
The correlation of synchronization and delay based on normalization sorting data is calculated by local similarity analysis (LSA), while generating class
It is similar to the related coefficient of Spearman sequence correlation analysis, network visualization is carried out by Cytopscape softwares and topology is tied
Structure is analyzed.
The result shows that 6 kinds of bacterium such as Meganema, Geminicoccus, Opitutus are the key that special degradation penicillin
Functional microorganism, the 34 kinds of bacterium occurred jointly with Meganema are penicillin wastewater processing function microorganism group core floras.In detail
See Fig. 1-Fig. 2.
Embodiment 2
(1) take returned sludge will first by strainer filtering sundries therein from city domestic sewage treatment plant secondary settling tank
It is inoculated into MBR reactors, a concentration of 3500~4500mg/L of seed sludge；The service condition of MBR is as follows, when hydraulic retention
Between (HRT) be 14 hours, dissolved oxygen be 3~6mg/L, pH value be 7~8, temperature be room temperature (25 ± 2 DEG C)；Water inlet is continuously improved
In load, COD (from 200mg/L to 1000mg/L) and ammonia nitrogen in stepping up first into water (from 20mg/L to 250mg/L)
Load, then each stage be continuously improved into salinity water (from 1% to 4%), the COD and ammonia of each operation phase monitoring Inlet and outlet water
Nitrogen concentration, operation a period of time, appropriate spoil disposal made sludge concentration in reactor maintain 4000mg/L~5000mg/L, to keep
The removal rate of the activity of sludge, COD and ammonia nitrogen reaches and is stably held in 90% and 99% or more, the activity that growth conditions are stablized
Sludge, as functional microorganism group；
(2) functional microorganism group is tamed into the sludge in process MBR reaction systems after each operation phase stable operation, from
Heart precipitation is used with enriched microorganismThe each stage sludge of Spin Kit for Soil kits carries out micro- life
The extraction of object total DNA；
(3) by above-mentioned DNA sample progress 16S rDNA hypervariable regions V3~V4 (341F and 806R) PCR amplification, (three flat
Row), the concentration of amplified production should be greater than 15ng/ μ L；
(4) Nanodrop2000 and agarose gel electrophoresis is used to carry out pcr amplification product purity and fragment length size
Measurement, using Qubit carry out pcr amplification product concentration measurement, sample P CR amplified productions are pressed according to measured concentration difference
Etc. quality mixed；
(5) mixed library uses 2100 biological analysers of Agilent and quantitative PCR to carry out library fragments big respectively
The accurate quantification of small and concentration, 0.1mol/LNaOH surely measured mixing library be denaturalized formed it is single-stranded, finally by
The MiSeq microarray datasets of Illumina companies carry out high-flux sequence using the sequencing strategy of 2 × 300bp；
(6) initial data Quality Control and bioinformatic analysis are finally carried out：First, according to barcode pairs of different samples
Initial data is split；Secondly, " primer sequence " in every sequence and its front end are used for adjusting using tagcleaner softwares
Equal " heterogeneous intervening sequence " sequence is not removed 0~7 bp of section laser signal；Furthermore by two terminal sequences according to weight
It closes and carries out quality of data filtering using Mothur platforms after part (overlap) is spliced, noise reduction, remove chimera, and go
Unless the Quality Controls such as bacterial sequences operate, and OTUs calculating and classification annotation are carried out with QIIME softwares；Finally, to time series
The OTU data of sampling and the monitoring water quality data that is corresponding to it have carried out the extension local similarity analysis (eLSA) of time series,
The correlation of synchronization and delay based on normalization sorting data is calculated by local similarity analysis (LSA), while generating class
It is similar to the related coefficient of Spearman sequence correlation analysis, network visualization is carried out by Cytopscape softwares and topology is tied
Structure is analyzed.
Meganema is penicillin principal degradation bacterium under the conditions of result of study shows Low-salinity (1%), and 4% salinity is rushed
It is penicillin principal degradation bacterium to hit lower Geminicoccus and Opitutus.Refer to Fig. 3-Fig. 4.
The present invention overcomes the problems of high bio-diversity Bacterial community parsing in bio-augmentation treatment system, from life
State angle has parsed response characteristic of the functional microorganism to ambient water quality parameter, has cracked the "black box" mould of biological reinforced regulation and control
Formula, meeting the technology based on high throughput sequencing technologies applied to bio-augmentation treatment system functional flora structure optimization regulation and control needs
It asks.
