Granular sludge stability identification method based on microbial activity change characteristics

Abstract

The invention discloses a method for identifying the stability of granular sludge based on microbial activity change characteristics. The method comprises the following steps: taking a certain amount of sludge at the aeration tail end of an AGS device as a sample; the method comprises the following steps: putting a sample into a transparent measuring cylinder, judging the settling performance of granular sludge, quantitatively detecting the cell activity of microorganisms in the sludge by using a flow cytometer, and meanwhile, carrying out online monitoring on the sludge sample of an aerobic granular sludge system by using a respiration graph measuring device (LB-WBM-450PI type smart workstation). Through multi-dimensional analysis of sedimentation characteristics, microbial metabolic activity and flow cytometry activity of the aerobic granular sludge, characteristic information such as particle morphological structure integrity, dynamic change between the microbial metabolic activity and the flow cytometry activity and the like can be provided, and data support is provided for stability regulation and control research of the aerobic granular sludge.

Description

Technical Field

[0001] The invention belongs to the technical field of water treatment, and in particular relates to a granular sludge stability identification method based on microbial activity change characteristics. Background Art

[0002] Monitoring of physical indicators of aerobic granular sludge is of great significance for breaking through the technical bottleneck of stable operation control and its wide application. However, the indicators currently tested are: density, particle size, sedimentation velocity, SV5 / SV 30 The regulatory effect of the granular sludge system with characteristics such as detection by microscope is not obvious, and the single dimension of detection cannot fully reflect the changes in the stability of the electricity charge of the granular sludge.

[0003] The application of respiratory atlas technology in the water treatment industry is mainly concentrated in the research of activated sludge systems. This technology can effectively determine the respiratory activity of heterotrophic and autotrophic bacteria. And quantitatively detect the endogenous respiration process of activated sludge, which is an important metabolic process of activated sludge. By adding different substrates, the respiration rates of different species of microorganisms can be separated. In addition, some studies have found that the increase in the proportion of endogenous respiration rate reflects the deterioration of the respiratory metabolic activity of activated sludge, which is an important parameter to characterize the activity of activated sludge and can be used as a quantitative description indicator of the health status of activated sludge.

[0004] However, in the previous operation of sewage treatment plants, phenomena such as sludge bulking, sludge floating and sludge aging often occurred and could not be discovered in time. Nowadays, the application of respiration mapping technology can help to detect and deal with these problems in time, thereby optimizing the operation and management of sewage treatment plants. These research results have deepened the further understanding of the microbial respiration process in activated sludge and provided a theoretical basis for the operation and management of sewage treatment plants. In general, the application of respiration mapping technology in the water treatment industry has important theoretical and practical significance. Respiration mapping technology can monitor abnormal phenomena in activated sludge and reveal how microbial activity affects the structure and stability of activated sludge. In addition, by combining these data with advanced data analysis and modeling technologies, such as machine learning and artificial intelligence, we can develop predictive models that can predict and adjust system operation in real time to optimize treatment efficiency and reduce energy consumption. More comprehensively, if respiration mapping technology is integrated with other advanced monitoring technologies, such as online sensors and high-throughput sequencing, more comprehensive system operation information can be provided. This integrated approach can improve our understanding of the dynamics of microbial communities in activated sludge systems and provide more accurate control strategies. Through long-term experiments and continuous monitoring, the long-term stability of the activated sludge system can be evaluated while monitoring key parameters such as long-term changes in microbial activity. This long-term perspective is essential for achieving continuous and reliable wastewater treatment. In summary, respiration mapping technology shows great application potential in optimizing existing sewage treatment processes and providing support for the development of more efficient and environmentally friendly wastewater treatment technologies in the future.

[0005] Flow cytometry (FCM) is an analytical technique based on the single-cell level. It can analyze samples quickly, quantitatively and accurately, and can analyze thousands of biological particles within seconds. At present, the application of flow cytometry in basic laboratory research is very common, and it also has certain applications in aquatic microbiology. In recent years, flow cytometry has been widely used in many fields of sewage treatment, and has shown great potential in evaluating the total number of bacteria, bacterial activity and microbial community structure in activated sludge in sewage treatment plants. A research team used flow cytometry to study the physiological characteristics and morphological change mechanisms of stress response in activated sludge processes, which can obtain dynamic and real state information of microbial communities in activated sludge, thereby strengthening the control and management of sewage treatment processes.

[0006] Although the above technical solutions achieve accurate and real-time identification and analysis of the biological activity of the activated sludge process, there is currently no fast and accurate multi-dimensional stability identification method for aerobic granular sludge processes.

[0007] The patent application document with publication number CN114716007A discloses the application of Streptomyces in promoting the formation of aerobic granular sludge and / or improving the stability of aerobic granular sludge. By using this solution, aerobic granular sludge can be cultured within 6 to 12 days, and the aerobic granular sludge obtained by culture has a smooth surface, regular shape, dense texture, and good stability. While shortening the formation time of aerobic granular sludge, the stability of aerobic granular sludge can also be improved. However, since this solution only emphasizes the rapid granulation, there is no reliable data to prove that this method can improve its stability, resulting in incomplete research and difficulty in rapid application in the regulation of granular sludge stability.

