CN114686610A - Method for detecting microbial community structure change in SNAD process operation process - Google Patents
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Abstract
The invention discloses a method for detecting microbial community structure change in the SNAD process operation process, which comprises the following steps: respectively sampling for 1 time in an inoculation sludge period, a nitrosation stabilization period, an SNAD starting initial period, an SNAD stabilization period and an SNAD stabilization later period in the SNAD process operation process to obtain six sludge samples; standing the six mud samples for 10-15min, removing supernatant, centrifuging for at least 1 time for removing humus, and extracting total bacterial DNA from solids obtained by centrifuging to obtain six DNA solutions; and carrying out PCR amplification to obtain a PCR amplification product, carrying out denaturing gradient gel electrophoresis on the PCR amplification product to obtain a DGGE band map, and obtaining the change of the microbial community structure in the SNAD process operation process. The detection method can quickly and efficiently detect and know the dynamic change of the flora structure on each node in the experimental period.
Description
Technical Field
The invention belongs to the technical field of molecular biology, and particularly relates to a method for detecting structural change of a microbial community in an SNAD (selective non-catalytic reduction) process operation process.
Background
The traditional biological denitrification process for treating urban domestic sewage has the defects of large occupied area, high energy consumption, insufficient external carbon source and the like, so that the operation cost is increased, and the operation management difficulty is increased. In recent years, with intensive research on microbiology and operation of denitrification processes, novel biological denitrification processes with high efficiency and low consumption appear, wherein the anaerobic ammonia oxidation technology receives wide attention due to the advantages of small occupied space, low operation cost and the like. The CANNON (complex automatic ammonium removal over nitrate) process can realize the removal of total nitrogen through the mutual cooperation of AOB and anammox bacteria in a reactor, and is mainly used for treating high ammonia nitrogen wastewater. Recent studies have shown that CANNON processes can also be implemented in low-temperature low ammonia nitrogen wastewater. The high-concentration organic matter has a certain inhibiting effect on the growth of anaerobic ammonium oxidation, but nitrite bacteria, anaerobic ammonium oxidation bacteria and denitrifying bacteria can coexist in a reactor when the concentration of the organic matter is lower. Therefore, the SNAD process takes the SBR as a platform, combines the processes of nitrosation, anaerobic ammonia oxidation and denitrification, and finally forms synchronous nitrosation, anaerobic ammonia oxidation and denitrification coupling denitrification.
In 2009, Yanofenlin et al put forward a concept of synchronous nitrosation-anammox-denitrification (SNAD) based on the theoretical basis of the CANON process. The SNAD process is characterized in that nitrosation, anaerobic ammonia oxidation and denitrification processes are simultaneously completed in one reactor, and the nitrosation, anaerobic ammonia oxidation and denitrification bacteria are coordinated to perform functions together by controlling reaction conditions, so that the aim of simultaneous denitrification is fulfilled.
Because the traditional culture technology has serious limitations in the aspects of researching the diversity, dynamics and the like of environmental engineering microorganisms, the research on the relationship between the microorganisms and the treatment effect in the sewage treatment process is still in a 'black box' stage at present, and the problem is well solved by the molecular biology technology developed in recent years. The PCR technology has good specificity and sensitivity when applied to sewage treatment, so the treatment operation is simpler and more convenient than the traditional biological control technology, the technology has certain limitation, repetitive enzymatic reaction is carried out before sewage detection to obtain DNA amplification, and some correction or combined derivative technologies are used to ensure the accuracy of sewage microorganism detection when necessary. Meanwhile, the successful PCR experiment cannot be separated from the accurate experiment design and operation flow, and the implementation of the PCR experiment needs to be optimized to ensure that all reaction parameters are accurately set, so that an accurate result is obtained.
Disclosure of Invention
Aiming at the defects of the prior art, the invention aims to provide a rapid, sensitive and efficient detection method for microbial community structure change in the SNAD process operation process, which detects the microbial community structure change from the whole process of SNAD process starting and debugging to the whole process of stable operation of a base, directly extracts total DNA from granular sludge in the whole process of SNAD process operation, and then detects the composition of the microbial community in the granular sludge by adopting a PCR-DGGE (polymerase chain reaction-denaturing gradient gel electrophoresis) technology.
