Method for analyzing functional bacteria in activated sludge

Abstract

The embodiment of the invention provides a method for analyzing functional bacteria in activated sludge. Comprising the following steps: pretreating a collected activated sludge sample to be analyzed; carrying out dyeing treatment on the pretreated activated sludge sample by adopting a nucleic acid fluorescent dyeing agent to obtain a to-be-detected activated sludge sample; after the analysis parameters of the flow cytometer are determined, detecting the activated sludge sample to be detected by adopting the flow cytometer to obtain a first detection result; and determining the concentration and the particle size of the functional bacteria of the activated sludge sample to be analyzed according to predefined analysis result parameters of the standard microspheres and the first detection result, further determining the biomass of the functional bacteria, the biomass of the first bacterial group and the biomass of the second bacterial group of the activated sludge sample to be analyzed; therefore, the functional bacteria metabolic activity of the activated sludge sample to be analyzed is determined. The analysis efficiency and accuracy of the activated sludge functional bacteria are improved.

Description

Technical Field

[0001] The present application relates to the field of environmental engineering or biotechnology, and in particular to an analysis method for functional bacteria in activated sludge. Background Art

[0002] The activated sludge method is the most commonly used and effective method in wastewater treatment systems. It uses functional bacteria in activated sludge to degrade and remove pollutants in sewage. 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 activated sludge.

[0003] In the prior art, microscopy is the main method for detecting and analyzing the types and numbers of bacteria in activated sludge systems. By acquiring bacterial images through a microscope, the types and numbers of bacteria can be identified and counted. At the same time, combined with visual graphics and algorithm models, automatic identification and monitoring and analysis of microorganisms in activated sludge can be achieved. However, in the prior art, it is difficult to accurately detect functional bacteria with small particle size and low concentration; in addition, microscopy technology is slow in image acquisition and bacteria counting, resulting in low timeliness of data, which is not conducive to rapid monitoring and real-time feedback control of activated sludge systems, and lacks quantitative evaluation of its activity.

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

[0005] The embodiments of the present application provide an analysis method for activated sludge functional bacteria, so as to achieve the effect of improving the analysis efficiency and accuracy of activated sludge functional bacteria.

[0006] In a first aspect, the present application provides an analysis method for activated sludge functional bacteria, comprising:

[0007] Collecting activated sludge samples to be analyzed;

[0008] Pre-treating the activated sludge sample to obtain a pre-treated activated sludge sample;

[0009] The pretreated activated sludge sample is stained with a nucleic acid fluorescent stain to obtain an activated sludge sample to be tested;

[0010] Determine the analysis parameters for flow cytometry;

[0011] According to the analysis parameters, the activated sludge sample to be tested is tested by using a flow cytometer to obtain a first test result;

[0012] Obtaining analysis result parameters of predefined standard microspheres;

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

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

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

[0016] Based on the biomass of the first bacterial group and the biomass of the second bacterial group, the functional bacterial metabolic activity of the activated sludge sample to be analyzed is determined.

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

[0018] Using a fixing agent to fix the activated sludge sample to obtain a first pretreated sample;

[0019] Using an ultrasonic instrument to perform ultrasonic treatment on the first pre-treated sample, and diluting the ultrasonically treated sample to obtain a second pre-treated sample;

[0020] The second pretreated sample is filtered using an organic nylon filter membrane to obtain a pretreated activated sludge sample.

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

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

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

[0024] In a possible implementation, a nucleic acid fluorescent dye is used to dye the pretreated activated sludge sample to obtain an activated sludge sample to be tested, including:

[0025] Use flow cytometry sheath fluid to dilute the original nucleic acid fluorescent stain 100 times to make the stain working solution;

[0026] The pretreated activated sludge sample is added to the dye working solution according to a preset volume ratio, and incubated in dark conditions to obtain the activated sludge sample to be tested.