[0008] The patent application document with publication number CN114853294A discloses a method for quickly solving the problem of sludge bulking caused by non-filamentous bacteria in granular sludge. This method can successfully solve the problem of sludge bulking caused by non-filamentous bacteria in different types of granular sludge, maintain the stability of the granular sludge system, and significantly enhance the reactor's treatment performance for wastewater. However, since this method only emphasizes the rapid solution to the sludge bulking problem of the granular sludge system, although sludge bulking affects the stability of the granular sludge, it is difficult to achieve a multi-dimensional solution to the phenomenon or problem of system instability by only quickly solving the single problem of sludge bulking. Summary of the invention

[0009] In order to overcome the shortcomings of the above-mentioned existing aerobic granular sludge stability detection technology, the purpose of the present invention is to provide a granular sludge stability identification method based on the characteristics of microbial activity changes. Through multi-dimensional analysis of the morphological stability, structural stability, and microbial metabolic stability of aerobic granular sludge, it is concluded that the aerobic granular sludge system has different stability change characteristics under different states; and it is stabilized by strengthening or suppressing certain performance. The present invention utilizes the qualitative analysis of microscope sludge morphology combined with the quantitative and microscopic analysis of flow cytometry and respiratory spectrum to make the detection results more accurate and objective, which supplements the shortcomings of the existing characterization methods for granular sludge stability changes in the water treatment industry, and has the advantages of simplicity, intuitiveness, accuracy, efficiency and low cost.

[0010] In order to achieve the above object, the technical solution adopted by the present invention is:

[0011] A method for identifying granular sludge stability based on the characteristics of microbial activity changes comprises the following steps:

[0012] Step 1, taking the aerobic granular sludge mixed liquid at the end of aeration as a sample, controlling the sludge concentration of the sample at 3-4 g / L, so that the sample is in a completely mixed state;

[0013] Step 2: Perform microscopic examination on the sample in step 1 to obtain the morphological characteristics of the sample; when the sample is completely mixed, measure the sedimentation performance, which includes: SV and SVI data; use the smart workstation to measure the respiratory spectrum to obtain the respiratory rate of the granular sludge, which includes the autotrophic specific respiratory rate SOUR A , heterotrophic bacteria specific respiration rate SOUR H , on-site specific respiration rate SOUR S , endogenous specific respiration rate SOUR e and maximum specific respiratory rate SOUR T ;

[0014] Step 3: Calculate the sludge health index RI according to the respiration rate of the granular sludge obtained in step 2, RI = SOUR e / SOUR T ;

[0015] Step 4, taking the mud-water mixture at the end of aeration as a sample, placing the sample in a 4±1°C environment and settling for at least 30 minutes, collecting the supernatant, and filtering the supernatant using a 5 μm nylon filter. After collecting the supernatant, the remaining sludge sample is resuspended to the original liquid level with a buffer solution to obtain a sludge mixed solution A;

[0016] Step 5, subjecting the sludge mixed liquor A obtained in step 4 to ultrasonic crushing, with an ultrasonic frequency of 20kHz, an ultrasonic power of 4.5W to 7.5W, a power ratio of 3% to 5%, and an intermittent ultrasonic crushing process, ultrasonicating for 2 to 3s, stopping for 2 to 4s, and the ultrasonic process lasting 5 to 10min. The sample after ultrasonication is filtered using a 5μm nylon filter to obtain a sludge mixed liquor B;

[0017] Step 6, use Green I (SGI) and propidium iodide (PI) are combined to prepare the dye. First, dimethyl sulfoxide (DMSO) is used as the solvent. Green I (SGI) standard stock solution was diluted to 100 times, and the diluted Green I (SGI): propidium iodide (PI) = 50:1;

[0018] Step 7, dilute the supernatant filtered in step 4 by 150 times with PBS to obtain a supernatant sample; dilute the sludge mixed solution B obtained in step 5 by 6×100 times with PBS to obtain a diluted sludge sample; fully mix the diluted supernatant sample and the sludge sample with the dye prepared in step 6 in a vortex oscillator, and dye in a constant temperature metal bath at 35° C. in the dark for 15 minutes to obtain a measurement sample; according to the volume ratio, the diluted sludge sample: dye = 100:1, the diluted supernatant sample: dye = 100:1;

[0019] Step 8, measuring the microbial cell activity of the measurement sample obtained in step 7, wherein the microbial cell activity includes: total bacterial concentration, live-dead bacterial rate, HNA bacteria and LNA bacteria, and cell size distribution;

[0020] Step 9, based on the sample morphological characteristics, sedimentation performance and respiration rate of the granular sludge obtained in step 2, the sludge health index RI obtained in step 3 and the microbial cell activity in the sample obtained in step 8, combined with the sample morphological characteristics and sedimentation performance, determine whether the state of the aerobic granular sludge system is stable in multiple dimensions.

[0021] The buffer solution is a phosphate buffer solution with a pH of 7.2.

[0022] In step 9, the indicators for judging whether the state of the aerobic granular sludge system tends to be stable are: the health index RI of the granular sludge is 7%-10%, the mortality rate is stable at 20%-30%, the proportion of high nucleic acid bacteria (HNA) bacteria is stable at more than 85%, and the autoheterotrophic respiration rate does not fluctuate significantly.

