Sewage pipe network microorganism in-situ monitoring method and device
By installing biomimetic biological ropes inside sewage pipe network inspection wells for microbial culture and DNA sequencing, the problems of difficult and low-accuracy microbial sampling in sewage pipe networks have been solved, enabling multi-time period, long-term tracking analysis and highly representative microbial detection.
Patent Information
- Application Number
- CN202511449010.8
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-10-11
- Publication Date
- 2026-01-13
AI Technical Summary
Existing sewage pipe network microbial sampling devices cannot effectively isolate different water layers, causing the test data to deviate from the true value. In addition, conventional sampling methods are labor-intensive, dangerous, and difficult to obtain representative samples.
The method involves installing an in-situ microbial culture device, including multiple biomimetic biological ropes, inside the inspection well of the sewage pipe network to culture, enrich, and separate microorganisms, which are then analyzed using DNA sequencing technology.
It enables multi-period, long-term tracking and analysis of sewage pipe networks, reduces manpower and material costs, improves the representativeness of microbial samples and the accuracy of analysis, and ensures worker safety.
Smart Images
Figure CN121320583A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of sewage pipeline microbial monitoring technology, and in particular to a method and device for in-situ monitoring of microorganisms in sewage pipeline networks. Background Technology
[0002] As the carrier of urban sewage, the sewage pipe network is a hierarchical water conveyance network composed of multiple pipelines and ancillary facilities. Sewage flows through branch pipes, trunk pipes, and finally to the main pipes, converging at each stage before being transported to sewage treatment facilities. The sewage within the pipe network contains multiphase substances, such as light floating matter that migrates with the water flow; suspended particulate matter that exhibits a dynamic distribution depending on hydraulic conditions; and heavy matter that settles at the bottom of the pipes or adheres to the pipe walls. Therefore, the pipe network system is not only a sewage transport channel but also a continuous biological transformation reactor, and its internal biological processes have a significant regulatory effect on water quality evolution.
[0003] Wastewater is prone to vertical stratification due to the physical properties of colloidal substances (such as density differences). This stratification is typically determined based on optical indicators (such as transmittance and turbidity). Accompanying this differentiation in water layer structure, microbial communities at different depths exhibit spatial heterogeneity. To investigate the vertical distribution patterns of microbial communities, precise sampling at different layers is necessary. However, conventional sampling devices (such as open-structure or mechanically valve-controlled sampling heads) allow surface water to seep into the collection unit through the inlet during descent, causing cross-contamination between deeper samples and the upper water layer, resulting in data deviations from the true values. This technical deficiency stems from the inability of existing equipment to effectively isolate non-target water layers, directly affecting the accuracy of microbial community analysis.
[0004] Arranging for professionals to regularly sample sewage microorganisms in each pipe network requires a lot of manpower, and the operation is dangerous and difficult. On the other hand, representative microbial water samples inside the pipe network cannot be easily obtained through conventional well sampling. Representative samples must be combined with long-term tracking and analysis over multiple time periods. Summary of the Invention
[0005] The purpose of this invention is to provide a method and device for in-situ monitoring of microorganisms in sewage pipe networks, which solves the problems of difficult and low sampling accuracy of sewage in pipe networks.
[0006] To achieve this objective, the present invention adopts the following technical solution:
[0007] A method for in-situ monitoring of microorganisms in a sewage pipe network, wherein the sewage pipe network is equipped with inspection wells; the method includes the following steps:
[0008] Step 1: Install a microbial in-situ culture device in the inspection well of the sewage pipe network. The microbial in-situ culture device includes multiple biomimetic biological ropes, and at least a portion of the biomimetic biological ropes are submerged in the sewage of the sewage pipe network.
[0009] Step 2: Maintain the in-situ microbial culture device for microbial culture within the specified culture time;
[0010] Step 3: Remove the in-situ microbial culture device and collect the biomimetic rope;
[0011] Step 4: Microbial enrichment and separation from the biomimetic rope:
[0012] Step 5, Sequencing: Extract DNA from the microorganism using a DNA kit, amplify the DNA using polymerase chain reaction, perform electrophoresis to examine and purify it, and construct a sequencing library and sequencing data.
