CN108486238B - High-throughput sequencing-based bioaugmentation functional flora analysis method - Google Patents
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Abstract
The invention discloses a high-throughput sequencing-based method for analyzing a bioaugmentation functional flora, which comprises the following operation steps: adopting a membrane bioreactor to domesticate functional microorganism groups by continuously increasing the load in the inlet water; collecting sludge which stably runs in each operation stage in the membrane bioreactor, carrying out centrifugal precipitation to enrich microorganisms, and extracting total DNA of the microorganisms from a sludge sample in each operation stage; amplifying the target fragment by PCR and purifying the amplification product; identifying the quality of the amplified products, and mixing the PCR amplified products of different samples according to the equal quality; performing fragment size and quantitative detection on the mixed library, and performing high-throughput sequencing; and finally, performing quality control and bioinformatics analysis on the original data. The analytic method can systematically analyze the structural characteristics of the microbial community in the wastewater biochemical treatment process from the ecological perspective, determine the core functional community in the wastewater treatment system, and screen key water quality parameters influencing the microbial community structure.
Description
Technical Field
The invention relates to a high-throughput sequencing-based analysis method for a bioaugmentation functional flora, belonging to the technical field of microbial bioaugmentation treatment.
Background
The environmental pollution caused by pharmaceutical wastewater is becoming more serious, and typical pharmaceutical wastewater such as antibiotics, vitamins, pesticides and the like causes serious pollution to the living environment of human beings. At present, biological methods are still the mainstream technology for pharmaceutical wastewater treatment. However, the biological treatment effect is easily affected due to the large fluctuation of the quality and the quantity of the pharmaceutical wastewater. The previous researches mostly focus on the treatment process and the determination of parameters thereof, but the information of key core flora and characteristic flora in the pharmaceutical wastewater treatment system is rarely known, and key factors influencing the flora structure and the treatment effect of the system are not clear, so the regulation and control of the treatment process are mainly guided by experience. Therefore, the deep analysis of the structure of the key functional flora in the biochemical enhanced treatment process of the pharmaceutical wastewater has great significance for improving the biochemical treatment efficiency.
Although technologies such as clone library, DGGE, TGGE and T-RFLP can also monitor and provide information of population change, due to the limitation of detection flux and analysis technology, the 'black box' of the pharmaceutical wastewater biological enhancement treatment system always troubles environmental protection researchers and first-line engineering technicians, and in recent years, a new generation of high-throughput sequencing technology and bioinformatics analysis method with the advantages of large flux, high accuracy, low cost and the like, which are rapidly developed, can realize rapid analysis of bacterial microbial community structure and diversity and enable the biological enhancement 'black box' to be transparent. The method has the advantages that the structural characteristics of the microbial community in the pharmaceutical wastewater biochemical treatment process are systematically analyzed from the ecological perspective, the core function microbial community in the pharmaceutical wastewater treatment system is determined, the key water quality parameters influencing the microbial community structure are screened, the microbial mechanism in the pharmaceutical wastewater treatment process is clarified, and the method has great significance for the optimal regulation and control of the pharmaceutical wastewater biological strengthening process and the development of the special-effect functional microbial inoculum.
Through searching, research on microbial community structure analysis has been published. For example, the invention patent with chinese patent application No. 201010132091.6 discloses a method for analyzing microbial community structure, which comprises extracting RNA from an environmental sample, separating small subunit (16S/18S) rRNA from the RNA, reverse transcribing the small subunit with random primers, then directly performing high-throughput sequencing and performing correlation analysis, wherein the method is used for studying microorganisms with activity in the environmental sample and avoiding the influence of PCR amplification on microbial community structure analysis, but because the environmental sample contains a large amount of humic acid, heavy metal ions and other substances, the extraction of total RNA and subsequent molecular manipulation can be severely inhibited, which often results in poor experimental repeatability; because the method needs to recycle the cut rubber, the operation is complicated, the workload is large, and the practical application of the method is limited; in addition, this method can only be used for studying microbial community structures, and cannot analyze the stress effect of functional microbial communities on environmental water quality parameters from an ecological perspective. Therefore, it is important to develop an analysis method for analyzing the structure of the bioaugmentation functional flora, which is simple and flexible to operate, has high flux, high accuracy and low cost, and can reveal the response characteristics of the functional microflora to the environmental water quality parameters.