[0023] Compared with the existing stability identification and regulation technology, the beneficial effects of the present invention are:

[0024] 1. Multi-dimensional innovation: The present invention provides a method for regulating the stability of granular sludge based on respiratory spectrum and flow cell activity change characteristics. Through multi-dimensional analysis of the morphological stability, structural stability, and microbial metabolic stability of aerobic granular sludge, it can provide characteristic information such as the dynamic changes between particle micromorphology, structural integrity, and microbial metabolic activity, providing support for the study of aerobic granular sludge stability.

[0025] 2. Online early warning function: Based on the respiratory spectrum early warning of activated sludge, the present invention combines the sedimentation performance of granular sludge and flow cell activity to detect abnormal situations in advance and provide users with early warning functions, which is conducive to preventing the occurrence of production accidents and improving the treatment efficiency and stability of the aerobic granular sludge system.

[0026] 3. High level of intelligence: The image processing and respiratory spectrum detection processing involved in the present invention can be automated and implemented in online monitoring equipment, which is conducive to the intelligent operation of the water plant.

[0027] 4. Advanced technology: The present invention adopts advanced flow cytometry and respiratory mapping technology to accurately analyze the change process of sludge particle size, which improves the accuracy and reliability of detection and can timely adjust the system's operating parameters according to the detection data to ensure stable operation of the system.

[0028] In summary, the present invention has the advantages of online early warning, multi-dimensional judgment and fast and efficient decision-making through multi-dimensional analysis and monitoring of the morphological stability, structural stability and microbial metabolic stability of aerobic granular sludge. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] FIG1 is a schematic diagram and a physical diagram of a device used in an embodiment of the present invention; wherein FIG1(a) is a schematic diagram of the device, and FIG1(b) is a physical diagram of the device.

[0030] Figure 2 Graph showing the operating condition changes of the device in an embodiment of the present invention.

[0031] FIG3 is a diagram showing the removal effects of major pollutants; among them, FIG3(a) is a diagram showing the removal effects of COD, and FIG3(b) is a diagram showing the removal effects of ammonia nitrogen.

[0032] Figure 4 This is a graph showing changes in physical and chemical parameters of aerobic granular sludge.

[0033] Figure 5 This is a graph showing the changes in flow cell parameters of granular sludge.

[0034] Figure 6 This is a graph showing changes in the respiration spectrum of aerobic granular sludge.

[0035] Figure 7 This is a representative microscopic image of aerobic granular sludge.

[0036] Figure 8 Flow chart for multidimensional identification and regulation based on microbial activity. DETAILED DESCRIPTION

[0037] The technical solution of the present invention is further described below through specific embodiments.

[0038] In view of the shortcomings of the existing technical analysis routes, the present invention proposes a granular sludge stability identification method based on the characteristics of microbial activity changes, focusing on the particle stability change process, analyzing the changing characteristics of particles from the perspectives of sedimentation performance, respiratory spectrum and sludge cell activity, and providing guidance for the stability regulation of aerobic granular sludge.

[0039] A method for identifying granular sludge stability based on the characteristics of microbial activity changes comprises the following steps:

[0040] Step 1, take the aerobic granular mud mixed liquid at the end of aeration as the experimental sample, measure its sludge concentration (can be measured manually offline or by online sludge concentration sensor), take 300mL of mud-water mixture as the measurement sample, control the sludge concentration of the sample to about 3-4g / L, so that the sample is in a completely mixed state;

[0041] In step 1, the time of sludge sampling is controlled at the aeration end of the system operation and the sludge volume is controlled to be about 300 mL (to meet the detection requirements).

[0042] Step 2, taking a small amount of sludge from the sample in step 1 and examining it under a common optical microscope to obtain the morphological characteristics of the sample;

[0043] When the samples are completely mixed, immediately use a digital camera to set the same shooting parameters to start image acquisition of the samples. The shooting time is not less than 30 minutes. When the sedimentation is completed, stop shooting to obtain a complete video of the sludge sedimentation, from which the sedimentation performance, i.e., SV and SVI data, etc., is obtained;

[0044] If the sedimentation image shooting time in step 2 cannot be continuously shot for 30 minutes, it should be greater than 5 minutes. However, the SV value must be recorded at the 30th minute of sedimentation.

[0045] Place the experimental sample in step 1 in the LB-WBM-450PI smart workstation measurement device (in actual use, the workstation can be used to automatically absorb the sample) for respiration spectrum measurement. The LB-WBM-450PI smart workstation mainly measures the respiration rate of granular sludge, including: autotrophic bacteria specific respiration rate SOUR A , heterotrophic bacteria specific respiration rate SOUR H , on-site specific respiration rate SOUR S , endogenous specific respiration rate SOUR e and maximum specific respiratory rate SOUR T ;

[0046] In actual applications, the respiratory spectrum data can be viewed online and needs to be checked regularly to understand the system status in a timely manner.