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

[0028] In a possible embodiment, the first detection result includes the total number of functional bacteria cells, the analysis time of the functional bacteria, the forward scattered light detection value, the side scattered light detection value and the purple side angle scattered light detection value, and 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, according to the predefined analysis result parameters of the standard microspheres and the first detection result, the concentration of the functional bacteria and the particle size of the functional bacteria in the activated sludge sample to be analyzed are determined, including:

[0030] The concentration of functional bacteria of the activated sludge sample to be analyzed is calculated 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 the standard microspheres and the analysis time of the standard microspheres;

[0031] The particle size of the functional bacteria of the activated sludge sample to be analyzed is determined according to the forward scattered light detection value, the side scattered light detection value and the purple side angle scattered light detection value of the functional bacteria and the predefined standard microspheres.

[0032] In one possible embodiment, determining the functional bacterial metabolic activity of the activated sludge sample to be analyzed based on the biomass of the first bacterial group and the biomass of the second bacterial group comprises:

[0033] The percentage of the biomass of the first bacterial group to the biomass of the second bacterial group is determined as the functional bacterial metabolic activity of 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 according to the first detection result includes:

[0035] Obtaining a preset nucleic acid content threshold;

[0036] According to the first detection result, a preset nucleic acid content threshold is used to determine the first bacterial group and the second bacterial group;

[0037] The biomass of the first bacterial group and the biomass of the second bacterial group in the activated sludge sample to be analyzed are determined 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 functional bacteria in activated sludge provided in the embodiment of the present application comprises the following steps: collecting activated sludge samples to be analyzed and performing pretreatment to obtain pretreated activated sludge samples; reducing the operational differences of activated sludge samples through standardized pretreatment and staining treatment, and effectively improving the accuracy of treating activated sludge samples; staining the pretreated activated sludge samples with nucleic acid fluorescent stain to obtain activated sludge samples to be detected, thereby enhancing the visibility and detectability of bacteria in the activated sludge samples to be detected; determining the analysis parameters of the flow cytometer to reduce the detection error of the flow cytometer; and detecting the activated sludge samples to be detected with the flow cytometer according to the analysis parameters to obtain The first detection result is obtained, thereby improving the detection efficiency; according to the obtained predefined analysis result parameters of the standard microspheres and the first detection result, the concentration of the functional bacteria of the activated sludge sample to be analyzed and the particle size of the functional bacteria are determined to determine the biomass of the functional bacteria of the activated sludge sample to be analyzed; according to the first detection result, the biomass of the first bacterial group and the biomass of the second bacterial group in the activated sludge sample to be analyzed are determined; according to the biomass of the first bacterial group and the biomass of the second bacterial group, the metabolic activity of the functional bacteria in the activated sludge sample to be analyzed is determined, and at the same time, rapid analysis of the concentration, biomass and activity of the functional bacteria is achieved, so that the analysis of the functional bacteria of the activated sludge is more comprehensive. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate embodiments consistent with the present application and, together with the description, serve to explain the principles of the present application.

[0040] Figure 1 Schematic diagram of the process of analyzing the functional bacteria of activated sludge provided in this application Figure 1 ;

[0041] Figure 2 Schematic diagram of functional bacterial analysis provided for this application Figure 1 ;

[0042] Figure 3 Schematic diagram of the process of analyzing the functional bacteria of activated sludge provided in this application Figure 2 ;

[0043] Figure 4 Schematic diagram of the process of analyzing the functional bacteria of activated sludge provided in this application Figure 3 ;

[0044] Figure 5 Schematic diagram of the process of analyzing the functional bacteria of activated sludge provided in this application Figure 4 ;

[0045] Figure 6Schematic diagram of the process of analyzing the functional bacteria of activated sludge provided in this application Figure 5 ;

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

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

[0048] The above drawings have shown clear embodiments of the present application, which will be described in more detail later. These drawings and text descriptions are not intended to limit the scope of the present application in any way, but to illustrate the concept of the present application to those skilled in the art by referring to specific embodiments. DETAILED DESCRIPTION

[0049] Exemplary embodiments will be described in detail herein, examples of which are shown in the accompanying drawings. When the following description refers to the drawings, the same numbers in different drawings represent the same or similar elements unless otherwise indicated. The implementations described in the following exemplary embodiments do not represent all implementations consistent with the present application. Instead, they are merely examples of devices and methods consistent with some aspects of the present 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 the user or fully authorized by all parties, and the collection, use and processing of relevant data must comply with relevant laws, regulations and standards, and provide corresponding operation entrances for users to choose to authorize or refuse.

