Method for culturing short-cut nitrification sludge

Abstract

This invention relates to a method for cultivating short-cut nitrifying sludge, belonging to the field of wastewater treatment. The method comprises the following steps: first, a certain amount of simulated wastewater is introduced into an upflow reactor, and sludge is inoculated; then, the simulated wastewater in the upflow reactor is aerated; next, the sludge is allowed to settle; finally, the upper part of the water in the upflow reactor is pushed out of the reactor. This method for cultivating short-cut nitrifying sludge falls between continuous flow and intermittent flow processes, enabling rapid short-cut nitrification and promoting the formation of AOB-rich short-cut nitrifying sludge particles. The method is simple to operate, economical, and requires a very short time to achieve stable short-cut nitrification. The sludge remains stable, and the generated nitrite provides substrate for anaerobic ammonia-oxidizing bacteria, achieving autotrophic denitrification and further improving the denitrification effect.

Description

Technical Field

[0001] This invention relates to a method for cultivating short-cut nitrifying sludge, belonging to the field of wastewater treatment. Background Technology

[0002] The traditional nitrification-denitrification biological nitrogen removal process works as follows: Under aerobic conditions, ammonia nitrogen is first oxidized to nitrite by ammonia-oxidizing bacteria (AOB), and then nitrite is oxidized back to nitrate by nitrite-oxidizing bacteria (NOB). Afterwards, in the anoxic stage, denitrifying bacteria (DNB) use organic matter to first reduce nitrate to nitrite, and then reduce nitrite back to nitrogen gas, thus achieving nitrogen removal. However, the high aeration volume required for the oxidation process under aerobic conditions and the need for an external carbon source for the reduction process under anoxic conditions contradict current advocacy for resource conservation. Short-cut nitrification-denitrification and short-cut nitrification-anaerobic ammonia oxidation processes can save energy compared to traditional nitrification-denitrification, but the key is to first achieve short-cut nitrification to accumulate nitrite. Therefore, short-cut nitrification processes have been developed. Short-cut nitrification utilizes the survival differences between AOB and NOB to suppress the activity of NOB and control the nitrification reaction only at the stage of generating nitrite nitrogen. It can provide nitrite for short-cut nitrification-denitrification and anaerobic ammonium oxidation, thereby achieving efficient and energy-saving biological nitrogen removal.

[0003] Short-cut nitrification is often the result of the combined effects of multiple factors, including free ammonia (FA), free nitrite (FNA), pH, temperature, alternating anaerobic and aerobic operation, and micro-aeration. However, since both AOB and NOB are aerobic microorganisms, and their growth requires similar temperatures, pH, and dissolved oxygen, these conditions are difficult to control. AOB cultivation can only be achieved by leveraging the small survival differences between AOB and NOB, making rapid and effective AOB cultivation challenging. Summary of the Invention

[0004] The purpose of this invention is to provide a method for cultivating short-cut nitrification sludge, which solves the problem that AOB is difficult to cultivate quickly and effectively in current short-cut nitrification.

[0005] To achieve the above objectives, the technical solution adopted in the short-cut nitrification sludge cultivation method of the present invention is as follows:

[0006] A method for cultivating short-cut nitrification sludge includes the following steps:

[0007] (1) Inoculate sludge in an upflow reactor and introduce a certain amount of simulated wastewater; the sludge inoculated is aerobic sludge with nitrification and denitrification capabilities;

[0008] (2) Aerate the simulated wastewater in the upflow reactor. Stop aeration and allow the sludge to settle when the ammonia nitrogen concentration drops to the set value or reaches the set aeration time.

[0009] (3) After the sludge settles, simulated wastewater is introduced from the bottom of the upflow reactor to push the water in the upper part of the upflow reactor out of the upflow reactor; the simulated wastewater is introduced in a continuous upflow push flow manner, and the amount of water pushed out from the top of the upflow reactor is 1 / 4 to 1 / 6 of the total amount of water in the upflow reactor.

[0010] (4) Repeat steps (2) and (3);

[0011] The simulated wastewater contains ammonia nitrogen and bicarbonate; the concentration of nitrogen in the simulated wastewater is 400-500 mg / L, and the concentration of bicarbonate in the simulated wastewater is 4800-5900 mg / L, wherein the bicarbonate is sodium bicarbonate.

