Analysis method for functional bacteria in activated sludge

Abstract

An embodiment of the present application provides a method for analyzing functional bacteria in activated sludge. The method includes: preprocessing the collected activated sludge sample to be analyzed; using a nucleic acid fluorescent staining agent to stain the preprocessed activated sludge sample to obtain the activated sludge sample to be detected; after determining the analysis parameters of the flow cytometer, using the flow cytometer to detect the activated sludge sample to be detected to obtain a first detection result; according to the analysis result parameters of the predefined standard microspheres and the first detection result, determining the concentration and particle size of the functional bacteria in the activated sludge sample to be analyzed, and further determining the biomass of the functional bacteria, the biomass of the first type of bacterial group, and the biomass of the second type of bacterial group in the activated sludge sample to be analyzed; thereby determining the metabolic activity of the functional bacteria in the activated sludge sample to be analyzed. To improve the analysis efficiency and accuracy of the functional bacteria in activated sludge.

Description

Technical Field

[0001] This application relates to the field of environmental engineering or biotechnology, and particularly to a method for analyzing functional bacteria in activated sludge. Background Art

[0002] The activated sludge process is the most commonly used and effective method in wastewater treatment systems. Pollutants in sewage are degraded and removed by using functional bacteria in the activated sludge. Among them, the concentration and metabolic activity of functional bacteria directly affect the efficiency and effect of sewage treatment. Therefore, it is necessary to detect the concentration and metabolic activity of functional bacteria in the activated sludge.

[0003] In the prior art, microscopy is the main method for detecting and analyzing the types and quantities of bacteria in an activated sludge system. Bacterial images are obtained through a microscope, and the types and quantities of bacteria can be identified and counted. At the same time, combined with visual graphics and algorithm models, automatic identification and monitoring analysis of microorganisms in the activated sludge can be achieved. However, in the prior art, it is difficult to accurately detect functional bacteria with small particle sizes and low concentrations. In addition, the microscopy technique is slow in the process of image acquisition and bacterial counting, resulting in low timeliness of data, which is not conducive to the rapid monitoring and real-time feedback regulation of the activated sludge system, and lacks a quantitative evaluation of its activity.

[0004] Based on this, in the prior art, there are problems of insufficient comprehensiveness, low efficiency, and poor accuracy in the analysis of functional bacteria in activated sludge. Summary of the Invention

[0005] Embodiments of this application provide a method for analyzing functional bacteria in activated sludge, so as to achieve the effect of improving the analysis efficiency and accuracy of functional bacteria in activated sludge.

[0006] In a first aspect, embodiments of this application provide a method for analyzing functional bacteria in activated sludge, including:

[0007] Collect an activated sludge sample to be analyzed;

[0008] Perform pretreatment on the activated sludge sample to obtain a pretreated activated sludge sample;

[0009] Use a nucleic acid fluorescent staining agent to stain the pretreated activated sludge sample to obtain an activated sludge sample to be detected;

[0010] Determine the analysis parameters of the flow cytometer;

[0011] According to the analysis parameters, use the flow cytometer to detect the activated sludge sample to be detected to obtain a first detection result;

[0012] Obtain the analysis result parameters of a predefined standard microsphere;

[0013] Determine the concentration and particle size of the functional bacteria in the activated sludge sample to be analyzed based on the analysis result parameters of the predefined standard microspheres and the first detection result;

[0014] Determine the biomass of the functional bacteria in the activated sludge sample to be analyzed based on the concentration and particle size of the functional bacteria in the activated sludge sample to be analyzed;

[0015] Determine the biomass of the first bacterial group and the biomass of the second bacterial group in the activated sludge sample to be analyzed based on the first detection result;

[0016] Determine the metabolic activity of the functional bacteria in the activated sludge sample to be analyzed based on the biomass of the first bacterial group and the biomass of the second bacterial group;

[0017] In a possible implementation, pretreat the activated sludge sample to obtain a pretreated activated sludge sample:

[0018] Use a fixing reagent to fix the activated sludge sample to obtain a first pretreated sample;

[0019] Use an ultrasonic instrument to ultrasonically treat the first pretreated sample and dilute the ultrasonically treated sample to obtain a second pretreated sample;

[0020] Filter the second pretreated sample with an organic nylon filter membrane to obtain a pretreated activated sludge sample.

[0021] In a possible implementation, the fixing reagent is a glutaraldehyde solution with a final concentration of 0.5% to 1.5%.

[0022] In a possible implementation, the processing power of the ultrasonic instrument is 50 kw, the ultrasonic time is 30 seconds, and the interval time is 1 min.

[0023] In a possible implementation, the pore size of the organic nylon filter membrane is 60 μm to 65 μm.

[0024] In a possible implementation, use a nucleic acid fluorescent staining agent to stain the pretreated activated sludge sample to obtain an activated sludge sample to be detected, including:

[0025] Dilute the original nucleic acid fluorescent staining agent 100 times with flow cytometry sheath fluid to obtain a staining agent working solution;

[0026] Add the pretreated activated sludge sample to the staining agent working solution according to a preset volume ratio and incubate it under dark conditions to obtain an activated sludge sample to be detected.

[0027] In a possible implementation, the analysis parameters of the flow cytometer include the gain value of the forward scatter light channel, the gain value of the side scatter light channel, and the reception threshold of the fluorescence intensity.

[0028] In a possible implementation, the first detection result includes the total number of cells of the functional bacteria, the analysis time of the functional bacteria, the forward scatter light detection value, the side scatter light detection value, and the purple side scatter light detection value. The analysis result parameters of the standard microspheres include the concentration of the standard microspheres, the total number of the standard microspheres, and the analysis time of the standard microspheres.

[0029] Accordingly, based on the analysis result parameters of the predefined standard microspheres and the first detection result, determining the concentration and particle size of the functional bacteria in the activated sludge sample to be analyzed includes:

[0030] Calculating the concentration of the functional bacteria in the activated sludge sample to be analyzed based on the total number of cells of the functional bacteria, the analysis time of the functional bacteria, the concentration of the standard microspheres, the total number of the standard microspheres, and the analysis time of the standard microspheres.