Disclosure of Invention
The purpose of the invention is as follows: aiming at the problems that key core flora and characteristic flora information in a pharmaceutical wastewater biological enhanced treatment system are lacked, and key factors influencing the system flora structure and treatment effect are not clear, and the existing analysis method is low in flux, complicated to operate, large in workload, high in cost and the like, the invention aims at realizing the black box transparency of the pharmaceutical wastewater biological enhanced treatment system, and provides a method suitable for deep analysis of functional flora in the wastewater biological enhanced treatment system by adopting a bioinformatics analysis means based on new-generation high-flux sequencing. The method can systematically analyze the structural characteristics of the microbial community in the (pharmaceutical wastewater) biochemical treatment process from the ecological perspective, determine the core function microbial community in the (pharmaceutical wastewater) treatment system, and screen key water quality parameters influencing the microbial community structure.
In order to solve the technical problems, the technical scheme adopted by the invention is as follows:
a method for analyzing a biological enhanced functional flora based on high-throughput sequencing comprises the steps of collecting sludge at each stage in a sludge acclimatization process, extracting total DNA of microorganisms, carrying out high-throughput sequencing based on 16S rDNA, finally carrying out deep analysis on the biological enhanced functional flora obtained by acclimatization by using bioinformatics means such as time sequence analysis, network analysis and the like, analyzing the structural characteristics of the microbial community in a biochemical treatment process from an ecology perspective, determining the core functional flora in the biochemical treatment process, and screening key water quality parameters influencing the structure of the functional microbial community, thereby realizing the guidance of the accurate regulation and control of a biological enhanced treatment system.
The bioaugmentation functional flora analysis method based on high-throughput sequencing specifically comprises the following operation steps:
step 5, performing fragment size and quantitative detection on the mixed library, and performing high-throughput sequencing;
and 6, finally, performing quality control on the original data and bioinformatics analysis.
Wherein, in the step 1, the operating conditions of the membrane bioreactor are as follows: the hydraulic retention time is 14 hours, the dissolved oxygen is 3-6 mg/L, the pH value is 7-8, and the temperature is 25 +/-2 ℃; by continuously increasing the load in the inlet water, monitoring the COD and ammonia nitrogen concentration of the inlet water and the outlet water in each operation stage, operating for a period of time and properly discharging sludge to ensure that the sludge concentration in the reactor is maintained at 4000 mg/L-5000 mg/L so as to keep the activity of the sludge, wherein the removal rate of the COD and the ammonia nitrogen reaches and stably keeps more than 90 percent and 99 percent, and the active sludge-functional microorganism group with stable growth state is obtained.
Wherein, in the step 1, the membrane material of the membrane component used by the membrane bioreactor is a hollow polyvinylidene fluoride fiber membrane, the aperture of the membrane is 0.03 mu m, the outer diameter is 2.2mm, the inner diameter is 1.0mm, and the membrane area is 0.235m2。
In the step 2, the sample is obtained from sludge after stable operation in each operation stage in an MBR reaction system in the functional microbiome domestication process, and is centrifugally precipitated to enrich microorganisms.
Wherein, in step 2, use is made of
And extracting the total DNA of the microorganisms from the sludge sample obtained in each operation stage by using a Spin Kit for Soil Kit, wherein the purity of the extracted DNA is A260/A280-1.8-2.0.
In step 3, the PCR amplification sequences of the target fragment are 16S rDNA hypervariable regions V3-V4 (341F and 806R), and the concentration of the amplification product is more than 15 ng/. mu.L.
In step 4, the quality identification of the amplified product comprises the determination of the concentration, purity and fragment length of the PCR product.
In step 5, fragment size and quantitative detection are carried out on the mixed library by using an Agilent2100 bioanalyzer and quantitative PCR respectively.
In step 5, the high-throughput sequencing instrument is a MiSeq sequencing platform of Illumina, and is used for 2 × 300bp two-end sequencing.