[0047] Step 3: Calculate the sludge health index RI according to the respiration rate of the granular sludge obtained in step 2, RI = SOUR e / SOUR T ;

[0048] Step 4, taking the mud-water mixture at the end of aeration as a sample, placing the sample in a 4°C environment and settling for 30 minutes (generally not less than 30 minutes, but if the time is too long, it is necessary to ensure that the sample is not contaminated during the settling process, and the settling time needs to be controlled the same each time), and then collecting the supernatant, and filtering the supernatant with a 5 μm disposable nylon filter head. After collecting the supernatant, the remaining sludge sample is resuspended to the original liquid level with a buffer solution to obtain a sludge mixed solution A;

[0049] Step 5, the bacteria in the sludge mixture are dispersed by using the ultrasound of an ultrasonic cell disruptor to disperse the flocs and particles in the water, that is, the sludge mixture A obtained in step 4 is ultrasonically disrupted by using an ultrasonic cell disruptor, the ultrasonic frequency of the ultrasonic cell disruptor is 20kHz, the ultrasonic power is 4.5W (power ratio 3%), and the ultrasonic disruption process is completed intermittently, ultrasonic for 2s, and stopped for 3s. The ultrasonic process lasts for 5 to 10min. The time will be adjusted according to the particle size of the sludge sample to ensure that the bacteria in the sludge can be completely extracted without destroying the cells. The sample after ultrasound is also filtered using a 5μm nylon filter head to obtain a sludge mixture B; the utensils, filter heads and syringes used in the experiment are sterilized at 121°C for 2h using a high-temperature sterilizer;

[0050] The ultrasonic frequency and ultrasonic power settings of the sludge mixed liquid cells cannot be greater than the requirements described in step 5 above. The time of the ultrasonic process can be adjusted according to the particle size of the sludge sample.

[0051] Step 6, use Green I (SGI) and propidium iodide (PI) are used to prepare a stain for the determination of live-death rate, high nucleic acid bacteria (HNA) and low nucleic acid bacteria (LNA) and cell size distribution. First, dimethyl sulfoxide (DMSO) is used as a solvent. Green I (SGI) standard stock solution was diluted to 100 times, and the diluted Green I (SGI): propidium iodide (PI) = 50:1;

[0052] Step 7, dilute the supernatant filtered in step 4 by 150 times with PBS in a 1.5 mL centrifuge tube to obtain a supernatant sample; dilute the sludge mixed solution B obtained in step 5 by 6×100 times with PBS in a 1.5 mL centrifuge tube to obtain a diluted sludge sample; fully mix the diluted supernatant sample and the sludge sample with the dye prepared in step 6 in a vortex oscillator, respectively, by volume ratio, the diluted sludge sample: dye = 100:1, the diluted supernatant sample: dye = 100:1, and then dye in a constant temperature metal bath at 35°C in the dark for 15 minutes to obtain the final machine measurement sample;

[0053] The time for metal bath light-proof dyeing in step 7 cannot be more than 15 minutes, and the temperature and time need to be strictly controlled (dyeing at 35° C. for 15 minutes).

[0054] Step 8, after the staining is completed, the measurement sample obtained in step 7 is measured using a flow cytometer BD Accuri C6, with fixed wavelengths of 488nm and 640nm, respectively, and green fluorescence signals (SGI signals, RSG signals) are collected at FL1=530nm and red fluorescence signals (PI signals) are collected at FL3>670nm to determine the activity of microbial cells in the sample, including total bacterial concentration, live-dead bacteria rate, HNA bacteria and LNA bacteria and cell size distribution. Three parallel samples are set for each sample, and the collected relevant data are processed using BD Accuri CFlow software. The morphological stability of the granular sludge in the aerobic granular sludge system is determined by microscopic examination;

[0055] Step 9: Based on the sample morphological characteristics, sedimentation performance and respiration rate of the granular sludge obtained in step 2, the sludge health index RI obtained in step 3 and the microbial cell activity in the sample obtained in step 8, combined with the sample morphological characteristics and sedimentation performance, a multi-dimensional judgment is made as to whether the state of the aerobic granular sludge system is stable. If it is unstable, the hydraulic retention time, aeration volume, sedimentation time and aeration time are adjusted accordingly according to the results.

[0056] The SVI of ungranulated flocculent sludge is much higher than that of aerobic granulated sludge. The SVI5 of inoculated flocculent sludge is greater than 200 mL / g. 30 Greater than 100mL / g, its SV5 / SV 30 Greater than 2. Before granulation, the proportion of high nucleic acid bacteria (HNA) in the sludge is less than 80%. The bacterial death rate of the sludge is greater than 40%. The specific respiration rate of autotrophic bacteria (SOUR A ) and heterotrophic specific respiration rate (SOUR H ) are at a low level, far lower than normal aerobic granular sludge. At the same time, its sludge health index (RI index) is above 30%, far greater than 16% of healthy flocculent sludge. At the same time, the Res / t index (Res / t = SOURCE s / SOUR T ) is also at a high level, and the pollutant concentration in the system effluent is high at this time.

[0057] In the early stage of granular sludge formation, due to the effect of hydraulic selective pressure, sludge with poor settling performance was quickly discharged from the system, and the sludge concentration in the system showed a large fluctuation trend at this stage. However, as the research continued, the settling performance of the sludge was gradually improved. When the sedimentation time of the aerobic granular sludge system was shortened to 5 minutes, the system gradually entered a relatively stable operating state. With the progress of aerobic sludge granulation, the mortality rate in the mature aerobic granular sludge in the reactor gradually decreased to about 25%, and the proportion of high nucleic acid bacteria (HNA) in the sludge increased to more than 90%. The respiration index (RI) of the untamed floc sludge and the mature aerobic granular sludge were 31% and 8%, respectively. The efficiency of the granulated sludge in the biochemical process has been significantly improved, such as the respiration rate of heterotrophic bacteria (SOUR H ) rises rapidly.