[0051] The activated sludge method is a commonly used method in wastewater treatment. It relies 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 method of functional bacteria concentration in activated sludge includes microscopic examination technology, which analyzes the types and quantity of bacteria through image recognition and counting. However, the detection accuracy of microscopic examination technology for bacteria with a particle size below 10 μm is significantly reduced, and it is impossible to accurately detect functional bacteria with small particle size and low concentration. In addition, the image acquisition and counting process is slow, resulting in low data timeliness and 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 activated sludge functional bacteria.

[0054] In order to solve the above problems, the core concept of the present application is: by pretreating the activated sludge samples to be analyzed and staining them with nucleic acid fluorescent dyes, the consistency of the activated sludge samples to be analyzed and the detectability of functional bacteria are ensured; the functional bacteria of the activated sludge samples are detected by flow cytometry to obtain the first detection result, which improves the detection accuracy of small particle size and low concentration bacteria, and determines the biomass of different bacterial groups according to the predefined analysis result parameters of standard microspheres and the first detection result, so as to calculate the metabolic activity of the functional bacteria in the activated sludge samples to be analyzed, thereby realizing a comprehensive analysis of the functional bacteria in the activated sludge.

[0055] The technical solution of the present application and how the technical solution of the present application solves the above-mentioned technical problems are described in detail below with specific embodiments. The following specific embodiments can be combined with each other, and the same or similar concepts or processes may not be repeated in some embodiments. The embodiments of the present application will be described below in conjunction with the accompanying drawings.

[0056] Figure 1 A schematic diagram of the process of analyzing the functional bacteria of activated sludge provided in this application, such as Figure 1 As shown, the method includes:

[0057] S101. Collect activated sludge samples to be analyzed.

[0058] In this embodiment, the activated sludge samples to be analyzed can be collected from aeration tanks, secondary sedimentation tanks, return sludge pipelines, and other locations.

[0059] S102. Pre-treating the activated sludge sample to obtain a pre-treated activated sludge sample.

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

[0061] S103, using a nucleic acid fluorescent dye to dye the pretreated activated sludge sample to obtain an activated sludge sample to be tested.

[0062] In this embodiment, the nucleic acid fluorescent stain includes SYBR Green I, wherein SYBR Green I is a fluorescent dye commonly used for nucleic acid staining, and the SYBR Green I stock solution has a concentration of 10000×. Through the characteristic of the nucleic acid fluorescent stain to specifically bind to the nucleic acid of biological cells, the biological cells and inorganic particles in the pretreated activated sludge sample can be distinguished to improve the accuracy of the detection of functional bacteria.

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

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

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

[0066] The gain values ​​of the forward angle scatter light channel and the side angle scatter light channel are calibrated and analyzed using flow cytometer standard analysis microspheres so that the preset standard microsphere sample data is located at the center of the two-dimensional coordinate graph to determine the analysis voltage and gain value of the forward angle scatter light channel and the side angle scatter light channel, thereby distinguishing different particle size ranges in the flow cytometric scatter plot.

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

[0068] Since the flow cytometer will generate background noise when performing analysis, thus affecting the accuracy of the analysis results, ultrapure water is needed as a blank control group to collect the purple side angle scattered light fluorescence parameters to remove the noise interference of the flow cytometer.

[0069] For example, based on the analysis results of ultrapure water, the signal intensity of nucleic acid fluorescence in the purple side scatter channel was set to less than 1×10 3 The signal is used as the background noise of the flow cytometer to obtain the receiving threshold of the fluorescence intensity. Furthermore, in the forward angle scattering light channel and the side angle scattering light channel, the signal intensity of the nucleic acid fluorescence is less than 1×10 3 The signal is inorganic particles, and the signal intensity of nucleic acid fluorescence is not less than 1×10 3 The signal is for microbial cells.

[0070] S105. According to the analysis parameters, the activated sludge sample to be tested is tested using a flow cytometer to obtain a first test result.

[0071] After determining the analysis parameters of the flow cytometer, the analysis parameters are saved, and all activated sludge samples to be tested are tested using the same analysis parameters.

