Method for determining optimum preservation temperature of sulfur autotrophic denitrifying bacteria biofilm
11473119 · 2022-10-18
Assignee
Inventors
Cpc classification
International classification
Abstract
The present disclosure discloses to a method for determining optimum preservation temperature of a sulfur autotrophic denitrifying bacteria biofilm, and belongs to the technical field of environment engineering. The method of the present disclosure comprises: determining the cell activity state of a sulfur autotrophic denitrifying bacteria biofilm preserved at different temperatures by flow cytometry, and determining the preservation temperature of the cell activity state closest to the cell activity state of the sulfur autotrophic denitrifying bacteria in pilot operation as the optimum preservation temperature. The cell activity state and performance effect are verified to be reliable after activity recovery by the test data. The method of the present disclosure can simplify the microbial activity recovery process of the sulfur autotrophic denitrifying bacteria biofilm, quickly start the sulfur autotrophic denitrifying bacteria biofilm sewage treatment, enable the removal rate of nitrate nitrogen and total nitrogen in a sewage treatment plant to reach 96% and 88% or above respectively, achieve the effects of energy saving and consumption reduction at the same time, and have very high industrial feasibility.
Claims
1. A method for determining an optimum preservation temperature of a sulfur autotrophic denitrifying bacteria biofilm, comprising: determining a cell activity state of the sulfur autotrophic denitrifying bacteria biofilm preserved at different temperatures by flow cytometry; and determining a preservation temperature of the cell activity state closest to the cell activity state of the sulfur autotrophic denitrifying bacteria in a pilot operation as the optimum preservation temperature, wherein the determining the cell activity state of the sulfur autotrophic denitrifying bacteria biofilm comprises determining contents of living cells, early apoptotic cells, late apoptotic cells and dead cells.
2. The method according to claim 1, wherein the determining the cell activity state of the sulfur autotrophic denitrifying bacteria biofilm by flow cytometry comprises the following steps: (1) preparing a test sample solution of the sulfur autotrophic denitrifying bacteria biofilm: diluting a sample of the sulfur autotrophic denitrifying bacteria biofilm with a first buffer; uniformly mixing and then passing through a hydrotalcite-containing filter device; centrifuging the filtered sample, and retaining a supernatant; then washing the cells with a pre-cooled second buffer; centrifuging and washing twice; and taking the supernatant as a sample and uniformly mixing with an appropriate amount of 10× Annexin V Binding Buffer; and (2) determining the cell activity state of each sample solution by flow cytometry.
3. The method according to claim 2, wherein a pH value of the first buffer is 6.2-6.8.
4. The method according to claim 2, wherein a dilution volume ratio of the first buffer to the sulfur autotrophic denitrifying bacteria biofilm is (8-10):1.
5. The method according to claim 2, wherein the filter device further comprises a nylon membrane with a pore size of 15-25 μm.
6. The method according to claim 2, wherein the first buffer comprises a phosphate buffer.
7. The method according to claim 3, wherein the first buffer comprises a phosphate buffer.
8. The method according to claim 5, wherein the first buffer comprises a phosphate buffer.
9. The method according to claim 2, wherein the first buffer comprises 39% v/v sodium dihydrogen phosphate and 61.0% v/v disodium hydrogen phosphate.
10. The method according to claim 3, wherein the first buffer comprises 39% v/v sodium dihydrogen phosphate and 61.0% v/v disodium hydrogen phosphate.
11. The method according to claim 5, wherein the first buffer comprises 39% v/v sodium dihydrogen phosphate and 61.0% v/v disodium hydrogen phosphate.
12. A method for rapidly starting a sulfur autotrophic denitrifying bacteria biofilm project, comprising: determining an optimum preservation temperature by the method of claim 2; placing a cultured mature sulfur autotrophic denitrifying bacteria biofilm in a preservation medium for preservation at the optimum preservation temperature; and after the activity is recovered, starting the sulfur autotrophic denitrifying bacteria biofilm project.