The purpose of the invention is realized by the following technical scheme.
A method for detecting microbial community structure change in the SNAD process operation process comprises the following steps:
1) respectively sampling for 1 time in an inoculation sludge period, a nitrosation stabilization period, an SNAD starting initial period, an SNAD stabilization period and an SNAD stabilization later period in the SNAD process operation process to obtain six sludge samples;
in the step 1), the sampling time of the sludge inoculation period is the 1 st day of the operation of the SNAD process, the sampling time of the nitrosation stabilization period is the 24 th to 26 th days of the operation of the SNAD process, the sampling time of the initial SNAD starting period is the 39 th to 41 th days of the operation of the SNAD process, the sampling time of the initial SNAD stabilization period is the 49 th to 51 th days of the operation of the SNAD process, the sampling time of the SNAD stabilization period is the 59 th to 61 th days of the operation of the SNAD process, and the sampling time of the later SNAD stabilization period is the 73 th to 75 th days of the operation of the SNAD process.
2) Standing the six mud samples obtained in the step 1) for 10-15min, removing supernatant, centrifuging for at least 1 time for removing humus, and extracting total bacterial DNA from solids obtained by centrifuging to obtain six DNA solutions;
in the step 2), the time of the centrifugation is 5-6min, and the rotation speed of the centrifugation is 15000-16000 rpm.
In the step 2), the centrifugation time is 4-5 times.
In the step 2), the temperature of the centrifugation is 4-5 ℃.
3) Performing PCR amplification to obtain PCR amplification product, wherein the primer is
F341-GC (5'-CGCCCGCCGCGCCCCGCGCCCGTCCCGCCGCCCCCGCCCGCCTACGGG AGGCAGCAG-3') and EU500(5'-GTATTACCGCGGCTGCTGG-3'), wherein the PCR amplification adopts a falling type amplification program, and the specific reaction conditions are as follows: pre-denaturation at 94 ℃ for 5 min; denaturation at 94 ℃ for 1min, annealing at 65 ℃ for 1min, and extension at 72 ℃ for 1min, for 20 cycles, each cycle being 0.5 ℃ lower; denaturation at 94 ℃ for 1min, annealing at 55 ℃ for 1min, and extension at 72 ℃ for 1min for 3 cycles; extending for 10min at 72 ℃;
in the step 3), the PCR reaction system is as follows: 5 parts by volume of 10 XPCR buffer, 4 parts by volume of dNTP, 1 part by volume of F341-GC, 1 part by volume of EU500, 0.5 part by volume of Taq DNA polymerase and 2 parts by volume of DNA solution were mixed, and distilled water was added to 50 parts by volume.
4) And (3) performing Denaturing Gradient Gel Electrophoresis (DGGE) on the PCR amplification product to obtain a DGGE band map, so as to obtain the change of the microbial community structure in the SNAD process operation process.
The detection method of the microbial community in the SNAD process operation process is established by adopting the PCR-DGGE molecular biology technology, can quickly and efficiently detect and understand the dynamic change of the community structure on each node in the experiment period, and can analyze the target sequence in a targeted manner, thereby avoiding the limitation that the traditional culture technology cannot separate and identify, obtaining the experiment result in a short time, greatly reducing the workload and the working practice, and ensuring the true and credible experiment result.
The succession of the composition of a microflora and the structure of a microflora in the SNAD reactor is researched, the dominant bacteria and the change of the microflora in normal operation are found out, and an important theoretical guidance can be provided for the operation of the SNAD granular sludge process.