In step 6, the quality control and bioinformatics analysis of the original data comprises the following steps: firstly, splitting original data according to the barcode of different samples; secondly, removing a primer sequence and a heterogeneous spacer sequence with the front end used for adjusting 0-7 bp inequality of the laser signal in each sequence by using tagCleaner software; thirdly, splicing the sequences at the two ends according to a superposition part (overlap), then performing data quality filtering, noise reduction, chimera removal and non-bacterial sequence quality control removal operations by using a Mothur platform, and performing OTUs calculation and classified annotation by using QIIME software; and finally, performing time-series extended local similarity analysis (eLSA) on the OTU data sampled by the time series and the monitored water quality data corresponding to the OTU data, calculating the correlation of synchronization and delay based on normalized sequencing data through the Local Similarity Analysis (LSA), simultaneously generating a correlation coefficient similar to the Spireman sequencing correlation analysis, and performing network visualization and topological structure analysis through cytopscope software.
The method for analyzing the bioaugmentation functional flora based on the high-throughput sequencing is applied to the analysis of the microbial community structure and the analysis of the response characteristics of the microbial community to the environmental water quality parameters.
Compared with the prior art, the technical scheme of the invention has the beneficial effects that:
firstly, the analysis method adopts the DNA extracted from the environmental sample rather than the RNA easy to degrade as a research object, so that the repeatability and the comparability of the experimental result can be ensured;
secondly, the analysis method is combined with a new generation sequencing technology, so that the problem that the existing method needs complicated steps such as culture separation, gel cutting recovery and the like is solved, and the analysis method has the advantages of simplicity, convenience and flexibility in operation, large flux, high accuracy and low cost;
finally, the analysis method provided by the invention adopts a time sequence analysis (eLSA) and network analysis method, not only can be used for analyzing the microbial community structure, but also can deeply analyze the response characteristics of the functional microbial community to the environmental water quality parameters from the ecological perspective, can realize accurate and efficient regulation and control of the pharmaceutical wastewater biological enhanced treatment system, and has a wide application prospect.
Drawings
FIG. 1 is a network analysis of the response characteristics of microorganisms to changes in environmental parameters (A) and penicillin concentration (B);
FIG. 2 is a plot of the co-occurrence (solid line) and co-elimination (dashed line) analysis of microorganisms in a penicillin wastewater MBR treatment system;
FIG. 3 is the distribution of microorganisms in each sludge sample at genus level under different salinity conditions;
FIG. 4 is a network analysis of response characteristics of microorganisms to changes in environmental parameters (salinity).
Detailed Description
The technical solutions of the present invention are further described below with reference to the accompanying drawings, but the scope of the claimed invention is not limited thereto.
Example 1
(1) Taking flowing sludge back from a secondary sedimentation tank of an urban domestic sewage treatment plant, filtering impurities in the flowing sludge through a filter screen, inoculating the impurities into an MBR (membrane bioreactor), wherein the concentration of the inoculated sludge is 3500-4500 mg/L; the operating conditions of the MBR are as follows: the Hydraulic Retention Time (HRT) is 14 hours, the dissolved oxygen is 3-6 mg/L, the pH value is 7-8, and the temperature is normal temperature (25 +/-2 ℃); continuously increasing the load in the inlet water, firstly gradually increasing the load of COD (from 200mg/L to 1000mg/L) and ammonia nitrogen (from 20mg/L to 250mg/L) in the inlet water, then continuously increasing the inlet water concentration of penicillin (from 100mg/L to 1000mg/L) in each stage, and simultaneously reducing the concentration of glucose (from 1000mg/L to 0mg/L) in the inlet water until the carbon source is completely replaced by penicillin, monitoring the COD and ammonia nitrogen concentration of inlet and outlet water in each operation stage, operating for a period of time to properly discharge sludge so that the sludge concentration in a reactor is maintained at 4000 mg/L-5000 mg/L to keep the activity of the sludge, wherein the removal rate of the COD and the ammonia nitrogen reaches and stably keeps above 90% and 99%, and the activated sludge with stable growth state is the functional microorganism group;
(2) the sludge after stable operation of each operation stage in the MBR reaction system in the functional microorganism group domestication process is centrifugally precipitated to enrich the microorganisms, and the method adopts
Extracting total DNA of the microorganisms from the sludge at each stage by a Spin Kit for Soil Kit;
(3) carrying out PCR amplification (three parallels) on the 16S rDNA hypervariable regions V3-V4 (341F and 806R) of the DNA sample, wherein the concentration of an amplification product is more than 15 ng/. mu.L;
(4) using Nanodrop2000 and agarose gel electrophoresis to measure the purity and fragment length of PCR amplification products, using Qubit to measure the concentration of the PCR amplification products, and mixing the PCR amplification products of the samples according to the equal mass according to different measurement concentrations;