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

[0073] For example, microbial cells containing nucleic acids will bind fluorescent dyes such as SYTOX® Green or SYBRGreen I and appear positive on the SYBR B525-A channel, while inorganic particles without nucleic acids will appear negative.

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

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

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

[0077] S106, obtaining predefined analysis result parameters of 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 the functional bacteria in the activated sludge sample to be analyzed according to the predefined analysis result parameters of the 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 and the activated sludge sample to be tested are the same. Therefore, the ratio of the total number of standard microspheres detected to the total number of functional bacteria in the activated sludge sample to be tested is the same as the ratio of the concentration of the standard microspheres to the concentration of functional bacteria in the activated sludge sample to be tested. The concentration of 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 detection value of forward scattered light, and the particle size of the functional bacteria can be determined by comparing the forward angle scattered light detection value 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 of the functional bacteria in the activated sludge sample to be analyzed and the particle size of the functional bacteria.

[0084] In this embodiment, based on the particle size of the functional bacteria, partitioning is performed to calculate the absolute biomass of the functional bacteria in different particle size ranges, and finally a summary analysis is performed, wherein the calculation formula for the biomass of each functional bacterium is as follows:

[0085]

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

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

[0088] In this embodiment, the first bacterial group refers to high nucleic acid content bacteria (HNA), and the second bacterial group refers to low nucleic acid content bacteria (LNA); and the biomass of the first bacterial group and the biomass of the second bacterial group are calculated.

[0089] S110, determining the functional bacterial metabolic activity of the activated sludge sample to be analyzed based on the biomass of the first bacterial group and the biomass of the second bacterial group.

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

[0091]

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

[0093] Among them, if the BMA value is higher than the preset threshold, it indicates that the higher the proportion of the first type of bacterial group in the activated sludge sample to be analyzed, the activated sludge sample to be analyzed is in a rapid active and metabolic stage. If the BMA value is lower than the preset threshold, it indicates that the activated sludge sample to be analyzed has begun to age, the high metabolic active bacteria have decreased, and the proportion of old and weak residual bacteria has increased, and external measures are needed for regulation.

[0094] The analytical method for functional bacteria in activated sludge provided by the present application reduces the operational variability of activated sludge samples by pre-treating and staining the collected activated sludge samples to be analyzed, effectively improves the accuracy of processing activated sludge samples, and enhances the visibility and detectability of bacteria in the activated sludge samples to be detected; determines the analytical parameters of the flow cytometer to reduce the detection error of the flow cytometer; according to the analytical parameters, uses the flow cytometer to detect the activated sludge samples to be detected to obtain the first detection result, thereby improving the detection efficiency; according to the obtained analysis results of the predefined standard microspheres The parameters and the first test result are used to determine the concentration of the functional bacteria in the activated sludge sample to be analyzed and the particle size of the functional bacteria, so as to determine the biomass of the functional bacteria in the activated sludge sample to be analyzed; according to the first test result, the biomass of the first bacterial group and the biomass of the second bacterial group in the activated sludge sample to be analyzed are determined; according to the biomass of the first bacterial group and the biomass of the second bacterial group, the metabolic activity of the functional bacteria in the activated sludge sample to be analyzed is determined, and at the same time, rapid analysis of the concentration, biomass and activity of the functional bacteria is achieved, thereby improving the comprehensiveness of the analysis of the functional bacteria in the activated sludge.

[0095] Figure 3 Schematic diagram of the process of analyzing the functional bacteria of activated sludge provided in this application Figure 2 ,like Figure 3 As shown, in this embodiment Figure 1Based on the embodiment, the above step S102 of pre-treating the activated sludge sample to obtain the pre-treated activated sludge sample is described in detail, and the method includes:

[0096] S301. Use a fixing agent to fix the activated sludge sample to obtain a first pretreated sample.

[0097] Optionally, the fixative agent 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 agent can be 1% glutaraldehyde solution. By using the fixing agent to fix the activated sludge sample, the functional bacteria in the activated sludge sample can be effectively prevented from continuing metabolic activities, resulting in changes in the number and metabolic state of the functional bacteria, thereby improving the stability of the activated sludge sample.