Drawings
FIG. 1 is an agarose gel electrophoresis of a DNA solution, wherein M is Marker;
FIG. 2 is an agarose gel electrophoresis of the PCR amplification product, wherein 0 is blank;
FIG. 3 is a DGGE electrophoretogram of PCR amplification products;
FIG. 4 shows a comparison of the similarity of electrophoresis bands of total bacteria;
FIG. 5 is a gel electrophoresis test of PCR amplification products after gel cutting and recovery, wherein M is GM331 Marker, and 0 is negative control;
FIG. 6 is a phylogenetic tree of the major flora in sludge based on the 16S rDNA sequence;
FIG. 7 shows nitrogen removal characteristics of an SBR reactor in which domestic sewage is sampled;
FIG. 8 is a COD removing characteristic of the SBR reactor in which the domestic sewage was sampled.
Detailed Description
The technical scheme of the invention is further explained by combining specific examples.
Drug purchase sources in the following examples: shanghai Bioengineering technology, Inc.
The model and manufacturer of the instrument involved in the following examples:
a clean bench (VS-1300L-U), a constant temperature biochemical incubator (SHP-250 type, Shanghai), a full automatic sterilization pot (MLS-3750 type), an ultra pure water instrument (century, Milli-Q), a high speed centrifuge (1-14ED, SIGMA), a PCR instrument (MyiQ Real-time, Bio-Rad), a water bath constant temperature oscillator (DSHZ-300A, Jiangsu), a horizontal electrophoresis tank (DYCP-31DN, six instruments factory of Beijing city), a Gel imaging system (Gel DocTM XR +, Bio-Rad), an analytical balance (AUY220 Shimadzu), -a low temperature refrigerator (Hell) of 20 ℃, an ultraviolet instrument (WB-9403B, six instruments factory of Beijing city).
Example 1
A method for detecting microbial community structure change in the SNAD process operation process comprises the following steps:
1) sampling for 1 time respectively in an inoculation sludge period, a nitrosation stabilization period, an SNAD starting initial period, an SNAD stabilization period and an SNAD stabilization later period in the running process of the SNAD process to obtain six sludge samples, wherein the six sludge samples are numbered from 1 to 6 respectively, the sampling time of the inoculation sludge period is the 1 st day of the running of the SNAD process, the sampling time of the nitrosation stabilization period is the 25 th day of the running of the SNAD process, the sampling time of the SNAD starting initial period is the 40 th day of the running of the SNAD process, the sampling time of the SNAD stabilization initial period is the 50 th day of the running of the SNAD process, the sampling time of the SNAD stabilization period is the 60 th day of the running of the SNAD process, and the sampling time of the SNAD stabilization later period is the 74 th day of the running of the SNAD process.
2) The humus is an organic substance formed by decomposing dead organisms in soil through microorganisms, in order to better remove the humus in the sludge, 1g of sludge sample is collected, the supernatant is discarded after the sludge is statically precipitated in a centrifugal tube for 10min, the ultra-pure water is used for supplementing to 40mL, the sludge is centrifuged at 15000rpm for 5min at 4 ℃, the supernatant is discarded, the centrifugation is repeated for 5 times, the obtained solid is centrifuged by adopting an Ezup column type genome DNA extraction kit (Shanghai Biotechnology Co., Ltd.) to extract total bacterial DNA, six DNA solutions are obtained, 5 mu L of each DNA solution is detected by 1.2% agarose gel, the agarose gel electrophoresis chart is shown in figure 1, the DNA fragment size of the sample is about 23kb, the purity and the brightness are good, the DNA extraction is proved to be successful, and a good template is provided for the next PCR.
