CN117757905A - Gene level-based wastewater toxicity detection method - Google Patents
Gene level-based wastewater toxicity detection method Download PDFInfo
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- CN117757905A CN117757905A CN202311796541.5A CN202311796541A CN117757905A CN 117757905 A CN117757905 A CN 117757905A CN 202311796541 A CN202311796541 A CN 202311796541A CN 117757905 A CN117757905 A CN 117757905A
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- 239000002351 wastewater Substances 0.000 title claims abstract description 76
- 231100000419 toxicity Toxicity 0.000 title claims abstract description 50
- 230000001988 toxicity Effects 0.000 title claims abstract description 50
- 108090000623 proteins and genes Proteins 0.000 title claims abstract description 28
- 238000001514 detection method Methods 0.000 title claims abstract description 27
- 238000000034 method Methods 0.000 claims abstract description 75
- 244000005700 microbiome Species 0.000 claims abstract description 60
- 230000000694 effects Effects 0.000 claims abstract description 27
- 239000010802 sludge Substances 0.000 claims abstract description 25
- 238000004065 wastewater treatment Methods 0.000 claims abstract description 13
- 230000000813 microbial effect Effects 0.000 claims abstract description 10
- 239000000523 sample Substances 0.000 claims abstract description 10
- 241000894006 Bacteria Species 0.000 claims description 13
- 238000012544 monitoring process Methods 0.000 claims description 9
- 238000010839 reverse transcription Methods 0.000 claims description 7
- 238000004458 analytical method Methods 0.000 claims description 5
- 238000003753 real-time PCR Methods 0.000 claims description 5
- 108020004999 messenger RNA Proteins 0.000 claims description 3
- 239000010865 sewage Substances 0.000 abstract description 15
- 238000013518 transcription Methods 0.000 abstract description 2
- 230000035897 transcription Effects 0.000 abstract description 2
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 6
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 5
- 238000011529 RT qPCR Methods 0.000 description 4
- 230000002159 abnormal effect Effects 0.000 description 4
- 238000006243 chemical reaction Methods 0.000 description 4
- 230000001546 nitrifying effect Effects 0.000 description 4
- 239000000126 substance Substances 0.000 description 4
- XKMRRTOUMJRJIA-UHFFFAOYSA-N ammonia nh3 Chemical compound N.N XKMRRTOUMJRJIA-UHFFFAOYSA-N 0.000 description 3
- 229910052757 nitrogen Inorganic materials 0.000 description 3
- ZKHQWZAMYRWXGA-KQYNXXCUSA-J ATP(4-) Chemical compound C1=NC=2C(N)=NC=NC=2N1[C@@H]1O[C@H](COP([O-])(=O)OP([O-])(=O)OP([O-])([O-])=O)[C@@H](O)[C@H]1O ZKHQWZAMYRWXGA-KQYNXXCUSA-J 0.000 description 2
- ZKHQWZAMYRWXGA-UHFFFAOYSA-N Adenosine triphosphate Natural products C1=NC=2C(N)=NC=NC=2N1C1OC(COP(O)(=O)OP(O)(=O)OP(O)(O)=O)C(O)C1O ZKHQWZAMYRWXGA-UHFFFAOYSA-N 0.000 description 2
- QAOWNCQODCNURD-UHFFFAOYSA-L Sulfate Chemical compound [O-]S([O-])(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-L 0.000 description 2
- 229960000074 biopharmaceutical Drugs 0.000 description 2
- 239000003245 coal Substances 0.000 description 2
- 239000002299 complementary DNA Substances 0.000 description 2
- 238000010586 diagram Methods 0.000 description 2
- 238000010790 dilution Methods 0.000 description 2
- 239000012895 dilution Substances 0.000 description 2
- 230000000696 methanogenic effect Effects 0.000 description 2
- 230000001105 regulatory effect Effects 0.000 description 2
- 230000035945 sensitivity Effects 0.000 description 2
- 102100034343 Integrase Human genes 0.000 description 1
- 241001453382 Nitrosomonadales Species 0.000 description 1
- 108010092799 RNA-directed DNA polymerase Proteins 0.000 description 1
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 1
- 230000004071 biological effect Effects 0.000 description 1
- 238000006555 catalytic reaction Methods 0.000 description 1
- 238000007405 data analysis Methods 0.000 description 1
- 238000003935 denaturing gradient gel electrophoresis Methods 0.000 description 1
- 230000007613 environmental effect Effects 0.000 description 1
- 239000003344 environmental pollutant Substances 0.000 description 1
- 231100000086 high toxicity Toxicity 0.000 description 1
- 238000012165 high-throughput sequencing Methods 0.000 description 1
- 239000010842 industrial wastewater Substances 0.000 description 1
- 239000012528 membrane Substances 0.000 description 1
- 230000002906 microbiologic effect Effects 0.000 description 1
- 230000003647 oxidation Effects 0.000 description 1
- 238000007254 oxidation reaction Methods 0.000 description 1
- 239000001301 oxygen Substances 0.000 description 1
- 229910052760 oxygen Inorganic materials 0.000 description 1
- 230000020477 pH reduction Effects 0.000 description 1
- 231100000614 poison Toxicity 0.000 description 1
- 231100000719 pollutant Toxicity 0.000 description 1
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- 238000004393 prognosis Methods 0.000 description 1
