Patent Application
20200140301
The present disclosure relates to a method for determining optimal preservation temperature of biofilm in wastewater treatment, and belongs to the technical field of environmental engineering.
The low content of organic pollutants in the inflow water of the wastewater treatment plant is always a technical difficulty for restricting the standard emission of total nitrogen, and simultaneously, along with the improvement of the emission standard of wastewater treatment, a large amount of soil is occupied by reconstruction and extension projects, a large amount of organic carbon source substances and phosphorus removal agents are added into a water body, so that the investment construction and operation cost are remarkably increased, the traditional activated sludge process cannot meet the emission requirement of pollutants, and thus the energy saving and consumption reducing effects of the wastewater treatment plant are seriously influenced. Therefore, wastewater denitrification technologies based on land-saving objectives and low carbon source utilization are receiving increasing attention. The effective wastewater treatment process in wastewater treatment comprises aerobic granular sludge, nitrifying-denitrifying biofilm process, wherein the aerobic granular sludge process has good settling performance, low operation cost, high biomass and treatment efficiency and thus has great development potential; the nitrifying-denitrifying biofilm process can realize synchronous nitrification and denitrification, and has important significance for removing ammonia nitrogen and total nitrogen in wastewater.
However, in the engineering example, the film forming period of the nitrifying-denitrifying biofilm is about 30-40 d, the culture period of the aerobic granular sludge is as long as 70-120 d, and the culture conditions are relatively harsh. If the aerobic granular sludge and the nitrifying-denitrifying biofilm can be cultured, matured and stored, the wastewater treatment plant with low carbon source in inflow water and short land resources can be effectively helped to start running in a short time, and pollutants can be discharged under the standard. The temperature is an important parameter influencing the activity of the biofilm, and the temperature which is most suitable for storing the biofilm such as aerobic granular sludge and nitrifying-denitrifying biofilm is determined, so that the activity recovery process is simplified, the starting time of engineering application is shortened, and the energy saving and consumption reducing effects are realized. However, in the existing method, the optimum preservation temperature needs to be determined by re-inoculating the aerobic granular sludge and the nitrifying-denitrifying biofilm into the bioreactor, and the time for determining the activity recovery effect of the aerobic granular sludge and the nitrifying-denitrifying biofilm is about 8-35 d, and thus the long time consumption becomes important for restricting the engineering application of the method.
In order to simplify the activity recovery process of the biofilm such as aerobic granular sludge and nitrifying-denitrifying biofilm, allow the pollutant indexes of the wastewater treatment plant to reach the standard for discharge in a short time and achieve the land saving, energy saving and consumption reducing effects at the same time, the present disclosure characterizes cell activity states in the biofilms stored under different temperature conditions based on the flow cytometry, verifies the characterization result of the flow cytometry according to the cell activity states after recovering the activity of the biofilm and the removal effect of pollutants, and finally establishes a method for determining the optimum preservation temperature of the biofilm based on the flow cytometry, and provides technical support for high-standard pollutant discharge and energy saving and consumption reducing operation of the wastewater treatment plant.
A first object of the present disclosure is to provide a method for determining an optimum preservation temperature of a wastewater treatment biofilm, which comprises measuring the cell activity state of the wastewater treatment biofilm based on flow cytometry, comparing the measured results of the cell activity states of the biofilm stored at different temperatures with those of the biofilm before preservation, and taking the preservation temperature closest to the cell activity state of the biofilm before preservation as the optimum preservation temperature.
In one embodiment of the present disclosure, the measurement of the cell activity state of the biofilm comprises the measurement of the content of living cells, early apoptotic cells, late apoptotic cells and dead cells.
In one embodiment of the present disclosure, the biofilm comprises aerobic granular sludge and a nitrifying-denitrifying biofilm.
In one embodiment of the present disclosure, the step of determining the optimum temperature in the flow cytometry comprises:
(1) preparing a biofilm test sample solution: diluting a biofilm sample with a buffer, shaking evenly, filtering, centrifuging, leaving the supernatant, purging the cells with a pre-cooled phosphate buffer, repeating centrifugation and wash twice, then taking the supernatant as a sample, and mixing well with an appropriate amount of 10× Annexin V Binding Buffer;
(2) placing in a flow cytometer for measuring the cell activity state of each sample solution.
In one embodiment of the present disclosure, the phosphate buffer is used as a buffer for dilution.