(5) the size and concentration of the library fragments of the mixed library are accurately quantified by respectively using an Agilent2100 bioanalyzer and quantitative PCR, the quantified mixed library is denatured into a single chain by 0.1mol/LNaOH, and finally high-throughput sequencing is carried out by adopting a sequencing strategy of 2 multiplied by 300bp through a Miseq sequencing platform of Illumina company;
(6) and finally, performing quality control and bioinformatics analysis on the original data: firstly, splitting original data according to the barcode of different samples; secondly, removing a primer sequence and a heterogeneous spacer sequence with the front end used for adjusting 0-7 bp inequality of the laser signal in each sequence by using tagCleaner software; thirdly, splicing the sequences at the two ends according to a superposition part (overlap), performing quality control operations such as data quality filtering, noise reduction, chimera removal, non-bacterial sequence removal and the like by using a Mothur platform, and performing OTUs calculation and classified annotation by using QIIME software; and finally, performing time-series extended local similarity analysis (eLSA) on the OTU data sampled by the time series and the monitored water quality data corresponding to the OTU data, calculating the correlation between synchronization and delay based on normalized sequencing data through the Local Similarity Analysis (LSA), simultaneously generating a correlation coefficient similar to the Spireman sequencing correlation analysis, and performing network visualization and topological structure analysis through cytopscope software.
The results show that 6 kinds of bacteria such as Meganema, Geminicocus, Opituus and the like are key functional microorganisms specially degrading penicillin, and 34 kinds of bacteria which appear together with Meganema are core flora of a functional microorganism group for treating penicillin wastewater. See figures 1-2 for details.
Example 2
(1) Taking flowing sludge back from a secondary sedimentation tank of an urban domestic sewage treatment plant, filtering impurities in the flowing sludge through a filter screen, inoculating the impurities into an MBR (membrane bioreactor), wherein the concentration of the inoculated sludge is 3500-4500 mg/L; the MBR has the following operating conditions that the Hydraulic Retention Time (HRT) is 14 hours, the dissolved oxygen is 3-6 mg/L, the pH value is 7-8, and the temperature is normal temperature (25 +/-2 ℃); continuously increasing the load in the inlet water, firstly gradually increasing the load of COD (from 200mg/L to 1000mg/L) and ammonia nitrogen (from 20mg/L to 250mg/L) in the inlet water, then continuously increasing the salinity of the inlet water (from 1% to 4%) in each stage, monitoring the COD and ammonia nitrogen concentration of the inlet water and the outlet water in each operation stage, operating for a period of time to properly discharge sludge so as to maintain the sludge concentration in the reactor at 4000mg/L to 5000mg/L and keep the activity of the sludge, wherein the removal rate of the COD and the ammonia nitrogen reaches and stably keeps more than 90% and 99%, and the activated sludge with stable growth state is the functional microbiome;
(2) the sludge after stable operation of each operation stage in the MBR reaction system in the functional microorganism group domestication process is centrifugally precipitated to enrich the microorganisms, and the method adopts
Extracting total DNA of the microorganism from sludge at each stage of the Spin Kit for Soil Kit;
(3) carrying out PCR amplification (three parallels) on the 16S rDNA hypervariable regions V3-V4 (341F and 806R) of the DNA sample, wherein the concentration of an amplification product is more than 15 ng/. mu.L;
(4) using Nanodrop2000 and agarose gel electrophoresis to measure the purity and fragment length of PCR amplification products, using Qubit to measure the concentration of the PCR amplification products, and mixing the PCR amplification products of the samples according to the equal mass according to different measurement concentrations;
(5) the size and concentration of the library fragments of the mixed library are accurately quantified by respectively using an Agilent2100 bioanalyzer and quantitative PCR, the quantified mixed library is denatured into a single chain by 0.1mol/LNaOH, and finally high-throughput sequencing is carried out by adopting a sequencing strategy of 2 multiplied by 300bp through a Miseq sequencing platform of Illumina company;
(6) and finally, performing quality control and bioinformatics analysis on the original data: firstly, splitting original data according to the barcode of different samples; secondly, removing a primer sequence and a heterogeneous spacer sequence with the front end used for adjusting 0-7 bp inequality of the laser signal in each sequence by using tagCleaner software; thirdly, splicing the sequences at the two ends according to a superposition part (overlap), performing quality control operations such as data quality filtering, noise reduction, chimera removal, non-bacterial sequence removal and the like by using a Mothur platform, and performing OTUs calculation and classified annotation by using QIIME software; and finally, performing time-series extended local similarity analysis (eLSA) on the OTU data sampled by the time series and the monitored water quality data corresponding to the OTU data, calculating the correlation between synchronization and delay based on normalized sequencing data through the Local Similarity Analysis (LSA), simultaneously generating a correlation coefficient similar to the Spireman sequencing correlation analysis, and performing network visualization and topological structure analysis through cytopscope software.