13. The method according to claim 12, wherein a COD concentration of the preservation medium is 40-50 mg/L, a concentration of NH.sub.4.sup.+—N is 0.3-0.5 mg/L, and a concentration of NO.sub.3.sup.−—N is 8-12 mg/L.
Description
BRIEF DESCRIPTION OF FIGURES
(1)
(2)
DETAILED DESCRIPTION
(3) In order to more clearly understand the technical content of the present disclosure, the following examples are specifically described, and the purpose is to better understand the present disclosure rather than limiting the scope of the present disclosure.
Example 1
(4) Preservation culture of sulfur autotrophic denitrifying bacteria biofilm:
(5) The preservation temperature of the sulfur autotrophic denitrifying bacteria biofilm was set to −20° C., 4° C. and 20° C. About 90 sulfur autotrophic denitrifying bacteria biofilms in a pilot reaction device were taken out and averaged into three equal parts to be respectively placed in a 500 ml serum bottle containing 250 ml of a preservation medium (the serum bottle was prefilled with N.sub.2 to discharge O.sub.2 in the air). The preservation medium is the effluent from a secondary sedimentation tank of a sewage treatment plant, and has a COD concentration of 40-50 mg/L, an NH.sub.4.sup.+—N concentration of 0.3-0.5 mg/L, and an NO.sub.3.sup.−—N concentration of 8-12 mg/L. The serum bottles (3 parallel samples at each preservation temperature) were placed at −20° C., 4° C. and 20° C. respectively, and preserved in static shading.
(6) During the commissioning operation of a pilot system, the pH of the effluent fluctuated between 6.2 and 7.0. After stable operation, the pH of the effluent was generally at 6.4-6.8.
(7) Cell state test of preserved sulfur autotrophic denitrifying bacteria biofilm:
(8) (1) 100 ml of sulfur autotrophic denitrifying bacteria biofilm mixed liquid was taken, diluted to 1 L with a phosphate buffer of pH 6.6, and stirred on a magnetic stirrer for 5 min to break the biofilm into flocs and ensure uniform distribution;
(9) (2) after the sample passed through a hydrotalcite filter layer to remove the sulfate in a liquid phase, the floc sample was filtered through a nylon membrane with the pore size of 20 μm and centrifuged at 8000 rpm for 5 min;
(10) (3) the precipitate was placed in a 50 ml centrifuge tube and the sample was centrifuged at 8000 rpm for 5 min;
(11) (4) the centrifuged sample supernatant was pipetted with a pipette, about 0.1 ml of sample was left, and the cells were blow washed with a pre-cooled phosphate buffer (pH 7.8), centrifuged and washed twice;
(12) (5) the supernatant of the centrifuged sample was pipetted, and about 0.1 ml of sample was left and mixed with 0.3 ml of 10× Annexin V Binding Buffer; and
(13) (6) 0.5 μl of PI stain was added to a control FITC Annexin V group, 0.5 μl of FITC Annexin V was added to a control PI group, 0.5 μl of FITC Annexin V and 0.5 μl of PI were added to a test group, after uniformly mixing, incubation was performed at room temperature in the dark for 15 min, and then detection was performed by flow cytometry.
(14) The selection of filter pore size when preparing samples is especially important. If the pore size is too large, more biological flocs will be introduced, resulting in uneven staining and affecting the final result. If the pore size is too small, biological flocs cannot be effectively obtained.