[0031] Determining the particle size of the functional bacteria in the activated sludge sample to be analyzed based on the forward scatter light detection value, the side scatter light detection value, and the purple side scatter light detection value of the functional bacteria and the predefined standard microspheres.

[0032] In a possible implementation, determining the metabolic activity of the functional bacteria in the activated sludge sample to be analyzed based on the biomass of the first bacterial group and the biomass of the second bacterial group includes:

[0033] Determining the percentage of the biomass of the first bacterial group and the biomass of the second bacterial group as the metabolic activity of the functional bacteria in the activated sludge sample to be analyzed.

[0034] In a possible implementation, determining the biomass of the first bacterial group and the biomass of the second bacterial group in the activated sludge sample to be analyzed based on the first detection result includes:

[0035] Obtaining a preset nucleic acid content threshold;

[0036] Based on the first detection result and using the preset nucleic acid content threshold, determining the first bacterial group and the second bacterial group;

[0037] Determining the biomass of the first bacterial group and the biomass of the second bacterial group in the activated sludge sample to be analyzed based on the concentration and particle size of the first bacterial group, and the concentration and particle size of the second bacterial group.

[0038] The analysis method of activated sludge functional bacteria provided by the embodiments of the present application pre-treats the collected activated sludge samples to be analyzed to obtain the pre-treated activated sludge samples. Through standardized pre-treatment and staining treatment, the operation differences of the activated sludge samples are reduced, and the accuracy of treating the activated sludge samples is effectively improved. A nucleic acid fluorescent staining agent is used to stain the pre-treated activated sludge samples to obtain the activated sludge samples to be detected, enhancing the visibility and detectability of bacteria in the activated sludge samples to be detected. The analysis parameters of the flow cytometer are determined to reduce the detection error of the flow cytometer. According to the analysis parameters, the flow cytometer is used to detect the activated sludge samples to be detected to obtain the first detection result, improving the detection efficiency. According to the analysis result parameters of the pre-defined standard microspheres and the first detection result obtained, the concentration and particle size of the functional bacteria in the activated sludge samples to be analyzed are determined to determine the biomass of the functional bacteria in the activated sludge samples to be analyzed. According to the first detection result, the biomass of the first type of bacterial group and the biomass of the second type of bacterial group in the activated sludge samples to be analyzed are determined. According to the biomass of the first type of bacterial group and the biomass of the second type of bacterial group, the metabolic activity of the functional bacteria in the activated sludge samples to be analyzed is determined, and at the same time, the rapid analysis of the concentration, biomass and activity of the functional bacteria is realized, making the analysis of the activated sludge functional bacteria more comprehensive. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] The accompanying drawings are incorporated herein and form a part of this specification, showing embodiments consistent with the present application and, together with the specification, are used to explain the principles of the present application.

[0040] Figure 1 Flow chart of the analysis method of activated sludge functional bacteria provided by the present application Figure 1 ;

[0041] Figure 2 Schematic diagram of functional bacteria analysis provided by the present application Figure 1 ;

[0042] Figure 3 Flow chart of the analysis method of activated sludge functional bacteria provided by the present application Figure 2 ;

[0043] Figure 4 Flow chart of the analysis method of activated sludge functional bacteria provided by the present application Figure 3 ;

[0044] Figure 5 Flow chart of the analysis method of activated sludge functional bacteria provided by the present application Figure 4 ;

[0045] Figure 6Flow schematic of the analysis method for functional bacteria in activated sludge provided by this application Figure 5 ;

[0046] Figure 7 Distribution map of the first type of bacterial group and the second type of bacterial group provided by this application;

[0047] Figure 8 Flow schematic diagram of a method for quantitative analysis and activity evaluation of functional bacteria in activated sludge provided by this application.

[0048] Through the above-mentioned drawings, specific embodiments of this application have been shown, and there will be more detailed descriptions hereinafter. These drawings and textual descriptions are not intended to limit the scope of the concept of this application in any way, but to illustrate the concept of this application to those skilled in the art by referring to specific embodiments. Detailed description of specific embodiments

[0049] Here, the exemplary embodiments will be described in detail, and the examples are shown in the drawings. When the following description refers to the drawings, unless otherwise indicated, the same numbers in different drawings represent the same or similar elements. The embodiments described in the following exemplary embodiments do not represent all embodiments consistent with this application. On the contrary, they are merely examples of devices and methods consistent with some aspects of this application as detailed in the appended claims.

[0050] It should be noted that the information and data involved in this application (including but not limited to sludge sample data and various parameter data) are all information and data authorized by users or fully authorized by all parties, and the collection, use, and processing of relevant data need to comply with relevant laws, regulations, and standards, and corresponding operation entrances are provided for users to choose to authorize or reject.

[0051] The activated sludge process is a commonly used method in wastewater treatment, relying on functional bacteria to degrade pollutants in sewage. The concentration and metabolic activity of functional bacteria directly affect the treatment efficiency. Therefore, it is necessary to detect the concentration and metabolic activity of functional bacteria in activated sludge.

[0052] In the prior art, the detection methods for the concentration of functional bacteria in activated sludge include microscopy techniques, which analyze the types and quantities of bacteria through image recognition and counting. However, the detection accuracy of microscopy techniques for bacteria with a particle size below 10 μm is significantly reduced, and it is impossible to accurately detect functional bacteria with small particle sizes and low concentrations. Moreover, the processes of image acquisition and counting are slow, resulting in low data timeliness, and there is also a lack of quantitative evaluation of bacterial metabolic activity.

[0053] Therefore, in the prior art, there are problems such as insufficient comprehensiveness, low efficiency, and poor accuracy in the analysis of functional bacteria in activated sludge.