[0058] When the system completes aerobic granulation, the parameters of the autoheterotrophic respiration rate in the respiration spectrum also tend to be stable, and the RI value enters a low level stage of about 10%. The bacterial death rate of the sludge is also stable at about 20%, and the proportion of high nucleic acid bacteria (HNA) in the sludge is also stable at more than 85%.

[0059] When the structure of particles in the system is broken or their shape is uneven, their sedimentation performance deteriorates significantly. 30 The ratio also gradually increased. This indicates that the structural stability of the particles began to be damaged, affecting the settling efficiency of the entire aerobic granular sludge system. At this time, the respiration spectrum of the sludge in the system also changed significantly, specifically in the heterotrophic bacteria specific respiration rate (SOUR H ) dropped significantly. The RI index of the sludge also increased to outside the healthy range of 7% to 12% of granular sludge. At the same time, the bacterial death rate in the sludge rose rapidly to 30%, while the proportion of HNA bacteria in the sludge dropped below 90%.

[0060] When the settling performance of the sludge in the system is very poor, it can be basically determined that the system has not yet produced aerobic granular sludge, and it is possible that the sludge in the system has not been granulated. If the respiration spectrum test results show that its RI index is greater than 12%, and its autotrophic bacteria activity is much lower than that of heterotrophic bacteria, and at the same time, the flow cell parameters of floc sludge and granular sludge are also very different, then it can be further determined that the sludge is in the floc stage without granulation.

[0061] When the sedimentation performance of the sludge in the system is good, and the proportion of HNA bacteria in the sludge is about 85% and the mortality rate is about 20%, it can be determined that the main body of the system is mature and stable aerobic granular sludge. At this time, the respiratory spectrum test results show that its RI index is about 8%, and the respiratory activity of its autotrophic and heterotrophic bacteria is very good.

[0062] When the settling performance of the sludge in the system is good, but the respiratory spectrum test results show that its RI index is greater than 14%, and the proportion of HNA bacteria in the flow results decreases slightly and its mortality rate increases slightly to about 30%, it can be determined that the aerobic granular sludge in the system is polarized, and some particles continue to grow rapidly into loose particles with a large particle size of 2 to 3 mm, and the remaining particles begin to gradually disintegrate. The proportion of mature and stable particles in the system decreases, and the gap between autotrophic bacteria and heterotrophic bacteria gradually increases. The respiratory activity of autotrophic bacteria decreases, and the respiratory activity of heterotrophic bacteria first increases and then decreases to a level lower than the mature and stable period.

[0063] Implementation Cases

[0064] As shown in Figure 1- Figure 2 As shown, step 1, inoculate the activated sludge from a municipal sewage treatment plant in Shaanxi Province into a laboratory-scale aerobic granular sequencing batch reactor (AGSBR). The effective volume of this reactor is about 12.4L, the total height is 0.8m, the effective height is 0.7m, and the inner diameter is 0.15m. This reaction device uses a bottom sand core aeration disk for aerobic aeration, controls the aeration volume through a rotor flowmeter, and adopts a bottom water inlet method. The reactor uses PLC to realize automatic control of the operating conditions. According to the multi-dimensional data feedback, the switches and operating times of the water inlet, aeration, sedimentation and drainage processes are adjusted in time to make the system run as stably as possible. The entire experimental process was carried out at room temperature (20-25°C), and the AGSBR device continued to operate for about 300 days;

[0065] Step 2: Take the aerobic granular mud mixed liquid at the aeration end of the AGSBR device at the same time every day as the experimental sample, measure its sludge concentration (offline manual measurement or MLSS online sensor measurement can be selected), take 300mL of the mud-water mixture as the measurement sample, control the sludge concentration of the sample to about 3-4g / L, and make the sample in a completely mixed state;

[0066] Step 3, taking a small amount of sludge from the sample in step 2 and examining it under a common optical microscope to obtain the morphological characteristics of the sample;

[0067] Step 4, when the sample is completely mixed, immediately use a digital camera to set the same shooting parameters to start image acquisition of the sample, the shooting time is not less than 30 minutes, and the shooting is stopped when the sedimentation is completed, so as to obtain a complete video of the sludge sedimentation, from which the sedimentation performance, i.e., SV and SVI and other data, is obtained;

[0068] Step 5: Place the experimental sample obtained in step 2 in the LB-WBM-450PI smart workstation measuring device (in actual use, the workstation can be used to directly take samples for self-priming) to measure the respiration spectrum. The LB-WBM-450PI smart workstation mainly measures the respiration rate of granular sludge, including: autotrophic bacteria specific respiration rate SOURA , heterotrophic bacteria specific respiration rate SOUR H , on-site specific respiration rate SOUR S , endogenous specific respiration rate SOUR e and maximum specific respiratory rate SOUR T ;

[0069] Step 6: Calculate the sludge health index RI according to the respiration rate of the granular sludge obtained in step 5, RI = SOUR e / SOUR T ;