[0013] In some embodiments, the biomimetic rope includes a first biomimetic rope located underwater and a second biomimetic rope located above water, the first biomimetic rope and the second biomimetic rope being spaced apart on a stabilizing frame, the stabilizing frame being disposed within the inspection well and extending into the sewage of the sewage pipe network.
[0014] In some embodiments, the microbial enrichment and separation specifically refers to:
[0015] The biofilm on the surface of the biomimetic biological rope was eluted with an acid salt buffer solution, and then the microorganisms in the eluent were enriched onto the aqueous filter membrane using a vacuum filter.
[0016] In some embodiments, the filtration accuracy of the aqueous filter membrane is 0.22 μm.
[0017] In some embodiments, the pH of the phosphate buffer solution is 7.2, and the phosphate buffer solution is prepared by dissolving sodium chloride, anhydrous sodium dihydrogen phosphate and disodium hydrogen phosphate in ultrapure water at a mass ratio of 138.0:9.0:35.8, adjusting the pH to 7.2 and then bringing the volume to 1L.
[0018] The amplification described in step 5 is as follows: first, initial denaturation at 95°C for 3 min, then 35 cycles, and finally extension at 72°C for 10 min. The specific cycle procedure is as follows: first denature at 95°C for 30 s, then anneal at 55°C for 30 s, and then extend at 72°C for 1 min.
[0019] In some embodiments, primer pairs 515F and 806R are used during the amplification process.
[0020] In some embodiments, the in-situ monitoring method for microorganisms in sewage pipe networks further includes step 6, performing data analysis, using bioinformatics tools to compare and analyze the sequencing data with a database, and obtaining sequencing results.
[0021] In step 3, after removing the in-situ microbial culture device, steps 1-2 need to be repeated to place the new in-situ microbial culture device in the inspection well of the sewage pipe network and carry out microbial culture within the specified culture time.
[0022] The present invention also provides a sewage pipe network microbial in-situ monitoring device, including a microbial in-situ culture device. The microbial in-situ culture device includes multiple biomimetic biological ropes set on a stabilizing frame. The multiple biomimetic biological ropes extend into the sewage pipe network along the inspection well, so that at least a portion of the biomimetic biological ropes can be submerged in the sewage of the sewage pipe network.
[0023] In some embodiments, at least a portion of the biomimetic ropes are located inside the inspection well and above the sewage within the sewage network.
[0024] The beneficial effects of this invention are:
[0025] This invention provides a method for in-situ monitoring of microorganisms in sewage pipe networks. A microbial in-situ culture device is lowered into the inspection well of the sewage pipe network. This device includes multiple biomimetic biological ropes, at least partially submerged in the sewage. This allows for the acquisition of in-situ microbial samples from the sewage pipe network within a monitoring period, enabling multi-time-period and long-term tracking analysis of microorganisms in the sewage pipe network. This significantly reduces the manpower and material costs of sewage microbial detection and ensures worker safety. Different layers of microbial in-situ culture samples can be obtained as needed. This invention significantly improves the representativeness of microbial samples and enhances the accuracy of analyzing changes in the structure of the pipe network's microbial community, possessing significant scientific and applied value for studying the microbial community in sewage pipe networks. Attached Figure Description
[0026] Figure 1 This is a schematic diagram of the structure of the in-situ microbial monitoring device for sewage pipe networks provided in an embodiment of the present invention;
[0027] Figure 2 This is a schematic diagram illustrating the principle of the in-situ microbial monitoring method for sewage pipe networks provided in this embodiment of the invention;
[0028] Figure 3 This is a schematic diagram of the test results of microbial samples in a sewage pipe network detected by the in-situ monitoring method for microorganisms in sewage pipe networks provided in this embodiment of the invention.
[0029] In the picture:
[0030] 1. Stabilizing frame; 2. Inspection well; 3. Bionic biological rope; 31. First bionic biological rope; 32. Second bionic biological rope; 4. Sewage pipe network. Detailed Implementation
[0031] The present invention will now be described in further detail with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and not intended to limit it. Furthermore, it should be noted that, for ease of description, the accompanying drawings show only the parts relevant to the present invention, and not all of the structures.