The results of the study show that under the condition of low salinity (1%), Meganema is the main degrading bacteria of penicillin, and Geminicocus and Opituus are the main degrading bacteria of penicillin under the condition of 4% salinity impact. See figures 3-4 for details.
The method provided by the invention overcomes the problem of structural analysis of high biodiversity flora in a bioaugmentation treatment system, analyzes the response characteristics of functional microorganisms to environmental water quality parameters from an ecological perspective, breaks a 'black box' mode of bioaugmentation regulation and control, and meets the technical requirement of applying a high-throughput sequencing technology to the structural optimization regulation and control of functional flora in the bioaugmentation treatment system.
Claims (2)
1. A bioaugmentation functional flora analysis method based on high-throughput sequencing is characterized by comprising the following operation steps:
(1) inoculating activated sludge into an MBR (membrane bioreactor), wherein the concentration of the inoculated sludge is 3500-4500 mg/L; the operating conditions of the MBR are as follows: the hydraulic retention time is 14 hours, the dissolved oxygen is 3-6 mg/L, the pH value is 7-8, and the temperature is 25 +/-2 ℃; continuously increasing the load in the inlet water, and firstly gradually increasing the COD and ammonia nitrogen load in the inlet water, wherein the concentration of the COD in the inlet water is increased from 200mg/L to 1000mg/L, and the concentration of the ammonia nitrogen in the inlet water is increased from 20mg/L to 250 mg/L; continuously increasing the inlet water concentration of penicillin in each stage, increasing the concentration of penicillin in inlet water from 100mg/L to 1000mg/L, simultaneously reducing the concentration of glucose in inlet water from 1000mg/L to 0mg/L until a carbon source is completely replaced by penicillin, monitoring COD (chemical oxygen demand) and ammonia nitrogen concentration of inlet and outlet water in each operation stage, operating for a period of time to discharge sludge so that the concentration of sludge in the reactor is maintained at 4000 mg/L-5000 mg/L to keep the activity of the sludge, and keeping the removal rate of COD and ammonia nitrogen to be more than 90% and 99% stably to obtain a functional microorganism group with a stable growth state;
(2) performing centrifugal precipitation on sludge which stably runs in each running stage in an MBR reaction system in a functional microbiome domestication process to enrich microorganisms, and extracting total DNA of the microorganisms from the sludge in each stage by adopting FastDNA Spin Kit for Soil Kit;
(3) carrying out PCR amplification on the 16S rDNA high variable region V3-V4 on the DNA sample, wherein the concentration of an amplification product is more than 15 ng/mu L;
(4) using Nanodrop2000 and agarose gel electrophoresis to measure the purity and fragment length of PCR amplification products, using Qubit to measure the concentration of the PCR amplification products, and mixing the PCR amplification products of the samples according to the equal mass according to different measurement concentrations;
(5) the size and concentration of the library fragments of the mixed library are accurately quantified by respectively using an Agilent2100 bioanalyzer and quantitative PCR, the quantified mixed library is denatured into a single chain by 0.1mol/LNaOH, and finally high-throughput sequencing is carried out by adopting a sequencing strategy of 2 multiplied by 300bp through a Miseq sequencing platform of Illumina company;
(6) and finally, performing quality control and bioinformatics analysis on the original data: firstly, splitting original data according to the barcode of different samples; secondly, removing a primer sequence and a heterogeneous spacer sequence with the front end used for adjusting 0-7 bp inequality of the laser signal in each sequence by using tagCleaner software; thirdly, splicing the sequences at the two ends according to the overlapped part, performing quality control operations of data quality filtering, noise reduction, chimera removal and non-bacterial sequence removal by using a Mothur platform, and performing OTUs calculation and classified annotation by using QIIME software; and finally, performing time-series extended local similarity analysis on the OTU data sampled by the time series and the water quality monitoring data corresponding to the OTU data, calculating the synchronization and delay correlation based on the normalized sequencing data through the local similarity analysis, simultaneously generating a correlation coefficient similar to the Spireman sequencing correlation analysis, and performing network visualization and topological structure analysis through cytopscope software.