- 238000005496 tempering Methods 0.000 description 1
- 239000003440 toxic substance Substances 0.000 description 1
- 239000010891 toxic waste Substances 0.000 description 1
Classifications
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02W—CLIMATE CHANGE MITIGATION TECHNOLOGIES RELATED TO WASTEWATER TREATMENT OR WASTE MANAGEMENT
- Y02W10/00—Technologies for wastewater treatment
- Y02W10/10—Biological treatment of water, waste water, or sewage
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- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention discloses a gene level-based wastewater toxicity detection method, and belongs to the technical field of wastewater treatment. The invention analyzes the activity of the total microorganism and the activity of the functional microorganism of the sewage treatment plant on the transcription level, can accurately judge the toxicity condition of the wastewater, and reduces the impact on the subsequent biological treatment reactor. The method used by the invention selects proper specific primers/probes according to the detected microorganism types, can quantitatively analyze the relative expression quantity or abundance of total microorganisms and microorganisms of various functional types, and can be suitable for any reactor type and sludge type. The method can accurately, specifically and sensitively indicate the microbial activity, has universality and practicability, can be applied to any wastewater type and scene, and can provide guidance for operation debugging and sludge acclimation.
Description
Technical Field
The invention belongs to the technical field of sewage treatment, and relates to a gene-level-based wastewater toxicity detection method which is used for detecting total microorganism or functional microorganism activity in sewage so as to judge the toxicity condition of the wastewater and is suitable for various types of wastewater.
Background
Biological treatment is one of the treatment methods commonly used in sewage treatment so far, as a novel technology at the end of the 19 th century. However, in industrial wastewater, wastewater produced in different industries and workshops of the same factory is different, the components are complex, and certain wastewater may have toxicity, so that the subsequent biological treatment is greatly damaged. In addition, different operation and debugging methods can generate different environmental conditions, so that the microbial activity is influenced, and the biological treatment process can lose effect.
Microbiological studies can provide guidance for wastewater treatment, which is increasingly important in wastewater biological treatment, but traditional microorganism detection has many limitations, such as: the invention patent 202110372744.6 discloses a front-end wastewater toxicity early warning and disposing device and an application method thereof, wherein tATP (total adenosine triphosphate) and dATP (dissolved adenosine triphosphate) are measured to judge the toxicity condition of wastewater, and the method can detect the microbial activity, but can not judge the functional microbial activity condition aiming at all microorganisms in a biochemical system, and can only be used in an activated sludge system. The invention patent 201110310407.0 discloses a biological activity early warning marking method for chemical wastewater treatment, which combines a PCR-DGGE technology and a RAPD technology on a DNA level, and the method comprises microorganisms, dead bacteria, dormant bacteria and the like which do not play a role, and has poor accuracy, specificity and sensitivity. Therefore, the detection of microorganisms in wastewater, particularly wastewater with toxicity, is needed to be combined and applied to the current molecular biology technology, and guidance is further provided for wastewater treatment.
The reverse transcription real-time fluorescent quantitative PCR technology is characterized in that mRNA is used as a template, cDNA is synthesized under the catalysis of reverse transcriptase, and then the cDNA is used as a template to carry out real-time fluorescent quantitative PCR, the microorganism is detected from the transcription level, the concentration of the microorganism is linearly related to the activity of the microorganism, and the activity of the microorganism can be accurately, specifically and sensitively indicated.
Aiming at the situation, the invention provides a gene level-based wastewater toxicity detection method, which can better predict the toxicity of wastewater by detecting the activity of total microorganisms or functional microorganisms, provide guidance for the selection of subsequent treatment methods and processes, and lighten the impact on subsequent biological treatment reactors.
Disclosure of Invention
The invention provides a gene level-based wastewater toxicity detection method. The method has the characteristics of accuracy, specificity, simplicity and wide application range.