[0032] In the description of this invention, unless otherwise explicitly specified and limited, the terms "connected," "linked," and "fixed" should be interpreted broadly. For example, they can refer to a fixed connection, a detachable connection, or an integral part; they can refer to a mechanical connection or an electrical connection; they can refer to a direct connection or an indirect connection through an intermediate medium; they can refer to the internal communication of two components or the interaction between two components. Those skilled in the art can understand the specific meaning of the above terms in this invention based on the specific circumstances.
[0033] In this invention, unless otherwise explicitly specified and limited, "above" or "below" the second feature can include direct contact between the first and second features, or contact between the first and second features through another feature between them. Furthermore, "above," "over," and "on top" of the second feature includes the first feature directly above or diagonally above the second feature, or simply indicates that the first feature is at a higher horizontal level than the second feature. "Below," "below," and "under" the second feature includes the first feature directly below or diagonally below the second feature, or simply indicates that the first feature is at a lower horizontal level than the second feature.
[0034] In the description of this embodiment, the terms "upper," "lower," "left," and "right," etc., refer to the orientation or positional relationship shown in the accompanying drawings. They are used only for ease of description and simplification of operation, and do not indicate or imply that the device or element referred to must have a specific orientation, or be constructed and operated in a specific orientation. Therefore, they should not be construed as limitations on the present invention. In addition, the terms "first" and "second" are used only for distinction in description and have no special meaning.
[0035] This invention first provides a method for in-situ monitoring of microorganisms in sewage pipe networks, such as... Figures 1-2 As shown, sewage pipe network 4 is equipped with inspection well 2; the in-situ microbial monitoring method for sewage pipe network includes the following steps:
[0036] Step 1: Install a microbial in-situ culture device in the inspection well 2 of the sewage pipe network 4. The microbial in-situ culture device includes multiple biomimetic biological ropes 3, and at least some of the biomimetic biological ropes 3 are submerged in the sewage of the sewage pipe network 4.
[0037] The biomimetic rope 3 can be made of conventional fiber material. However, depending on the sewage environment within the sewage network 4, the material and biomimetic form of the biomimetic rope 3 can be specifically designed. The biomimetic rope 3 can serve as a microbial carrier in the sewage environment, facilitating microbial detection. In this embodiment, as... Figure 1 and Figure 2 The biomimetic biological rope 3 includes a first biomimetic biological rope 31 located underwater and a second biomimetic biological rope 32 located above water. The first biomimetic biological rope 31 is submerged in the sewage of the sewage network 4 for in-situ microbial cultivation, while the second biomimetic biological rope 32 is suspended above the sewage surface, not in contact with the sewage, allowing for microbial detection and monitoring of the gaseous environment above the sewage, facilitating the monitoring and maintenance of the gaseous environment space of the sewage network 4 and the inspection well 2. Unless otherwise specified, the following steps for treating the biomimetic biological rope 3 involve treating the first biomimetic biological rope 31 and the second biomimetic biological rope 32 separately.
[0038] Step 2: Maintain the in-situ microbial culture device for microbial culture within the specified culture time. Typically, the biomimetic biological rope 3 needs to be kept in the wastewater for microbial culture in a cycle of one month, such as 30 days. When obvious abnormalities are observed in the microorganisms in the wastewater, the cycle number can be increased or decreased to reduce or increase the number of monitoring sessions.
[0039] Step 3: After the culture cycle is completed, remove the in-situ microbial culture device and collect the biomimetic rope 3;
[0040] Step 4: Microbial enrichment and separation of the biomimetic rope 3:
[0041] Step 5, Sequencing: Extract microbial DNA using a DNA kit, amplify the DNA using polymerase chain reaction, perform electrophoresis to test and purify it, construct a sequencing library and sequencing data, and easily obtain the species and content of microorganisms based on the sequencing data.