[0070] Step 7, taking the mud-water mixture at the end of aeration as a sample, placing the sample in a 4°C environment to settle for 30 minutes, collecting the supernatant, and filtering it with a 5 μm disposable nylon filter to remove non-microbial impurities in the mixed solution. After collecting the supernatant, the remaining sludge sample is resuspended to the original liquid level with a buffer solution to obtain a sludge mixed solution A;

[0071] Step 8, the bacteria in the sludge mixture are dispersed by using the ultrasound of an ultrasonic cell disruptor to disperse the flocs and particles in the water, that is, the sludge mixture A obtained in step 7 is ultrasonically disrupted by using an ultrasonic cell disruptor, the ultrasonic frequency of the ultrasonic cell disruptor is 20kHz, the ultrasonic power is 4.5W (power ratio 3%), and the ultrasonic disruption process is completed intermittently (ultrasonic 2s, stop 3s), the ultrasonic process lasts for 5 to 10 minutes, and the time will be adjusted according to the particle size of the sludge sample to ensure that the bacteria in the sludge can be completely extracted without destroying the cells. The sample after ultrasound is also filtered with a 5μm nylon filter head to obtain a sludge mixture B; the utensils, filter heads and syringes used in the experiment are sterilized at 120°C for 2h using a high-temperature sterilizer;

[0072] Step 9, use Green I (SGI) and propidium iodide (PI) are used to prepare a stain for the determination of live-death rate, high nucleic acid bacteria (HNA) and low nucleic acid bacteria (LNA) and cell size distribution. First, dimethyl sulfoxide (DMSO) is used as a solvent. Green I (SGI standard stock solution diluted to 100 times, according to the volume ratio, the diluted Green I (SGI: propidium iodide (PI) = 50:1;

[0073] Step 10, dilute the supernatant filtered in step 7 by 150 times with PBS in a 1.5 mL centrifuge tube to obtain a supernatant sample; dilute the sludge mixed solution B obtained in step 8 by 6×100 times with PBS in a 1.5 mL centrifuge tube to obtain a diluted sludge sample; fully mix the diluted supernatant sample and the sludge sample with the dye prepared in step 9 in a vortex oscillator, respectively, by volume ratio, the diluted sludge sample: dye = 100:1, the diluted supernatant sample: dye = 100:1, and then dye in a constant temperature metal bath at 35°C in the dark for 15 minutes to obtain the final machine measurement sample;

[0074] Step 11, after the staining is completed, the measurement sample obtained in step 10 is measured using a flow cytometer BD Accuri C6, with fixed wavelengths of 488nm and 640nm, respectively, and green fluorescence signals (SGI signals, RSG signals) are collected at FL1=530nm and red fluorescence signals (PI signals) are collected at FL3>670nm, and the activity of microbial cells in the sample is measured, including total bacterial concentration, live and dead bacterial rate, HNA bacteria and LNA bacteria, and cell size distribution. Each sample has three parallel samples, and the collected relevant data are processed using BD Accuri CFlow software. The morphological stability of the granular sludge in the aerobic granular sludge system is determined by microscopic examination;

[0075] Step 12, based on the sample morphological characteristics obtained in step 3, the sedimentation performance obtained in step 4, the respiration rate of the granular sludge obtained in step 5, the sludge health index RI obtained in step 6, and the microbial cell activity in the sample obtained in step 11, judge whether the state of the aerobic granular sludge system is stable from multiple dimensions of morphological characteristics, sedimentation performance, respiration spectrum characteristics and microbial cell activity of the aerobic granular sludge, and then make corresponding strategy adjustments based on the results.

[0076] Example analysis: This example divides the entire experimental stage into the following main stages based on the granular sludge settling performance status, respiratory spectrum changes and flow cell activity.

[0077] Phase I is mainly the acclimatization phase, during which the activated sludge gradually granulates and stabilizes for more than 20 days. This phase lasts for 3 months. In the first few weeks, the reactor sedimentation time is reduced from 30 minutes to 5 minutes. At the same time, the volume exchange ratio (VER) is increased from 0.428 to 0.714 on the 68th day of Phase I to accelerate the aerobic granulation process.

[0078] In phase 2, the reactor operating conditions were frequently adjusted and changed. In the first two weeks of phase 2, the ammonia nitrogen load was gradually increased to prepare for subsequent regulation and enrich nitrifying bacteria. In the middle of phase 2, the system was adjusted to anoxic aerobic anoxic aerobic (AOAO) to achieve enhanced anoxic denitrification and inhibit the rapid growth of particle size. In the late stage of phase 2, in order to increase the particle size and observe the change trend of the respiration spectrum, denitrification was further enhanced.

[0079] In stage three, the hydraulic retention time was shortened from 4 hours to 3 hours in the first few weeks to increase the processing load to accelerate the rapid growth of sludge particles and increase basic data to improve the control method. Subsequently, the hydraulic retention time was shortened from 3 hours to 4 hours. At the same time, no other control system was performed and the granular sludge gradually disintegrated quickly.