In one aspect, the invention provides a method for detecting toxicity of wastewater based on gene level, comprising the following steps:
factory floor wastewater collected through the pipeline first flows into a buffer Chi Zancun, and buffer pool effluent enters a side stream reactor. After the effluent of the buffer pool enters the side flow reactor, partial sludge flows into the side flow reactor from the main flow bioreactor through a pipeline, after the waste water and the sludge are mixed and react by 0.5-5. 5h, the sludge in the side flow reactor and a normal main flow reactor sludge sample are taken to perform total microorganism or functional microorganism activity detection, if the activity detection is qualified, the waste water of the buffer pool directly enters the main flow reactor, if the activity detection is unqualified, the waste water of the buffer pool enters a treatment pool to be treated, and the treated waste water returns to a toxicity detection system again to perform toxicity detection; the wastewater in the buffer tank can independently flow to the main flow reactor, the side flow reactor or the treatment tank, the sludge in the main flow reactor can flow back to the side flow reactor through a pipeline, and the wastewater in the treatment tank can independently flow to the main flow reactor and the side flow reactor.
The method for detecting the activity of the total microorganisms or the functional microorganisms is based on mRNA level, adopts reverse transcription real-time fluorescent quantitative PCR technology, selects proper specific primers or probes according to the type of the microorganisms to be detected, and quantitatively analyzes the relative expression quantity or abundance of functional genes of one or more microorganisms which play roles, such as the total microorganisms, denitrifying bacteria, nitrifying bacteria, methanogenic bacteria, fermenting and acidifying microorganisms, sulfate reducing bacteria, anaerobic ammoxidation bacteria and the like; the method used in the detection technology can be prepared into a standard commercial kit, and can be monitored on line.
The method can detect the activity of total microorganisms or functional microorganisms in various reactors such as an anaerobic reactor, an anoxic reactor, an aerobic reactor and the like.
The method can detect the activity of total microorganisms or functional microorganisms in various sludge types such as activated sludge, granular sludge, biological membranes and the like, and the sample size for analysis is 0.5-2 mL.
The method can detect the toxicity of the wastewater at the front end of the wastewater treatment process, can detect the toxicity of the wastewater before or after a certain process in the wastewater treatment process, is set according to actual requirements, and has flexibility.
According to the method for detecting the activity of the total microorganisms or the functional bacteria, when the multiple change of the relative expression quantity of the functional genes of the total microorganisms in the sludge of the side flow reactor is more than or equal to 1 or the abundance of the functional genes is reduced by 1/10-1/2, the toxicity of the wastewater is in an acceptable range, and the effluent of the buffer pool directly enters the main flow bioreactor for treatment; when the fold change of the relative expression quantity of the functional genes of the side flow reactor is between 0 and 1 or the abundance of the functional genes is more than or equal to 1/10-1/2, the wastewater needs to be treated or diluted.
In addition, the invention provides a method for detecting the activity of total microorganisms or functional microorganisms in the reactor, namely, the activity of the total microorganisms or the functional microorganisms in the anaerobic reactor, the anoxic reactor or the aerobic reactor is detected by applying the gene level-based wastewater toxicity detection method. Further, the sample size for analysis in this method is 0.5-2 mL.
In addition, the invention provides a wastewater treatment process, which comprises the step of detecting the toxicity of wastewater by applying the gene level-based wastewater toxicity detection method at the front end or in the process of the process.
In addition, the invention provides a standard kit for on-line monitoring, which is prepared according to the method for detecting the activity of the total microorganisms or the functional microorganisms.
The innovation point of the invention is as follows:
by the reverse transcription real-time quantitative PCR method, not only can the total microbial activity be judged, but also the functional microbial activity of the process can be judged, so that the toxicity condition of the wastewater is judged, the impact of toxic substances on a bioreactor is reduced, and the process operation is controlled better;
the reverse transcription real-time quantitative PCR method adopted by the invention judges the toxicity condition of the wastewater by analyzing the relative expression quantity or abundance of total microbial activity or functional microorganisms, and provides guidance for the subsequent treatment method and process selection;
the method used in the invention can also be used in the sewage treatment process debugging stage, and the most appropriate process parameters are selected through analyzing the relative expression quantity or abundance of the functional microorganisms;
the method can detect the toxicity of the wastewater at the front end of the wastewater treatment process, can detect the toxicity of the wastewater before or after a certain process in the wastewater treatment process, and has flexibility according to actual requirements. The method can also be used for the prognosis of the toxicity condition of the advanced oxidation effluent.