[0054] To solve the above problems, the core concept of this application lies in: by preprocessing the activated sludge sample to be analyzed and performing staining treatment with a nucleic acid fluorescent staining agent, the consistency of the activated sludge sample to be analyzed and the detectability of functional bacteria are ensured; using a flow cytometer to detect the functional bacteria in the activated sludge sample to obtain a first detection result, improving the detection accuracy of small particle size and low concentration bacteria, and determining the biomass of different bacterial groups according to the analysis result parameters of predefined standard microspheres and the first detection result, so as to calculate the metabolic activity of functional bacteria in the activated sludge sample to be analyzed and achieve a comprehensive analysis of the functional bacteria in the activated sludge.

[0055] The following uses specific embodiments to elaborate in detail on the technical solution of this application and how the technical solution of this application solves the above technical problems. These several specific embodiments below can be combined with each other, and the same or similar concepts or processes may not be repeated in some embodiments. The embodiments of this application will be described below in conjunction with the accompanying drawings.

[0056] Figure 1 It is a schematic flowchart of the analysis method for functional bacteria in activated sludge provided by this application, as Figure 1 shown, this method includes:

[0057] S101. Collect the activated sludge sample to be analyzed.

[0058] In this embodiment, the activated sludge sample to be analyzed can be collected from positions such as the aeration tank, secondary sedimentation tank, and return sludge pipeline.

[0059] S102. Preprocess the activated sludge sample to obtain a preprocessed activated sludge sample.

[0060] In this embodiment, by preprocessing the activated sludge sample, the stability and accuracy of the activated sludge sample are improved.

[0061] S103. Use a nucleic acid fluorescent staining agent to perform staining treatment on the preprocessed activated sludge sample to obtain an activated sludge sample to be detected.

[0062] In this embodiment, the nucleic acid fluorescent staining agent includes SYBR Green I. Among them, SYBR Green I is a fluorescent dye commonly used for nucleic acid staining. The SYBR Green I stock solution has a concentration of 10000×. By using the characteristic of specific binding of the nucleic acid fluorescent staining agent to the nucleic acid of biological cells, the biological cells and inorganic particles in the preprocessed activated sludge sample are distinguished to improve the detection accuracy of functional bacteria.

[0063] S104. Determine the analysis parameters of the flow cytometer.

[0064] Optionally, the analysis parameters of the flow cytometer include the gain value of the forward scatter light channel, the gain value of the side scatter light channel, and the reception threshold of the fluorescence intensity.

[0065] In this embodiment, the gain value refers to a parameter for amplifying or adjusting a signal. The flow cytometer sequentially collects optical and laser information of the particulate matter passing through the laser, and presents the collected particulate matter information to a flow scatter plot.

[0066] Use a standard analysis microsphere of the flow cytometer to calibrate and analyze the gain values of the forward scatter light channel and the side scatter light channel, so that the preset standard microsphere sample data is located at the center position of the two-dimensional coordinate plot, to determine the analysis voltage and gain value of the forward scatter light channel and the side scatter light channel, so as to distinguish different particle size intervals in the flow scatter plot.

[0067] Through standard calibration, the results of each standard calibration are kept within the same range, determining the analysis parameters of the flow cytometer and ensuring the stability of the optical system and the flow system of the flow cytometer.

[0068] Since the instrument will generate background noise during the analysis of the flow cytometer, which affects the accuracy of the analysis results, therefore, ultrapure water is used as a blank control group to collect the fluorescence parameters of the purple side scatter light to remove the noise interference of the flow cytometer.

[0069] For example, based on the analysis results of ultrapure water, the signal with the signal intensity of nucleic acid fluorescence in the purple side scatter light channel lower than 1×10 3 is used as the background noise of the flow cytometer, so as to obtain the reception threshold of the fluorescence intensity. Further, in the forward scatter light channel and the side scatter light channel, the signal with the signal intensity of nucleic acid fluorescence lower than 1×10 3 is an inorganic particulate matter, and the signal with the signal intensity of nucleic acid fluorescence not lower than 1×10 3 is a microbial cell.

[0070] S105. According to the analysis parameters, use a flow cytometer to detect the activated sludge sample to be detected to obtain a first detection result.

[0071] After determining the analysis parameters of the flow cytometer, save the analysis parameters, and use the same analysis parameters to detect all activated sludge samples to be detected.

[0072] In the activated sludge sample to be detected, as Figure 2 shown, the fluorescence detection channels include the SYBR B525-A channel and the VSSC V450-A channel. Figure 2The ordinate SYBR B525-A therein represents the signal intensity of nucleic acid fluorescence in the SYBR B525-A channel of a flow cytometer, with the unit of AU (Arbitrary Units, used to represent the relative change of signal intensity), and is used to detect the total number of cells of functional bacteria. Figure 2 The abscissa VSSC V450-A therein represents the microbial cells in the activated sludge sample to be detected, and the signal intensity of the reflected light generated in the VSSC V450-A channel of the flow cytometer, with the unit of AU, and is used to detect the particle size of functional bacteria.

[0073] For example, microbial cells containing nucleic acids will bind to the fluorescent dye and show positive on the SYBR B525-A channel, while inorganic particles without nucleic acids show negative. Among them, the fluorescent dyes include SYTOX® Green or SYBR Green I.

[0074] Optionally, the first detection result includes the total number of cells of functional bacteria, the analysis time of functional bacteria, the forward scatter light detection value, the side scatter light detection value, and the purple side scatter light detection value.

[0075] In this embodiment, for example, the total number of cells detected of functional bacteria is 100,000, and the analysis time of functional bacteria is 30 seconds.

[0076] Optionally, if the concentration of functional bacteria in the activated sludge sample to be detected is lower than the preset functional bacteria concentration threshold, the analysis time of functional bacteria can be increased to 2 to 5 minutes.

[0077] S106. Obtain the analysis result parameters of the predefined standard microspheres.

[0078] Optionally, the analysis result parameters of the standard microspheres include the concentration of the standard microspheres, the total number of the standard microspheres, and the analysis time of the standard microspheres.