The method for extracting the total bacterial DNA comprises the following steps: weighing 200mg of solid obtained by centrifugation, adding 400 μ L of Buffer SCL preheated at 65 ℃, shaking and mixing uniformly, and placing in a water bath at 65 ℃ for 5 min; centrifuge at 12000rpm for 3min at room temperature. Sucking the supernatant into a clean 1.5mL centrifuge tube; adding equal volume of Buffer SP, reversing and mixing uniformly, and carrying out ice bath for 10 min; centrifuge at 12000rpm for 3min at room temperature. Sucking the supernatant into a clean 1.5mL centrifuge tube; adding 200 μ L chloroform, mixing well, and centrifuging at 12000rpm for 5 min. Absorbing the upper aqueous phase into a clean centrifugal tube with the volume of 1.5 mL; add 1.5 times volume (900 μ L) of Buffer SB, mix well and add it to the adsorption column with a pipette, and let stand at room temperature for 2 min. Centrifuging at 12000rpm for 30s, and pouring out waste liquid in the collecting pipe; putting the adsorption column back into the collection tube, adding 700 mu L of Wash Solution, centrifuging at 12000rpm for 30s, and pouring off waste liquid in the collection tube; putting the adsorption column back into the collection tube, adding 300 μ L Wash Solution, centrifuging at 12000rpm for 1min, and pouring off the waste liquid in the collection tube; placing the adsorption column back into the collection tube, and centrifuging at 12000rpm for 2 min; taking out the adsorption column, placing into a new 1.5mL centrifuge tube, adding 75 μ L TE Buffer into the center of the adsorption membrane, standing for 3min, centrifuging at 12000rpm for 2min, and storing the obtained DNA solution at-20 deg.C or directly using in subsequent experiment.
3) 0.3g of each DNA solution is taken for PCR amplification to obtain a PCR amplification product, the PCR amplification product is detected by 1.2 percent agarose gel electrophoresis, the agarose gel electrophoresis picture is shown in figure 2, the figure shows that the size of an amplification fragment is about 240bp, the amplification fragment is proved to be an amplified target band, and the band is single and has no non-specific amplification. Wherein, the primers are F341-GC (5'-CGCCCGCCGCGCCCCGCGCCCGTCCCGCCGCCCCCGCCCGCCTACGGG AGGCAGCAG-3') and EU500(5'-GTATTACCGCGGCTGCTGG-3') which have specificity to the V3 region of most bacteria 16S rDNA genes, the PCR amplification adopts a fall-down amplification program, and the specific reaction conditions are as follows: pre-denaturation at 94 ℃ for 5 min; denaturation at 94 ℃ for 1min, annealing at 65 ℃ for 1min, and extension at 72 ℃ for 1min, for 20 cycles, each cycle being 0.5 ℃ lower; denaturation at 94 ℃ for 1min, annealing at 55 ℃ for 1min, and extension at 72 ℃ for 1min for 3 cycles; extending for 10min at 72 ℃;
the PCR reaction system is as follows: mu.L of 10 XPCR buffer and 4. mu.L of dNTP (the concentrations of dATP, dTTP, dCTP and dGTP in dNTP are each 2.5mmol L-1) 1. mu.L of F341-GC (20. mu. molL)-1) 1 μ L of EU500 (20 μmolL)-1) 0.5. mu.L of Taq DNA polymerase (5U) and 2. mu.L of the DNA solution were mixed, and the mixture was added with distilled water to 50. mu.L.
4) And (3) performing Denaturing Gradient Gel Electrophoresis (DGGE) on the PCR amplification product by adopting a Bio-Rad DCodeTM DGGE system to obtain a DGGE band map, wherein the DGGE electrophoresis map is shown in figure 3, so that the change of the microbial community structure in the running process of the SNAD process is obtained.
The method for performing denaturing gradient gel electrophoresis on the PCR amplification product comprises the following steps: 8% polyacrylamide gel is prepared by a gradient mixing device, and the denaturation range of the polyacrylamide gel is 35% -55% (7 mol. L for 100% of denaturant)-1Urea and 40% formamide). Wherein the concentrations of the denaturant and the polyacrylamide are gradually increased from the upper part to the lower part of the glue. After the polyacrylamide gel is completely solidified, putting the rubber plate into the containerIn the electrophoresis tank containing the electrophoresis buffer, 20. mu.L of the amplification product from LPCR was mixed with 10. mu.L of 6 × loading buffer, added to the loading well, and electrophoresed in 1 × TAE electrophoresis buffer for 8 hours (60 ℃, 130V). After the electrophoresis was completed, the Gel Red nucleic acid Gel dye was stained for 30min and photographed in a Gel imaging system (Gel DocTMXR +, Bio-Rad).