[0012] The short-cut nitrification sludge cultivation method of this invention is between continuous flow and intermittent flow processes, including three stages: aerobic aeration, sludge settling, and anaerobic influent / effluent. During the anaerobic influent / effluent stage, simulated wastewater is introduced from the bottom of the upflow reactor. The volume of water pushed out from the top of the upflow reactor is 1 / 4 to 1 / 6 of the total volume. With the continuous repetition of the aerobic aeration, anaerobic settling, and anaerobic influent / effluent stages, a large amount of nitrite produced by short-cut nitrification during the aerobic period accumulates in the system, accounting for up to nine-tenths of the total nitrogen, with the remainder being nitrate and ammonia nitrogen concentration extremely low. Subsequently, a perfect system is formed within the reactor. The ammonia nitrogen entering the reactor during the anaerobic stage undergoes short-cut nitrification after being completely mixed with the simulated wastewater from the previous aerobic stage during the initial aerobic phase, resulting in further short-cut nitrification. The short-cut nitrification process has shifted from inhibiting NOB with free ammonia produced by high ammonia nitrogen to inhibiting NOB with free nitrite produced by high nitrite. The short-cut nitrification sludge cultivation method of this invention is simple to operate, economical, and feasible. It requires no additional agents specifically added to promote short-cut nitrification, and the time required to achieve stable short-cut nitrification is extremely short, enabling rapid short-cut nitrification. Furthermore, it promotes the formation of AOB-rich short-cut nitrification sludge particles. The short-cut nitrification sludge maintains a stable state, and the generated nitrite provides substrate for anaerobic ammonia-oxidizing bacteria, achieving autotrophic denitrification and further improving the denitrification effect.

[0013] In the short-cut nitrification sludge cultivation method of the present invention, the raw water used in the aerobic aeration stage is a weakly alkaline simulated wastewater with a high ammonia nitrogen concentration. Ammonia nitrogen can participate in the short-cut nitrification reaction as a substrate required for AOB growth, and can also dissolve in water to generate free ammonia. Free ammonia within a certain concentration range will inhibit the activity of NOB, thereby accelerating the realization of short-cut nitrification. Short-cut nitrification is a biochemical reaction process that consumes alkalinity. Sufficient bicarbonate can provide enough alkalinity to cause ammonia oxidation of all ammonia nitrogen in the system. The sludge settling stage can ensure the effective retention of biomass. During the anaerobic influent and effluent stages, the activities of both AOB and NOB, as aerobic microorganisms, are inhibited. However, when the aerobic stage is repeated, the activity of AOB can recover quickly, but the activity of NOB recovers more slowly. Therefore, repeating the aerobic and anaerobic processes in steps (2) and (3) can eliminate NOB on the one hand and maintain the activity of AOB on the other hand, ultimately realizing the cultivation of short-cut nitrification sludge.

[0014] In this invention, sludge can be inoculated first in an upflow reactor, and then a certain amount of simulated wastewater can be introduced; alternatively, a certain amount of simulated wastewater can be introduced first in an upflow reactor, and then sludge can be inoculated; or sludge can be inoculated and a certain amount of simulated wastewater can be introduced simultaneously in an upflow reactor.

[0015] Preferably, in step (3), the amount of water pushed out from the top of the upflow reactor is 1 / 6 of the total amount of water in the upflow reactor.

[0016] Preferably, the molar ratio of ammonia nitrogen to bicarbonate in the simulated wastewater is 1:2. Theoretically, when the molar ratio of ammonia nitrogen to bicarbonate is 1:2, all ammonia nitrogen in the system can undergo ammonia oxidation. The biochemical reaction equation for ammonia nitrogen and bicarbonate is as follows: 55NH4 + +76O2+109HCO3 - →C5H7O2N+54NO2 - + 57H2O+104H2CO3.