[0079] In this embodiment, for example, the concentration of the standard microspheres is 5000 / μL (±10%), the particle size is 1 μm, and the excitation wavelength is 488 nm.

[0080] S107. Determine the concentration of functional bacteria and the particle size of functional bacteria in the activated sludge sample to be analyzed according to the analysis result parameters of the predefined standard microspheres and the first detection result.

[0081] In this embodiment, for example, the analysis flow rate pressure and analysis time of the standard microspheres are the same as those of the activated sludge sample to be detected. Therefore, the ratio of the total number of detected standard microspheres to the total number of functional bacteria in the activated sludge sample to be detected is the same as the ratio of the concentration of the standard microspheres to the concentration of the functional bacteria in the activated sludge sample to be detected. The concentration of the functional bacteria in the activated sludge sample is obtained by calculation.

[0082] In this embodiment, for example, cells and microspheres of the same particle size have the same detected value of forward scattered light. The particle size of the functional bacteria can be determined by comparing the detected value of the forward angle scattered light of the standard microspheres.

[0083] S108. Determine the biomass of the functional bacteria in the activated sludge sample to be analyzed according to the concentration and particle size of the functional bacteria in the activated sludge sample to be analyzed.

[0084] In this embodiment, based on the particle size of the functional bacteria, zoning processing is carried out, the absolute biomass of the functional bacteria in different particle size scale intervals is calculated, and finally summary analysis is carried out. Among them, the calculation formula for the biomass of each functional bacteria is as follows:

[0085]

[0086] In the formula, is the biomass of the functional bacteria, with the unit of μg / L; is the cell radius of the functional bacteria; is the empirical density of the functional bacteria, which is 1×10 9 μg / L; is the concentration of the functional bacteria in the corresponding particle size range.

[0087] S109. Determine the biomass of the first type of bacterial group and the biomass of the second type of bacterial group in the activated sludge sample to be analyzed according to the first detection result.

[0088] In this embodiment, the first type of bacterial group refers to bacteria with high nucleic acid content (HNA, High Nucleic Acid content bacteria), and the second type of bacterial group refers to bacteria with low nucleic acid content (LNA, Low Nucleic Acid content bacteria); and calculate the biomass of the first type of bacterial group and the biomass of the second type of bacterial group.

[0089] S110. Determine the metabolic activity of the functional bacteria in the activated sludge sample to be analyzed according to the biomass of the first type of bacterial group and the biomass of the second type of bacterial group.

[0090] In this embodiment, the first type of bacterial group represents functional bacteria with relatively high metabolic activity and in the rapid metabolism stage; the second type of bacterial group represents functional bacteria with relatively low metabolic activity and in the initial stage of division or senescence; the functional bacteria metabolic activity (Bacterial Metabolic Activity, BMA) of the activated sludge sample to be analyzed is the ratio of the biomass of the first type of bacterial group to the biomass of the second type of bacterial group, and the calculation formula is as follows:

[0091]

[0092] In the formula, represents the biomass of the first type of bacterial group, represents the biomass of the second type of bacterial group;

[0093] Among them, when the BMA value is higher than the preset threshold, it indicates that the proportion of the first type of bacterial group in the activated sludge sample to be analyzed is higher, and the activated sludge sample to be analyzed is in the rapid active and metabolic stage. When the BMA value is lower than the preset threshold, it indicates that the activated sludge sample to be analyzed begins to transform into aging, the high-metabolic-activity bacteria decrease, and the proportion of old, weak, and residual bacteria increases, and external measures are needed for regulation.

[0094] The analysis method of activated sludge functional bacteria provided by this application reduces the operational differences of the activated sludge sample through preprocessing and staining of the collected activated sludge sample to be analyzed, effectively improves the accuracy of processing the activated sludge sample, and enhances the visibility and detectability of bacteria in the activated sludge sample to be detected; determines the analysis parameters of the flow cytometer to reduce the detection error of the flow cytometer; according to the analysis parameters, uses the flow cytometer to detect the activated sludge sample to be detected to obtain the first detection result, improving the detection efficiency; according to the analysis result parameters of the predefined standard microspheres and the first detection result obtained, determines the concentration and particle size of the functional bacteria in the activated sludge sample to be analyzed, so as to determine the biomass of the functional bacteria in the activated sludge sample to be analyzed; according to the first detection result, determines the biomass of the first type of bacterial group and the biomass of the second type of bacterial group in the activated sludge sample to be analyzed; according to the biomass of the first type of bacterial group and the biomass of the second type of bacterial group, determines the functional bacteria metabolic activity of the activated sludge sample to be analyzed, and simultaneously realizes the rapid analysis of the functional bacteria concentration, biomass, and activity, improving the comprehensiveness of the analysis of activated sludge functional bacteria.

[0095] Figure 3 is the process schematic of the analysis method of activated sludge functional bacteria provided by this application Figure 2 , as Figure 3 shown, this embodiment is in Figure 1Based on the embodiments, the pretreatment of the activated sludge sample in the above step S102 to obtain a pretreated activated sludge sample is described in detail. The method includes:

[0096] S301. Fix the activated sludge sample with a fixing reagent to obtain a first pretreated sample.

[0097] Optionally, the fixing reagent is a glutaraldehyde solution with a final concentration of 0.5% to 1.5%.

[0098] In this embodiment, for example, the final concentration of the fixing reagent can be a 1% glutaraldehyde solution. By fixing the activated sludge sample with the fixing reagent, it can effectively prevent the functional bacteria in the activated sludge sample from continuing to carry out metabolic activities, resulting in changes in the quantity and metabolic state of the functional bacteria; and improve the stability of the activated sludge sample.

[0099] Optionally, the fixing reagent can also include formaldehyde, methanol, ethanol or acetic acid.

[0100] S302. Ultrasonically treat the first pretreated sample with an ultrasonic instrument and dilute the ultrasonically treated sample to obtain a second pretreated sample.