In one embodiment of the present disclosure, the phosphate buffer comprises sodium dihydrogen phosphate and disodium hydrogen phosphate.
In one embodiment of the present disclosure, when the biofilm is aerobic granular sludge, the preparation of the test sample solution is obtained by diluting aerobic granular sludge with a buffer of pH 7.0-8.0.
In one embodiment of the present disclosure, when the biofilm is a nitrifying-denitrifying biofilm, the preparation of the test sample solution is obtained by diluting the nitrifying-denitrifying biofilm with a buffer of pH 6.6-7.0.
In one embodiment of the present disclosure, the dilution volume ratio of the buffer to the biofilm is 8-10:1.
In one embodiment of the present disclosure, when the biofilm is aerobic granular sludge, a nylon membrane having a pore size of 5-15 μm is used for filtration.
In one embodiment of the present disclosure, when the biofilm is a nitrifying-denitrifying biofilm, a nylon membrane having a pore size of 6-8 μm is used for filtration.
In one embodiment of the present disclosure, the centrifugation speed is 5000-10000 rpm.
In one embodiment of the present disclosure, the mixed volume ratio of the sample supernatant to the 10× Annexin V Binding Buffer is 1:2-4.
In one embodiment of the present disclosure, the measurement of the cell activity state of each sample solution by the flow cytometer is carried out by adding 0.5 μl PI staining agent to the control FITC Annexin V group, adding 0.5 μl FITC Annexin V to the control PI group, adding 0.5 μl FITC Annexin V and 0.5 μl PI to the test group, mixing well, incubating in the dark at room temperature, and then testing on a flow cytometer.
In one embodiment of the present disclosure, the incubation time is 10-20 min.
A second object of the present disclosure is to provide a method for rapidly initiating the wastewater treatment biofilm engineering, which comprises preliminarily culturing and maturing the biofilm, placing in a preservation medium, storing at an optimum preservation temperature, and using for wastewater treatment after recovering the activity; the optimum temperature is determined by the above method.
In one embodiment of the present disclosure, the preservation medium of the aerobic granular sludge has a COD of 250 to 350 mg/L, NH4+—N of 55-65 mg/L, and PO43−—P of 6-10 mg/L.
In an embodiment of the present disclosure, the activity recovery of the aerobic granular sludge comprises inoculating the aerobic granular sludge into a sequencing batch reactor (SBR) with an effective volume of 10.0 L, a water drainage ratio of 45-60%, a reaction period of 2.5-5 h, a static water inflow period of 1-1.5 h, an aeration reaction period of 1.5-2.5 h, a sludge settling period of 2-6 min, and a rapid drainage period of 2-6 min; controlling the air and nitrogen content and proportion to ensure the anaerobic state of the water inflow section and the aerobic state of the reaction section by a real-time control system; and controlling SRT to be 25 days.
In one embodiment of the present disclosure, the preservation medium of the nitrifying-denitrifying biofilm has a COD of 180-220 mg/L, NH4+—N of 25-35 mg/L, NO3−—N of 18-25 mg/L and PO43−—P of 6-10 mg/L.
In one embodiment of the present disclosure, the activity recovery of the nitrifying-denitrifying biofilm comprises inoculating a nitrifying-denitrifying biofilm into a bioreactor, based on an anoxic-oxic process, setting HRT as 10-15 h, the nitrification and denitrification filling ratio as 40%-60%, and the nitrifying liquid reflux ratio as 70%-85%.
A third object of the present disclosure is to apply the above method to wastewater treatment.
The present disclosure characterizes the proportion of living cells, early apoptotic cells, late apoptotic cells and dead cells of various biofilms through flow cytometry, determines the optimum preservation temperature within a few hours, performs correlation analysis on the characteristic indexes of the biofilm activity recovery process, and establishes the method for determining the optimum preservation temperature of the biofilm based on the flow cytometry. By using the method, the step of recovering the biofilm activity can be omitted, the wastewater treatment plant which intends to adopt the biofilm process technology to discharge the pollutants (ammonia nitrogen, total nitrogen, total phosphorus) under the standard is effectively helped to realize the land saving, energy saving and consumption reducing operation, and simultaneously, the starting time of engineering application of the biofilm process can be effectively shortened, the long-term stable operation of the biofilm process is maintained, and the method has high industrial feasibility.