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

[0100] S302, using an ultrasonic instrument to perform ultrasonic treatment on the first pre-treated sample, and diluting the ultrasonically treated sample to obtain a second pre-treated 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, bacterial aggregates may exist in the first pretreated sample. The processing power of the ultrasonic instrument is set to 50 kW, the ultrasonic time is set to 30 s, and the interval time is set to 1 min. The first pretreated sample is repeatedly ultrasonically treated 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. Ultrapure water is used to dilute the first pretreated sample 1000 to 10000 times to obtain a second pretreated sample. Bacterial aggregates refer to aggregate structures formed by the combination of bacteria and other microorganisms or particulate matter.

[0103] Preferably, the bacterial concentration of the second pre-treated sample is between 10 6 Up to 10 9 cell / mL.

[0104] S303, filtering the second pretreated sample using 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, the second pre-treated sample is filtered using an organic nylon filter membrane, which can remove large inorganic particles and plankton in the second pre-treated sample and prevent the second pre-treated sample from clogging the nozzle and pipeline of the flow cytometer.

[0107] The analysis method of functional bacteria in activated sludge provided in the present application improves the stability and accuracy of activated sludge samples by sequentially subjecting activated sludge samples to fixation treatment, ultrasonic treatment and filtration treatment.

[0108] Figure 4 Schematic diagram of the process of analyzing the functional bacteria of activated sludge provided in this application Figure 3 ,like Figure 4 As shown, in this embodiment Figure 1 Based on the embodiment, the method of dyeing the pretreated activated sludge sample with a nucleic acid fluorescent dye in step S103 to obtain an activated sludge sample to be detected is described in detail. The method includes:

[0109] S401. Use flow cytometry sheath fluid to dilute the original nucleic acid fluorescent stain 100 times to make the stain working solution.

[0110] In this embodiment, the flow cytometry sheath fluid includes PBS (Phosphate-Buffered Saline), and 10000× concentration of SYBR Green I stock solution is diluted 100 times with PBS to obtain a dye working solution, wherein the dye working solution can be stored for one month in an environment of 2°C to 8°C.

[0111] S402, adding the pretreated activated sludge sample to the dye working solution according to a preset volume ratio, and incubating in the dark to obtain an activated sludge sample to be tested.

[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, the volume of the dye working solution is 1 mL; the pretreated activated sludge sample and the dye working solution mixed according to the preset volume ratio are vortexed for 10 seconds by a vortex mixer to allow the pretreated activated sludge sample and the dye working solution to be fully mixed; the fully mixed pretreated activated sludge sample and the dye working solution are incubated under dark conditions for at least 10 minutes to obtain the activated sludge sample to be tested.

[0113] The analysis method of activated sludge functional bacteria provided in the present application realizes the distinction between inorganic particles and bacterial cells in the activated sludge sample by staining the pretreated activated sludge sample, so as to improve the detection accuracy of activated sludge functional bacteria.

[0114] Figure 5 Schematic diagram of the process of analyzing the functional bacteria of activated sludge provided in this application Figure 4 ,like Figure 5 As shown, in this embodiment Figure 1 Based on the embodiment, the above step S107 is described in detail for determining the concentration of functional bacteria and the particle size of functional bacteria in the activated sludge sample to be analyzed according to the predefined analysis result parameters of the standard microspheres and the first detection result. The method includes:

[0115] S501. Calculate the concentration of functional bacteria of 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 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 functional bacteria in the activated sludge sample to be analyzed is as follows:

[0117]

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

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

[0120] In this embodiment, the forward scattered light detection value is used to distinguish the functional bacteria with micron-sized particles; the purple side angle scattered light is used to distinguish the functional bacteria with nanometer-sized particles; and the side scattered 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 of the activated sludge sample to be analyzed, including:

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

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

[0124] Figure 6 Schematic diagram of the process of analyzing the functional bacteria of activated sludge provided in this application Figure 5 ,like Figure 6 As shown, in this embodiment Figure 1 Based on the embodiment, the above step S109 of determining the biomass of the first bacterial group and the biomass of the second bacterial group in the activated sludge sample to be analyzed according to the first detection result is described in detail. The method includes:

[0125] S601, obtaining 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 of the functional bacteria detected in the activated sludge sample to be analyzed is proportional to the nucleic acid content, and the preset nucleic acid content threshold is a preset fluorescence intensity of 5×10 3 Up to 8×10 3 .

