Method for determining optimal preservation temperature of aerobic denitrifiers in wastewater treatment for total nitrogen removal

Abstract

The present disclosure relates to a method for determining optimal preservation temperature of aerobic denitrifiers in wastewater treatment for total nitrogen removal, and belongs to the technical field of environmental engineering. The method of the present disclosure comprises measuring the cell activity state of the aerobic denitrifier stored at different temperatures based on flow cytometry, and taking the preservation temperature closest to the cell activity state of the aerobic denitrifier during the pilot operation as the optimum preservation temperature, and the data obtained from the test uses the cell activity state and performance effects after activity recovery to verify reliability. By using the method of the present disclosure, the step of recovering the aerobic denitrifier activity can be omitted, and the wastewater treatment plant, which intends to adopt the aerobic denitrifier process technology to achieve efficient removal of nitrate and total nitrogen, is effectively helped to realize the energy saving, consumption reducing operation, and the removal rate of nitrate and total nitrogen can reach 90% and 88% respectively. At the same time, the starting time of engineering application of the aerobic denitrifier process can be effectively shortened, the long-term stable operation of the aerobic denitrifier process is maintained, and the method has high industrial feasibility.

Claims

1. A method for determining an optimum preservation temperature of an aerobic denitrifier, comprising: measuring a cell activity state of the aerobic denitrifier stored at different temperatures based on flow cytometry, and taking a preservation temperature closest to the cell activity state of the aerobic denitrifier during a pilot operation as the optimum preservation temperature, wherein the measuring the cell activity state comprises measuring contents of living cells, early apoptotic cells, late apoptotic cells and dead cells.

2. The method according to claim 1, wherein the measuring the cell activity state of the wastewater treatment biofilm based on the flow cytometry comprises: (1) preparing a test sample solution of the aerobic denitrifier: diluting an aerobic denitrifier sample with a buffer, mixing evenly, filtering, centrifuging, leaving a 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; and (2) placing in a flow cytometer for measuring a cell activity state of each sample solution.

3. The method according to claim 2, wherein a pH value of the buffer is 7.3 -7.6.

4. The method according to claim 2, wherein the buffer includes a phosphate buffer.

5. The method according to claim 3, wherein the buffer comprises (8-28)% v/v sodium dihydrogen phosphate and (72-92)% v/v disodium hydrogen phosphate.

6. The method according to claim 3, wherein a dilution volume ratio of the buffer to the aerobic denitrifier is 4-10:1.

7. The method according to claim 3, wherein a nylon membrane having a pore size of 6-15 m is used for filtration.

8. The method according to claim 4, wherein a nylon membrane having a pore size of 6-15 m is used for filtration.

9. A method for rapidly initiating aerobic denitrifier engineering, comprising: using the method according to claim 1 to determine an optimum preservation temperature; and placing a mature aerobic denitrifier in a preservation medium for storage at the optimum preservation temperature, and using for the aerobic denitrifier engineering after recovering activity.

10. The method according to claim 9, wherein the preservation medium has 100 to 150 mg/L of COD, 27.5-38.5 mg/L of NH.sub.4.sup.+N, and 4.5-8.0 mg/L of NO.sub.3.sup.-N.

Description

BRIEF DESCRIPTION OF FIGURES

[0023] FIG. 1 shows the removal rate of nitrate nitrogen in aerobic denitrifiers after activity recovery.

[0024] FIG. 2 shows the removal rate of total nitrogen in aerobic denitrifiers after activity recovery.

DETAILED DESCRIPTION

Example 1: Determination of the Optimum Preservation Temperature of Aerobic Denitrifiers

Preservation Conditions of Aerobic Denitrifiers

[0025] The preservation temperature of the aerobic denitrifier was set to 20 C., 4 C. and 20 C. About 180 of the polyurethane filler enriched with the aerobic denitrifiers were taken out, divided into three equal portions and placed in 1000 mL serum bottles containing 600 mL of preservation medium, respectively. The preservation medium was the effluent from the anoxic tank of the sewage treatment plant, and had a COD of 100-150 mg/L, NH.sub.4.sup.+N of 27.5-38.5 mg/L, NO.sub.3.sup.-N of 4.5-8.0 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 Characterization of Aerobic Denitrifiers

[0026] Aerobic denitrifiers stored at 20 C., 4 C. and 20 C. were stored for more than 60 days, and then used to determine the aerobic denitrifier cell status. Cell state test conditions by flow cytometry were as follows:

[0027] (1) Crushed sample preparation 180 of the polyurethane filler enriched with the aerobic denitrifier (volume was about 200-300 ml) was taken, diluted to 1 L with phosphate buffer of pH 7.5, and used an ultrasonic breaker (ultrasonic energy density 90 kJ/g) to release the aerobic denitrifier in the polyurethane suspension filler into the phosphate buffer and ensure its uniform distribution;