The wastewater of the wastewater treatment plant of the present disclosure includes domestic water in residential areas and a small part of industrial wastewater in the upstream, and the annual average of inflow water is COD of 236 mg/L, ammonia nitrogen of 30.1 mg/L, total nitrogen of 37.8 mg/L, and total phosphorus of 4.5 mg/L. The nitrate nitrogen content is less than 1.0 mg/L.
Preservation Conditions of Aerobic Granular Sludge:
The preservation temperature of the aerobic granular sludge was set to −20° C., 4° C. and 20° C. The 900 mL of the aerobic granular sludge and wastewater mixture in the aerobic granular sludge pilot plant was taken out, divided into three equal portions and placed in 1000 mL serum bottles containing 500 mL of preservation medium, respectively. The components of preservation medium were as follows: NaAc of 4200 mg/L, NH4Cl of 1100 mg/L, K2HPO4 of 370 mg/L, KH2PO4 of 140 mg/L, MgSO4 of 440 mg/L, KCl of 170 mg/L, and trace element solution of 1 ml/L; the components of trace element liquid composition were as follows: FeCl3.6H2O of 1.5 g/L, H3BO3 of 0.15 g/L, CuSO4.5H2O of 0.03 g/L, KI of 0.03 g/L, MnCl2.4H2O of 0.12 g/L, Na2MoO4.2H2O of 0.06 g/L, ZnSO4.7H2O of 0.12 g/L, and CoCl2.6H2O of 0.15 g/L. The preservation medium had a COD of 300 mg/L, NH4+—N of 60 mg/L, and PO43−—P of 8 mg/L. Serum bottles (3 parallel samples at each preservation temperature) were placed at −20° C., 4° C. and 20° C., and stored statically in the dark for 3 months.
Cell State Characterization of Stored Aerobic Granular Sludge:
Cell State Test Conditions by Flow Cytometry were as Follows:
(1) A mixture of aerobic granular sludge stored in a 10 mL pilot system at each temperature for 3 months was taken, respectively, diluted to 100 mL with phosphate buffer of pH 7.2, and shaken for 2 min in a vortexer to break the sludge into flocs and ensure a uniform distribution;
(2) The crushed sample was filtered through a nylon membrane having a pore size of 10 μm, and 1.5 mL was placed in a 1.5 mL sharp-bottomed centrifuge tube;
(3) The sample was centrifuged at 8000 rpm for 5 min;
(4) The supernatant of the sample after centrifugation was pipetted with leaving about 0.1 mL of sample, the cells were purged with pre-cooled phosphate buffer, and the centrifugation and wash were repeated twice;
(5) The supernatant of the sample after centrifugation was pipetted with leaving about 0.1 mL of sample, and mixed well with 0.3 mL of 10× Annexin V Binding Buffer;
(6) 0.5 μL of PI staining agent was added to the control FITC Annexin V group, 0.5 μL of FITC Annexin V was added to the control PI group, 0.5 μL of FITC Annexin V and 0.5 μL of PI were added to the test group, which were mixed well and incubated for 15 min at room temperature in the dark, and then tested on a flow cytometer.
The cell state results of aerobic granular sludge were shown in Table 1. The living cell proportion of aerobic granular sludge in the pilot system was higher, indicating that the pilot system works well. The aerobic granular sludge stored at −20° C. had the lowest living cell content and the highest dead cell content, indicating that it was not suitable to store aerobic granular sludge at −20° C. The aerobic granular sludge stored at 4° C. had a proportion of late apoptotic cells and dead cells of about 25.2%, indicating that the 4° C. condition can be used for the preservation of aerobic granular sludge. However, when the preservation temperature was 20° C., the living cell proportion of aerobic granular sludge was as high as 68.5%, which was only 20.0% lower than that of the aerobic granular sludge in the pilot system, indicating that the preservation condition at 20° C. was more suitable for storing aerobic granular sludge, at the same time, since it was necessary to consume more energy to control and maintain the low temperature condition of 4° C., it was preliminarily determined that 20° C. was the optimum temperature for storing aerobic granular sludge.
Activity Recovery Conditions of the Stored Aerobic Granular Sludge:
The aerobic granular sludge derived from different serum bottles was inoculated into a sequencing batch reactor (SBR) for the activity recovery of aerobic granular sludge; the aerobic granular sludge stored at −20° C., 4° C. and 20° C. was placed in R1, R2 and R3, respectively. The SBR had an effective volume of 10.0 L, a water drainage ratio of 50%, a reaction period of 3 h, a static water inflow period of 60 min, an aeration reaction period of 112 min, a sludge settling period of 3 min, and a rapid drainage period of 5 min. The air and nitrogen content and proportion were controlled by a real-time control system to ensure the anaerobic state of the water inflow section and the aerobic state of the reaction section; and SRT was controlled to be 25 days.