Claims (10)
1. a kind of biological reinforced functional flora analytic method based on high-flux sequence, it is characterised in that：It is tamed and dociled by acquiring sludge
The sludge in each stage during change extracts its microorganism total DNA, and based on the high-flux sequence of 16S rDNA, finally transport
The biological reinforced functional flora obtained to domestication with Time-Series analysis and network analysis bioinformatics means carries out deep analysis, from
The structure feature of microbiologic population in ecological point system analysis biochemical processing process, determines Core Feature flora therein,
And screen the crucial water quality parameter for influencing functional microorganism structure of community.
2. the biological reinforced functional flora analytic method according to claim 1 based on high-flux sequence, which is characterized in that
Specifically include following operating procedure：
Step 1, with membrane bioreactor, the domestication of functional microorganism group is carried out by the way that load in water inlet is continuously improved；
Step 2, the sludge after each operation phase stable operation in membrane bioreactor is acquired, centrifugation is to be enriched with micro- life
Object, and the extraction of microorganism total DNA is carried out to the mud sample of each operation phase；
Step 3, target fragment PCR amplification and amplified production is purified；
Step 4, to amplified production Quality Identification, and by different sample P CR amplified productions by etc. quality mix；
Step 5, clip size and quantitative detection, and high-flux sequence are carried out to mixing library；
Step 6, initial data Quality Control and bioinformatic analysis are finally carried out.
3. the biological reinforced functional flora analytic method according to claim 2 based on high-flux sequence, it is characterised in that：
In step 1, the service condition of membrane bioreactor is：Hydraulic detention time is 14 hours, and dissolved oxygen is 3~6mg/L, and pH value is
7~8, temperature is 25 ± 2 DEG C；By the way that the load in intaking, the COD and ammonia nitrogen of each operation phase monitoring Inlet and outlet water is continuously improved
Concentration, operation a period of time, appropriate spoil disposal made sludge concentration in reactor maintain 4000mg/L~5000mg/L, to keep dirty
The removal rate of the activity of mud, COD and ammonia nitrogen reaches and is stably held in 90% and 99% or more, obtains the work(of growth conditions stabilization
It can microorganism group；Wherein, the membrane material of membrane module used in membrane bioreactor is hollow polyvinylidene fluoride film, and aperture is
0.03 μm, outer diameter 2.2mm, internal diameter 1.0mm, membrane area 0.235m2。
4. the biological reinforced functional flora analytic method according to claim 2 based on high-flux sequence, it is characterised in that：
In step 2, the mud sample comes from each operation phase stabilization in functional microorganism group domestication process membrane bioreactor
Postrun sludge, centrifugation is with enriched microorganism.
5. the biological reinforced functional flora analytic method according to claim 4 based on high-flux sequence, it is characterised in that：
In step 2, useSpin Kit for Soil kits carry out mud sample that each operation phase obtains into
The extraction of row microorganism total DNA, DNA purity is A260/A280=1.8~2.0 after extraction.
6. the biological reinforced functional flora analytic method according to claim 2 based on high-flux sequence, it is characterised in that：
In step 3, target fragment PCR amplification sequence is 16S rDNA hypervariable region V3~V4, and the concentration of amplified production is more than 15ng/ μ
L。
7. the biological reinforced functional flora analytic method according to claim 2 based on high-flux sequence, it is characterised in that：
In step 4, the Quality Identification of amplified production includes measuring concentration, purity and the fragment length size of PCR product.
8. the biological reinforced functional flora analytic method according to claim 2 based on high-flux sequence, it is characterised in that：
In step 5,2100 biological analysers of Agilent and quantitative PCR is used to carry out clip size and quantitative inspection to mixing library respectively
It surveys.
9. the biological reinforced functional flora analytic method according to claim 2 based on high-flux sequence, it is characterised in that：
In step 5, high-flux sequence instrument is the MiSeq microarray datasets of Illumina companies, is sequenced for the both ends 2 × 300bp.
10. the biological reinforced functional flora analytic method according to claim 2 based on high-flux sequence, feature exist
In：In step 6, initial data Quality Control and bioinformatic analysis include the following steps：First, according to different samples
Barcode splits initial data；It secondly, will be " primer sequence " and its preceding in every sequence using tagcleaner softwares
End is removed for adjusting " heterogeneous intervening sequence " sequence that 0~7 bp of laser signal is not waited；Furthermore by both ends sequence
It arranges and carries out quality of data filtering, noise reduction using Mothur platforms after being spliced according to intersection, go chimera, and removal
Non-bacterial sequence Quality Control operation, and carry out OTUs calculating and classification annotation with QIIME softwares；Finally, to time sequential sampling
OTU data and be corresponding to it monitoring water quality data carry out time series extension local similarity analysis, by part it is similar
Property analysis calculate based on normalization sorting data synchronization and delay correlation, while generate be similar to Spearman sort phase
The related coefficient of closing property analysis carries out network visualization and Analysis of Topological Structure by Cytopscape softwares.
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