In order to understand the dynamic change condition of the total bacterial community structure in the SNAD process culture domestication process, the similarity of the total bacterial community at each period in the DGGE band map is analyzed, and the figure is shown in figure 4. As can be seen from the sequence distribution diagram of the similarity, the similarity value between each lane and lane 6 basically conforms to the process of culture and acclimation, which indicates that the sludge culture and acclimation is a step-by-step and the microorganisms gradually evolve to enter the normal operation state.
The main independent 16S rDNA band on DGGE gel was cut with a sterile blade and placed in a 1.5mL centrifuge tube, 50. mu.L of sterilized ultrapure water was added, the centrifuge tube was allowed to stand overnight at 4 ℃ and 2. mu.L of the leachate was used as a template for PCR amplification. The primers used for PCR amplification after gel cutting recovery are as follows: f357: 5'-CCTACGGGAGGCAGCAG-3' and R518: 5'-ATTACCGCGGCTGCTGG-3', the fragment of the amplified product is about 200bp long. The PCR reaction program is: pre-denaturation at 94 ℃ for 5 min; then 30 cycles: 94 ℃ for 1min, 55 ℃ for 1min and 72 ℃ for 1 min; finally, extension was carried out at 72 ℃ for 8 min. The PCR amplification products after cutting and recovery were detected by electrophoresis on 1.2% agarose gel as shown in FIG. 5.
Cloning PCR products of DNA obtained after gel cutting recovery of bands of a DGGE map, delivering clones to a biotechnology company for sequencing, sequencing 10 samples (bands L1, L2, L3, L4, L6, L7, L9, L14, L16 and L22) of 23 DNA samples, inputting gene sequences obtained by sequencing into an NCBI website, performing comparison analysis by using BLAST (BLAST program) and sequences existing in a database, and constructing an evolutionary tree by using MEGA4 software, wherein the algorithm is a neighbor-join method (neighbor-join-analysis), and the obtained evolutionary tree is shown in figure 6.
Partial dominant bacteria of the total bacteria are subjected to gel cutting recovery cloning sequencing and phylogenetic tree analysis, and the dominant bacteria mainly comprise beta-Proteobacteria (beta-Proteobacteria), gamma-Proteobacteria (gamma-Proteobacteria), Firmicutes (Firmicutes), bacteroides (bacteroides) and Uncultured bacteria (Uncultured bacteria). The distribution of microorganisms is wide, main dominant strains are distributed in different classes, detected strains contain a little more beta-proteobacteria and keep a stable preferential status, secondary populations of sclerenchyma bacteria, gamma-proteobacteria, bacteroides and the like are enhanced along with the operation of the reactor to form a new dominant community, and a certain theoretical basis is provided for improving the SNAD granular sludge culture domestication efficiency and optimizing operation parameters.
Comparative example 1
Steps 1) to 3) of comparative example 1 are substantially the same as steps 1) to 3) of the detection method of example 1, the only difference being that the PCR amplification procedure in step 3) of comparative example 1 is: pre-denaturation at 94 ℃ for 5 min; denaturation at 94 ℃ for 1min, annealing at 65 ℃ for 1min, and extension at 72 ℃ for 1min for 25 cycles; extension at 72 ℃ for 10 min.
The PCR amplification product of comparative example 1 was electrophoretically detected in a Bio-Rad horizontal electrophoresis apparatus using a 1.2% agarose gel, and the PCR amplification product was run gel without bands.
The samples taken in the step 1) of the above examples and comparative examples were domestic sewage, and the water quality index of the domestic sewage is shown in Table 1. The sludge sample is taken from anammox granular sludge at the lower part of the anammox fixed bed reactor.