Characteristics of Aerobic Granular Sludge after Activity Recovery:
After the activity recovery, all of the aerobic granular sludge in R1, R2 and R3 had good performance. As shown in Table 2, after recovering the aerobic granular sludge activity, the density and particle size of aerobic granular sludge at different preservation temperatures were close to those of aerobic granular sludge before preservation. Although the biomass of aerobic granular sludge (MLSS) at different preservation temperatures was only 91% of the biomass before preservation, on average, the biomass was 37.9% higher than the biomass of aerobic granular sludge after preservation, indicating that aerobic granular sludge re-adapted to the environment and the biomass was stably increased. Generally, the average denitrification rate and phosphorus release rate of activated sludge in the wastewater treatment plant were 3.0 mg/g MLSS·h and 2.2 mg/g MLSS·h, respectively. The domesticated aerobic granular sludge in the pilot plant will respectively take 25 d and 29 d to reach the same denitrification rate and phosphorus release rate. After the activity of the stored aerobic granular sludge was recovered, the aerobic granular sludge in R1 will respectively take 12 d and 13 d to reach the same denitrification rate and phosphorus release rate, the aerobic granular sludge in R2 will respectively take 10 d and 11 d to reach the same denitrification rate and phosphorus release rate, and the aerobic granular sludge in R3 will respectively take 8 d and 7 d to reach the same denitrification rate and phosphorus release rate, indicating that the aerobic granular sludge after the activity recovery all had better nitrogen and phosphorus removal effects, wherein the aerobic granular sludge stored at the temperature of 20° C. has the shortest activity recovery time and the condition at 20° C. was more suitable for storing the aerobic granular sludge.
Settling Performance and Stability of Aerobic Granular Sludge after Activity Recovery:
After the activity recovery, the aerobic granular sludge in R1, R2 and R3 had good settling performance, as shown in
Removal Efficiency of Pollutants by Aerobic Granular Sludge after Activity Recovery:
After the activity recovery process, the removal rates of total nitrogen and total phosphorus by aerobic granular sludge at different preservation temperatures were gradually increased (
Correlation Between Aerobic Granular Sludge Characteristics and Sludge Cell States after Activity Recovery:
After 30 d of aerobic granular sludge activity recovery, flow cytometry was used to analyze the aerobic granular sludge cell states (as shown in Table 3). The living cell content in aerobic granular sludge at different preservation temperatures was basically the same as the content of living cells in the aerobic granular sludge of the pilot system, indicating that all of the aerobic granular sludge after the activity recovery can play the role of pollutant removal. Among them, the proportion of aerobic granular sludge living cells in R3 was the highest (86.5%±3.5%), and the proportion of late apoptotic cells (3.8%±1.0%) and the proportion of dead cells (3.3%±0.3%) were the lowest, indicating the aerobic granular sludge cells stored at 20° C. had the highest cell activity and 20° C. was more suitable as a condition for storing aerobic granular sludge.
According to Correl correlation analysis, it was found that the denitrification rate and the phosphorus release rate of aerobic granular sludge had a very high correlation with the proportion of aerobic granular sludge live cells (as shown in Table 4), and the correlation coefficients were 0.9940 and 0.9954, respectively, indicating that the use of the proportion of aerobic granular sludge living cells as a method for evaluating the activity of aerobic granular sludge was extremely feasible. At the same time, in the stored aerobic granular sludge, the proportion of aerobic granular sludge live cells was the highest under the preservation condition of 20° C., which was consistent with results for the proportion of aerobic granular sludge living cells in R3 after activity recovery.
Therefore, it was determined that 20° C. was the most suitable condition for storing aerobic granular sludge, and flow cytometry can be used as the basis for determining the optimum preservation temperature of aerobic granular sludge. Flow cytometry is easy to operate, the data are fast and easy to obtain, accurate and reliable, and the aerobic granular sludge activity recovery process can be omitted, which is of great significance for the preservation and activity recovery of aerobic granular sludge.