The invention is characterized in that: the reverse transcription real-time quantitative PCR method is used for detecting the toxicity of the wastewater, so that the toxicity condition of the wastewater to organisms is accurately predicted; the method can select proper specific primers/probes according to the detected microorganism types, can quantitatively analyze the relative expression quantity or abundance of total microorganisms, and can quantitatively analyze the relative expression quantity or abundance of microorganisms with functions such as denitrifying bacteria, nitrifying bacteria, methanogenic bacteria, fermenting acidification microorganisms, sulfate reducing bacteria, anaerobic ammonia oxidizing bacteria and the like, and the specificity and the sensitivity are high; compared with high-throughput sequencing, the method shortens the detection and analysis time, and obtains the result 2-10h at the fastest speed and 4d at the slowest speed. In addition, the requirements on the expertise of experimental analysts are low, the operation or the on-line control can be carried out according to the experimental operation instruction, the data processing is simple, the processing is not needed by large-scale software or special software, the data analysis does not need the expertise knowledge, and the universality and the practicability are realized; the adopted method for detecting the activity of the total microorganisms or the functional microorganisms can be prepared into a kit mode and is directly used for on-line monitoring; the method provided by the invention can be applied to any reactor type and sludge type; the method can accurately, specifically and sensitively indicate the microbial activity condition, has universality and practicability, and can be applied to any wastewater type.
Drawings
FIG. 1 is a schematic diagram of a gene level-based wastewater toxicity detection method
Description of the embodiments
The invention is further illustrated by way of example with reference to the accompanying drawings in which:
a schematic diagram shown in fig. 1 is established, and a method for detecting toxicity of wastewater based on gene level is described as follows:
collecting factory wastewater or wastewater possibly with toxicity in a buffer pool; the effluent of the buffer pool enters a side flow reactor of a toxicity detection system, then part of sludge flows into the side flow reactor from the main flow bioreactor through a pipeline, after the wastewater reacts with the sludge for 0.5 to 5 hours, the sludge in the side flow reactor and a normal main flow reactor sludge sample are subjected to reverse transcription real-time fluorescence quantitative PCR technology to detect the activity of microorganisms or functional microorganisms; when the multiple variation range of the relative expression quantity of the functional genes of the side flow reactor is 0-1 or the abundance of the functional genes is reduced by 1/10-1/2, the toxicity of the wastewater is in an acceptable range, and the effluent of the buffer pool directly enters the main flow bioreactor for treatment; when the fold change of the relative expression quantity of the functional genes of the side flow reactor is more than or equal to 1 or the abundance of the functional genes is more than 1/10-1/2, the wastewater needs to be pretreated/diluted. The effluent of the buffer pool enters the treatment pool for treatment or dilution, the wastewater after treatment/dilution continuously enters the side flow reactor for reaction, the sludge/biological film after reaction is detected again by using the real-time quantitative PCR technology, and the wastewater after detection is qualified can enter the main flow reactor for subsequent treatment. If the detection is unqualified, the wastewater is returned to the treatment tank again for treatment.
Examples
The method provided by the invention is used for detecting and early warning the toxicity of the wastewater in a pharmaceutical factory. The whole biochemical property of the wastewater in the factory is better, the COD of the effluent is 150-200 mg/L and TN is less than or equal to 25 mg/L in most of the time. But when a certainWhen the discharge amount of the waste water is increased, the COD, total nitrogen and ammonia nitrogen of the effluent are increased. Analysis of experimental data shows that the main problem is in the aerobic treatment stage. By adopting the system to monitor the toxicity of the wastewater of the plant, anaerobic effluent firstly enters the regulating tank for temporary storage, then enters the side flow reactor, and simultaneously the sludge of the main flow reactor is pumped into the side flow reactor for mixed reaction for 1h. Normally, nitrifying bacteriaamoThe Ct value of the A gene is between 10 and 30, and the abundance is 10 1 -10 4 copies ng genomic DNA -1 . After the system is adopted, the toxicity condition of the wastewater can be predicted successfully, anaerobic effluent can be treated in time, and effluent COD, ammonia nitrogen and total nitrogen can reach the standard. The monitoring data of the abnormal condition before the method is adopted and the running condition after the method is adopted are shown in the table.