[0028] (2) The crushed sample was filtered through a nylon membrane having a pore size of 10 m, and then centrifuged at 8000 rpm for 5 min;

[0029] (3) Placed the sediment in a 50 ml centrifuge tube, and centrifuged at 8000 rpm for 5 min;

[0030] (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 (pH was 7.8), and the centrifugation and wash were repeated twice;

[0031] (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;

[0032] (6) 0.5 L of PI staining agent was added to the control FITC Annexin V group, 0.5 L of FITC Annexin V was added to the control PI group, 0.5 L of FITC Annexin V and 0.5 L 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.

[0033] The cell state results were shown in Table 1. The living cell proportion of aerobic denitrifiers in the pilot system was higher, indicating that the aerobic denitrifier pilot system works well. The aerobic denitrifier stored at 20 C. had the lowest living cell content, indicating that it was not suitable to store aerobic denitrifiers at 20 C. The aerobic denitrifier stored at 4 C. had a proportion of living cell of about 57.7%, late apoptotic cells and dead cells of about 29.0%. The higher ratio of late apoptotic cells to dead cells indicated that 4 C. is not suitable for the preservation of aerobic denitrifiers. However, when the preservation temperature was 20 C., the living cell proportion of aerobic denitrifiers was as high as 66.0%, which was only 25.4% lower than that of the aerobic denitrifier in the pilot system, and the dead cells proportion was about 14.5%, indicating that 20 C. is the storage temperature with the lowest proportion of dead cells. Therefore, it was preliminarily determined that 20 C. was the optimum temperature for storing aerobic denitrifiers.

TABLE-US-00001 TABLE 1 Cell activity states (%) of aerobic denitrifiers after storage Early Late aerobic Living apoptotic apoptotic denitrifiers cells cells cells Dead cells Pilot system 88.5 4.9 4.7 0.1 3.3 0.1 3.5 0.1 Stored at 20 C. 45.5 2.2 12.5 0.8 9.9 0.5 32.1 1.7 Stored at 4 C. 57.7 3.0 13.3 0.8 12.5 0.7 16.5 0.5 Stored at 20 C. 66.0 3.5 12.5 0.7 7.0 0.3 14.5 0.4

Example 2: Verification of the Tested Optimum Temperature Results of Aerobic Denitrifiers

Activity Recovery Conditions of the Stored Aerobic Denitrifiers

[0034] The polyurethane filler enriched with the aerobic denitrifier derived from different serum bottles was inoculated into a bioreactor for the activity recovery of aerobic denitrifiers; the aerobic denitrifier stored at 20 C., 4 C. and 20 C. was placed in R1 (bioreactor 1), R2 (bioreactor 2) and R3 (bioreactor 3), respectively. The bioreactor had an effective volume of 10.0 L, and the DO of the bioreactor was controlled to 4-5 mg/L, the HRT was controlled to 4-8 h, and the filling ratio of the polyurethane suspension filler was 35%.

Characteristics of Aerobic Denitrifiers After Activity Recovery

[0035] After 30 days of activity recovery, the characteristics of aerobic denitrifiers in R1, R2 and R3 were shown in Table 2. As shown in Table 2, after recovering the aerobic denitrifier activity, the nitrate removal rate and total nitrogen removal rate in R1 and R2 are lower than the nitrate removal rate and total nitrogen removal rate of the aerobic denitrifier before storage, only the nitrate removal rate and total nitrogen removal rate of the aerobic denitrifier stored at 20 C. were more consistent with those before storage. The denitrification rate and simultaneous nitrification and denitrification rate of aerobic denitrifiers stored at different storage temperatures were lower than before storage. After activity recovery, the denitrification rate and simultaneous nitrification and denitrification rate of aerobic denitrifiers in R2 and R3 were closer to those before storage, but the denitrification rate and simultaneous nitrification and denitrification rate of aerobic denitrifiers in R1 were relatively low.

[0036] Generally, the denitrification rate and simultaneous nitrification and denitrification rate of aerobic denitrifiers were 6.0 mg/(m2.Math.h) and 1.5 mg/(m2.Math.h), respectively, and the domesticated aerobic denitrifiers in the pilot plant will respectively take 21 d and 30 d to reach the same denitrification rate and simultaneous nitrification and denitrification rates.