Preservation and Culture of Nitrifying-Denitrifying Biofilm:
The preservation temperature of the nitrifying-denitrifying biofilm was set to −20° C., 4° C. and 20° C. 180 nitrifying-denitrifying biofilms in the biochemical reaction tank of the wastewater treatment plant were taken out, divided into three equal portions and placed in 1000 mL serum bottles containing 500 mL of preservation medium to maintain the nitrification and denitrification capacity of the biofilm, respectively. The components of preservation medium were as follows: NaAc of 240 mg/L, NH4Cl of 110 mg/L, KNO3 of 80 mg/L, K2HPO4 of 30 mg/L, KH2PO4 of 15 mg/L, MgSO4 of 40 mg/L, and KCl of 70 mg/L. The preservation medium had a COD of 200 mg/L, NH4+—N of 30 mg/L, NO3−—N of 20 mg/L, and PO43−—P of 8 mg/L. Serum bottles (3 parallel samples at each preservation temperature) were placed at −20° C., 4° C. and 20° C., and stored statically in the dark.
Cell State Test of Stored Nitrifying-Denitrifying Biofilm:
The nitrifying-denitrifying biofilms stored at −20° C., 4° C. and 20° C. for more than 120 d were used to determine the cell state of nitrifying-denitrifying biofilms. The cell state test conditions of flow cytometry were as follows:
(1) 10 mL nitrifying-denitrifying biofilm was taken, diluted to 100 mL with phosphate buffer of pH 7.0, and shaken for 2 min in a vortexer to break the biofilm into flocs and ensure a uniform distribution;
(2) The crushed sample was filtered through a nylon membrane having a pore size of 6 μm, and 1.5 mL was placed in a 1.5 mL sharp-bottomed centrifuge tube;
(3) The sample was centrifuged at 8000 rpm for 5 min;
(4) The supernatant of the sample after centrifugation was pipetted with leaving about 0.1 mL of sample, the cells were purged with pre-cooled phosphate buffer, and the centrifugation and wash were repeated twice;
(5) The supernatant of the sample after centrifugation was pipetted with leaving about 0.1 mL of sample, and mixed well with 0.3 mL of 10× Annexin V Binding Buffer;
(6) 0.5 μl of PI staining agent was added to the control FITC Annexin V group, 0.5 μL of FITC Annexin V was added to the control PI group, 0.5 μL of FITC Annexin V and 0.5 μL of PI were added to the test group, which were mixed well and incubated for 15 min at room temperature in the dark, and then tested on a flow cytometer.
The selection of the filtration pore size in the preparation of the sample was particularly important. If the pore size was too large, more biological flocs will be introduced, uneven dyeing will be produced, which will affect the final result; if the pore size was too small, the biological flocs cannot be effectively obtained.
The cell state results of nitrifying-denitrifying biofilm were shown in Table 5. The living cells proportion of nitrifying-denitrifying biofilm in the biochemical reaction tank of the wastewater treatment plant was higher, indicating that the wastewater treatment plant works well. The nitrifying-denitrifying biofilm stored at −20° C. had the lowest living cell content and the highest dead cell content, indicating that it was not suitable to store nitrifying-denitrifying biofilm at −20° C. The nitrifying-denitrifying biofilm stored at 4° C. had the highest living cell proportion of 68.0% and a proportion of late apoptotic cells and dead cells of about 19.8%, indicating that the 4° C. condition can be used for the preservation of nitrifying-denitrifying biofilm. When the preservation temperature was 20° C., the living cell proportion of nitrifying-denitrifying biofilm was as high as 59.4%, which was only 12.6% lower than that of the nitrifying-denitrifying biofilm stored at 4° C., but the proportion of late apoptotic cells and dead cells for such nitrifying-denitrifying biofilm was about 31.6%, indicating that the preservation condition at 20° C. was not suitable for storing nitrifying-denitrifying biofilm, either. Therefore, it was preliminarily determined that 4° C. was the optimum temperature for storing nitrifying-denitrifying biofilm.
Activity Recovery Conditions of the Stored Nitrifying-Denitrifying Biofilm:
The operation mode of sequencing batch reactor was used: nitrifying-denitrifying biofilm derived from different serum bottles was inoculated into a bioreactor (effective volume 10.0 L) for the activity recovery of nitrifying-denitrifying biofilm. The nitrifying-denitrifying biofilm stored at −20° C., 4° C. and 20° C. were placed in R1, R2 and R3, respectively. Based on the anoxic-oxic (AO) process, the bioreactor achieved simultaneous nitrification and denitrification in the sequencing batch reaction. The HRT was set to 12 h, the nitrification and denitrification filling ratio was 50%, and the nitrification liquid reflux ratio was 80%.