[0127] S602: According to the first detection result, a preset nucleic acid content threshold is used to determine the first bacterial group and the second bacterial group.

[0128] In this embodiment, the functional bacteria whose fluorescence intensity is higher than the preset nucleic acid content threshold are the first type of bacterial group, and the functional bacteria whose fluorescence intensity is not higher than the preset nucleic acid content threshold are the second type of bacterial group. The distribution of the first type of bacterial group and the second type of bacterial group is as follows: Figure 7 As shown, Figure 7 The vertical axis SYBR B525-A in the flow cytometer represents the fluorescence intensity of the SYBR B525-A channel in the flow cytometer, and the unit is AU. Figure 7 The abscissa VSSC V450-A in the figure represents the signal intensity of the reflected light generated by the microbial cells in the activated sludge sample to be detected in the VSSC V450-A channel in the flow cytometer, with the unit being AU.

[0129] S603. Determine the biomass of the first bacterial group and the biomass of the second bacterial group in the activated sludge sample to be analyzed according to the concentration and particle size of the first bacterial group and the concentration and particle size of the second bacterial group.

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

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

[0132] Alternatively, if Figure 8 As shown, the present application also provides a method for quantitative analysis and activity evaluation of activated sludge functional bacteria, comprising:

[0133] Step 1: functional bacteria sampling and pretreatment, 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 samples were ultrasonically dispersed in an intermittent-circulating manner;

[0136] The activated sludge samples after ultrasonic dispersion were pre-filtered using a 60 μm filter membrane;

[0137] The pre-filtered activated sludge samples were stained with nucleic acid dyes and incubated in the dark.

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

[0139] Ultrapure water was used to determine the noise threshold;

[0140] Fluorescence thresholds were determined using pre-set unstained samples;

[0141] Obtain standard microspheres to determine concentration and preset particle size analysis templates;

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

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

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

[0145] Biomass estimation was performed based on bacterial size and concentration.

[0146] Step 4: Functional bacterial activity assessment, including:

[0147] The metabolic activity index of activated sludge functional bacteria was proposed based on the relationship between nucleic acid content and metabolism;

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

[0149] The present application provides an analysis device for activated sludge functional bacteria, comprising:

[0150] The collection module is used to collect activated sludge samples to be analyzed.

[0151] The pretreatment module is used to pretreat the activated sludge sample to obtain the pretreated activated sludge sample.

[0152] The dyeing module is used to dye the pretreated activated sludge sample with a nucleic acid fluorescent dye to obtain an activated sludge sample to be tested.

[0153] The first determination module is used to determine the analysis parameters of the flow cytometer.

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

[0155] The detection module is used to detect the activated sludge sample to be detected by using 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 bacterial cells, the analysis time of functional bacteria, the forward scattered light detection value, the side scattered light detection value and the purple side angle scattered light, and the microsphere parameters of the standard microspheres include the concentration of the standard microspheres, the total number of standard microspheres and the analysis time of the standard microspheres.

[0157] The acquisition module is used to acquire the microsphere parameters of predefined standard microspheres.

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

[0159] The third determination module is used to determine the biomass of the functional bacteria in the activated sludge sample to be analyzed according to the concentration of the functional bacteria in the activated sludge sample to be analyzed and the particle size of the functional bacteria.

[0160] The fourth determination module is used to determine the biomass of the first bacterial group and the biomass of the second bacterial group in the activated sludge sample to be analyzed according to the first detection result.

[0161] The fifth determination module is used to determine the functional bacterial metabolic activity of the activated sludge sample to be analyzed according to the biomass of the first bacterial group and the biomass of the second bacterial group.

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

[0163] The activated sludge sample is subjected to a fixation treatment using a fixation agent to obtain a first pre-treated sample.

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

[0165] The first pre-treated sample is ultrasonically treated using a sonicator, and the ultrasonically treated sample is diluted to obtain a second pre-treated 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] The second pretreated sample is filtered using an organic nylon filter membrane to obtain a pretreated activated sludge sample.