TABLE 1
NH in domestic sewage SNAD granular sludge process starting domestication process4 +-N、NO3 --N、NO2 -The changes of the inlet and outlet water concentrations and the removal rates of-N and COD are shown in FIGS. 7 and 8. And (3) 1-20 days, the nitrosation effect of the granular sludge is gradually increased, and when the time reaches 20 days, the concentration of the ammonia nitrogen in the effluent is reduced to 3mg/L, the concentration of the nitrite in the effluent is increased to 58mg/L, the ammonia nitrogen removal rate is increased to 93%, and the nitrite accumulation rate reaches 95%. 21-29 days, the nitrosation effect of the granular sludge is kept stable, and the average ammonia nitrogen removal rate and the nitrous nitrogen accumulation rate are respectively kept stable96% and 95%. 30-48 days later, the denitrification performance of the reactor is gradually enhanced, the ammonia nitrogen in the effluent is gradually reduced from 46mg/L to below 10mg/L, and the total nitrogen removal rate is increased from 22.6% to 83.9%. 49-74d, the reactor keeps higher total nitrogen removal capacity, the ammonia nitrogen concentration of effluent is below 5mg/L, the nitrate nitrogen concentration and the nitrite concentration of effluent are kept stable, the average values are 3mg/L and 4mg/L respectively, and the average total nitrogen removal rate is 85%.
The invention has been described in an illustrative manner, and it is to be understood that any simple variations, modifications or other equivalent changes which can be made by one skilled in the art without departing from the spirit of the invention fall within the scope of the invention.
Claims (6)
1. A method for detecting microbial community structure change in the SNAD process operation process is characterized by comprising the following steps:
1) respectively sampling for 1 time in an inoculation sludge period, a nitrosation stabilization period, an SNAD starting initial period, an SNAD stabilization period and an SNAD stabilization later period in the SNAD process operation process to obtain six sludge samples;
2) standing the six mud samples obtained in the step 1) for 10-15min, removing supernatant, centrifuging for at least 1 time for removing humus, and extracting total bacterial DNA from solids obtained by centrifuging to obtain six DNA solutions;
3) carrying out PCR amplification to obtain a PCR amplification product, wherein primers are F341-GC (5'-CGCCCGCCGCGCCCCGCGCCCGTCCCGCCGCCCCCGCCCGCCTACGGGAGGCAGCAG-3') and EU500(5'-GTATTACCGCGGCTGCTGG-3'), the PCR amplification adopts a falling type amplification program, and the specific reaction conditions are as follows: pre-denaturation at 94 deg.C for 5 min; denaturation at 94 ℃ for 1min, annealing at 65 ℃ for 1min, and extension at 72 ℃ for 1min, for 20 cycles, each cycle being 0.5 ℃ lower; denaturation at 94 ℃ for 1min, annealing at 55 ℃ for 1min, and extension at 72 ℃ for 1min for 3 cycles; extending for 10min at 72 ℃;
4) and performing denaturing gradient gel electrophoresis on the PCR amplification product to obtain a DGGE band map, and obtaining the change of the microbial community structure in the SNAD process operation process.
2. The detection method according to claim 1, wherein in the step 1), the sampling time of the sludge inoculation period is day 1 of the SNAD process operation, the sampling time of the nitrosation stabilization period is days 24-26 of the SNAD process operation, the sampling time of the SNAD initial start period is days 39-41 of the SNAD process operation, the sampling time of the SNAD initial stabilization period is days 49-51 of the SNAD process operation, the sampling time of the SNAD stabilization period is days 59-61 of the SNAD process operation, and the sampling time of the SNAD post-stabilization period is days 73-75 of the SNAD process operation.
3. The detection method according to claim 2, wherein in the step 2), the centrifugation time is 5-6min, and the centrifugation rotation speed is 15000-16000 rpm.
4. The detection method according to claim 3, wherein in the step 2), the number of the centrifugation is 4 to 5.
5. The detection method according to claim 4, wherein the temperature of the centrifugation in the step 2) is 4 to 5 ℃.
6. The detection method according to claim 5, wherein in the step 3), the PCR reaction system is: 5 parts by volume of 10 XPCR buffer, 4 parts by volume of dNTP, 1 part by volume of F341-GC, 1 part by volume of EU500, 0.5 part by volume of Taq DNA polymerase and 2 parts by volume of DNA solution were mixed, and distilled water was added to 50 parts by volume.
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