[0017] Preferably, the simulated wastewater is prepared by mixing water, ammonia nitrogen, trace element solution, and sodium bicarbonate; the volume of trace element solution used per 1000L of simulated wastewater is 1L; the trace element solution includes solution A and solution B, with a volume ratio of 1:1; solution A is prepared by mixing water, EDTA, and FeSO4·7H2O, with each 1L of water corresponding to 5g of EDTA and 5g of FeSO4·7H2O; solution B is prepared by mixing water, EDTA, H3BO3, and... The simulated wastewater was prepared by mixing MnCl₂·4H₂O, CuSO₄·5H₂O, ZnSO₄·7H₂O, NiCl₂·6H₂O, Na₂MoO₄·2H₂O, and CoCl₂·6H₂O. The corresponding masses of EDTA, H₃BO₃, MnCl₂·4H₂O, CuSO₄·5H₂O, ZnSO₄·7H₂O, NiCl₂·6H₂O, Na₂MoO₄·2H₂O, and CoCl₂·6H₂O per 1 L of water were 15 g, 0.014 g, 0.99 g, 0.25 g, 0.43 g, 0.19 g, 0.22 g, and 0.24 g, respectively. The trace elements in the simulated wastewater primarily provide essential nutrients for microbial growth.

[0018] Preferably, after wastewater is introduced into the upflow reactor and sludge is inoculated, the concentration of sludge in the upflow reactor is 4000–5000 mg / L. When the sludge concentration in the upflow reactor is within the above range, the number of microorganisms in the system is relatively high. A relatively high microbial quantity is conducive to the rapid conversion of nutrients such as ammonia nitrogen, resulting in better biochemical performance of the system.

[0019] The inoculated sludge is mature aerobic granular sludge, i.e., granular sludge with good nitrification and denitrification performance. The short-cut nitrification sludge cultivation method of this invention is stable and effective. The high aeration rate in the aerobic stage helps maintain a stable granular state, and short-cut nitrification can still be stably achieved even under high aeration conditions. The granular sludge exhibits significant settling effect during the sludge settling stage, with extremely low sludge loss. In practical engineering applications, the use of a secondary settling tank is avoided, saving costs and reducing land area.

[0020] Preferably, the simulated wastewater also contains COD. Preferably, the COD concentration in the simulated wastewater is 80–120 mg / L. For example, the COD concentration in the simulated wastewater is 100 mg / L. The role of COD in the simulated wastewater is mainly to maintain the granulation of the sludge.

[0021] Preferably, the pH of the simulated wastewater is 7.5–8.0. For example, the pH of the simulated wastewater is 8. The alkaline compounds in the simulated wastewater are mainly carbonates. When the concentration of bicarbonate is 4800–5900 mg / L, the pH of the simulated wastewater is 7.5–8.0, which is conducive to the survival of AOBs.

[0022] Preferably, the volume of the upflow reactor is 2-3 L. For example, the volume of the upflow reactor is 2.4 L. The shape of the upflow reactor is preferably cylindrical, and when the shape of the upflow reactor is cylindrical, the inner diameter of the upflow reactor is preferably 5.3 cm.

[0023] Preferably, in step (3), the flow rate of the simulated wastewater introduced from the bottom of the upflow reactor is 0.4 to 0.8 L / h. For example, in step (3), the flow rate of the simulated wastewater introduced from the bottom of the upflow reactor is 0.40 L / h.

[0024] Preferably, when simulated wastewater is introduced from the bottom of the upflow reactor, the influent method is a continuous upflow plug flow. For example, simulated wastewater can be introduced from the bottom of the upflow reactor using a peristaltic pump. Using a peristaltic pump allows the influent and effluent of the reactor to occur simultaneously, ensuring that the influent volume equals the effluent volume. This also guarantees that the simulated wastewater remains within the upflow reactor, while only the water at the top of the reactor flows out, thus not affecting the effluent quality.

[0025] Preferably, in step (2), during aeration, the aeration rate per liter of wastewater in the reactor is 500-600 mL / min, and the aeration time is 1 h. When the aeration rate and aeration time are within the above range, a strong gas shear force can be generated, which can maintain the strength, density, stability, and activity of sludge particles at a good level. At the same time, it stimulates the microorganisms in the sludge particles to secrete extracellular polymers (EPS), which then adhere to the surrounding flocs and promote the formation of granular sludge.

[0026] Preferably, the sludge settling time is 5 to 10 minutes. For example, the sludge settling time is 5 minutes. When the sludge settling time is within the above range, sufficient settling time can be given to sludge particles and sludge flocs, ensuring effective retention of biomass.

[0027] Preferably, the time for step (3) is 50 to 55 minutes. For example, the time for step (3) is 55 minutes. Step (3) is the anaerobic influent and effluent stage. When the time for step (3) is within the above range, the activities of both AOB and NOB, as aerobic microorganisms, are inhibited. However, when the aerobic stage is passed again, the activity of AOB can be quickly restored, but the activity of NOB is restored more slowly.