Table 1 monitoring data in case of abnormal situation
Table 2 monitoring data after this method was used
Examples
The method provided by the invention is used for detecting and early warning the toxicity of wastewater in an industrial park. The industrial park is mainly a toughening plant and also comprises 1 biopharmaceutical and coal chemical plant. The sewage plant mainly receives domestic sewage and a tempering plant, the sewage quality and the water quantity are generally stable, the water quality is not complex, and the treatment is better. However, the sewage discharge amount of the biopharmaceutical and coal chemical plants is increased or the pollutant concentration of the sewage is increased, and the sewage is difficult to treat and has high toxicity, so that the treatment effect of the sewage plant is poor and the effluent does not reach the standard. By adopting the system to monitor the toxicity of the wastewater in the industrial park, the wastewater entering the sewage plant is temporarily stored in the regulating tank firstly and then enters the side flow reactor, and meanwhile, the sludge in the main flow reactor is pumped into the side flow reactor through a pipeline to carry out mixed reaction 1h. After the toxic waste water is discharged into the reactor,A 2 the relative expression quantity multiple change of the total microorganism of the O process relative to the normal condition is between 0 and 0.9. Normally, nitrifying bacteriaamoThe Ct value of the A gene is between 10 and 30, and the abundance is 10 1 -10 4 copies ng genomic DNA -1 Denitrifying genenirK andnirthe Ct value of S is between 10 and 30, and the abundance is 10 2 -10 5 copies ng genomic DNA -1 . After the system is adopted, the toxicity condition of the wastewater can be predicted successfully, the inflow water of a sewage plant is treated in time, and the COD (chemical oxygen demand), ammonia nitrogen and total nitrogen of the outflow water can reach the standard. The monitoring data of the abnormal condition before the method is adopted and the running condition after the method is adopted are shown in the table.
TABLE 3 monitoring data in case of abnormal situation
Table 4 monitoring data after this method was used
The method provided by the invention is used for detecting the toxicity of wastewater in pharmaceutical factories and industrial park sewage treatment plants, so that the toxicity of wastewater is accurately and successfully predicted, and the impact of wastewater toxicity on a subsequent bioreactor is reduced. The detection method can detect the toxicity of the wastewater at the front end of the wastewater treatment process, can detect the toxicity of the wastewater before or after a certain process in the wastewater treatment process, has flexibility, can accurately, specifically and sensitively indicate the microbial activity, has universality and practicability, and can be applied to any wastewater type.
Claims (7)
1. A method for detecting toxicity of wastewater based on gene level is characterized in that: the method comprises the following steps: the factory wastewater collected through a pipeline flows into a buffer Chi Zancun, the effluent of the buffer pool enters a side flow reactor, then partial sludge flows into the side flow reactor from the main flow bioreactor through the pipeline, after the wastewater and the sludge react in a mixed mode by 0.5-5: 5h, the total microorganism or functional microorganism activity detection is carried out on the sludge in the side flow reactor and a normal main flow reactor sludge sample, if the activity detection is qualified, the wastewater of the buffer pool directly enters the main flow reactor, if the activity detection is unqualified, the wastewater of the buffer pool enters a treatment pool for treatment, and the treated wastewater returns to a toxicity detection system for toxicity detection; the wastewater in the buffer tank can flow to the main flow reactor, the side flow reactor or the treatment tank independently, and the wastewater in the treatment tank can flow to the main flow reactor and the side flow reactor independently.
2. The method according to claim 1, characterized in that: the method for detecting the activity of the total microorganism or the functional microorganism is based on the mRNA level and specifically comprises the following steps: and selecting a proper specific primer or probe according to the type of the microorganism to be detected, and analyzing the relative expression quantity or absolute abundance of functional genes of the total microorganism or functional microorganism by using a reverse transcription real-time fluorescent quantitative PCR technology.
3. The method according to claim 1, characterized in that: the method for detecting the activity of total microorganisms or functional bacteria comprises the steps that when the multiple change of the relative expression quantity of total microorganism functional genes in sludge in a side flow reactor is more than or equal to 1 or the abundance of the functional genes is reduced by 1/10-1/2, the toxicity of wastewater is in an acceptable range, and the effluent of a buffer pool directly enters a main flow bioreactor for treatment; when the fold change of the relative expression quantity of the functional genes of the side flow reactor is between 0 and 1 or the abundance of the functional genes is more than or equal to 1/10-1/2, the wastewater needs to be treated or diluted.
4. A method for detecting total or functional microbial activity in a reactor, characterized by: use of the method of any one of claims 1-3 for detecting the activity of total or functional microorganisms in an anaerobic, anoxic or aerobic reactor.
5. The method according to claim 4, wherein: the sample size for analysis was 0.5-2 mL.
6. A wastewater treatment process is characterized in that: the wastewater treatment process comprising the step of detecting toxicity of wastewater at the front end of the process or during the process using the method of any one of claims 1-3.
7. A standard kit for on-line monitoring, characterized in that: the kit is prepared according to the method for detecting the activity of total microorganisms or functional microorganisms according to claim 2.
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