[0042] This invention provides a method for in-situ monitoring of microorganisms in sewage pipe networks. An in-situ microbial culture device is lowered into the inspection well 2 of the sewage pipe network 4. This device includes multiple biomimetic biological ropes 3, at least partially submerged in the sewage. This allows for the acquisition of in-situ microbial samples in both the aqueous and gaseous phases of the sewage pipe network 4 during the monitoring period. This enables multi-period and long-term tracking analysis of microorganisms in the sewage pipe network 4, significantly reducing the manpower and material costs of sewage microbial detection and ensuring worker safety. Different layers of in-situ microbial culture samples can be obtained as needed. This invention significantly improves the representativeness of microbial samples and enhances the accuracy of analyzing changes in the structure of the pipe network microbial community, possessing significant scientific and applied value for studying the microbial community in the sewage pipe network 4.
[0043] In some embodiments, the biomimetic rope 3 includes a first biomimetic rope 31 located underwater and a second biomimetic rope 32 located above water. The first biomimetic rope 31 and the second biomimetic rope 32 are spaced apart on the stabilizer 1, which is located inside the inspection well 2 and extends into the sewage of the sewage pipe network 4.
[0044] In this embodiment, multiple biomimetic ropes 3 are spaced apart on the stabilizer 1, which facilitates the adjustment and control of the positional relationship between the multiple biomimetic ropes 3 and the sewage through the stabilizer 1. This allows some of the biomimetic ropes 3 to be located inside the sewage for detecting microorganisms in the liquid phase environment inside the sewage, while others can be located above the sewage without contacting it for monitoring microorganisms in the gas phase environment above the sewage. This improves the comprehensive monitoring of the sewage pipe network 4 and enhances the safety of the surrounding environment.
[0045] In some embodiments, microbial enrichment and isolation specifically include:
[0046] The biofilm on the surface of the biomimetic biological rope 3 was eluted with an acid salt buffer solution, and then the microorganisms in the eluent were enriched onto the aqueous filter membrane using a vacuum filter.
[0047] Specifically, the removed biomimetic rope 3 can be placed in a plastic bottle containing a phosphate buffer solution. The bottle is then placed in a shaker and vibrated for 2 hours. The resulting suspension is then enriched onto an aqueous filter membrane with a filtration precision of 0.22 μm. The phosphate buffer solution has a pH of 7.2 and is prepared as follows: sodium chloride, anhydrous sodium dihydrogen phosphate, and disodium hydrogen phosphate are dissolved in ultrapure water at a mass ratio of 138.0:9.0:35.8. After adjusting the pH to 7.2, the volume is brought to 1 L.
[0048] Preferably, the aqueous filter membrane is a hybrid fiber microporous membrane (MF-CA-CN / MCE). The pH value can be adjusted using sodium hydroxide or hydrochloric acid solution, and ultrapure water is used for final volume determination.
[0049] The amplification in step 5 is as follows: first, initial denaturation at 95℃ for 3 min, then 35 cycles, and finally extension at 72℃ for 10 min. The specific cycle procedure is as follows: first denature at 95℃ for 30 s, then anneal at 55℃ for 30 s, and then extend at 72℃ for 1 min.
[0050] The primer pair used during amplification was 515F (5'-GTGYCAGCMGCCGCGGTAA-3') and 806R (5'-GGACTACNNGGGTWTCTAAT-3').
[0051] The in-situ monitoring method for microorganisms in wastewater pipe networks also includes step 6, which involves data analysis of the sequencing data obtained in step 5. This involves using bioinformatics tools to compare and analyze the sequencing data with a database to obtain sequencing results, which can provide reliable data support for the analysis of the microbial community structure of wastewater pipe network 4. The data analysis includes the allocation of operational taxa, species identification, and assessment of microbial diversity. The processed sequences are assigned OTUs (operational taxa) using the reproducible interactive microbiome analysis platform QIIME2 or UPARSE software, with a threshold of 97% similarity introduced, and species-level annotation of microorganisms is performed. Species annotation is performed using the SILVA database, and QIIME2 is used for Alpha and Beta diversity analysis of the microbial community, including calculating the Shannon diversity index, richness index, etc.
[0052] In step 3, after each removal of the in-situ microbial culture device, steps 1-2 need to be repeated to place the new in-situ microbial culture device in the inspection well 2 of the sewage pipe network 4 and carry out microbial culture within the specified culture time.