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

[0169] Optionally, the dyeing module can also be used for:

[0170] Use flow cytometry sheath fluid to dilute the original nucleic acid fluorescent stain 100 times to make the stain working solution;

[0171] The pretreated activated sludge sample is added to the dye working solution according to a preset volume ratio, and incubated in dark conditions to obtain the activated sludge sample to be tested.

[0172] Optionally, the second determining module may also be specifically used for:

[0173] The concentration of functional bacteria of the activated sludge sample to be analyzed is calculated 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 the standard microspheres and the analysis time of the standard microspheres;

[0174] The particle size of the functional bacteria of the activated sludge sample to be analyzed is determined according to the forward scattered light detection value, the side scattered light detection value and the purple side angle scattered light.

[0175] Optionally, the fourth determining module may also be specifically configured to:

[0176] Obtaining a preset accounting content threshold;

[0177] According to the first detection result, a preset bacterial content threshold is used to determine the first bacterial group and the second bacterial group;

[0178] The biomass of the first bacterial group and the biomass of the second bacterial group in the activated sludge sample to be analyzed are determined based on the concentration and particle size of the first bacterial group and the concentration and particle size of the second bacterial group.

[0179] Optionally, the fifth determining module may also be specifically configured to:

[0180] The percentage of the biomass of the first bacterial group to the biomass of the second bacterial group is determined 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. Its implementation principle and technical effect are similar, and this embodiment will not be described in detail here.

[0182] The present 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 comprises a communication component, wherein the processor, the memory and the communication component are connected via a bus.

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

[0185] The specific implementation process of the processor can be found in the above method embodiment, and its implementation principle and technical effect are similar, so this embodiment will not be repeated here.

[0186] In the above embodiments, it should be understood that the processor can be a central processing unit (CPU), or other general-purpose processors, digital signal processors (DSP), application-specific integrated circuits (ASIC), etc. A general-purpose processor can be a microprocessor or any conventional processor. The steps of the method disclosed in the invention can be directly implemented as a hardware processor, or can be implemented by a 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 (NVM), such as at least one disk storage.

[0188] The bus can be an Industry Standard Architecture (ISA) bus, a Peripheral Component Interconnect (PCI) bus, or 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 ease of representation, the bus in the drawings of this application is not limited to only one bus or one type of bus.

[0189] The present application also provides a computer-readable storage medium, in which computer-executable instructions are stored. When a processor executes the computer-executable instructions, the above 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, magnetic disk or optical disk. The readable storage medium can be any available medium that can be accessed by a general or special-purpose computer.

[0191] An exemplary readable storage medium is coupled to a 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 (Application Specific Integrated Circuits, referred to as: ASIC). Of course, the processor and the readable storage medium can also exist in the device as discrete components.

[0192] The division of units is only a logical function division, and there may be other divisions in actual implementation, such as 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 mutual coupling or direct coupling or communication connection shown or discussed can be an indirect coupling or communication connection through some interface, device or unit, which can be electrical, mechanical or other forms.

[0193] The units described as separate components may or may not be physically separated, and the components shown as units may or may not be physical units, that is, they may be located in one place or distributed on multiple network units. Some or all of the units may be selected according to actual needs to achieve the purpose of the solution of this embodiment.

[0194] In addition, each functional unit in each embodiment of the present invention may be integrated into one processing unit, or each unit may exist physically separately, or two or more units may be integrated into 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 this understanding, the technical solution of the present invention, or the part that contributes to the prior art, or the part of the technical solution, can be embodied in the form of a software product. The computer software product is stored in a storage medium, including several instructions for a computer device (which can be a personal computer, server, or network device, etc.) to perform all or part of the steps of the methods of each embodiment of the present invention. The aforementioned storage medium includes: U disk, mobile hard disk, read-only memory (ROM, Read-Only Memory), random access memory (RAM, Random Access Memory), disk or optical disk, etc. Various media that can store program codes.

[0196] Those skilled in the art can understand that all or part of the steps of implementing the above-mentioned 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, the steps of the above-mentioned method embodiments are executed; and the aforementioned storage medium includes: ROM, RAM, disk or optical disk and other media that can store program codes.