(15) The cell state test results of the sulfur autotrophic denitrifying bacteria biofilm are shown in Table 1. The proportion of living cells of the sulfur autotrophic denitrifying bacteria biofilm in a biochemical reaction tank of the sewage treatment plant is high, indicating that the sewage treatment plant has a good operation effect. The sulfur autotrophic denitrifying bacteria biofilm preserved at −20° C., 4° C. and 20° C. was used for determining the cell state of the sulfur autotrophic denitrifying bacteria biofilm after being preserved for more than 100 d, the results are shown in Table 1. The proportion of living cells of the sulfur autotrophic denitrifying bacteria biofilm in the pilot reaction device was high, indicating that the sulfuric autotrophic denitrification effect of the pilot system was good. The content of living cells used for determining the sulfur autotrophic denitrifying bacteria biofilm preserved at 20° C. was the lowest, indicating that 20° C. was not suitable for preserving the sulfur autotrophic denitrifying bacteria biofilm. The proportion of living cells of the sulfur autotrophic denitrifying bacteria biofilm preserved at 4° C. was 47.1%, and the proportion of late apoptotic cells and dead cells was about 48.3%. The relatively high proportion of late apoptotic cells and dead cells indicates that 4° C. is also not suitable for preserving the sulfur autotrophic denitrifying bacteria biofilm. When the preservation temperature was −20° C., the proportion of living cells of the sulfur autotrophic denitrifying bacteria biofilm reached 60.8%, which was only 23.4% lower than the proportion of living cells of the sulfur autotrophic denitrifying bacteria biofilm in the pilot reaction device, and meanwhile, the proportion of dead cells was about 20.4%, which is close to the proportion of dead cells of the sulfur autotrophic denitrifying bacteria biofilm in the pilot reaction device. Therefore, it was preliminarily determined that −20° C. was the optimum temperature for preserving the sulfur autotrophic denitrifying bacteria biofilm.
(16) TABLE-US-00001 TABLE 1 Cell activity state of preserved sulfur autotrophic denitrifying bacteria biofilm (%) Sulfur autotrophic Early Late denitrifying bacteria Living apoptotic apoptotic biofilm cells cells cells Dead cells Pilot operation 79.4 ± 3.8 0.1 ± 0.1 0.1 ± 0.1 20.5 ± 1.5 system Preserved at −20° C. 60.8 ± 3.2 3.0 ± 0.2 15.8 ± 1.0 20.4 ± 1.7 Preserved at 4° C. 47.1 ± 3.0 4.6 ± 0.9 23.7 ± 1.8 24.6 ± 2.2 Preserved at 20° C. 36.3 ± 3.0 9.2 ± 1.2 30.4 ± 2.7 24.1 ± 0.8
Example 2 Conditions for Activity Recovery of Preserved Sulfur Autotrophic Denitrifying Bacteria Biofilm
(17) Sulfur autotrophic denitrifying bacteria biofilms from different serum bottles were inoculated into bioreactors (with an effective volume of 20.0 L and an effective height of 150 cm) for carrying out activity recovery on the sulfur autotrophic denitrifying bacteria biofilms. The sulfur autotrophic denitrifying bacteria biofilms preserved at −20° C., 4° C. and 20° C. were placed in R1, R2 and R3 respectively. Coarse sand and stones were laid at 20 cm of the bottom of the bioreactor to support the upper elemental sulfur particles, the upper part of the coarse sand and the stones were 100 cm of elemental sulfur particles, and the porosity was about 40%.
Example 3 Characteristics of Sulfur Autotrophic Denitrifying Bacteria Biofilm after Activity Recovery
(18) After 30 days of activity recovery, the characteristics of the sulfur autotrophic denitrifying bacteria biofilms in R1, R2 and R3 are shown in Table 2. After activity recovery of the sulfur autotrophic denitrifying bacteria biofilm, the nitrate nitrogen removal rate of the sulfur autotrophic denitrifying bacteria biofilm preserved at 4° C. and 20° C. is lower than that of the sulfur autotrophic denitrifying bacteria biofilm before preservation, and only the nitrate nitrogen removal rate of the sulfur autotrophic denitrifying bacteria biofilm preserved at −20° C. is the same as that before preservation. The total nitrogen removal rate of the sulfur autotrophic denitrifying bacteria biofilm at different preservation temperatures is all lower than that before preservation. After activity recovery, the total nitrogen removal rate of the sulfur autotrophic denitrifying bacteria biofilm preserved at 20° C. and −20° C. is relatively close to that of the sulfur autotrophic denitrifying bacteria biofilm before preservation, but the total nitrogen removal rate of the sulfur autotrophic denitrifying bacteria biofilm preserved at 4° C. is relatively low.