[0053] like Figure 3As shown, the relative abundance of dominant phyla in the microbial community obtained by the microbial monitoring method of sewage network 4 provided in this embodiment of the invention is compared with the relative abundance of dominant phyla in the microbial community after traditional sewage sampling and analysis. Samples A1-A21 represent the microbial species detected in sewage samples by traditional sewage sampling, while samples A22-A33 represent the microbial species monitored by this invention. At the phylum level, the microbial community of sewage network 4 is mainly dominated by several key phyla. Among them, Pseudomonadota has a high proportion in all microbial samples, reaching a maximum of 85.25% in sample A33, making it the main dominant phylum. This is followed by Bacteroidetes, Campylobacterota, and Bacillota, with relative contents of 4.05-40.52%, 0.86%-53.40%, and 1.93%-29.44%, respectively. The average percentage of Pseudomonas in samples A1-A21 was 27.40%, and the average percentage of Pseudomonas in samples A22-A23 was 31.65%. In the sewage network 4 samples, the average percentage of Bacteroidetes was 13.15% for samples A1-A21 and 22.13% for samples A22-A23. The average percentage of Campylobacteria was 19.50% for samples A1-A21 and 3.83% for samples A22-A23. The average percentage of Firmicutes was 5.92% for samples A1-A21 and 13.08% for samples A22-A23. Therefore, among the top four dominant phyla, except for Campylobacteria, in situ monitoring... The proportions of Pseudomonas, Bacteroidetes, and Firmicutes in the microbial samples were higher than those in traditional single-time instantaneous sewage samples. This indicates that, compared to traditional single-time instantaneous sampling, the monitoring results obtained using the in-situ microbial monitoring method for sewage pipe networks provided by this invention can not only monitor the instantaneous and long-term microbial community within sewage pipe network 4, but also further demonstrate the strong anaerobic treatment capacity of sewage pipe network 4. This invention can more comprehensively evaluate the true effectiveness of sewage pipe network 4 as a "pretreatment bioreactor," significantly improve the representativeness of microbial samples, and enhance the accuracy of analyzing changes in the microbial community structure of sewage pipe network 4. It has important scientific and applied value for studying the microbial community in sewage pipe network 4.
[0054] This invention also provides an in-situ monitoring device for microorganisms in sewage pipe networks, such as... Figure 1 and Figure 2As shown, it includes an in-situ microbial culture device, which includes multiple biomimetic ropes 3 set on a stabilizing frame 1. The multiple biomimetic ropes 3 extend into the sewage pipe network 4 along the inspection well 2, so that at least part of the biomimetic ropes 3 can be submerged in the sewage in the sewage pipe network 4.
[0055] The stabilizer 1 can be fixed to the wall of the inspection well 2 with bolts. The stabilizer 1 adopts a segmented structure, such as a telescopic pipe structure. The two pipe segments are telescopically connected and can be locked with screws, which makes it easy to adjust the length of the stabilizer 1. This ensures that the first biomimetic biological rope 31 below can be submerged in sewage during monitoring, and is used to monitor in-situ microorganisms in the liquid phase (sewage) of the sewage network 4 and collect microbial samples.
[0056] In some embodiments, at least a portion of the biomimetic ropes 3 are located inside the inspection well 2 and above the sewage in the sewage network 4.
[0057] like Figure 2 The second biomimetic biological rope 32, located above the water surface on the stabilizing frame 1, is used to monitor in-situ microorganisms in the gas phase of the sewage pipe network 4 and to collect microbial samples. For the microbial samples on the second biomimetic biological rope 32, sequencing data can be obtained using the same processing method as the first biomimetic biological rope 31, thereby revealing the distribution of the microbial community.
[0058] Obviously, the above embodiments of the present invention are merely examples for clearly illustrating the present invention, and are not intended to limit the implementation of the present invention. Those skilled in the art will be able to make various obvious changes, readjustments, and substitutions without departing from the scope of protection of the present invention. It is neither necessary nor possible to exhaustively describe all embodiments here. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the scope of protection of the claims of the present invention.