[0101] Optionally, the processing power of the ultrasonic instrument is 50 kw, the ultrasonic time is 30 s, and the interval time is 1 min.

[0102] In this embodiment, there may be bacterial aggregates in the first pretreated sample. Set the processing power of the ultrasonic instrument to 50 kw, the ultrasonic time to 30 s, and the interval time to 1 min, and repeat the ultrasonic treatment of the first pretreated sample until the bacterial aggregates are dispersed to make the first pretreated sample more uniform; for example, the number of cycles of ultrasonic treatment can be 3 times; and use ultrapure water to dilute the first pretreated sample by 1000 to 10000 times to obtain a second pretreated sample; wherein, the bacterial aggregate refers to an aggregated structure formed by the combination of bacteria with other microorganisms or particulate matters.

[0103] Preferably, the bacterial concentration of the second pretreated sample is between 10 6 and 10 9 cell / mL.

[0104] S303. Filter the second pretreated sample with an organic nylon filter membrane to obtain a pretreated activated sludge sample.

[0105] Optionally, the pore size of the organic nylon filter membrane is 60 μm to 65 μm.

[0106] In this embodiment, an organic nylon filter membrane is used to filter the second pretreated sample, which can remove large particulate inorganic matter and plankton in the second pretreated sample, and prevent the second pretreated sample from clogging the nozzles and pipelines of the flow cytometer.

[0107] The method for analyzing activated sludge functional bacteria provided by this application improves the stability and accuracy of the activated sludge sample by successively performing fixation treatment, ultrasonic treatment, and filtration treatment on the activated sludge sample.

[0108] Figure 4 It is a schematic flow of the method for analyzing activated sludge functional bacteria provided by this application Figure 3 , as Figure 4 shown, based on the Figure 1 embodiment, the step S103 of using a nucleic acid fluorescent stain to stain the pretreated activated sludge sample to obtain the activated sludge sample to be detected is described in detail. The method includes:

[0109] S401. Dilute the original nucleic acid fluorescent stain 100 times with the flow cytometer sheath fluid to obtain a stain working solution.

[0110] In this embodiment, the flow cytometer sheath fluid includes PBS (Phosphate-Buffered Saline). Dilute the SYBR Green I stock solution with a concentration of 10,000× 100 times with PBS to obtain a stain working solution. Among them, the stain working solution can be stored for one month in an environment of 2 °C to 8 °C.

[0111] S402. Add the pretreated activated sludge sample to the stain working solution according to a preset volume ratio, and incubate it under dark conditions to obtain the activated sludge sample to be detected.

[0112] In this embodiment, the preset volume ratio is 100:1. For example, if the volume of the pretreated activated sludge sample is 100 mL, then the volume of the stain working solution is 1 mL; vortex the pretreated activated sludge sample and the stain working solution mixed according to the preset volume ratio with a vortex mixer for 10 seconds to make the pretreated activated sludge sample and the stain working solution fully mixed; incubate the fully mixed pretreated activated sludge sample and the stain working solution under dark conditions for at least 10 minutes to obtain the activated sludge sample to be detected.

[0113] The method for analyzing activated sludge functional bacteria provided by this application differentiates inorganic particulate matter and bacterial cells in the pretreated activated sludge sample through staining treatment, so as to improve the detection accuracy of activated sludge functional bacteria.

[0114] Figure 5 Flow schematic of the analysis method for the activated sludge functional bacteria provided by this application Figure 4 , such as Figure 5 shown, based on the Figure 1 embodiment, the steps in S107 for determining the concentration and particle size of the functional bacteria in the activated sludge sample to be analyzed according to the analysis result parameters of the predefined standard microspheres and the first detection result are described in detail. The method includes:

[0115] S501. Calculate the concentration of the functional bacteria in the activated sludge sample to be analyzed according to the total number of functional bacteria cells, the analysis time of the functional bacteria, the concentration of the standard microspheres, the total number of standard microspheres, and the analysis time of the standard microspheres.

[0116] In this embodiment, the calculation formula for the concentration of the functional bacteria in the activated sludge sample to be analyzed is as follows:

[0117]

[0118] In the formula, is the concentration of the functional bacteria in the activated sludge sample to be analyzed, with the unit of number / μL; is the total number of functional bacteria cells; is the concentration of the standard microspheres, which is 5000 number / μL; is the analysis time of the standard microspheres; is the total number of standard microspheres; is the analysis time of the functional bacteria.

[0119] S502. Determine the particle size of the functional bacteria in the activated sludge sample to be analyzed according to the forward scatter light detection value, the side scatter light detection value, and the purple side scatter light.

[0120] In this embodiment, the forward scatter light detection value is used to distinguish the functional bacteria with a particle size in the micron range; the purple side scatter light is used to distinguish the functional bacteria with a particle size in the nanometer range; the side scatter light detection value is used to evaluate the internal structure of the functional bacteria.

[0121] Optionally, a particle size analysis template is constructed based on the standard microspheres on the machine to determine the particle size of the functional bacteria in the activated sludge sample to be analyzed, including:

[0122] The particle size analysis template constructed by the standard microspheres on the machine includes 500 nm, 1 μm, 2 μm, 4 μm, 10 μm, and 15 μm; under the same analysis conditions, the functional bacteria that appear in the corresponding area of the flow cytometry scatter plot have the same particle size as the standard microspheres that appear in the same area.

[0123] The analysis method of activated sludge functional bacteria provided by the present application improves the comprehensiveness of the functional bacteria in the activated sludge sample to be analyzed by calculating the concentration of the functional bacteria in the activated sludge sample to be analyzed and statistically analyzing the particle size of the functional bacteria in the activated sludge sample to be analyzed.

[0124] Figure 6 It is a schematic flow of the analysis method of activated sludge functional bacteria provided by the present application Figure 5 , such as Figure 6 shown. Based on the Figure 1 embodiment, the biomass of the first type of bacterial group and the biomass of the second type of bacterial group in the activated sludge sample to be analyzed are determined according to the first detection result in step S109 above, and the method includes:

[0125] S601. Obtain a preset nucleic acid content threshold.