(19) Generally, the nitrate nitrogen removal rate and total nitrogen removal rate of the sulfur autotrophic denitrifying bacteria biofilm are 140 gNO.sub.3.sup.−-N/(m.sup.3.Math.h) and 125 gTN/(m.sup.3.Math.h), respectively. The time of the sulfur autotrophic denitrifying bacteria biofilm acclimated in the pilot operation system reaching the same nitrate nitrogen removal rate and total nitrogen removal rate are 65 d and 70 d, respectively. After activity recovery of the preserved sulfur autotrophic denitrifying bacteria biofilm, the time of the sulfur autotrophic denitrifying bacteria biofilm in R1 reaching the same nitrate nitrogen removal rate and total nitrogen removal rate were 12 d and 15 d, respectively, the time of the sulfur autotrophic denitrifying bacteria biofilm in R2 reaching the same nitrate nitrogen removal rate and total nitrogen removal rate were 21 d and 22 d, respectively, and the time of the sulfur autotrophic denitrifying bacteria biofilm in R3 reaching the same nitrate nitrogen removal rate and total nitrogen removal rate were 18 d and 20 d, respectively. It is indicated that the sulfur autotrophic denitrifying bacteria biofilms after activity recovery all have good denitrification effect, wherein the sulfur autotrophic denitrifying bacteria biofilm preserved at −20° C. has the shortest recovery time of microbial activity and −20° C. is suitable for preserving the sulfur autotrophic denitrifying bacteria biofilm.
(20) TABLE-US-00002 TABLE 2 Characteristics of sulfur autotrophic denitrifying bacteria biofilm after preservation and activity recovery Required time Required Nitrate (d) when nitrate time (d) nitrogen nitrogen when total removal Total removal rate is nitrogen rate nitrogen greater than removal rate gNO.sub.3.sup.−- removal rate 140 gNO.sub.3.sup.−- exceeds 125 N/(m.sup.3 .Math. h) gTN/(m.sup.3 .Math. h) N/(m.sup.3 .Math. h) gTN/(m.sup.3 .Math. h) Pilot 164.1 145.0 65 70 operation system After preservation of sulfur autotrophic denitrifying bacteria biofilm After 103.5 90.9 — — preservation at −20° C. After 75.5 66.4 — — preservation 4° C. After 93.9 65.9 — — preservation 20° C. After activity recovery of sulfur autotrophic denitrifying bacteria biofilm Sulfur 165.0 137.5 12 15 autotrophic denitrifying bacteria biofilm preserved at −20° C. Sulfur 144.5 125.1 21 22 autotrophic denitrifying bacteria biofilm preserved at 4° C. Sulfur 147.1 136.8 18 20 autotrophic denitrifying bacteria biofilm preserved at 20° C.
Example 4 Removal Efficiency of Sulfur Autotrophic Denitrifying Bacteria Biofilm to Pollutants after Activity Recovery
(21) After the activity recovery process, the nitrate nitrogen and total nitrogen removal rate of the sulfur autotrophic denitrifying bacteria biofilm at different preservation temperatures gradually increased (
Example 5 Correlation Between Sulfur Autotrophic Denitrifying Bacteria Biofilm Characteristics and Sludge Cell State after Activity Recovery
(22) After 30 days of activity recovery of the sulfur autotrophic denitrifying bacteria biofilm, the cell state of the sulfur autotrophic denitrifying bacteria biofilm was analyzed by flow cytometry, the results are shown in Table 3. The content of living cells in the sulfur autotrophic denitrifying bacteria biofilm at different preservation temperatures is basically the same as that in the sulfur autotrophic denitrifying bacteria biofilm in the pilot operation system, indicating that after activity recovery, the sulfur autotrophic denitrifying bacteria biofilm can achieve stable nitrate nitrogen and total nitrogen removal effects. Where, the proportion of living cells in the sulfur autotrophic denitrifying bacteria biofilm in R1 was the highest (79.5%±4.0%), and the proportion of late apoptotic cells (5.6%±1.0%) and dead cells (11.1%±1.1%) were the lowest, indicating that the sulfur autotrophic denitrifying bacteria biofilm has the highest activity under the preservation condition of −20° C., and the condition of −20° C. is suitable for preserving the sulfur autotrophic denitrifying bacteria biofilm.