[0197] Finally, it should be noted that those skilled in the art will readily conceive of other embodiments of the present invention after considering the specification and practicing the invention disclosed herein. The present invention is intended to cover any variations, uses or adaptations of the present invention, which follow the general principles of the present invention and include common knowledge or customary technical means in the art not disclosed by the present invention, are not limited to the precise structure described above and shown in the drawings, and may be modified and changed in various ways without departing from the scope thereof. The scope of the present invention is limited only by the appended claims.

Claims

1. A method for analyzing functional bacteria in activated sludge, characterized in that: include: Collecting activated sludge samples to be analyzed; Pre-treating the activated sludge sample to obtain a pre-treated activated sludge sample; Using a nucleic acid fluorescent dye to dye the pretreated activated sludge sample to obtain an activated sludge sample to be tested; Determine the analysis parameters for flow cytometry; According to the analysis parameters, the activated sludge sample to be detected is detected using the flow cytometer to obtain a first detection result; Obtaining analysis result parameters of predefined standard microspheres; Determining 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; Determining the biomass of the functional bacteria in the activated sludge sample to be analyzed according to the concentration of the functional bacteria in the activated sludge sample to be analyzed and the particle size of the functional bacteria; Determining the biomass of the first bacterial group and the biomass of the second bacterial group in the activated sludge sample to be analyzed according to the first detection result; The functional bacterial metabolic activity of the activated sludge sample to be analyzed is determined based on the biomass of the first bacterial group and the biomass of the second bacterial group.

2. The method according to claim 1, characterized in that The activated sludge sample is pretreated to obtain a pretreated activated sludge sample: Using a fixing agent to fix the activated sludge sample to obtain a first pretreated sample; Using an ultrasonic instrument to perform ultrasonic treatment on the first pre-treated sample, and diluting the ultrasonically treated sample to obtain a second pre-treated sample; The second pretreated sample is filtered using an organic nylon filter membrane to obtain a pretreated activated sludge sample.

3. The method according to claim 2, characterized in that The fixing agent is a glutaraldehyde solution with a final concentration of 0.5% to 1.5%.

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

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

6. The method according to claim 1, characterized in that The method of using a nucleic acid fluorescent dye to dye the pretreated activated sludge sample to obtain an activated sludge sample to be tested includes: Use flow cytometry sheath fluid to dilute the original nucleic acid fluorescent stain 100 times to make the stain working solution; The pretreated activated sludge sample is added to the dye working solution according to a preset volume ratio, and incubated in the dark to obtain an activated sludge sample to be tested.

7. The method according to any one of claims 1 to 6, characterized in that: The analysis parameters of the flow cytometer include a gain value of a forward angle scattered light channel, a gain value of a side angle scattered light channel and a receiving threshold value of fluorescence intensity.

8. The method according to any one of claims 1 to 6, characterized in that: The first detection result includes the total number of functional bacteria cells, the analysis time of functional bacteria, the forward scattered light detection value, the side scattered light detection value and the purple side angle scattered light detection value, and 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; Accordingly, the method of determining 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 includes: Calculating the concentration of functional bacteria of the activated sludge sample to be analyzed according to 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; The particle size of the functional bacteria of the activated sludge sample to be analyzed is determined according to the forward scattered light detection value, the side scattered light detection value and the purple side angle scattered light detection value of the functional bacteria and the predefined standard microspheres.

9. The method according to any one of claims 1 to 6, characterized in that: The step of determining the functional bacterial metabolic activity of the activated sludge sample to be analyzed based on the biomass of the first bacterial group and the biomass of the second bacterial group comprises: The percentage of the biomass of the first bacterial group and the biomass of the second bacterial group is determined as the functional bacterial metabolic activity of the activated sludge sample to be analyzed.

10. The method according to claim 9, characterized in that Determining the biomass of the first bacterial group and the biomass of the second bacterial group in the activated sludge sample to be analyzed according to the first detection result includes: Obtaining a preset nucleic acid content threshold; According to the first detection result, the preset nucleic acid content threshold is used to determine the first bacterial group and the second bacterial group; The biomass of the first bacterial group and the biomass of the second bacterial group in the activated sludge sample to be analyzed are determined according to the concentration and particle size of the first bacterial group and the concentration and particle size of the second bacterial group.

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