[0126] In this embodiment, after the activated sludge sample to be analyzed is stained with a nucleic acid fluorescent stain, the fluorescence intensity detected in the functional bacteria in the activated sludge sample to be analyzed is proportional to the nucleic acid content. The preset nucleic acid content threshold is a preset fluorescence intensity of 5×10 3 to 8×10 3 .

[0127] S602. According to the first detection result and using the preset nucleic acid content threshold, determine the first type of bacterial group and the second type of bacterial group.

[0128] In this embodiment, the functional bacteria with a fluorescence intensity higher than the preset nucleic acid content threshold are the first type of bacterial group, and the functional bacteria with a fluorescence intensity not higher than the preset nucleic acid content threshold are the second type of bacterial group. Among them, the distribution of the first type of bacterial group and the second type of bacterial group is as Figure 7 shown, where Figure 7 the ordinate SYBR B525-A in Figure 7 represents the fluorescence intensity in the SYBR B525-A channel in the flow cytometer, with the unit of AU, and

[0129] the abscissa VSSC V450-A in

[0130] In this embodiment, based on the concentration and particle size of the first type of bacterial population, and the concentration and particle size of the second type of bacterial population, the biomass of the first type of bacterial population and the biomass of the second type of bacterial population are calculated respectively.

[0131] The analysis method of activated sludge functional bacteria provided by this application classifies the functional bacteria in the activated sludge sample to be analyzed based on the nucleic acid content, providing an accurate detection standard for the metabolic activity of subsequent functional bacteria.

[0132] Optionally, as Figure 8 shown, this application also provides a method for quantitative analysis and activity evaluation of activated sludge functional bacteria, including:

[0133] Step 1: Sampling and pretreatment of functional bacteria, including:

[0134] After uniformly sampling the functional bacteria, an activated sludge sample is obtained, and the activated sludge sample is fixed with a glutaraldehyde solution;

[0135] The activated sludge sample is ultrasonically dispersed in an intermittent-circulation manner;

[0136] The ultrasonically dispersed activated sludge sample is pre-filtered using a 60 μm filter membrane;

[0137] The pre-filtered activated sludge sample is stained with a nucleic acid dye and incubated in the dark.

[0138] Step 2: Parameter analysis and result identification, including:

[0139] Determine the noise threshold using ultrapure water;

[0140] Determine the fluorescence threshold using a preset unstained sample;

[0141] Obtain standard microspheres to determine the concentration and a preset particle size analysis template;

[0142] Collect and identify the bacterial particles in the activated sludge sample.

[0143] Step 3: Quantitative analysis of functional bacteria, including:

[0144] Calculate the bacterial concentration based on the concentration determined by the standard microspheres and the preset particle size analysis template;

[0145] Estimate the biomass based on the particle size and concentration of the bacteria.

[0146] Step 4: Activity evaluation of functional bacteria, including:

[0147] Propose a metabolic activity index for the activated sludge functional bacteria based on the nucleic acid content and metabolic relationship;

[0148] Evaluate the activity of functional bacteria in activated sludge and the aging condition of sludge.

[0149] This application provides an analysis device for functional bacteria in activated sludge, including:

[0150] A sampling module for sampling the activated sludge sample to be analyzed.

[0151] A pretreatment module for pretreating the activated sludge sample to obtain a pretreated activated sludge sample.

[0152] A staining module for staining the pretreated activated sludge sample with a nucleic acid fluorescent stain to obtain an activated sludge sample to be detected.

[0153] A first determination module for determining the analysis parameters of a flow cytometer.

[0154] Optionally, the analysis parameters of the flow cytometer include the gain value of the forward scatter light channel, the gain value of the side scatter light channel, and the reception threshold of the fluorescence intensity.

[0155] A detection module for detecting the activated sludge sample to be detected with a flow cytometer according to the analysis parameters to obtain a first detection result.

[0156] Optionally, the first detection result includes the total number of functional bacteria cells, the analysis time of functional bacteria, the forward scatter light detection value, the side scatter light detection value, and the purple side scatter light. The microsphere parameters of the standard microspheres include the concentration of the standard microspheres, the total number of the standard microspheres, and the analysis time of the standard microspheres.

[0157] An acquisition module for acquiring the microsphere parameters of predefined standard microspheres.

[0158] A second determination module for determining the concentration and particle size of the functional bacteria in the activated sludge sample to be analyzed according to the microsphere parameters of the predefined standard microspheres and the first detection result.

[0159] A third determination module for determining the biomass of the functional bacteria in the activated sludge sample to be analyzed according to the concentration and particle size of the functional bacteria in the activated sludge sample to be analyzed.

[0160] A fourth determination module for determining the biomass of the first type of bacterial group and the biomass of the second type of bacterial group in the activated sludge sample to be analyzed according to the first detection result.

[0161] A fifth determination module for determining the metabolic activity of the functional bacteria in the activated sludge sample to be analyzed according to the biomass of the first type of bacterial group and the biomass of the second type of bacterial group.

[0162] Optionally, the preprocessing module may specifically further be used for:

[0163] Fix the activated sludge sample with a fixing reagent to obtain a first preprocessed sample.

[0164] Optionally, the fixing reagent is a glutaraldehyde solution with a final concentration of 0.5% to 1.5%.

[0165] Ultrasonically treat the first preprocessed sample with an ultrasonic instrument, and dilute the sample after ultrasonic treatment to obtain a second preprocessed sample.

[0166] Optionally, the processing power of the ultrasonic instrument is 50 kw, the ultrasonic time is 30 s, and the interval time is 1 min.

[0167] Filter the second preprocessed sample with an organic nylon filter membrane to obtain a preprocessed activated sludge sample.

[0168] Optionally, the pore size of the organic nylon filter membrane is 60 μm to 65 μm.