Characteristics of Nitrifying-Denitrifying Biofilm after Activity Recovery:
After the activity recovery, all of the nitrifying-denitrifying biofilm in R1, R2 and R3 had good performance. As shown in Table 6, after recovering the nitrifying-denitrifying biofilm activity, the density and thickness of nitrifying-denitrifying biofilm stored at 4° C. and 20° C. were close to those of nitrifying-denitrifying biofilm before preservation, and only the density and thickness of nitrifying-denitrifying biofilm stored at −20° C. slightly decreased. The biomass of nitrifying-denitrifying biofilm at different preservation temperatures all decreased, but after activity recovery, the biomass of nitrifying-denitrifying biofilm stored at 4° C. and 20° C. has achieved the biomass level of nitrifying-denitrifying biofilm before preservation, indicating that nitrifying-denitrifying biofilm re-adapted to the environment and the biomass was stably increased. Generally, the average nitrification rate and denitrification rate of biofilm in the wastewater treatment were 4.5 g NO3−—N/m2·d and 5.0 g NO3−-N/m2·d, respectively. The domesticated nitrifying-denitrifying biofilm in the wastewater treatment plant will respectively take 25 d and 21 d to reach the same nitrification rate and denitrification rate. After the activity of the stored nitrifying-denitrifying biofilm was recovered, the nitrifying-denitrifying biofilm in R1 will respectively take 19 d and 17 d to reach the same nitrification rate and denitrification rate, the nitrifying-denitrifying biofilm in R2 will respectively take 8 d and 6 d to reach the same nitrification rate and denitrification rate, and the nitrifying-denitrifying biofilm in R3 will respectively take 13 d and 10 d to reach the same nitrification rate and denitrification rate. The biofilm thickness L of R1 was significantly reduced before and after the activity recovery, but a higher thickness can be maintained in both R2 and R3, so that a concentration gradient of oxygen was produced in the biofilm, which was beneficial to denitrification. It is indicated that all of the nitrifying-denitrifying biofilms after activity recovery had good denitrification effect. The nitrifying-denitrifying biofilm stored at 4° C. had the shortest activity recovery time and the condition at 4° C. was suitable for storing nitrifying-denitrifying biofilm.
Stability of Nitrifying-Denitrifying Biofilm after Activity Recovery:
Extracellular polymer was an important factor in the formation of nitrifying-denitrifying biofilm, and the ratio (PN/PS) of protein (PN) substance to polysaccharide (PS) substance in extracellular polymer was an important index for measuring the structural stability of the nitrifying-denitrifying biofilm. The changes in the extracellular polymer PN/PS during activity recovery process of the nitrifying-denitrifying biofilm were shown in
Removal Efficiency of Pollutants by Nitrifying-Denitrifying Biofilm after Activity Recovery:
After the activity recovery process, the removal rates of ammonia nitrogen and total nitrogen by nitrifying-denitrifying biofilm at different preservation temperatures were gradually increased (
Correlation Between Nitrifying-Denitrifying Biofilm Characteristics and Sludge Cell States after Activity Recovery:
After the nitrifying-denitrifying biofilm activity recovery, flow cytometry was used to analyze the nitrifying-denitrifying biofilm cell states as shown in Table 7. The living cell content in nitrifying-denitrifying biofilm at different preservation temperatures was basically the same as the content of living cells in the nitrifying-denitrifying biofilm of the wastewater treatment plant, indicating that all of the nitrifying-denitrifying biofilm after the activity recovery can play the role of pollutant removal. Among them, the proportion of nitrifying-denitrifying biofilm living cells in R2 was the highest (84.3%±3.0%), and the proportion of late apoptotic cells (6.2%±1.5%) and the proportion of dead cells (4.3%±0.3%) were the lowest, indicating the nitrifying-denitrifying biofilm cells stored at 4° C. had the highest cell activity and 4° C. was more suitable as a condition for storing nitrifying-denitrifying biofilm.