[0037] After the activity of the stored aerobic denitrifier was recovered, the aerobic denitrifier in R1 will respectively take 18 d and 25 d to reach the same denitrification rate and simultaneous nitrification and denitrification rates, the aerobic denitrifier in R2 will respectively take 15 d and 20 d to reach the same denitrification rate and simultaneous nitrification and denitrification rates, and the aerobic denitrifier in R3 will respectively take 11 d and 18 d to reach the same denitrification rate and simultaneous nitrification and denitrification rates, indicating that the aerobic denitrifier after the activity recovery all had better nitrogen removal effects, wherein the aerobic denitrifier 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 denitrifier.

TABLE-US-00002 TABLE 2 Properties of aerobic denitrifiers after preservation and activity recovery Time (d) Time (d) required required for for simultaneous nitrate total denitrifi- nitrification and nitrogen nitrogen cation denitrification removal removal rate to be rate to be more rate rate more than 6.0 than 1.5 (mg/h) (mg/h) mg/(m.sup.2 .Math. h) mg/(m.sup.2 .Math. h) Before 4.5 5.8 21 30 preservation After aerobic denitrifiers preservation After 2.0 2.7 preservation at 20 C. After 2.1 2.9 preservation at 4 C. After 2.5 3.1 preservation at 20 C. After activity recovery of the aerobic denitrifier Aerobic 3.5 4.5 18 25 denitrifiers stored at 20 C. Aerobic 4.1 5.1 15 20 denitrifiers stored at 4 C. Aerobic 4.3 5.7 11 18 denitrifiers stored at 20 C.

Removal Efficiency of Pollutants by the Aerobic Denitrifier After Activity Recovery

[0038] After the activity recovery process, the removal rates of nitrate and total nitrogen by aerobic denitrifiers at different preservation temperatures were gradually increased (FIG. 1 and FIG. 2), and the removal rates of nitrate and total nitrogen were over 90% and 88%, respectively. On the 18.sup.th day after the activity recovery, the aerobic denitrifier in R3 had the best removal effect on nitrate and total nitrogen, and the nitrate and total nitrogen removal rates showed a steady increase trend. This result also corresponded to the fastest recovery of the higher denitrification rate and simultaneous nitrification and denitrification rate by the aerobic denitrifier in R3 in Table 2, indicating that the condition at 20 C. was more suitable for storing aerobic denitrifiers and was highly feasible in practical applications.

Correlation Between Aerobic Denitrifiers Characteristics and Cell States After Activity Recovery

[0039] After 30 d of aerobic denitrifiers activity recovery, flow cytometry was used to analyze the aerobic denitrifier cell states (as shown in Table 3). The living cell content in aerobic denitrifiers at different preservation temperatures was basically the same as the content of living cells in the aerobic denitrifier of the pilot system, indicating that all of the aerobic denitrifier after the activity recovery can play the role of nitrate and total nitrogen removal. Among them, the proportion of aerobic denitrifiers living cells in R3 was the highest (80.3%4.2%), and the proportion of late apoptotic cells (7.5%0.3%) and the proportion of dead cells (6.7%0.1%) were the lowest, indicating the aerobic denitrifier cells stored at 20 C. had the highest cell activity and 20 C. was more suitable as a condition for storing aerobic denitrifiers.

TABLE-US-00003 TABLE 3 Cell activity states (%) of aerobic denitrifier cells after activity recovery (30 d) Early Late Aerobic Living apoptotic apoptotic denitrifier cells cells cells Dead cells Pilot system 87.0 4.5 4.5 0.1 3.6 0.1 4.9 0.2 Stored at 20 C. 78.0 4.3 6.0 0.1 7.1 0.1 8.9 0.3 Stored at 4 C. 78.5 4.3 6.0 0.1 7.5 0.3 9.0 0.3 Stored at 20 C. 80.3 4.2 5.5 0.1 7.5 0.3 6.7 0.1

[0040] According to Correl correlation analysis, it was found that the denitrification rate and simultaneous nitrification and denitrification rate of aerobic denitrifier had a very high correlation with the proportion of aerobic denitrifier live cells (as shown in Table 4), and the correlation coefficients were 0.9088 and 0.9507, respectively, indicating that the use of the proportion of aerobic denitrifier living cells as a method for evaluating the activity of aerobic denitrifier was extremely feasible. At the same time, in the stored aerobic denitrifier, the proportion of aerobic denitrifier live cells was the highest under the preservation condition of 20 C., which was consistent with results for the proportion of aerobic denitrifier living cells in R3 after activity recovery.