[0028] Preferably, in step (4), when repeating steps (2) and (3), the amount of simulated wastewater entering and the amount of water pushed out from the top of the upflow reactor in step (3) are both 1 / 4 to 1 / 6 of the total amount of water in the upflow reactor.

[0029] More preferably, in step (4), when repeating steps (2) and (3), in step (3), the amount of simulated wastewater entering and the amount of water pushed out from the top of the upflow reactor are both 1 / 6 of the total amount of water in the upflow reactor.

[0030] Preferably, in step (4), steps (2) to (3) are repeated for 30 consecutive days until the ratio of the mass concentration of nitrite in the water pushed out from the top of the upflow reactor in step (3) to the sum of the mass concentrations of nitrite and nitrate in the water pushed out from the top of the upflow reactor is not less than 80%. After the culture is completed, short-cut nitrifying sludge is obtained. The obtained short-cut nitrifying sludge can be taken out and used to oxidize ammonia nitrogen to nitrite nitrogen, and then the nitrite nitrogen can be converted into nitrogen gas by denitrifying bacteria to complete denitrification. Alternatively, on the basis of the original reactor, wastewater containing ammonia nitrogen can continue to be introduced into the reactor to oxidize ammonia nitrogen to nitrite nitrogen, and then the wastewater rich in nitrite nitrogen can be denitrified by the action of denitrifying bacteria. Attached Figure Description

[0031] Figure 1 The diagram shows the upflow reactor used in the short-cut nitrification sludge cultivation method of Embodiment 1 of the present invention. The reference numerals are as follows: 1-reactor body, 2-inlet, 3-outlet, 4-sampling port;

[0032] Figure 2 This is a schematic diagram illustrating the stages of the short-cut nitrification sludge cultivation method according to Example 1 of the present invention, wherein... Figure 2 (a) is the aerobic aeration stage. Figure 2 (b) is the anaerobic sedimentation stage. Figure 2 (c) is the anaerobic influent / effluent stage; Figure 2 The reference numerals in (a) are as follows: 11-simulated wastewater, 22-sludge particles, 33-water surface at the outlet;

[0033] Figure 3 This is a schematic diagram showing the nitrite accumulation rate obtained from daily sampling and testing during days 0 to 61 in the short-cut nitrification sludge cultivation method of the embodiment.

[0034] Figure 4 This is a schematic diagram showing the test results of the short-range nitrification performance of sludge particles in the experimental example;

[0035] Figure 5 This is a schematic diagram of the microbial composition at the genus level in granular sludge before and after cultivation. Detailed Implementation

[0036] The technical solution of the present invention will be further described below with reference to specific embodiments.

[0037] The method for calculating the nitrite accumulation rate in this embodiment of the invention is as follows:

[0038] Nitrite accumulation rate (%) = Mass concentration of nitrite in wastewater at the outlet of the upflow reactor / Sum of mass concentrations of nitrite and nitrate in wastewater at the outlet of the upflow reactor.

[0039] Example

[0040] The short-path nitrification sludge cultivation method in this embodiment uses an upflow reactor, such as... Figure 1 As shown, it includes a reactor body 1 and an outlet 3 and a sampling port 4 disposed on the side wall of the reactor body 1; the reactor body 1 is a tubular structure with the bottom end sealed by an end cap, the end cap having an inlet 2 and an opening at the top, the inner diameter of the upflow reactor is 5.3 cm, and the effective volume is 2.4 L.

[0041] The method for cultivating short-cut nitrifying sludge in this embodiment specifically includes the following steps:

[0042] (1) Inoculate an aerobic sludge with nitrification and denitrification capabilities into an upflow reactor, and then introduce simulated wastewater. The volume of the aerobic sludge and simulated wastewater is 2.4L.