[0080] As shown in Figure 3, during the entire test process, the influent COD concentration was around 500 mg / L, the effluent concentration was basically stable at below 50 mg / L, and the influent ammonia nitrogen concentration was 18 mg / L from 106 days to 130 days. After 130 days, the influent ammonia nitrogen concentration stabilized at around 45 mg / L. Except for the low ammonia nitrogen removal rate in stage three, the effluent ammonia nitrogen was generally less than 3 mg / L.

[0081] like Figure 4 As shown, about 20 days after inoculation with floc sludge, particles began to appear in the system, and the proportion of particles larger than 300μm increased significantly. On the 50th day, granulation was completed in the reactor, with an average particle size of about 300μm. In the early stage of stage II, the number of small particles smaller than 300μm decreased, while the proportion of particles larger than 300μm, especially larger than 500μm, increased significantly. In the middle and late stages of stage II, the growth of large particles was destroyed by enhanced anoxic denitrification. In a short period of time, enhanced anoxic denitrification controlled the growth trend of large-size particles, especially the growth of particles >500μm, and also controlled the reduction of particles <300μm. However, enhanced anoxic denitrification of large particles changed the sludge sedimentation properties in the system, resulting in a significant increase in effluent SS and a decrease in the system's biomass. After particle formation, SVI 30 Below 20mL / g, SVI5 / SVI 30 Maintained at a low level close to 1. In the late stage of stage 2 when the effluent suspended solids concentration is high, SVI5 / SVI 30 A certain degree of increase indicates that the sedimentation performance of the sludge has deteriorated.

[0082] like Figure 5As shown, in the aerobic granular sludge system, the research results of the present invention found that specific functional microorganisms (such as HNA) are crucial to the treatment effect. The HNA proportion of sludge flocs is lower than that of granular sludge. At the same time, when the granular sludge disintegrates, its HNA proportion does not drop as suddenly as the particle size. At the same time, the disintegrated granular sludge seems to have many similarities with the stable granular sludge. When the particle size of the granular sludge increases rapidly, the proportion of its HNA functional microorganisms does not increase but decreases to a certain extent. This also confirms the view that large-size granular sludge is not necessarily beneficial to the system. At the same time, flow cytometry can measure the physiological activity parameters of microbial cells in the AGS system, such as the live and dead bacteria rate. These parameters reflect the activity and health status of the microorganisms, which help to judge the overall operating efficiency and potential hidden dangers of the AGS system, and provide early warning to avoid system collapse and disintegration affecting water treatment efficiency.

[0083] like Figure 6 As shown, the SOUR of the untamed flocculent sludge H is 8.75mgO2 / gSS / h, while the SOUR of aerobic granular sludge is H The RI index of untamed floc sludge and aerobic granular sludge was 31% and 8%, respectively. It can be found that the autotrophic respiration rate (SOUR) of granular sludge under low carbon-nitrogen ratio is 22.37 mgO2 / gSS / h, which is significantly higher than that of untamed floc sludge. In addition, the RI index of untamed floc sludge and aerobic granular sludge is 31% and 8%, respectively. A ), heterotrophic bacterial respiration rate (SOUR H ) and RI index were 0.68mgO2 / gSS / h, 19.45mgO2 / gSS / h and 7% respectively. The autotrophic respiration rate SOUR of granular sludge with high carbon-nitrogen ratio A 、SOUR H The RI index was 0.36 mgO2 / gSS / h, 21.76 mgO2 / gSS / h and 5% respectively. This shows that the mature aerobic granular sludge system can quickly adapt to the low carbon-nitrogen ratio environment, and the reduction of carbon-nitrogen ratio will not reduce the metabolic activity of the sludge in the short term, which also reflects the advantages of the aerobic granular sludge system. A 、SOUR H and RI index were 0.66mgO2 / gSS / h, 20.96mgO2 / gSS / h and 5% respectively. A 、SOUR H and RI index were 1.37mgO2 / gSS / h, 23.16mgO2 / gSS / h and 16% respectively. This is because the AOAO process and the decrease in biomass increased the pollutant load, causing the microbial activity to increase significantly compared to before. When the particles increased abnormally, the RI index also increased abnormally, mainly because of the SOURH The RI value of the sludge in the system is significantly reduced due to system instability. Therefore, the present invention uses the respiration spectrum technology to timely detect the process of sludge from domestication to mature aerobic particles, and establishes a set of basic respiration spectrum data. For example, when the RI value of the sludge in the system is significantly reduced, it means that the aerobic granular sludge begins to form and enters a mature and stable state. If the RI value increases from a low value and the sedimentation performance of the sludge deteriorates, the system is very likely to collapse due to the polarization of particle size, and large particle size and floc sludge need to be screened out in time.

[0084] like Figure 7 As shown in the figure, the morphology of the reactor inoculated sludge is significantly different from that of the granulated sludge. After the sludge is granulated, the aerobic granular sludge formed in the reactor is visible to the naked eye. The sludge is taken out from the sampling port and placed in a glass culture dish. The small granular sludge with regular shapes can be seen more clearly. The particles observed under the microscope are darker in color and tightly combined, with smooth edges, and no filamentous microorganisms attached around the granular sludge. When the reactor runs for about three months, the proportion of large particles in the system increases significantly. At this time, the sludge in the system has a more regular morphology and larger particles. Particles of different particle sizes can be clearly seen in the culture dish, and there is basically no floc sludge in the system at this time. Only a small amount of small granular sludge can be observed under the microscope. The particle size of the granular sludge in the reactor began to gradually increase after the 150th day, and the morphology of the sludge also changed significantly as the particle size increased. However, when the particle size rapidly increased to more than 600μm, as the particles continued to increase, some sludge was attached to a large number of filamentous microorganisms, and the color in the culture dish was slightly lighter, and some were even whitish. When the particle size of aerobic granular sludge reaches a certain size, the stability of the system may be affected.