(23) TABLE-US-00003 TABLE 3 Cell activity state of sulfur autotrophic denitrifying bacteria biofilm after activity recovery (%) Sulfur autotrophic Early Late denitrifying apoptotic apoptotic Dead bacteria biofilm Living cells cells cells cells Pilot operation 82.5 ± 4.1 2.5 ± 0.5 4.3 ± 0.8 10.7 ± 1.0 system Preserved at −20° C. 79.5 ± 4.0 3.8 ± 0.5 5.6 ± 1.0 11.1 ± 1.1 Preserved at 4° C. 75.0 ± 4.2 4.9 ± 0.5 7.2 ± 0.8 12.9 ± 1.2 Preserved at 20° C. 77.8 ± 4.0 5.0 ± 0.7 6.0 ± 0.8 11.2 ± 1.2
(24) According to Correl correlation analysis, as shown in Table 4, there is a high correlation between the nitrate nitrogen removal rate and total nitrogen removal rate of the sulfur autotrophic denitrifying bacteria biofilm and the proportion of living cells of the sulfur autotrophic denitrifying bacteria biofilm, and the correlation coefficients are respectively 0.9577 and 0.9450, indicating that use of the proportion of living cells of the sulfur autotrophic denitrifying bacteria biofilm as a method for evaluating the activity of the sulfur autotrophic denitrifying bacteria biofilm has high feasibility. Meanwhile, in the preserved sulfur autotrophic denitrifying bacteria biofilm, the proportion of living cells of the sulfur autotrophic denitrifying bacteria biofilm at the preservation condition of −20° C. is the highest, which is consistent with the results of the proportion of living cells of the sulfur autotrophic denitrifying bacteria biofilm in R1 after activity recovery.
(25) TABLE-US-00004 TABLE 4 Correlation between sulfur autotrophic denitrifying bacteria biofilm characteristics and cell activity state after activity recovery Sulfur Sulfur autotrophic Sulfur autotrophic denitrifying autotrophic denitrifying bacteria biofilm denitrifying bacteria biofilm preserved bacteria biofilm preserved at at −20° C. preserved at 4° C. 20° C. Nitrate nitrogen 165.0 144.5 147.1 removal rate gNO.sub.3.sup.−- N/(m.sup.3 .Math. h) Total nitrogen 137.5 125.1 136.8 removal rate gTN/(m.sup.3 .Math. h) Proportion of living 79.5 ± 4.0 75.0 ± 4.2 76.8 ± 4.0 cells (%) Correlation between 0.9577 nitrate nitrogen removal rate and proportion of living cells Correlation between 0.9450 total nitrogen removal rate and proportion of living cells
(26) Therefore, it is determined that −20° C. is the optimum temperature for preserving the sulfur autotrophic denitrifying bacteria biofilm. Flow cytometry can be used as a basis for determining the optimum preservation temperature of the sulfur autotrophic denitrifying bacteria biofilm. Flow cytometry is easy to operate and fast and easy to obtain accurate and reliable data, can omit the activity recovery process of the sulfur autotrophic denitrifying bacteria biofilm, and is of great significance for the preservation and activity recovery of the sulfur autotrophic denitrifying bacteria biofilm.