According to Correl correlation analysis, as shown in table 8, the nitrification rate and the denitrification rate of nitrifying-denitrifying biofilm had a very high correlation with the proportion of nitrifying-denitrifying biofilm live cells, and the correlation coefficients were 0.9286 and 0.9819, respectively, indicating that the use of the proportion of nitrifying-denitrifying biofilm living cells as a method for evaluating the activity of nitrifying-denitrifying biofilm was extremely feasible. At the same time, in the stored nitrifying-denitrifying biofilm, the proportion of nitrifying-denitrifying biofilm live cells was the highest under the preservation condition of 4° C., which was consistent with results for the proportion of nitrifying-denitrifying biofilm living cells in R2 after activity recovery.
Therefore, it was determined that 4° C. was the most suitable condition for storing nitrifying-denitrifying biofilm, and flow cytometry can be used as the basis for determining the optimum preservation temperature of nitrifying-denitrifying biofilm. Flow cytometry is easy to operate, the data are fast and easy to obtain, accurate and reliable, and the nitrifying-denitrifying biofilm activity recovery process can be omitted, which is of great significance for the preservation and activity recovery of nitrifying-denitrifying biofilm.
Preservation and Culture of Nitrifying-Denitrifying Biofilm:
The preservation temperature of the nitrifying-denitrifying biofilm was set to −20° C., 4° C. and 20° C. 180 nitrifying-denitrifying biofilms in the biochemical reaction tank of the wastewater treatment plant were taken out, divided into three equal portions and placed in 1000 mL serum bottles containing 500 mL of preservation medium, respectively. The components of preservation medium were as follows: NaAc of 240 mg/L, NH4Cl of 110 mg/L, KNO3 of 80 mg/L, K2HPO4 of 30 mg/L, KH2PO4 of 15 mg/L, MgSO4 of 40 mg/L, and KCl of 70 mg/L. The preservation medium had a COD of 200 mg/L, NH4+—N of 30 mg/L, NO3—N of 20 mg/L, and PO43−—P of 8 mg/L. Serum bottles (3 parallel samples at each preservation temperature) were placed at −20° C., 4° C. and 20° C., and stored statically in the dark.
Cell State Test of Stored Nitrifying-Denitrifying Biofilm:
The nitrifying-denitrifying biofilms stored at −20° C., 4° C. and 20° C. for more than 120 d were used to determine the cell state of nitrifying-denitrifying biofilms. The cell state test conditions of flow cytometry were as follows:
(1) 10 mL nitrifying-denitrifying biofilm was taken, diluted to 100 mL with phosphate buffer of pH 7.2, and shaken for 2 min in a vortexer to break the biofilm into flocs and ensure a uniform distribution;
(2) The crushed sample was filtered through a nylon membrane having a pore size of 6 μm, and 1.5 mL was placed in a 1.5 mL sharp-bottomed centrifuge tube;
(3) The sample was centrifuged at 8000 rpm for 5 min;
(4) The supernatant of the sample after centrifugation was pipetted with leaving about 0.1 mL of sample, the cells were purged with pre-cooled phosphate buffer, and the centrifugation and wash were repeated twice;
(5) The supernatant of the sample after centrifugation was pipetted with leaving about 0.1 mL of sample, and mixed well with 0.3 mL of 10× Annexin V Binding Buffer;
(6) 0.5 μL of PI staining agent was added to the control FITC Annexin V group, 0.5 μL of FITC Annexin V was added to the control PI group, 0.5 μL of FITC Annexin V and 0.5 μL of PI were added to the test group, which were mixed well and incubated for 15 min at room temperature in the dark, and then tested on a flow cytometer.
The test results for cell state of nitrifying-denitrifying biofilm were shown in table 9.
From the results of Table 9, it was found that the samples prepared by using the phosphate buffer of pH 7.2 were selected to be tested, the proportion of the cell state in the living condition at each preservation temperature was relatively close, the beneficial results could not be obtained for analysis, and the data reliability was poor.
In addition, the inventors also investigated the effect of filter pore size on the sample test: the nitrifying-denitrifying biofilm samples were respectively prepared with pore sizes of 8 μm and 10 μm, and it was found that the analysis results of the sample prepared with pore size of 8 μm were consistent with the verification experiment, and the data was reliable; the corresponding data with 10 μm did not have analytical capacity and cannot be used to determine the optimum preservation temperature.
| Number | Date | Country | Kind |
|---|---|---|---|
| 2018109254213 | Aug 2018 | CN | national |
| 2018110613927 | Sep 2018 | CN | national |
| Number | Date | Country | |
|---|---|---|---|
| Parent | PCT/CN2018/114552 | Nov 2018 | US |
| Child | 16720243 | US |