[0169] Optionally, the staining module may specifically further be used for:

[0170] Dilute the original nucleic acid fluorescent stain 100 times with flow cytometry sheath fluid to obtain a working solution of the stain;

[0171] Add the preprocessed activated sludge sample to the working solution of the stain according to a preset volume ratio, and incubate it under dark conditions to obtain an activated sludge sample to be detected.

[0172] Optionally, the second determination module may specifically further be used for:

[0173] Calculate the concentration of functional bacteria in the activated sludge sample to be analyzed based on the total number of functional bacteria cells, the analysis time of functional bacteria, the concentration of standard microspheres, the total number of standard microspheres, and the analysis time of standard microspheres;

[0174] Determine the particle size of the functional bacteria in the activated sludge sample to be analyzed based on the forward scatter light detection value, the side scatter light detection value, and the purple side scatter light.

[0175] Optionally, the fourth determination module may specifically further be used for:

[0176] Obtain a preset nucleic acid content threshold;

[0177] Based on the first detection result and using the preset nucleic acid content threshold, determine the first type of bacterial group and the second type of bacterial group;

[0178] Determine the biomass of the first type of bacterial population and the biomass of the second type of bacterial population in the activated sludge sample to be analyzed according to the concentration and particle size of the first type of bacterial population, and the concentration and particle size of the second type of bacterial population.

[0179] Optionally, the fifth determination module may specifically be further configured to:

[0180] Determine the percentage of the biomass of the first type of bacterial population and the biomass of the second type of bacterial population as the functional bacterial metabolic activity of the activated sludge sample to be analyzed.

[0181] The analysis device for activated sludge functional bacteria provided in this embodiment can execute the method provided in the above method embodiment, and its implementation principle and technical effects are similar, which will not be elaborated here in this embodiment.

[0182] This application provides an analysis device for activated sludge functional bacteria. The analysis device for activated sludge functional bacteria provided in this embodiment includes: at least one processor and a memory.

[0183] Optionally, the analysis device for activated sludge functional bacteria further includes a communication component. Among them, the processor, the memory, and the communication component are connected through a bus.

[0184] In the specific implementation process, at least one processor executes the computer execution instructions stored in the memory, so that at least one processor executes the above method.

[0185] The specific implementation process of the processor can refer to the above method embodiment, and its implementation principle and technical effects are similar, which will not be elaborated here in this embodiment.

[0186] In the above embodiment, it should be understood that the processor may be a central processing unit (English: Central Processing Unit, abbreviated as: CPU), or other general-purpose processors, digital signal processors (English: Digital Signal Processor, abbreviated as: DSP), application specific integrated circuits (English: Application Specific Integrated Circuit, abbreviated as: ASIC), etc. The general-purpose processor may be a microprocessor or the processor may also be any conventional processor, etc. The steps of the method disclosed in combination with the invention can be directly implemented by the hardware processor to complete, or can be implemented by the combination of hardware and software modules in the processor.

[0187] The memory may include a high-speed memory (Random Access Memory, RAM), and may also include a non-volatile memory (Non-volatile Memory, NVM), such as at least one disk memory.

[0188] The bus can be an Industry Standard Architecture (ISA) bus, a Peripheral Component Interconnect (PCI) bus, an Extended Industry Standard Architecture (EISA) bus, etc. The bus can be divided into an address bus, a data bus, a control bus, etc. For the sake of convenience in representation, the buses in the accompanying drawings of this application are not limited to only one bus or one type of bus.

[0189] This application also provides a computer-readable storage medium, in which computer-executable instructions are stored. When the processor executes the computer-executable instructions, the above-mentioned method is implemented.

[0190] The above-mentioned readable storage medium can be implemented by any type of volatile or non-volatile storage device or a combination thereof, such as static random access memory (SRAM), electrically erasable programmable read-only memory (EEPROM), erasable programmable read-only memory (EPROM), programmable read-only memory (PROM), read-only memory (ROM), magnetic memory, flash memory, a magnetic disk or an optical disc. The readable storage medium can be any available medium accessible by a general-purpose or special-purpose computer.

[0191] An exemplary readable storage medium is coupled to the processor, so that the processor can read information from the readable storage medium and write information to the readable storage medium. Of course, the readable storage medium can also be a component of the processor. The processor and the readable storage medium can be located in an Application Specific Integrated Circuit (ASIC). Of course, the processor and the readable storage medium can also exist as discrete components in a device.

[0192] The division of units is only a logical function division. In actual implementation, there may be other division methods. For example, multiple units or components can be combined or integrated into another system, or some features can be ignored or not executed. Another point is that the couplings or direct couplings or communication connections shown or discussed with each other can be indirect couplings or communication connections through some interfaces, devices or units, and can be in electrical, mechanical or other forms.

[0193] The unit described as a separation component may or may not be physically separated. The component shown as a unit may or may not be a physical unit, that is, it may be located in one place or may be distributed over multiple network units. Some or all of the units can be selected according to actual needs to achieve the purpose of the solution of this embodiment.

[0194] In addition, each functional unit in various embodiments of the present invention may be integrated in a processing unit, may exist as individual physical units, or two or more units may be integrated in one unit.

[0195] If the function is implemented in the form of a software functional unit and sold or used as an independent product, it can be stored in a computer-readable storage medium. Based on such understanding, the technical solution of the present invention, in essence, or the part that contributes to the prior art, or part of this technical solution, can be embodied in the form of a software product. This computer software product is stored in a storage medium and includes several instructions for causing a computer device (which may be a personal computer, a server, or a network device, etc.) to execute all or part of the steps of the methods in various embodiments of the present invention. The aforementioned storage medium includes: USB flash drives, mobile hard disks, read-only memories (ROM, Read-Only Memory), random access memories (RAM, Random Access Memory), magnetic disks, or optical disks, etc., all kinds of media that can store program codes.