2. A bioaugmentation functional flora analysis method based on high-throughput sequencing is characterized by comprising the following operation steps:
(1) inoculating activated sludge into an MBR (membrane bioreactor), wherein the concentration of the inoculated sludge is 3500-4500 mg/L; the operating conditions of the MBR are as follows: the hydraulic retention time is 14 hours, the dissolved oxygen is 3-6 mg/L, the pH value is 7-8, and the temperature is 25 +/-2 ℃; continuously increasing the load in the inlet water, and firstly gradually increasing the COD and ammonia nitrogen load in the inlet water, wherein the concentration of the COD in the inlet water is increased from 200mg/L to 1000mg/L, and the concentration of the ammonia nitrogen in the inlet water is increased from 20mg/L to 250 mg/L; then continuously increasing the inlet water concentration of penicillin in each stage, increasing the concentration of penicillin in inlet water from 100mg/L to 1000mg/L, simultaneously reducing the concentration of glucose in inlet water from 1000mg/L to 0mg/L until a carbon source is completely replaced by penicillin, continuously increasing the salinity of inlet water in each stage, increasing the salinity of inlet water from 1% to 4%, monitoring COD (chemical oxygen demand) and ammonia nitrogen concentration of inlet and outlet water in each operation stage, operating for a period of time to discharge sludge so that the concentration of sludge in a reactor is maintained at 4000 mg/L-5000 mg/L to keep the activity of the sludge, and stably keeping the removal rate of COD and ammonia nitrogen at more than 90% and 99% to obtain a functional microorganism group with stable growth state;
(2) performing centrifugal precipitation on sludge which stably runs in each running stage in an MBR reaction system in a functional microbiome domestication process to enrich microorganisms, and extracting total DNA of the microorganisms from the sludge in each stage by adopting FastDNA Spin Kit for Soil Kit;
(3) carrying out PCR amplification on the 16S rDNA high variable region V3-V4 on the DNA sample, wherein the concentration of an amplification product is more than 15 ng/mu L;
(4) using Nanodrop2000 and agarose gel electrophoresis to measure the purity and fragment length of PCR amplification products, using Qubit to measure the concentration of the PCR amplification products, and mixing the PCR amplification products of the samples according to the equal mass according to different measurement concentrations;
(5) the size and concentration of the library fragments of the mixed library are accurately quantified by respectively using an Agilent2100 bioanalyzer and quantitative PCR, the quantified mixed library is denatured into a single chain by 0.1mol/LNaOH, and finally high-throughput sequencing is carried out by adopting a sequencing strategy of 2 multiplied by 300bp through a Miseq sequencing platform of Illumina company;
(6) and finally, performing quality control and bioinformatics analysis on the original data: firstly, splitting original data according to the barcode of different samples; secondly, removing a primer sequence and a heterogeneous spacer sequence with the front end used for adjusting 0-7 bp inequality of the laser signal in each sequence by using tagCleaner software; thirdly, splicing the sequences at the two ends according to the overlapped part, performing quality control operations of data quality filtering, noise reduction, chimera removal and non-bacterial sequence removal by using a Mothur platform, and performing OTUs calculation and classified annotation by using QIIME software; and finally, performing time-series extended local similarity analysis on the OTU data sampled by the time series and the water quality monitoring data corresponding to the OTU data, calculating the synchronization and delay correlation based on the normalized sequencing data through the local similarity analysis, simultaneously generating a correlation coefficient similar to the Spireman sequencing correlation analysis, and performing network visualization and topological structure analysis through cytopscope software.
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