[0043] In the simulated wastewater, the concentration of nitrogen in ammonia nitrogen was 500 mg / L, the concentration of sodium bicarbonate was 5900 mg / L, the concentration of COD was 100 mg / L, and the pH of the simulated wastewater was 8.0. The concentration of sludge inoculated in the upflow reactor was 4000 mg / L. The simulated wastewater was mainly prepared by mixing water, ammonia nitrogen, trace element solution, and sodium bicarbonate. The volume of trace element solution used for every 1000 L of simulated wastewater was 1 L. The trace element solution included solution A and solution B, with a volume ratio of 1:1. Solution A was prepared by mixing water, EDTA, and FeSO4·7H2O, with 5 g of EDTA and 5 g of FeSO4·7H2O used for every 1 L of water. Solution B is prepared by mixing water, EDTA, H3BO3, MnCl2·4H2O, CuSO4·5H2O, ZnSO4·7H2O, NiCl2·6H2O, Na2MoO4·2H2O, and CoCl2·6H2O. The masses of EDTA, H3BO3, MnCl2·4H2O, CuSO4·5H2O, ZnSO4·7H2O, NiCl2·6H2O, Na2MoO4·2H2O, and CoCl2·6H2O used per 1L of water are 15g, 0.014g, 0.99g, 0.25g, 0.43g, 0.19g, 0.22g, and 0.24g, respectively.

[0044] (2) The simulated wastewater in the upflow reactor was aerated for 1 hour at a flow rate of 600 mL / min. After aeration, the sludge particles were allowed to settle for 5 minutes, until all the sludge particles had settled to the bottom of the upflow reactor. Schematic diagrams of the aeration and settling stages are shown below. Figure 2 As shown in a and 2b. During the aeration stage, the sludge particles 22 are suspended in the simulated wastewater 11 under the action of gas shear force. During the settling stage, the liquid level 33 of the simulated wastewater in the reactor is located below the outlet 3 at the upper end of the reactor.

[0045] (3) Then, a peristaltic pump is used to introduce simulated wastewater (flow rate of 0.40 L / h) from the bottom of the upflow reactor, pushing the water from the top of the upflow reactor out of the reactor. The amount of water pushed out from the top of the upflow reactor is 1 / 6 of the total water volume in the upflow reactor. A schematic diagram of the anaerobic influent and effluent stages is shown below. Figure 2 As shown in c.

[0046] (4) Then repeat steps (2) and (3) continuously for 24 hours a day, taking samples from the water pushed out from the top of the upflow reactor every other day and testing the nitrite accumulation rate. The test results are as follows: Figure 3 As shown in Figure 3, the nitrite accumulation rate can reach up to 100%, and from day 3 to day 61, except for days 9 and 11, the nitrite accumulation rate is maintained above 80%, indicating that the short-cut nitrification performance can be maintained stably.

[0047] Experimental Example 1

[0048] After the short-cut nitrification sludge cultivation method described in the above embodiment is completed, 30 mL of wet sludge particles are taken from the bottom of the upflow reactor, washed, and placed in a serum bottle containing 400 mL of simulated wastewater for batch experiments. The batch experiments last for two hours, with high aeration throughout (aeration rate of 200 mL / min). During the experiment, water samples are taken every ten minutes, and then the water quality is tested. The concentrations of nitrogen in ammonia nitrogen, nitrogen in nitrite, and nitrogen in nitrate in the water samples are measured, and the short-cut nitrification performance of the sludge particles is judged by the concentration of nitrogen in nitrite. The simulated wastewater is prepared by mixing water, ammonia nitrogen, trace element solution, and sodium bicarbonate. The concentration of nitrogen in ammonia nitrogen in the simulated wastewater is 50 mg / L, and the concentration of sodium bicarbonate is 240 mg / L. The volume of trace element solution used is 1 L per 1000 L of simulated wastewater. The trace element solution includes solution A and solution B, with a volume ratio of 1:1. Solution A is prepared by mixing water, EDTA, and FeSO4·7H2O, with each 1L of water corresponding to 5g of EDTA and 5g of FeSO4·7H2O. Solution B is prepared by mixing water, EDTA, H3BO3, MnCl2·4H2O, CuSO4·5H2O, ZnSO4·7H2O, NiCl2·6H2O, Na2MoO4·2H2O, and CoCl2·6H2O. The masses of EDTA, H3BO3, MnCl2·4H2O, CuSO4·5H2O, ZnSO4·7H2O, NiCl2·6H2O, Na2MoO4·2H2O, and CoCl2·6H2O used per 1L of water are 15g, 0.014g, 0.99g, 0.25g, 0.43g, 0.19g, 0.22g, and 0.24g, respectively.