[0196] Those of ordinary skill in the art can understand that all or part of the steps of implementing the above method embodiments can be completed by hardware related to program instructions. The aforementioned program can be stored in a computer-readable storage medium. When the program is executed, it executes the steps including the above method embodiments; and the aforementioned storage medium includes: ROM, RAM, magnetic disks, or optical disks, etc., all kinds of media that can store program codes.

[0197] Finally, it should be noted that: After considering the specification and practicing the invention disclosed herein, those skilled in the art will easily think of other implementation schemes of the present invention. The present invention aims to cover any variations, uses, or adaptive changes of the present invention. These variations, uses, or adaptive changes follow the general principles of the present invention and include common general knowledge or conventional technical means in the technical field not disclosed in the present invention. It is not limited to the precise structures already described and shown in the drawings, and various modifications and changes can be made without departing from its scope. The scope of the present invention is only limited by the appended claims.

Claims

1. An analytical method for functional bacteria in activated sludge, characterized in that Comprising: Collecting an activated sludge sample to be analyzed; Performing pretreatment on the activated sludge sample to obtain a pretreated activated sludge sample; Using a nucleic acid fluorescent stain to stain the pretreated activated sludge sample to obtain an activated sludge sample to be detected; Determining the analysis parameters of a flow cytometer; According to the analysis parameters, using the flow cytometer to detect the activated sludge sample to be detected to obtain a first detection result; Obtaining the analysis result parameters of a predefined standard microsphere; According to the analysis result parameters of the predefined standard microsphere and the first detection result, determining the concentration and particle size of the functional bacteria in the activated sludge sample to be analyzed; According to the concentration and particle size of the functional bacteria in the activated sludge sample to be analyzed, determining the biomass of the functional bacteria in the activated sludge sample to be analyzed; According to the first detection result, determining the biomass of the first type of bacterial group and the biomass of the second type of bacterial group in the activated sludge sample to be analyzed; According to the biomass of the first type of bacterial group and the biomass of the second type of bacterial group, determining the metabolic activity of the functional bacteria in the activated sludge sample to be analyzed; The analysis parameters of the flow cytometer include the gain value of the forward scatter light channel, the gain value of the side scatter light channel, and the reception threshold of the fluorescence intensity; determining the analysis parameters of the flow cytometer includes: Using a flow cytometer standard analysis microsphere to calibrate and analyze the gain values of the forward scatter light channel and the side scatter light channel, so that the preset standard microsphere sample data is located at the center position of the two-dimensional coordinate graph, to determine the analysis voltage and gain value of the forward scatter light channel and the side scatter light channel; Ultra-pure water was used as a blank control group to collect the fluorescence parameters of purple side scatter light; based on the analysis results of ultra-pure water, the signal intensity of nucleic acid fluorescence in the purple side scatter light channel lower than 1×10 3 was used as the background noise of the flow cytometer, so as to obtain the reception threshold of fluorescence intensity; The first detection result includes the total number of functional bacteria cells, the analysis time of the functional bacteria, the forward scatter light detection value, the side scatter light detection value, and the purple side scatter light detection value, and the analysis result parameters of the standard microsphere include the concentration of the standard microsphere, the total number of the standard microsphere, and the analysis time of the standard microsphere; Accordingly, the determining the concentration and particle size of the functional bacteria in the activated sludge sample to be analyzed according to the analysis result parameters of the predefined standard microsphere and the first detection result includes: Calculating the concentration of the functional bacteria in the activated sludge sample to be analyzed according to the total number of functional bacteria cells, the analysis time of the functional bacteria, the concentration of the standard microsphere, the total number of the standard microsphere, and the analysis time of the standard microsphere; Determining the particle size of the functional bacteria in the activated sludge sample to be analyzed according to the forward scatter light detection value, the side scatter light detection value, and the purple side scatter light detection value of the functional bacteria and the predefined standard microsphere.

2. The method according to claim 1, wherein The performing pretreatment on the activated sludge sample to obtain a pretreated activated sludge sample: Using a fixing reagent to fix the activated sludge sample to obtain a first pretreated sample; Using an ultrasonic instrument to ultrasonically treat the first pretreated sample and diluting the ultrasonically treated sample to obtain a second pretreated sample; Filter the second pre-treated sample with an organic nylon filter membrane to obtain a pre-treated activated sludge sample.

3. The method according to claim 2, wherein The fixing reagent is a glutaraldehyde solution with a final concentration of 0.5% to 1.5%.

4. The method according to claim 2, wherein The processing power of the ultrasonic instrument is 50 kw, the ultrasonic time is 30 s, and the interval time is 1 min.

5. The method according to claim 2, wherein The pore size of the organic nylon filter membrane is 60 μm to 65 μm.

6. The method according to claim 1, wherein The method of using a nucleic acid fluorescent stain to stain the pre-treated activated sludge sample to obtain a to-be-detected activated sludge sample includes: Dilute the original nucleic acid fluorescent stain 100 times with flow cytometry sheath fluid to obtain a working solution of the stain; Add the pre-treated activated sludge sample to the working solution of the stain according to a preset volume ratio and incubate it under dark conditions to obtain a to-be-detected activated sludge sample.

7. The method according to any one of claims 1 to 6, characterized in that The method of determining the functional bacterial metabolic activity of the to-be-analyzed activated sludge sample according to the biomass of the first type of bacterial group and the biomass of the second type of bacterial group includes: Determine the percentage of the biomass of the first type of bacterial group and the biomass of the second type of bacterial group as the functional bacterial metabolic activity of the to-be-analyzed activated sludge sample.

8. The method according to claim 7, wherein The method of determining the biomass of the first type of bacterial group and the biomass of the second type of bacterial group in the to-be-analyzed activated sludge sample according to the first detection result includes: Obtain a preset nucleic acid content threshold; According to the first detection result, use the preset nucleic acid content threshold to determine the first type of bacterial group and the second type of bacterial group; According to the concentration and particle size of the first type of bacterial group and the concentration and particle size of the second type of bacterial group, determine the biomass of the first type of bacterial group and the biomass of the second type of bacterial group in the to-be-analyzed activated sludge sample.

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