[0085] like Figure 8 As shown, when the aerobic granular sludge process is actually operated, the present invention needs to use the sedimentation characteristics, morphological structure, respiratory spectrum and flow cell activity of the sludge to perform multi-faceted detection of the sludge state in order to determine the operating state of the sludge in this process and then regulate the system. If the sedimentation performance deteriorates and the respiratory spectrum and flow cell activity indicate that flocs and large-size sludge appear in the system, they need to be screened out in time.

[0086] The present invention provides a basis for quick monitoring methods such as sedimentation, respiratory spectrum technology and flow cytometry for early warning of stability changes in aerobic granular sludge systems.

[0087] The present invention is not limited to the above-mentioned implementation cases. On the basis of the technical solution disclosed in the present invention, personnel in this industry field can make some substitutions and deformations to some of the technical feature methods according to the disclosed technical content without creative labor, and these substitutions and deformations are all within the protection scope of the present invention.

Claims

1. A method for identifying granular sludge stability based on the characteristics of microbial activity changes. It is characterized in that The following steps are involved: Step 1, taking the aerobic granular sludge mixed liquid at the end of aeration as a sample, controlling the sludge concentration of the sample at 3-4 g / L, so that the sample is in a completely mixed state; Step 2, performing microscopic examination on the sample in step 1 to obtain the morphological characteristics of the sample; When the sample is completely mixed, the sedimentation performance is measured, including SV and SVI data; the respiration spectrum is measured using the smart workstation to obtain the respiration rate of the granular sludge, which includes the autotrophic specific respiration rate SOUR A , heterotrophic bacterial specific respiration rate SOUR H , on-site specific respiration rate SOUR S , endogenous specific respiration rate SOUR e and maximum specific respiratory rate SOUR T ; Step 3: Calculate the sludge health index RI according to the respiration rate of the granular sludge obtained in step 2, RI = SOUR e / SOUR T ; Step 4, taking the mud-water mixture at the end of aeration as a sample, placing the sample in a 4±1°C environment and settling for at least 30 minutes, collecting the supernatant, and filtering the supernatant using a 5 μm nylon filter. After collecting the supernatant, the remaining sludge sample is resuspended to the original liquid level with a buffer solution to obtain a sludge mixed solution A; Step 5, subjecting the sludge mixed liquor A obtained in step 4 to ultrasonic crushing, with an ultrasonic frequency of 20kHz, an ultrasonic power of 4.5W to 7.5W, a power ratio of 3% to 5%, and an intermittent ultrasonic crushing process, ultrasonicating for 2 to 3s, stopping for 2 to 4s, and the ultrasonic process lasting 5 to 10min. The sample after ultrasonication is filtered using a 5μm nylon filter to obtain a sludge mixed liquor B; Step 6, use Green I (SGI) and propidium iodide (PI) are combined to prepare the dye. First, dimethyl sulfoxide (DMSO) is used as the solvent. Green I (SGI) standard stock solution was diluted to 100 times, and the diluted GreenI (SGI): propidium iodide (PI) = 50:1; Step 7, dilute the supernatant filtered in step 4 by 150 times with PBS to obtain a supernatant sample; dilute the sludge mixed solution B obtained in step 5 by 6×100 times with PBS to obtain a diluted sludge sample; fully mix the diluted supernatant sample and the sludge sample with the dye prepared in step 6 in a vortex oscillator, and dye in a constant temperature metal bath at 35° C. in the dark for 15 minutes to obtain a measurement sample; according to the volume ratio, the diluted sludge sample: dye = 100:1, the diluted supernatant sample: dye = 100:1; Step 8, measuring the microbial cell activity of the measurement sample obtained in step 7, wherein the microbial cell activity includes: total bacterial concentration, live-dead bacterial rate, HNA bacteria and LNA bacteria, and cell size distribution; Step 9, based on the sample morphological characteristics, sedimentation performance and respiration rate of the granular sludge obtained in step 2, the sludge health index RI obtained in step 3 and the microbial cell activity in the sample obtained in step 8, combined with the sample morphological characteristics and sedimentation performance, determine whether the state of the aerobic granular sludge system is stable in multiple dimensions.

2. A method for identifying granular sludge stability based on the characteristics of microbial activity changes according to claim 1, It is characterized in that The buffer solution is a phosphate buffer solution with a pH of 7.

2.

3. A method for identifying granular sludge stability based on the characteristics of microbial activity changes according to claim 1, It is characterized in that In step 9, the indicators for judging whether the state of the aerobic granular sludge system tends to be stable are: the health index RI of the granular sludge is 7%-10%, the mortality rate is stable at 20%-30%, the proportion of high nucleic acid bacteria (HNA) bacteria is stable at more than 85%, and the autoheterotrophic respiration rate does not fluctuate significantly.

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