Comparative Example 1
(27) Preservation culture of sulfur autotrophic denitrifying bacteria biofilm:
(28) The preservation temperature of the sulfur autotrophic denitrifying bacteria biofilm was set to −20° C., 4° C. and 20° C. About 90 sulfur autotrophic denitrifying bacteria biofilms in the pilot reaction device were taken out and averaged into three equal parts to be respectively placed in a 500 ml serum bottle containing 250 ml of the preservation medium (the serum bottle was prefilled with N.sub.2 to discharge O.sub.2 in the air). The preservation medium is the effluent from a secondary sedimentation tank of a sewage treatment plant, and has a COD concentration of 40-50 mg/L, an NH.sub.4.sup.+—N concentration of 0.3-0.5 mg/L, and an NO.sub.3.sup.−—N concentration of 8-12 mg/L. The serum bottles (3 parallel samples at each preservation temperature) were placed at −20° C., 4° C. and 20° C. respectively, and preserved in static shading.
(29) Cell state test of preserved sulfur autotrophic denitrifying bacteria biofilm:
(30) (1) 100 ml of sulfur autotrophic denitrifying bacteria biofilm mixed liquid was taken, diluted to 1 L with a phosphate buffer of pH 6.6, and stirred on a magnetic stirrer for 5 min to break the biofilm into flocs and ensure uniform distribution;
(31) (2) the floc sample was filtered through a nylon membrane with the pore size of 20 μm and centrifuged at 8000 rpm for 5 min;
(32) (3) the precipitate was placed in a 50 ml centrifuge tube and the sample was centrifuged at 8000 rpm for 5 min;
(33) (4) the centrifuged sample supernatant was pipetted with a pipette, about 0.1 ml of sample was left, and the cells were blow washed with a pre-cooled phosphate buffer (pH 7.8), centrifuged and washed twice;
(34) (5) the supernatant of the centrifuged sample was pipetted, and about 0.1 ml of sample was left and mixed with 0.3 ml of 10× Annexin V Binding Buffer; and
(35) (6) 0.5 μl of PI stain was added to a control FITC Annexin V group, 0.5 μl of FITC Annexin V was added to a control PI group, 0.5 μl of FITC Annexin V and 0.5 μl of PI were added to a test group, after uniformly mixing, incubation was performed at room temperature in the dark for 15 min, and then detection was performed by flow cytometry.
(36) The cell state test results of the sulfur autotrophic denitrifying bacteria biofilm are shown in Table 5.
(37) TABLE-US-00005 TABLE 5 Cell activity state of preserved sulfur autotrophic denitrifying bacteria biofilm (without being adsorbed by hydrotalcite layer) Sulfur autotrophic Early Late denitrifying bacteria apoptotic apoptotic biofilm Living cells cells cells Dead cells Pilot operation 51.5% ± 4.9% 15.8% ± 1.9% 15.7% ± 1.8% 17.0% ± 1.9% system Preserved at −20° C. 58.8% ± 5.1% 13.5% ± 2.1% 13.7% ± 2.0% 14.0% ± 2.1% Preserved at 4° C. 45.7% ± 4.7% 18.0% ± 2.7% 18.5% ± 2.5% 17.8% ± 2.4% Preserved at 20° C. 43.3% ± 4.7% 19.0% ± 3.0% 19.2% ± 3.5% 18.5% ± 2.9%
(38) From the results of Table 5, it is found that the samples that have not been adsorbed by the hydrotalcite layer have similar changes in the test results. At different preservation temperatures, the sulfur autotrophic denitrifying bacteria biofilm preserved at −20° C. has a higher content of living cells. At the three preservation temperature conditions, the contents of the early apoptotic cells, the late apoptotic cells and the dead cells are very close. At the same time, in the pilot operation system with good denitrification effect, the proportion of living cells of the sulfur autotrophic denitrifying bacteria biofilm is only 51.5%±4.9%, which is lower than that of the sulfur autotrophic denitrifying bacteria biofilm preserved at −20° C. The above results indicate that if higher concentration of sulfate presents in the sample, the test results of the cell activity state are significantly affected, resulting in failure to determine the optimum preservation temperature.