[0049] Experimental results are as follows Figure 4 As shown in the figure, the results indicate that the vast majority of the oxidized ammonia nitrogen is converted into nitrite nitrogen, while only a small portion of the ammonia nitrogen undergoes complete nitration to produce nitrate nitrogen.

[0050] Experiment Example 2

[0051] Microbial diversity analysis was performed on the granular sludge obtained after the cultivation methods of the aerobic sludge inoculated in the upflow reactor in the examples and the short-cut nitrification sludge in the examples. The microbial composition and abundance at the genus level in the granular sludge before and after cultivation are shown in Table 1 and 2. Figure 5 As shown.

[0052] Table 1. Microbial types and abundance in granular sludge before and after culture.

[0053]

[0054]

[0055]

[0056] From Table 1 and Figure 5 It can be seen that the abundance of Nitrosomonas before cultivation was 0.01% and the abundance after cultivation was 8.01%, indicating that the cultivation method of short-cut nitrification sludge in the embodiment is effective.

Claims

1. A method for cultivating short-cut nitrification sludge, characterized in that, Includes the following steps: (1) Inoculate sludge in an upflow reactor and introduce a certain amount of simulated wastewater; the sludge inoculated is aerobic sludge with nitrification and denitrification capabilities; (2) Aerate the simulated wastewater in the upflow reactor. Stop aeration when the ammonia nitrogen concentration drops to the set value or the set aeration time is reached, and let the sludge settle. (3) Anaerobic influent and effluent: After the sludge settles, simulated wastewater is introduced from the bottom of the upflow reactor, and the water in the upper part of the upflow reactor is pushed out of the upflow reactor. The simulated wastewater is introduced in a continuous upflow plug flow manner, and the amount of water pushed out from the top of the upflow reactor is 1 / 4 to 1 / 6 of the total amount of water in the upflow reactor. (4) Repeat steps (2) and (3); The simulated wastewater contains ammonia nitrogen and bicarbonate; the concentration of nitrogen in the simulated wastewater is 400~500 mg / L, and the concentration of bicarbonate in the simulated wastewater is 4800~5900 mg / L, wherein the bicarbonate is sodium bicarbonate. In step (1), after sludge is inoculated into the upflow reactor and wastewater is introduced, the concentration of sludge in the upflow reactor is 4000~5000 mg / L.

2. The method for cultivating short-cut nitrifying sludge as described in claim 1, characterized in that, The simulated wastewater also contains COD; the concentration of COD in the simulated wastewater is 80~120 mg / L.

3. The method for cultivating short-cut nitrifying sludge as described in any one of claims 1-2, characterized in that, The pH of the simulated wastewater is 7.5~8.

0.

4. The method for cultivating short-cut nitrifying sludge as described in any one of claims 1-2, characterized in that, The volume of the upflow reactor is 2-3L; the inner diameter of the upflow reactor is 5.3cm.

5. The method for cultivating short-cut nitrifying sludge as described in claim 4, characterized in that, In step (3), the flow rate of simulated wastewater introduced from the bottom of the upflow reactor is 0.4~0.8L / h.

6. The method for cultivating short-cut nitrifying sludge as described in claim 4, characterized in that, In step (2), during aeration, the aeration rate of each liter of wastewater in the reactor is 500~600 mL / min, and the aeration time is 1 h.

7. The method for cultivating short-cut nitrifying sludge as described in any one of claims 1-2, characterized in that, In step (4), when repeating steps (2) and (3), in step (3), the amount of simulated wastewater entering and the amount of water pushed out from the top of the upflow reactor are both 1 / 4 to 1 / 6 of the total amount of water in the upflow reactor.

8. The method for cultivating short-cut nitrifying sludge as described in any one of claims 1-2, characterized in that, In step (4), when repeating steps (2) and (3), in step (3), the amount of simulated wastewater entering and the amount of water pushed out from the top of the upflow reactor are both 1 / 6 of the total amount of water in the upflow reactor.

9. The method for cultivating short-cut nitrifying sludge as described in any one of claims 1-2, characterized in that, In step (4), repeat steps (2) to (3) until the ratio of the mass concentration of nitrite in the water pushed out from the top of the upflow reactor in step (3) to the sum of the mass concentrations of nitrite and nitrate in the water pushed out from the top of the upflow reactor is not less than 80% for 30 consecutive days, and the cultivation is completed.

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