TABLE-US-00004 TABLE 4 Correlation between aerobic denitrifier characteristics and cell activity sates after activity recovery (30 d) Aerobic Aerobic Aerobic denitrifier denitrifier denitrifier stored stored stored at 20 C. at 4 C. at 20 C. Denitrification 5.5 5.8 6.0 rate mg/(m.sup.2 .Math. h) Simultaneous 1.0 1.2 1.2 nitrification and denitrification rate mg/(m.sup.2 .Math. h) Living cell 78.0 4.3 78.5 4.3 80.3 4.2 proportion (%) Correlation 0.9088 between denitrification rate and living cells Correlation between 0.9507 simultaneous nitrification and denitrification rate with living cells

[0041] Therefore, it was determined that 20 C. was the most suitable condition for storing aerobic denitrifier, 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 denitrifier activity recovery process can be omitted, which is of great significance for the preservation and activity recovery of aerobic denitrifier.

Comparative Example 1

Preservation and Culture of Nitrifying Denitrifying Biofilm

Preservation Conditions of Aerobic Denitrifiers

[0042] The preservation temperature of the aerobic denitrifier was set to 20 C., 4 C. and 20 C. About 180 of the polyurethane filler enriched with the aerobic denitrifier were taken out, divided into three equal portions and placed in 1000 mL serum bottles containing 600 mL of preservation medium, respectively. The preservation medium was the effluent from the anoxic tank of the sewage treatment plant, and had a COD of 100-150 mg/L, NH.sub.4.sup.+N of 27.5-38.5 mg/L, NO.sub.3.sup.-N of 4.5-8.0 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 Characterization of Aerobic Denitrifiers

[0043] Aerobic denitrifiers stored at 20 C., 4 C. and 20 C. were stored for more than 60 days, and then used to determine the aerobic denitrifier cell status. Cell state test conditions by flow cytometry were as follows:

[0044] (1) Crushed sample preparation 180 of the polyurethane filler enriched with the aerobic denitrifier (volume was about 200-300 ml) was taken, diluted to 1 L with phosphate buffer of pH 7.2 and 7.8, respectively, and used an ultrasonic breaker (ultrasonic energy density 90 kJ/g) to release the aerobic denitrifier in the polyurethane suspension filler into the phosphate buffer and ensure its uniform distribution;

[0045] (2) The crushed sample was filtered through a nylon membrane having a pore size of 10 m, and then centrifuged at 8000 rpm for 5 min;

[0046] (3) Placed the sediment in a 50 ml centrifuge tube, and centrifuged at 8000 rpm for 5 min;

[0047] (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 (pH was 7.8), and the centrifugation and wash were repeated twice;

[0048] (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;

[0049] (6) 0.5 L of PI staining agent was added to the control FITC Annexin V group, 0.5 L of FITC Annexin V was added to the control PI group, 0.5 L of FITC Annexin V and 0.5 L 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.

[0050] The cell state results were shown in Table 5 and Table 6.

TABLE-US-00005 TABLE 5 Cell activity states (%) of aerobic denitrifiers after storage (phosphate buffer of pH 7.2) Early Late aerobic Living apoptotic apoptotic denitrifiers cells cells cells Dead cells Pilot system 88.5 4.9 4.7 0.1 3.3 0.1 3.5 0.1 Stored at 20 C. 52.5 3.1 6.3 0.5 11.7 0.8 29.5 1.4 Stored at 4 C. 57.0 3.0 3.2 0.2 13.8 0.8 26.0 1.3 Stored at 20 C. 59.7 3.1 3.1 0.2 17.9 1.0 19.3 1.0

[0051] As shown in Table 5, aerobic denitrifiers stored at 20 C. had the highest proportion of live cells, but aerobic denitrifiers at three temperatures had relatively close viable cell content, and the total content of late apoptotic and dead cells was also relatively close, so when the value of pH is 7.2, the significance of flow cytometric analysis is poor, and it is not suitable as a suitable pH for determining the optimal storage temperature of aerobic denitrifiers.

TABLE-US-00006 TABLE 6 Cell activity states (%) of aerobic denitrifiers after storage (phosphate buffer of pH 7.8) Early Late aerobic Living apoptotic apoptotic denitrifiers cells cells cells Dead cells Pilot system 88.5 4.9 4.7 0.1 3.3 0.1 3.5 0.1 Stored at 20 C. 49.2 2.7 0.5 0.1 21.5 1.7 27.6 1.9 Stored at 4 C. 47.9 2.5 0.2 0.1 22.9 1.8 29.0 2.3 Stored at 20 C. 48.9 2.5 0.3 0.1 21.8 1.9 29.0 2.1

[0052] As shown in Table 6, the aerobic denitrifiers stored at three temperatures not only had a relatively close content of live cells, but also had a similar content of late apoptotic cells and dead cells. At the same time, the results based on the detection of early apoptosis cells were low, indicating that when the value of pH is 7.2, the results of flow cytometric analysis could not effectively determine the optimal storage temperature for aerobic denitrifiers.