Claims
1. A method for in-situ monitoring of microorganisms in a sewage pipe network, wherein the sewage pipe network (4) is equipped with inspection wells (2); characterized in that, The in-situ microbial monitoring method for sewage pipe networks includes the following steps: Step 1: Install a microbial in-situ culture device in the inspection well (2) of the sewage pipe network (4). The microbial in-situ culture device includes multiple biomimetic biological ropes (3), and at least part of the biomimetic biological ropes (3) are submerged in the sewage of the sewage pipe network (4). Step 2: Maintain the in-situ microbial culture device for microbial culture within the specified culture time; Step 3: Remove the in-situ microbial culture device and collect the biomimetic biological rope (3); Step 4: Microbial enrichment and separation from the biomimetic rope (3); Step 5: Extract DNA from the microorganism using a DNA kit, amplify the DNA using polymerase chain reaction, perform electrophoresis testing and purification, and construct a sequencing library and sequencing data.
2. The method for in-situ monitoring of microorganisms in sewage pipe networks according to claim 1, characterized in that, The biomimetic rope (3) includes a first biomimetic rope (31) located underwater and a second biomimetic rope (32) located above water. The first biomimetic rope (31) and the second biomimetic rope (32) are spaced apart on a stabilizing frame (1). The stabilizing frame (1) is located inside the inspection well (2) and extends into the sewage of the sewage pipe network (4).
3. The method for in-situ monitoring of microorganisms in sewage pipe networks according to claim 1, characterized in that, The microbial enrichment and isolation specifically involves: The biofilm on the surface of the biomimetic biological rope (3) was eluted with a phosphate buffer solution, and then the microorganisms in the eluent were enriched onto the aqueous filter membrane using a vacuum filter.
4. The method for in-situ monitoring of microorganisms in sewage pipe networks according to claim 3, characterized in that, The filtration accuracy of the water-based filter membrane is 0.22 μm.
5. The method for in-situ monitoring of microorganisms in sewage pipe networks according to claim 3, characterized in that, The pH of the phosphate buffer solution is 7.
2. The phosphate buffer solution is prepared by dissolving sodium chloride, anhydrous sodium dihydrogen phosphate and disodium hydrogen phosphate in ultrapure water at a mass ratio of 138.0:9.0:35.8, adjusting the pH to 7.2 and then bringing the volume to 1L.
6. The method for in-situ monitoring of microorganisms in sewage pipe networks according to claim 1, characterized in that, The amplification described in step 5 is as follows: first, initial denaturation at 95°C for 3 min, then 35 cycles, and finally extension at 72°C for 10 min. The specific cycle procedure is as follows: first denature at 95°C for 30 s, then anneal at 55°C for 30 s, and then extend at 72°C for 1 min.
7. The method for in-situ monitoring of microorganisms in sewage pipe networks according to claim 6, characterized in that, The primer pair used in the amplification process was 515F and 806R.
8. The method for in-situ monitoring of microorganisms in sewage pipe networks according to claim 1, characterized in that, It also includes step 6, which involves data analysis, using bioinformatics tools to compare and analyze the sequencing data with the database to obtain sequencing results.
9. The method for in-situ monitoring of microorganisms in sewage pipe networks according to claim 1, characterized in that, After removing the in-situ microbial culture device in step 3, steps 1-2 need to be repeated to place the new in-situ microbial culture device in the inspection well (2) of the sewage pipe network (4) and carry out microbial culture within the specified culture time.
10. A wastewater pipe network microbial in-situ monitoring device, characterized in that, The device is used to implement the in-situ microbial monitoring method for sewage pipe networks according to any one of claims 1-9; the in-situ microbial monitoring device for sewage pipe networks includes an in-situ microbial culture device, the in-situ microbial culture device includes a plurality of biomimetic biological ropes (3) set on a stabilizing frame (1), the plurality of biomimetic biological ropes (3) extending into the sewage pipe network (4) along the inspection well (2), such that at least a portion of the biomimetic biological ropes (3) can be submerged in the sewage of the sewage pipe network (4).
11. The in-situ microbial monitoring device for sewage pipe networks according to claim 10, characterized in that, Of the plurality of the biomimetic ropes (3), at least a portion of the biomimetic ropes (3) are located inside the inspection well (2) and above the sewage in the sewage pipe network (4).